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Vol. 70, No. 1, January 1990

Printed in US.A.

Role of Amino Acid Transport and Countertransport

in Nutrition and Metabolism
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan

I. PresentationoftheProblem ................................................................ 43
A. Molecular recognition within a class of analogues .......................................... 43
B. Specificity of amino acid transport across membranes is often but not always weak .......... 44
C. Summary picture of sharing of plasma membrane transport systems ....................... 44
D. Interaction among transport systems: consequences for distribution and flows of various
aminoacids ......................................................................... 47
E. Changes in amino acid transport in neoplastic cells need to be evaluated, understanding that
system L may serve for exodus more than for uptake .................................. 50
F. Brief examination of place of dispensable amino acids ..................................... 51
G. Recent observations on regulation of system A ............................................ 52
II. Selective Compositions of Amino Acid Flows Among Tissues: Factors Determining These Flows . 52
A. Epithelial brush borders versus plasma membrane: development of polarization of an
epithelium .......................................................................... 52
B. General considerations of makeup of amino acid flows ..................................... 55
C. Alanine and amino acid dispensability: conditional essentiality ............................. 55
D. Principalglutamineflows ................................................................ 56
E. Lysosomal proteolysis and lysosomal amino acid transport ................................. 61
F. Amino acid transport and protective function of glutathione ............................... 63
G. A redox cycle is somewhat analogously dependent on membrane transport .................. 66
H. Amino acid flows to and from the brain ................................................... 66
I. Dietary and other environmental influences on brain nutrition by amino acids ............... 69
J. Flows of amino acids across placental barrier between maternal and fetal organism ......... 70
III. Question of Attenuation of Transcription as a Basis for Regulation of Amino Acid Transport
AcrossBiomembranes .................................................................. 71
IV. Summary Statement ........................................................................ 71

I. PRESENTATION OF THE PROBLEM one point or another achieved sufficient detail of molec-
ular recognition to handle each amino acid with re-
markable discrimination from other amino acids. We
A. Molecular Recognition Within a Class of Analogues tend to take such delight in the finest catalytic discrim-
inations between pairs of analogues that we may tend
The amino acids represent our largest group of mu- to overlook that occasional enzymes, as well as trans-
tually analogous nutrients. Correspondingly wide is porters, discriminate only weakly among analogues,
their representation among metabolites and body com- scarcely more strongly, I suppose, than they need to,
ponents. The subtle gradations in molecular recognition within reasonable environmental limits. The degree of
that the analogous amino acid residues allow to be built overall economy achieved in the use of common and
into protein molecules are well appreciated. At the overlapping catalytic pathways must have been ad-
same time, the structural analogies within such a group justed during the evolutionary development of complex
undoubtedly contributed to evolutionary economies and functions to correspond to the statistical width of varia-
continue to serve as a basis for economy in the number tion in the pattern of analogues (here, of amino acids)
of catalytic steps needed to synthesize, degrade, and fit apt to be received from the environment and the conse-
the members together into macro- and micromolecular quences of wrong selections. If we consider then the
machinery, so as to serve in a regulated way the overall trade-offs among risks and economies inherent in evo-
biological needs of the organism. That economy has lutionary change, and also past and ongoing changes in
been achieved at a risk of competition among these ana- the environment and in our dietary preferences within
logues, a risk that we suppose has set limits on the the environment, it is not strange that the risks we
success of the evolutionary pursuit of that economy. encounter of imbalance among the proportions in which
As we survey amino acid metabolism, we can analogous nutrients (here, the amino acids) are con-
scarcely fail to conclude that the living organism has at sumed, although real, are generally not overwhelming,

0031-9333/90 $1.50 Copyright 0 1990 the American Physiological Society 43


at least until persons come to apply self-medication C. Summary Picture of Sharing of Plasma
with single amino acids. Membrane Transport Systems

Some of the oldest literature on transport of amino

B. SpeciJicity of Amino Acid Transport Across acids and sugars across epithelia spoke of a single
Membranes Is Often but Not Always Weak transport function for each of these classes. Various
dipolar amino acids were found to inhibit the transport
Initial studies of microorganisms and of subcellu- of each other, as though all might share the same route.
lar organelles showed that for catalysis of transport Gradually it became generally appreciated through
across membranes, the specificity of recognition of quantitative study, however, that several parallel sys-
substrates can on occasion be approximately as high as tems were shared and participated concurrently. In an
that for enzymatic catalysis. For the tissues of the odd contrast, transport systems specific to a single
higher animal, however, the catalysis of transport amino acid tended at first to be considered typical for
across biomembranes may show remarkably low speci- microorganisms. An early biochemical bias felt that
ficity in selection among analogues. Is it not astonish- membrane transport of each nutrient should be pro-
ing that a transporter would prefer L-methionine only duced by an adaptation of a special enzyme. Even when
fourfold over its D-antipode (129) or that an elevation of enzymes associated or coupled with the transport activ-
ambient phenylalanine could interfere with the inflow ity were identified, it tended to be overlooked that the
of leucine or even the outflow of glycine across a bio- transport step and the associated enzyme reaction often
membrane (31, 32)? The same membrane showing this appeared to be distinct catalytic events. The parallel
behavior might catalyze by another route the quite spe- lesson that a given metabolite may be transported si-
cific transport of another amino acid or perhaps be multaneously by two or three or perhaps even more
locked into transmembrane exchange for two others. independent transport systems was also accepted
slowly. In each of these systems a given amino acid
Two separate components of Na+-dependent D-ala-
shares its transport with a different group of amino
nine transport have been reported for the straight and
acids. I dealt elsewhere (34) with the inherent complex-
the convoluted section of the rabbit proximal renal tu-
ity encountered in codifying the various amino acids as
bule (92). Both of these components are inhibited by to which transport systems catalyze which movements,
L-alanine and are probably the ordinary systems serv- which systems tend to exchange which amino acids
ing for L-amino acids, although their relation to the
across the membrane, and which amino acids compete
Na+-dependent transport of other amino acids has not with which amino acids for catalysis of that movement.
yet been determined. The basis for the selectivity by the The attack on these questions by finding and counting
general transport system Xio serving for both aspar- apparent rectangular hyperbolas in plots of transport
tate (Asp-) and glutamate (Glu-), being minimal be- flux against substrate concentration proved in time to
tween the stereoisomers of Asp- but showing unexcep- be inadequate. Therefore that approach has had to be
tional selectivity in the case of L-Glu-, can be accounted supplemented with other methods, one being the wide
for by steric considerations (43). exploration of the competitive inhibition by analogues
I take my first responsibility to be to examine the under wide ranges of conditions, with these analogues
consequences for biological regulation of weak trans- specifically selected or designed to challenge the ini-
port specificity where it is weak. Can we understand at tially assumed homogeneity of the route of transport.
the present time how an often weak substrate specific- To examine how transport systems interact, how-
ity for membrane transport may be sufficient to mini- ever, we need first only to see in summary what a few of
mize the risks that might accompany an imprecision of the parallel transport systems are like. On that basis we
the regulation of metabolic flows among the amino hope to come to understand how the economy presum-
acids? What environmental change may bring forth ably inherent in the sharing of one or more systems by
risks inherent in a low-specificity transport step? as many as a dozen analogous amino acids can be at-
Beyond these two questions lies the central problem of tained without sacrifice of the needed biological regula-
this review: To what degree does membrane transport tion of the flow of each. In an earlier review in this
on the one hand and the rates of metabolic reactions on journal, I (29) treat the concept that flows (homeorhy-
the other hand figure in the modulation of interorgan sis) rather than concentrations (homeostasis, narrowly
amino acid flows? In considering the converse of this conceived) may be the more important target of such
question, one encounters expositions of metabolic regu- regulation. The literature pertinent to interorgan
lation, as a worthy example, that of nutritional sparing amino acid flows has grown considerably in the inter-
of methionine by cystine in the mammal in which by vening years, but I do not take my present assignment
chance no membrane transport step seems to be consid- to be simply its updating. Rather I should examine the
ered. How often will such explanations prove complete? still somewhat dubious proposition that a modest num-
Adaptive and endocrine regulatory influences on var- ber of irregularly shared transport systems, plus occa-
ious amino acid transport systems were reviewed by sional rather specific ones, along with the pertinent
Christensen and Kilberg in 1987 (40). metabolic reactions can lead to sufficiently regulated

flows. Were it not so, such an arrangement would not inherent in the inorganic ion gradients, incidentally
have survived, and we would scarcely expect to see the perhaps including those of K+, as for system A (14a,
various adaptive and hormonal modulations of trans- 64a), despite occasional dismissal of K+ gradients as
membrane flows (40). Thus the question I would like factors in transport energization. Because of that access
most to answer but probably cannot yet is: How is suf- these systems are capable of generating steep gradients
ficient regulation attained under the real arrange- of various amino acids in favor of the cytoplasmic rela-
ments? tive to the external environment. One of these, system
Table 1 lists 13 membrane transport systems but A, operates steeply uphill and hence shows weak revers-
refers to -10 other systems for comparisons. (A reader ibility. It is the target of endocrine regulation and also
who feels the several systems represent together a con- of regulation as an adaptation to cellular starvation
tinuum hardly worth discriminating may also feel that in its amino acid substrates, as reviewed by Guidotti
the amino acids themselves also represent a continuum, et al. (67).
although hardly a parallel one. If what we call systems System ASC has a narrower range than system A
A and L should each prove to be a continuum of similar as to the length of the amino acid side chains it finds
systems, then the major interruptions of such continua acceptable. A p-, y-, or b-hydroxyl or sulfhydryl group
remain to us fascinating features.) In this section, how- intensifies the cosubstrate action of Na+. In contrast to
ever, I focus on the reactivity of some 17 amino acids system A, system ASC regularly shows trans-stimula-
with three rather ubiquitous transport systems, A, tion. By trans.stimulation I refer to an acceleration of
ASC, and L. Two of these systems, A and ASC, are Na+ the initial rate of uptake of an amino acid substrate by
dependent, which gives them access to the energy stores preloading with such a substrate or its analogue or,

TABLE 1. Summary of so3ne known amino acid transport systems in tissues and cells of higher animals

Systems for Dipolar Amino Acids Somewhat Analogous Systems

Na+-dependent systems

GlY Widespread; Gly and Sar; variants known Imino, iminoglycine, and epithelial systems
Tolerance of
A Ubiquitous, serves for most dipolar amino
N-Me group
acids; often repressed; variants known I
ASC Ubiquitous, some variation in scope; excludes N-Me amino acids, but Na+ independent asc system in various
often includes prolines; on protonation accepts analogous anionic erythrocytes has somewhat analogous scope
amino acids; may accept Arg without Na+
B o,+ Wide-scope system, oocytes, blastocysts, probably renal and intestinal bs+, a Na+-independent analogue more sharply
brush borders; accepts cationic and bicyclic amino acids despite limited by positions of branching, in
their bulk preimplantation mouse blastocyts
N Gln, Asn, His; term so far restricted to hepatocyte N, a Gln transporter of muscle
P-System ,&Ala, Tau, 4-aminobutyrate System specific to 4-aminobutyrate

Na+-independent systems
L Ubiquitous; tolerates bulky and branched chains; high exchange Separate systems Ll, etc., low K, analogue
property; bicyclic amino acids as model substrates develops on incubating of hepatocytes
T Erythrocytes, hepatocyte; favors benzenoid amino acids System t in fibroblast lysosome

Na+-Independent Systems for Cationic Amino Acids

(systems specific each to Arg and Orn known)* Somewhat Analogous Systems

Y+ Ubiquitous; fails to discriminate between Arg and Lys and homologues Variants, as in lysosomes and mouse blastocysts,
do not accept (homoser + Na+)

Systems for Anionic Amino Acids (capital X where Na+ dependent)

Ubiquitous, includes neurons; Na dependent; similarly reactive with Asp- and Glu-
Corrected designation ASC, operating in its protonated form; largely excludes Glu- and longer analogues
Glu- and analogues, largely excluding Asp- and short analogues; Na+ independent
Like xo, except cystine competes and exchanges with Glu-; at least in some cases, locked into exchange; explains some CNS
Glu- binding

Note that analogous systems have led to similarities in designations, e.g., ASC and asc, Bv+ and bs+, L and Ll, also N and N. These
similarities should not be taken as decisions for identify or that one system is somehow the precursor to the other. Glu-, glutamate, whereas
usual abbreviation Glu does not specify whether glutamic acid or glutamate is meant. *For convention used here for discriminating Na+-de-
pendent from Na+-independent systems by capitalization, see Ref. 7a.






Inn1 ..
h&Ale AIB Pro Al0 Cys Thr Asn Gin Ser Met Phe ku Ile VOI His BCH

FIG. 1. A provisional division among 3 mediating systems for uptake of dipolar amino acids by CHO cell. NS, Na+-independent component
of entry that escaped inhibition by 10 mM BCH. See text for other details. *Statistically insignificant values. [From Shotwell et al. (1529.1

conversely, acceleration of its exodus by a high external regards: 1) in according system ASC its appropriate
level of such substrates. System A shows this phenome- relative place in relation to the usually repressed sys-
non only weakly and only for substrates of special tem A, which is much more conspicuous in neoplastic
structures (28). The transmembrane exchange of sub- cell lines and Z) in reemphasizing the often overlooked
strates characteristic of this phenomenon generally large overlap in the partition of transport of the various
does not, however, deplete the energy inherent in its dipolar amino acids between system L, on the one hand,
transmembrane gradients. As for system A, system and the Na+-dependent systems A and ASC, on the
ASC can generate steep amino acid gradients. The other hand. In view of charts of this sort, one cannot
quantitative role of system A relative to system ASC in remain satisfied to hear leucine or phenylalanine iden-
the in vivo context tended to be exaggerated at first tified or used as specific system L substrates or glu-
when early studies were made on neoplastic cells (e.g., tamine, threonine, or even alanine (with a larger sys-
the Ehrlich cell) or in isolated cells subjected to amino tem L component in some other cells) arbitrarily iden-
acid deprivation. In both of these cases, system A activ- tified as transported specifically by the Na+-dependent
ity is unusually high, whereas it tends to be somewhat routes.
repressed under physiological conditions. We should appreciate that the partition of influx
Figure 1 shows the partition of amino acid move- among the three mediated routes included in Figure 1
ments when presented one at a time into the Chinese for the CHO cell applies closely only for the rather
hamster ovary (CHO) cell among systems A, ASC, and well-selected concentration of 0.2 mM used for each
L, plus the unassigned nonsaturable component (159, amino acid. A companion chart for the partition ob-
and momentarily ignoring other known and unknown tained when each amino acid is tested at a concentra-
routes. These routes have been discriminated by noting tion of an order of magnitude lower should add to the
the effects of Na+ replacement by choline and by toler- instructive value of Figure 1. To warn ourselves of fur-
ance to competitive inhibition by either of two selective ther inherent complexities, we might also show for com-
analogues, a-(methylamino)isobutyric acid (MeAIB) or parison the entry rates for each amino acid presented in
Z-( -)-endoamino-bicycloheptane-Z-carboxylic acid turn in labeled form when all of the ordinary amino
(BCH). The procedure is likely to have assigned to sys- acids are presented together with it, each at a concen-
tem A glycine uptake by system Gly, cystine uptake tration characteristic of the blood plasma in the post-
assigned to system L by the then unknown exchange for absorptive state.
cellular glutamate (system XL), and to have left unas- Some of the more specific amino acid transport
signed histidine uptake as a cation. Nevertheless the systems, for example, those that select for amino acids
scope of the chart in confirming partitions observed of net charge, offer rather more obvious possibilities for
earlier has been highly influential, particularly in two specific biological regulation than do those illustrated

in Figure 1. In parallel, models have been proposed as to ways are known in which the charge differences can be
how an ion flow, e.g., of H+, drives the asymmetric ex- rectified.
change of Asp- for Glu- across the mitochondrial I) Certain dipolar amino acids show a Na+-depen-
membrane [for example, see Dierks et al. (55)]. In later dent transport by a system y+ that ordinarily carries
sections of this review, we encounter comparable speci- out Na+-independent transport and transmembrane ex-
ficities of transport interaction. change of cationic (tripolar) amino acids, e.g., lysine
On the other hand, we can sense how degrees of and arginine. In this case the structure of the dipolar
coordination are achieved in the transmembrane flows amino acid allows Na+ to serve as a surrogate for the
of groups of amino acids where these are transported distal cationic group of such an amino acid as arginine.
and exchanged (countertransported) by a single system. It appears that the terminal hydroxyl group of homo-
For example, the group of amino acids that can be ex- serine or the carboxamido group of glutamine, as exam-
changed across membranes by system L represents a ples, allows a bonding between Na+ and the dipolar
wide range of amino acids, wider than is ordinarily amino acid to allow a tripolar binding at the transport
supposed, and that concerns of course both their influx site. The possibility has been suggested that such amino
and efflux. Exchange within this system, among others, acids provided in excess can facilitate by exchange renal
allows the energy inherent in transmembrane gra- tubular resorption of cystine by a transport route re-
tained in cystinuria (33). A somewhat reciprocal Na+-
dients to be divided algebraically among the really nu-
independent reaction of arginine takes place with Na+-
merous members of the group, reaching all the way
dependent system AX, although in this case the cat-
from the very high affinity of phenylalanine and leu- ionic amino acids are not necessarily transported (166).
tine, to the quite low affinities of, for example, glycine, 2) At least one transport mediator for dipolar
serine, and even lysine, which is of course not 100% amino acids (AK) can on protonation also react with
cationic. Transport system A shows a weak trans-stim- anionic amino acids with otherwise analogous struc-
ulation of transport but may be made to show instead ture (108).
trans-inhibition by a change in structure of the amino I exclude from this list cases where the presumably
acid involved (Fig. 2). This phenomenon probably needs zwitterionic amino acid, cystine, is able to interact as an
also to be understood if an appreciation of a coordinated anion with glutamate transport (sect. IIF~) or presum-
regulation of amino acid flows is ultimately to be ably as a cation with lysine transport (see Ref. 29a).
reached. There are, however, further cases where dipolar and
The physiological role of transmembrane exchange cationic amino acids appear to interact with a common
of amino acids appears not to be limited to pairs of transport system in which no basis is yet established
molecules with the same electrical charge. Two special for them to assume an analogous distribution of
charged groups. These cases include the competition be-
tween alanine and cationic amino acids for transport
and also the stimulation of the efflux of alanine by
cationic amino acids via system asc in horse erythro-
cytes (59a) and the depletion of cellular arginine and
lysine from mouse blastocysts concurrent with methio-
nine accumulation (174). These flows are Na+ indepen-
dent; hence that cation seems unlikely to serve to bal-
ance the charge discrepancy. Therefore for the present
we cannot exclude the possible existence of transport
systems that ignore net charge differences in allowing
pairs of otherwise analogous substrates to interact. The
suspicion that system asc of the horse erythrocyte may
z 0.16 bear a generic relation to Na+-dependent system ASC
(59a) gains support perhaps from their both showing
interaction with analogous cationic and dipolar amino

D. Interaction Among Transport Systems:

Consequencesjbr Distribution and Flows
01 1 I 1 I 1 of Various Amino Acids
I 2 3 4 5
CAmino Acid I ,r,erno, = mru
To simplify, we may first consider how the com-
FIG. 2. External AIB trans-stimulates exodus of AIB or MeAIB bined operation of systems A and AX interacts with
from Ehrlich ascites tumor cell, whereas external MeAIB trans-in- the operation of system L. In the latter we have a ubiq-
hibits exodus of either of these 2 amino acids. [From Christensen uitous Na+-independent transport system or perhaps a
w.1 family of systems. Its measure in some cases may in-

elude overlapping contributions by analogous Na+-inde- ultimate answers but that kinetic tests meanwhile sug-
pendent systems of higher affinity, designated Ll, L2, gest the questions worth answering.
or similarly, and also in some cells by the rather differ- In exploring the function of system L, we tend to
ent system T, which favors the transport of benzenoid supply a selected external amino acid alone and to ob-
amino acids. System L typically generates rather small serve its uptake, while neglecting possible concurrent
gradients of the amino acid substrates it favors. Indeed, and possibly equivalent releases from the cell of the
its property of exchange or trans-stimulation is so same or other endogenous amino acids. Furthermore
strong that rather careful experiments were required to the tests often are not repeated at physiological levels
establish that it does produce net transport, inward or to see whether the net direction of the movement of the
outward, according to the direction of its substrate given amino acid may then be reversed. In other words,
gradient. For BCH, however, net uptake greatly exceeds we may tend to bias our experiments so that we see this
the concurrent net exodus of its various endogenous and other systems producing uptake. Because we tend
substrate amino acids (122). to be object minded, we therefore find it easy to think of
Even though system L can clearly produce net up- system L as simply another system for amino acid up-
take, the gradients maintained for many ordinary take. The proof of net direction for such a system is not
amino acids at in vivo steady states are small enough to simple because of its dependence on the structure of the
have generated doubts that system L considered alone amino acid under test and on the composition of the
is concentrating them. The unusually strong accumula- cellular amino acid pool. The Na+-dependent transport
tion of the bicyclic amino acid analogue BCH, at least by systems for many of their substrates will establish
the Ehrlich cell, no doubt relates to a substrate struc-
ture-dependent asymmetry in system L, since no other
system appears in certain instances to participate in its
transport. The vigor of trans-stimulation between a
pair of analogues can be intense if one of them (say Km VmJm
control 3-3 4.2
BCH) is inside the cell and the other (say thialysine) is loaded 6.3 7-l
on the outside and yet it can be very weak when their
positions are reversed (28; Fig. 3). No doubt the same BCH


asymmetry built into system L accounts for the espe-

cially strong accumulation of BCH. This asymmetry is
often less conspicuous among most ordinary amino Km 4n.s
acids but is undoubtedly biologically important.
may feel a tendency to look on this Na+-independent
transport systeml in its various occurrences as partic-

control O-8
ularly simple and uninteresting, when indeed these
puzzling evidences of asymmetry may make its study
especially informative as to the changes in tertiary
Km km Km Lx
protein structure that may occur during function. Di- control O-22 l-8
control O-25 2.4
rectional differences in the response of amino acid loaded O-8 7.4 loaded O-3 3.2
transport systems of hepatocytes and hepatoma cells to
N-ethylmaleimide and p-chloromercuriphenylbenzoate
have been observed (26,163). Striking inconsistencies in
the ability of amino acid analogues to inhibit transport
by specific systems, including system L, on the one L Km.,
hand, and their ability to protect the transporter from control l-6 l-2
loaded 2.8 1.9
reversible inhibition by p-chloromercuribenzenesulfo- ?
nate, on the other hand, have been noted by Tabb and GPA HA

Christensen (163). The orientation of the sulfhydryl FIG. 3. Sidedness in mutual acceleration of exodus and entry
group promises to be informative also for erythrocyte produced between 2 amino acids. Amino acid enclosed in a circle has
hexose transport (109). been introduced into Ehrlich ascites tumor cell, in one case in a la-
Differences among substrates in effective binding beled and in other case in an unlabeled form, in that case at 3-8 mM.
energies at the two surfaces of the membrane and hence L@2: unbarred arrows show that each of paired amino acids mutually
in relative rates of movement of the carrier, accord- accelerates transcellular migration of the other. Right: when posi-
ing to whether it is loaded or not and accordingly to tions of pair are reversed, bars show that exchange may be small or
unobservable. cis-dch, cis-1,4-diaminocyclo-hexane carboxylic acid;
which amino acid it carries in which direction, are in- GPA, 4-amino-1-guanylpiperidine-4-carboxylic acid; HA, L-homoar-
herently critical for trans-stimulation. Stein (162) has ginine; MPA, 4-amino-1-methylpiperidine-4-carboxylic acid; Tly, L-
discussed the kinetics of the asymmetry of cotransport thialysine. K,, concentration of external amino acid producing a half-
and countertransport in contrast to that for the simple maximal stimulation of exodus of internal partner. Other than HA,
carrier. He reached the conclusion that only structural these amino acids are transported mainly by system L. [From Chris-
studies on the participating molecules can provide the tensen (%).I

gradients too steep to be sustained by the weakly ener- some of the subjects by catheters in positions to record
gized systems, such as system L. A physiological or ex- effects on flows as follows: no effect was seen on leg
perimental steady state may be expected where concen- muscle release nor splanchnic uptake of aromatic
trative uptake by the Na+-dependent systems drives a amino acids. On the basis of leg muscle results, whole
net downhill efflux by at least one of the Na+-indepen- body muscle uptake of leucine occurred at roughly one-
dent systems. half the infusion rate. Renal clearance and tubular re-
During four decades, our predictions as to how the sorption of amino acids were not influenced. The uptake
pathological accumulation of an excess of one amino of isoleucine and methionine by the brain was inhibited
acid ought to influence the in vivo distribution of var- during leucine infusion. Plasma insulin rose no more
ious other amino acids have come only gradually to take than 25%. The authors suggested that with increased
into account the complexities inherent in the interac- intracellular leucine the L system may initiate a trans-
tions among parallel transport systems. For example, membrane exchange of amino acids, leucine outward
we tended at first to urge, in oral discussions if not in and the other amino acids inward, so as to influence
print, that experimental elevation of the plasma leucine their distribution with regard to muscle and liver. Even
should by transport inhibition elevate the circulating though this proposal gives little attention to contribu-
levels of isoleucine and valine, whereas that has proved tions of the transport systems operating in parallel
generally not the case (see Ref. 144a). The paradoxical with L, I find this insightful as a preliminary hypoth-
phenomenon of a stimulation of tryptophan uptake by esis. The effects seem quite pertinent to those obtained
the Ehrlich cell when excess methionine was added si- with hyperphenylalaninemia in rats (52; see sect. III).
multaneously was interpreted by Oxender and Chris- Although Hagenfeldt et al. (70) also considered the
tensen (130) as arising from the latter amino acid en- known effect of leucine on protein synthesis in skeletal
tering the cell quickly enough via system A, relative to muscle, they placed more emphasis on the high affinity
the entry of tryptophan, to make intracellular methio- of leucine for system L to explain its effect on the dis-
nine promptly available as a trans-stimulator of tryp- tribution of other amino acids. How are these transport
tophan influx by exchange via system L. Jacquez (90) effects then related to the recognized special regulatory
provided a neat confirmation that a Na+-dependent and action of leucine on protein turnover in muscle (24b) not
a Na+-independent system do indeed cooperate in this seen for a nonmetabolizable transport analogue? The
phenomenon. dehydrogenase for the branched-chain keto acids is
This behavior shows that the uptake of methionine stimulated in muscle and adipose of rats by leucine ele-
by system A presents to system L a sufficient outwardly vations, among other factors (110). Perhaps the meta-
directed gradient to drive an accelerated tryptophan bolic and transport effects of leucine will ultimately
uptake and hence that the two systems must operate prove% :have its particularly high transport affinity as
between the same two anatomic compartments. We a common explanation.
have called methionine the stimulator and tryptophan Although we emphasize here the effects of the
the stimulated in this context (39). We showed that pathological excesses of an amino acid on the distribu-
dipolar amino acids could be divided into two classes: I) tion and thereby on the fate of other amino acids shar-
the stimulators, the steady-state gradients of which ing common transport pathways, Young and co-workers
were increased when transport by system L is symmet- have emphasized the nutritional advantage of provid-
rically blocked; and 2) the stimulated, the gradients of ing a test amino acid at an intake needed to balance its
which instead were decreased. The results showed that irreversible oxidation under various conditions. On the
a much higher steady-state gradient of methionine was latter basis these investigators have urged increasing
maintained when system L was prevented from contrib- substantially the recommended intakes of four of the
uting to the methionine efflux. Phenylalanine used in indispensable amino acids, leucine, isoleucine, valine,
place of methionine failed to show that response. and threonine, and have recommended giving attention
Clearly these relations make complicated predic- to the intake of some of the dispensable amino acids,
tions of the results of interactions between transport including glycine, proline, and cysteine [see review by
systems in vivo, especially because fully parallel studies Young (193)]. A dictum by Crabtree and Newsholme
have not yet been carried out on the more abundant (47) is cited. High fluxes of individual amino acids
tissues, e.g., liver and muscle. Complexities in the rela- through given pathways provide the kinetic basis for
tions of the branched amino acids are evident from a precision and sensitivity of needed regulation. This dic-
study by Hagenfeldt et al. (70). In those studies leucine tum must not of course be taken to justify undue raising
was infused intravenously into postabsorptive volun- of intakes of each amino acid one by one. In the field of
teers for 2.5 h. Its arterial concentration rose four- to trauma, compensatory strengthening of the flows of
sixfold. Earlier findings were confirmed that the levels certain amino acids is receiving attention, as I discuss
declined progressively for other amino acids, especially in section IID for glutamine.
isoleucine and methionine (-55%), valine (-40%), and Allow me to insert here a familiar warning. Even
tyrosine and phenylalanine (-35% ). Although isoleu-
tine and valine reached high levels on similar infusions, Since completion of this review, a group of papers bearing
they failed to cause declines in plasma concentrations of heavily on the problems involved in these kinetic considerations has
the other amino acids (57). Blood samples were taken in aDDeared (see Ref. 16a and following: DaDers).

concentrative for the same substrates. In a recent pub-

lication (32), I summarize results indicating that inhibi-
G tion of system L by an amino acid analogue may in-
G crease rather than decrease the steady-state gradients
of glycine between the cellular phase of liver or muscle
GI G and the blood plasma in the intact weanling rat. We
t G
G have also found that administering an amino acid ana-
logue that specifically inhibits system L intensifies the
hepatic gradient of v;irious amino acids with respect to
G G the plasma in the rat. Hence, system L appears in vivo
to serve very widely for exodus of amino acids from the
G liver and muscle. System L, rather than the Na+-de-
G1 IG pendent routes, appears to serve largely inward for the
G postabsorptive glutamine nutrition of the rat small in-
testine via the basolateral surface (184a), and post-
prandially that route should dominate in amino acid
G exodus from mucosal cells.
In section III I consider the likely consequences of
FIG. 4. Diagram illustrating that an amino acid may be pumped the high accumulations of phenylalanine in untreated
into a cell mainly by a strongly energized system and released from phenylketonuria (PKU) and also of leucine in maple-
cell by a less strongly energized system. Accordingly, an analogue syrup urine disease in perturbing the flows and distri-
may selectively inhibit either system and therefore lower whichever bution of various amino acids to the special nutritional
flux dominates. One or more additional systems may also participate.
C, mediator; G, molecules of a transported amino acid. [From Chris-
disadvantage of the brain, with disastrous conse-
tensen (28a), (c) 1975, Addison-Wesley Publishing Co., Reading, Mas- quences to central nervous system (CNS) development
sachusetts. Reprinted with permission.] in childhood (also to the fetus in utero). The interac-
tions discussed in this section indicate that these effects
are not restricted to the BBB but also lead to seques-
tration of various amino acids in muscle, liver, and
for the three selected apparently ubiquitous transport other tissues and hence to intensified amino acid catab-
systems, their properties should not be taken a priori to olism and presumably to secondary handicaps to the
be exactly the same in every occurrence, a lesson taught nutrition of tissues beyond the BBB.
us early by the differences from Ehrlich cell transport
shown by erythrocytes and hepatocytes. In parallel, en-
zymologists have come to expect differences from tissue E. Changes in Amino Acid Transport in Neoplastic
to tissue in enzymes that may bear function and names Cells Need to Be Evaluated, Understanding
in common. A route transporting a model substrate That System L May Serve for Exodus
BCH is not necessarily system L in all occurrences More Than for Uptake
(175); it is not proved that the transport of bulky dipolar
amino acids at the blood-brain barrier (BBB) is an ex- The obvious importance to the acquisition of amino
pression of the gene determining system L in fibro- acids for growth led years ago to the proposition that
blasts and elsewhere. A system transporting MeAIB abnormally strong amino acid accumulation is charac-
and showing other usual traits of system A may not be teristic of neoplasia (27). The concept that neoplastic
exactly identical with other occurrences of system A. In diseases arise from membrane changes underlying the
particular, any transport system serving largely for a transport of nutrients was reformulated by Holley (82).
single amino acid of special metabolic significance in a With enhanced understanding of differences in the
given tissue may be expected to show degrees of func- roles of parallel plasma membrane transport systems,
tional specificity to that tissue. The designation, system we probably need to narrow our question to be: Is this
N, for a hepatic transport system serving for glutamine, enhanced accumulation by system A or by which trans-
histidine, and asparagine should not be applied to other port system? By transforming fetal hepatocytes with a
tissues without clear indications of functional parallel- temperature-sensitive mutant of SV-40, Handlogten
ism. The term Nm has been applied for a muscle trans- and Kilberg (72) were able to change the resultant cell
port system largely specific for glutamine, but the su- line at will from the rapidly growing cells at the per-
perscript m implies as yet no generic relation to hepatic missive temperature of 33OC to fully differentiated cells
system N. Furthermore, parallel transport systems at the growth restrictive temperature of 40C. The cells
that have concentrative activities that are quite dissim- at 33OC showed high rates of system A transport but
ilar are apt inevitably to enter into a cooperation of the the transfer to 40C at least halved the system A trans-
sort shown in Figure 4, whatever analogies their desig- port rate (&ax) within 24 h. Their results indicated that
nations may suggest. de novo synthesis of the system A carrier is regulated in
Figure 4 has been much used for presenting the correlation with the temperature-dependent growth.
idea of how strongly concentrative systems will interact Saier et al. (147) have recently reviewed system A as a
with parallel transport systems that are less strongly particular target of oncogene action and as a regulator

of cellular growth in the Madin-Darby canine kidney trition of the cell. The amino acids have been divided
(MDCK) epithelial cell line. Boerner and Saier (17) by into two familiar groups according to their dietary es-
their continued comparison of the MDCK cell line with sentiality for growth and maintenance or according to
its chemically induced, oncogenically transformed vari- their dietary dispensability when the essential amino
ant, MDCK-T1, show that the elevated expression of acids are provided in adequate amounts. The dispens-
system A activity on amino acid starvation or on trans- able amino acids are generally subject to ready meta-
formation or by 1%O-tetradecanoylphorbol-13-acetate bolic breakdown as well as synthesis, whereas the es-
(TPA) treatment is modulated by a route sensitive to sential amino acids accumulate more readily because
butyrate, an inhibitor of histone acetylation. In con- of catabolic rates inadequate to balance the supply. It is
trast the expression of system A seen in growing amino conceivable that system L has a special role in protect-
acid-repressed MDCK cells is modulated by a mecha- ing cells from undue accumulations of some of the es-
nism sensitive to azacytidine, an inhibitor of DNA sential amino acids. Conversely, our transport systems
methylation. Hence the specific expression of system A maintain the cellular levels of the metabolically impor-
is held to be regulated by at least two distinct control tant dispensable amino acids at levels higher than is
mechanisms. typical for the indispensable amino acids. Early studies
The activity of system L has been found to be selec- with the Ehrlich ascites tumor cell showed the very
tively diminished to about one-tenth normal in the B high accumulation of several amino acids, presumably
lymphocytes in chronic lymphocytic leukemia (CLL) because this cell is derepressed in system A activity.
(153,154). At the same time the property of trans-stim- This co ntrast suggests that our initial question might
ulation characteristic of that system was similarly de- well be exami ned more specifically for system A or for
creased (155). This transport loss is a paradoxical ob- its variants as seen in transformed and neoplastic cell
servation in view of the enhanced proliferation of these
cells at an immature stage. Once we accept the possibil-
ity developed above and elsewhere (31, 32) that system
L may serve more largely for the exodus than for the F. Brief Examination of Place of Dispensable
entry of various dipolar (neutral) amino acids from Amino Acids
cells, however, we see that it is an oversimplification to
predict as a consequence of this transport loss that the Already we have encountered, especially for the
lymphocyte will necessarily be starved in the various cases of alanine and glutamine, corrections to the view
amino acids typically most reactive with system L. It that the essential amino acids should receive most of
might well also be too simple to reverse the argument our attention in discussing nutrient flows between the
and attribute the defect solely to too large a cellular loss organs of the higher organism. The amino acids with
of the amino acids presumed ordinarily to leave the cell the larger roles in intermediary metabolism are the
via system L. amino acids for which the organism is likely to preserve
Exposure of the immature CLL cells to TPA led to reactions that al low synthes is, as is ill ustrated by glu-
maturation to a plasmacytoid phenotype and resump- tamine, alanine, and glycine in Figure 5. We would be
tion of immunoglobulin secretion (see Ref. 186). After illogical to suppose that the dispensable glutamine is
16-40 h of that treatment the maximum rate of the unimportant as a nutrient relative say to glucose, which
saturable uptake of BCH had increased by 8- to 14-fold, is a dispensable nutrient in the same sense that gluta-
paralleling the induction of cell maturation (186). The mine is. The information that an amino acid can be
rate attained was similar to that estimated for normal synthesized in the organism does not mean that a given
B lymphocytes, which only tripled under the conditions, tissue need not receive its supply of that amino acid
increasing uptake by CLL cells lo-fold. The property of from other tissues. Even if it can be synthesized by the
trans-stimulation reappeared, and the amino acid selec- very cell we are considering, appropriate transport may
tivity of system L was unchanged. Cycloheximide at a conceivably not maintain a concentration or a supply of
level inhibiting >90% of protein synthesis blocked the a dispensable amino acid sufficient to sustain its opti-
enhancement of system L in CLL cells. Leucine trans- mal utilization in that cell. Instead, sequestering of that
port also showed sharp declines in resting CLL cells; amino acid in another tissue by an aberration in mem-
much of the small residual leucine transport was not brane transport may conceivably lead there to a waste-
inhibited by BCH and hence was attributed to system ful rate of utilization that leaves one of the tissues nor-
ASC (154). Treatment with TPA for 12 h accelerated the mally sharing it deprived. The part played by transport
BCH-inhibitable leucine uptake to a degree similar to in the economy of a nutrient is more obvious when the
the increase observed for BCH uptake. The uptake of nutrient arises outside the cell under consideration.
MeAIB was not elevated in CLL cells over the normal Nevertheless the central question should be uniform: Is
B-cells, but TPA treatment led to its contrastingly an appropriate level of a given nutrient maintained in
rapid induction, which was attributed to the familiar or a sufficient flow of it sustained to the cellular com-
adaptation seen in an amino acid-impoverished envi- partment where it is needed?
ronment. Because of methodological limitations, just which
The paradoxical situation described indicates that tissue function may be suffering most from an inade-
we need to learn more about the relations among amino quate interorgan flow may not be obvious. The defect
acid fluxes to understand their significance for the nu- may well be recognized only as a global deficiency of

a Arterial - hcpotic venous difference

( splonchnic exchange)

0 Arterial - femoral venous diffe fence

(muscle exchange)

FIG. 5. Net exchanges of various

amino acids in course of blood circulation
in normal humans in postabsorptive state.
[From Felig (%a). Reproduced with per-
mission from the AnnuaZ Review of Bio-
chemistry, vol. 44. (c)1975 by Annual Re-
views Inc.]

-80 aNHe
Alo Gin Gly Ly8 Pro Thr His Lw Vol Arg Phe fyr Met I80 Tau Orn 6ut Cit Cys SW

nutrition, for example, failure to maintain nitrogen bal- activity, both for a CHO cell line and for its trans-
ance or to thrive. formed derivative. A correlation between system A ac-
tivity and ouabain-binding sites among several CHO
mutants was taken to show that these two respond to
G. Recent Observations on Regulation of System A increases of the same molecular species (136a). Such
results support the reserved position some of us have
A background of adaptive and regulatory influ- taken on the question of a role for K+ in the energiza-
ences on various amino acid transport systems was re- tion of amino acid transport by alkali ion gradients.
viewed by Christensen and Kilberg in 1987 (40). More Vadgama et al. (170) have used the ontogeny of
recently, Handlogten and Kilberg (72) found that the fetal erythroid cells of the rat liver to show the differ-
fetal hepatocyte line RLA209-15, which has been trans- ent schedules of appearance and regression of various
formed with a temperature-sensitive W-40 mutant, transport systems. These studies have now been ex-
behaves like fully differentiated cells at the growth-re- tended to the differentiation of human and murine
strictive temperature of 40C. Incubation at the erythroleukemic cell lines and associated changes in
growth-permissive temperature of 33OC led, however, to their proteins as stimulated by amino acid deprivation
a transformed phenotype characterized by rapid cell and as induced by butyrate, dimethyl sulfoxide
division and decreased production of liver-specific pro- (DMSO), and other agents (171). Accumulation of glo-
teins. The latter cells show high rates of system A bin mRNA was stimulated by these agents, and addi-
transport, but their transfer to 40C halved this activ- tion of hemin led to hemoglobin synthesis. These results
ity within 24 h. This decline was independent of cell associate changes in synthesis of system A protein with
density, although the basal rate of system A uptake was stages in differentiation of the cells.
inversely porportional to cell density in the rapidly
growing cells. The characteristic difference in transport
activity could still be shown in detergent-solubilized II. SELECTIVE COMPOSITIONS OF AMINO ACID FLOWS
vesicles and also after reconstitution of the vesicle pro- AMONG TISSUES: FACTORS DETERMINING THESE FLOWS
teins into artificial liposomes. These results together
indicate that the de novo synthesis of the system A
carrier is regulated in conjunction with the tempera- A. Epithelial Brush Borders Versus Plasma
ture-dependent growth of this fetal hepatocyte. Membrane: Development of Polarization
Dawson and Cook (50) attribute the early TPA of an Epithelium
stimulation of system A activity in the LLC-PK1 cell
line to a protein kinase C-dependent phosphorylation of The interactions among transport systems are in-
the transporter. The delayed stimulation appeared, herently more complicated when the barrier separating
however, to act on the synthesis of a glycoprotein, pre- two phases under consideration is a layer of cells, as in
sumably the transporter. Schenerman et al. (148a) an epithelium, or the endothelial layer of the microves-
found that ouabain treatment at low levels increased sels of the BBB. In the latter case, investigation has
together the system A activity and the Na+-K+-ATPase shown that the net direction of operation of the equiva-

lent of system L is toward the brain, which might well vening membranes, or by the drive of differences in
represent the outward direction of transport from the metabolism in the two cells, or by combinations of these
endothelial cells. Tests have raised doubts whether an differences. An ambitious hope of this review is to help
equivalent of system A actually serves in the BBB to set the stage for an examination as to whether and
transport model substrates of that transport system where a choice can yet be made between these two fac-
toward the brain (see Ref. 64). tors in determining interorgan flows.
To the nutritionist, the intestinal transport of nu- Degrees of asymmetry as to inward versus outward
trient molecules arising from digestion provides the transport are inherent characteristics of transport sys-
central example of a flow of amino acids directed from tems of plasma membranes as well as for transcellular
one organ compartment to another. The service of two movements across epithelia. This view has occasionally
anatomically distinct membranes and the presence of been accepted rather too readily by overlooking that for
the intracellular compartment intervening between the a given transport system both fluxes inward and out-
lumen and the plasma side are characteristic of epithe- ward persist together even though energization may
lial transport. At an earlier historic stage a tendency produce a considerable asymmetry between the two.
arose to suppose that molecules once concentrated into Also we impute biological function to the net direc-
the mucosal cell needed only to be released passively tionality of the catalysis of flow by a system and there-
across the basolateral membrane. This view overlooked fore may overlook that by no means all the molecules of
for the moment that boundary membranes more the solute are moved in that direction. We may, as I
usually show selectivity and asymmetry in handling already suggested, suppose that system L exists merely
solutes and also the necessity of maintaining nourish- so that cells may take up certain amino acids favored by
ing concentrations of various of the amino acids within it and fail to consider that an opposite role for system L
the mucosal cell, especially in the postabsorptive state. may often predominate.
If one remembers that almost all of the cells of the body Let me now return briefly to the contrasts between
concentrate many of the amino acids into their inte- amino acid transport at the brush border and at the
riors, some of them quite intensely, it becomes obvious basolateral poles of a cell in an epithelium. The set of
that if the small intestinal tissue fails to do likewise and transport systems found so far at the basolateral mem-
thus does not compete for its share, it may well suffer brane of the renal and intestinal epithelia have tended
impoverishment in this society of cells. Furthermore, in to identify it as highly analogous to plasma membranes
the postabsorptive state, as studied with ileal loops in in general (114, 160, 186a). In contrast, at least one
situ, the absorption of model, metabolism-resisting principal amino acid transport system of the intestinal
amino acids has been shown to be a reversible process, brush border is a conspicuous wide-range, Na+-depen-
coming to much the same concentration gradient in dent system quite unlike systems A or ASC; it accepts
favor of the plasma with respect to the luminal fluid no even cationic and bicyclic amino acids in addition to
matter whether one provides the amino acid in excess in almost all of the ordinary bipolar amino acids. Thus
the luminal or plasma compartment (37). this is a system rather like system Bp+ seen in blasto-
That behavior may help us to identify the principal cysts and certain oocytes (173-175). Differential regula-
means whereby amino acid flows arise between dissimi- tion of transport among the amino acids by such a sys-
lar cells, each with a plasma membrane totally sur- tem would present difficulties. Conceivably it may
rounding it, i.e., nonepithelial or nonendothelial cells, prove to be phylogenetically primitive. Because of api-
for which we do not recognize more than one distinct cal and basolateral differences, the usual epithelial
exterior phase faced by that membrane. The necessary function appears to transfer solutes, whether ionic or
establishment of an effective flow of a given amino acid not, across each of the two serial barriers by quite un-
from one type of cell to another appears to require that like routes. This feature when once analyzed may facili-
the intensity of its uptake by the target tissue overbal- tate the recognition of stages in the developmental po-
ances the intensity of its uptake by the tissue of origin. larization of an epithelium. Epithelial cells initially
We have argued that the characteristic fall in the placed into culture in a dispersion offer striking in-
plasma amino acid levels seen both in humans and in stances of the appearance of functional polarization as
guinea pigs early in pregnancy corresponds logically to they grow to contiguity as a monolayer.
the introduction into the organism of one more organ, A recent comprehensive review of the renal han-
the placenta, competing hungrily, although selectively, dling of amino acids by Silbernagl (159) provides im-
for the circulating amino acids (44). The imbalance of portant support in this area. Hence I am able to focus on
uptake needed to drive an interorgan flow could arise aspects that seem to me particularly pertinent to trans-
from net metabolic consumption at the former tissue port in interorgan nutrition, a subject treated only
outpacing net metabolic consumption at the latter tis- briefly by Silbernagl. The developers of the cell line
sue, or it could arise from steeper accumulation at the LLC-PK1 described its growth into monolayers and not
target tissue than at the tissue of origin, or finally the in free cell suspensions (83). Derived from the kidney of
imbalance could arise unevenly, that is between net di- a young pig, this cell line shows several differentiating
rectional transport being rate limiting at one of these characteristics of the straight segment of the renal
and the net of metabolic consumption and production proximal tubule. Its developers also described the for-
being rate limiting at the other. In short, the vectorial mation of domelike structures by the monolayers,
force could be applied differentially at one of the inter- which are not seen, however, when the monolayer is

grown on a permeable supporting surface. This behav- (177). Presumably only when junctions are largely
ior strongly suggested that the monolayer had taken an closed could even a preexisting directional pumping
orientation allowing a transporting function that produce an observable gradient, since back leakage
moves fluid and thereby lifts regions of the monolayer would otherwise reverse the pumping.
to form domes. Although a number of investigators Three marker transport systems appeared over-
have contributed to this history, I discuss next results whelmingly at one of the two poles of a LLC-PK1 clone
coming especially from the laboratory of Rabito (177) to in monolayer, as follows: ouabain-sensitive Rb+ uptake
illustrate features pertinent to this review. was basolateral, Na+-dependent ar-methylglucoside
Renal epithelial cells grown in monolayers and also transport was apical, and Na+-dependent D-ASP--L-
membrane vesicles isolated experimentally from the Glu- transport was apical (71). In contrast, however,
two opposing surfaces of an epithelium (114) have the monolayer showed the Na+-H+ antiporter at both
served to indicate in which of these two barriers its poles. These two activities could, however, be distin-
various transport systems lie. For example, the activi- guished pharmacologically, pointing to two distinct
ties of systems A, ASC, and L were found to take the forms of this antiporter under separate genetic control.
basolateral orientation. That study did not yet find a The defect in the intestinal and renal transport of
place for the quite different Na+-dependent amino acid cationic amino acids in lysinuric protein intolerance
transport system (perhaps Boy+) now recognized to be (LPI) also lies in the basolateral membranes of these
associated with the brush border as mentioned. An ac- epithelia. Not surprisingly then, this defect is expressed
tivity resembling the cationic amino acid transport also in the system y+ transport by skin fibroblasts cul-
system y+ of other plasma membranes was also asso- tured from several such patients (160). It is, remark-
ciated with the basolateral surface (157). In the MDCK ably, the efflux of lysine from these cells that is im-
cultured cell line, this activity resembles the usual sys- paired. Yrans-stimulation of that flux is correspond-
tem y+ even with regard to its transport of cationic ingly impaired. Hence abnormal sequestration of the
amino acids, but it also shows Na+-dependent transport basic amino acids arises. This finding could have been
reactivity with selected dipolar (neutral) amino acids, a interpreted simplistically to mean that one protein cat-
reactivity maximizing at the same distance between the alyzes the inward and another the outward movement
a-amino and a terminal hydroxyl or carboxamide of lysine, heretofore both attributed to system y+. Al-
group. This is a feature seen in system y+ of the various though I urged a separate identification of the catalysis
nonepithelial plasma membranes. of two amino acid fluxes between Na+-dependent and
The Na+-dependent glucose transporter was de- Na+-independent systems in section ID, that argument
tected mainly at the brush-border pole of the LLC-PK1 does not mean that I abandon the general view that
cells when they had grown to a contiguous membrane each transport system operates in both directions so
on a collagen-coated polycarbonate supporting filter. that its characteristics can often be described by study-
The inhibitory action of phlorizin was also exerted from ing either flux. Detailed identification of the genetic
the brush-border side (138). Evidence was seen that the defect in LPI may solve this riddle; perhaps it might
commitment of the cell surface into two regions as to point to an accessory protein that contributes asym-
which of the types of transporters it is to receive was metrically to the action of the carrier per se.
made only as contiguity is attained by the initially sepa- Could the resulting tissue sequestration of the cat-
rate cells of the monolayer. The enzymes, alkaline ionic amino acids, particularly as it applies to arginine
phosphatase and glutamyl transpeptidase, have also and ornithine, account for the ammonemia, which is
been shown to increase in activity at the stage when characteristically seen unless protein intake is re-
junctions were occluding, pointing to insertion of their stricted in LPI? Might not perturbations of alkali-metal
molecules mainly after anatomic limits for the two ion distribution be expected to arise from unusual se-
types of cell surface have been established (139). Wang questration of cationic amino acids in various cells?
and Nelson (178a) report that the cultured epithelial Might that sequestration of cationic amino acids be al-
MDCK cells, after they have established one polarity in leviated by exchange with such a dipolar amino acid
multicellular cysts, can be caused to reverse their polar- (plus Na+) as homoserine ? A gene dosage effect was
ity by placing the cysts in a collagen gel matrix, as observed (160) by comparison with subjects heterozy-
indicated histologically. gous with respect to LPI and also for the defect in in-
A clone of the pig kidney cells designated LLC- testinal absorption as measured by the failure of
PKIA served to demonstrate further the appearance of plasma lysine to rise to the usual degree in a tolerance
polarized transepithelial pumping. When placed in sus- test. How much might its widespread tissue sequestra-
pension, these cells showed little Na+-H+ antiport activ- tion contribute, beyond defective intestinal absorption,
ity, which was by necessity not measured transepithe- to the failure of the plasma lysine to rise as much as
lially. This activity increased when the cells were plated usual in this tolerance test?
at high density. The orientation ultimately taken by the The authors of that valuable paper point out that
antiporter was revealed by unidirectional Na+ influx LPI presents the first demonstration that skin fibro-
measured from the basolateral side. Rises in transepi- blasts can be used to study a corresponding transport
thelial electrical resistance, measuring the extent to defect in intestinal and renal membranes. Furthermore
which the junctions between cells had closed, showed the strong functional analogy they reemphasize be-
during growth a delay relative to the rise in Na+ influx tween the basolateral membrane of these epithelial

cells and the plasma membrane of parenchymal cells relative magnitude of these flows is summarized in an
suggests that other such findings may follow and that earlier review (see Ref. 29). In the following sections I
the full explanation of the differential expression of discuss some of these and other major flows. Initial
defective efflux may be achieved. The opportunity to emphasis here on certain quantitatively large interor-
study some of these questions in cultured cell lines jus- gan flows of amino acids should not of course divert us
tifies our considering nutrition of such cell lines in this from ultimately considering also the smaller interorgan
review [see Bettger and McKeehan (15)], namely the flows. Even where these represent only quantitively
discovery of such relations that may apply in vivo. We minor aspects of nitrogen metabolism, some of them
encounter other examples, especially in discussing cys- are undoubtedly of substantial overall importance.
tine and cysteine transport (see sect. IIF). Am I in danger of exaggerating the physiological
consequences of some perturbations of amino acid
flows? When Christensen and Cullen (35) fed weanling
B. General Considerations of Makeup rats 6% AIB in their normal ration for 21 days, we were
of Amino Acid Flows surprised to observe that, despite the large effect of this
analogue on amino acid distribution, the rats tripled
A possibly familiar background concept is outlined their 41-g initial weights. Their growth was no more
here. We fall under a temptation to suppose that the slowed than that of pair-fed litter mates receiving the
nourishing flow of amino acids from the gut as they are control diet. The AIB feeding lowered the analytically
released there by the digestion of proteins is simply determined levels of various dipolar amino acids in the
continued through the blood stream as a random flow liver, including the essential amino acids valine, isoleu-
through the branches of that stream from which the tine, leucine, tyrosine, and phenylalanine, by one-third
various tissues feed rather indiscriminately. Let us re- to one-half; however, spontaneous restriction of their
flect how far the actual picture departs from so simple a food intake by the rats appeared to account or compen-
concept. We do well to begin by thinking not in terms of sate for any growth-handicapping effects of these per-
the background composition of the blood plasma, amino turbations. Some of these amino acids may of course be
acid by amino acid, but rather in terms of the amino decreased as secondary responses to effects on other
acids for which there is an important net traffic from amino acids. The remarkable tolerance to this particu-
one organ to another. Figure 5 focuses attention on the lar manipulation of hepatic concentrations of amino
characteristic high flows of amino acids alanine, gluta- acids and also of their gradients with respect to the
mine, glycine, lysine, and serine, which in the postab- plasma leaves me with an unsolved puzzle. The circum-
sorptive state represents in the aggregate a major part stance that the perturbations of the organismal distri-
of the flows between the two organs presented. The flow bution of amino acids produced in this experiment were
of alanine is designated as part of the alanine-glucose somewhat balanced among the amino acids may have
cycle, whereby the amino groups released in muscle are allowed them to be tolerated better than highly imbal-
returned to the liver in the form of alanine. Gluconeo- anced perturbations would be. Hepatic Asp- and Glu-
genesis then occurs in the liver to complete the cycle. were, however, concurrently increased rather than de-
Even in the fed animal we could not expect protein creased. Acute effects in the opposite direction of AIB
digestion to yield proportionately so large a flow of ala- and MeAIB on certain hepatic amino acid gradients 2 h
nine from the intestine to the liver. Glutamine also par- after intraperitoneal injection were also reported in the
ticipates in this transport of amino groups from muscle, same study.
although much of the glutamine is removed from the
splanchnic circulation by kidney and intestine. The left-
over part of the glutamine molecule nourishing the in- C. Alanine and Amino Acid Dispensability:
testine tends to be passed on to the liver in the form of Conditional Essentiality
Although glutamic acid may well be the most Alanine is the main glucogenic amino acid flowing
abundant amino acid consumed in the diet, the flow of to the liver, the resultant glucose flows back to the
glutamate illustrated here obviously has largely a more muscle to receive an amino group, and then the alanine
complicated origin, even in the fed animal. The sum- flows to the liver to complete the alanine-glucose cycle.
mary diagram of Figure 5 helps us to see how imprecise Systems ASC, A, and L participate in its passage into
we would be to picture a nutrition whereby the various the liver cell, with probably the two Na+-dependent
cells are simply flooded with the direct products of di- systems producing net uptake. The rate of alanine
gestion. Of the quantities of the various amino acids transport into the liver limits its utilization even under
added to the portal plasma, the liver ultimately re- a high alanine load at which a coordinated acceleration
moves most, with the exception of the branched-chain of its catabolism allows alanine transport to remain
amino acids (BCAA), which it allows to proceed mainly rate limiting (58). Therefore the phenomenon of adap-
to muscle for transamination (for review see Ref. 75). tive regulation of system A transport can remain effec-
The liver uses in general more glutamine than is added tive even at high alanine loads. A 48-h fast in rats
to the portal plasma. It would be another huge over- caused the isolated hepatocyte treated in vitro with
simplification to think of fasting as reversing the direc- aminoxyacetate to double its alanine gradients (101).
tions of the major flows. Published evidence for the Both influx and efflux of alanine were accelerated by

the prior fast, which is a two-way action to be expected that one or another amino acid is undersupplied to one
for adaptive regulation of transport (183). The greater tissue or another, studies of interorgan nutrition are
action on influx might suggest that a disproportionate apt to be necessary, I believe, to indicate where the
part of alanine efflux occurs by a route not subject to deficiency strikes and why.
adaptive regulation but is no doubt better explained by
the net inward movement of alanine.
Numerous indications of nutritional advantage for D. Principal Glutamine Flows
the dietary presence of dispensable amino acids have
been reported, even though the classic nitrogen balance
methods may have failed to show any. Several of the I. GlutamineJZows within a cultured cell
dispensable amino acids clearly show conditional es-
sentiality, as in the cases of tyrosine or cystine in pa- On incubation of fibroblasts in a buffer containing
tients with certain defects in hepatic metabolism. Jack- 0.7 mM [15N]Gln and no Glu-, intracellular [lN]Gln
son et al. (89) reported that glycine supplementation is reached a plateau at 80% of isotope enrichment within
necessary for a satisfactory rate of lean tissue growth 20 min. In contrast, intracellular [15N]G1u- rose only to
in preterm infants. Beliveau and Brusilow (14) showed 40% isotope enrichment after 4 h in the same buffer
that when sodium benzoate is injected into rats on a containing 0.5 mM pure [15N]Gl~- and no glutamine
diet devoid of glycine and serine, growth restriction and (49). The free glutamine concentration of the cell was
biochemical changes result. Infants with one of the con- firmly dependent on the extracellular glutamine level,
genital defects in the catabolism of the BCAA may whereas the free cellular Glu- level remained steady
show a deficiency in the rate at which alanine is made irrespective of the extracellular level of this amino acid.
available from muscle for hepatic gluconeogenesis. Despite the large difference in rates, the cells took up
These infants need to be maintained on lowered intakes both amino acids against concentration gradients. Glu-
of BCAA. This purpose is served by bringing the dietary tamine uptake accounted for 90% of the cell glutamine
content of protein as low as is compatible with satisfac- turnover, whereas only one-fourth of the intracellular
tory growth, given supplementation with limiting es- glutamine was derived from exchangeable Glu- when
sential amino acids. Results with two such cases showed the environment was glutamine free. These results in-
that dietary supplementation of these diets with ala- dicated that free Glu- must be divided between pools in
nine was more effective than the addition of a variety of these cultured cells.
other dispensable amino acids in decreasing the amount
of protein needed for growth (185). This result would be
a logical consequence of the compensation by exogenous 2. GlutamineJEows within the intact organism:
alanine for a presumably inadequate flow of alanine to renal contribution
liver, which is dependent on the breakdown of the
branched amino acids in muscle. The optimum supple- Regulation of the organisms H+ economy in
ment of glycine (again allegedly dispensable) in isova- chronic acidosis depends on the flow of glutamine from
leric acidemia, either during stable periods of leucine organs capable of releasing this amino acid to organs
restriction or during leucine-loaded periods, has re- capable of taking it up and eliminating from the organ-
cently been studied (120). ism the H+ inevitably extracted when excretory NH: is
These examples illustrate the dependence for an substituted for an equivalent amount of excretory urea.
important function of the supply of a dispensable amino The normal flow of glutamine is from the musculature
acid on the metabolism of other amino acids that yield to the splanchnic bed, whereas in the liver it provides a
that amino acid. At the same time we see the necessity principal fuel and an amido group for urea formation.
for an interorgan flow, in this case from muscle to liver. As metabolic acidosis develops, the glutamine flow is
Amino acids that can be synthesized by the whole or- diverted to the kidneys. How this rerouting is regulated
ganism are by no means sure to be synthesizable by any stands as a central question of this section. The diver-
and every tissue in that organism. The hepatic arginine sion from the splanchnic bed to the kidneys is asso-
economy (182) of the rat may be so well isolated from ciated with a sharp decline in the arterial glutamine
the arginine economy of the rest of the organism by a concentration; conversely, the opposed diversion is as-
transport limitation as to allow a general deficiency sociated with elevated arterial levels. Thus it appears
despite continuous rapid synthesis in the liver. Allow that the homeostasis of concentration is sacrificed in
me to repeat: just because an amino acid can be synthe- producing the physiologically appropriate reversal of
sized in a given cell does not even guarantee that the destination. How is the interorgan flow of glutamine
transport processes of that cell will retain an adequate directed to the appropriate organ, with possibly some-
concentration of it to sustain its normal utilization what different answers in various species? This ques-
there. Furthermore, an amino acid may be accumulated tion is much discussed and has been treated in a recent
in the organism to degrees such that its transport com- review by Welbourne (180). Multiple mechanisms of
petition may cause wasteful accumulation and degrada- regulation need to be considered, including modifica-
tion of other amino acids, say in muscle or liver, with tion of enzymatic and transmembrane fluxes in the sev-
resulting deprivation of other tissues, e.g., the brain (31, eral involved organs. Included in the latter list are mod-
32). Although overall nutritional deficiency may reveal ified release from muscle; modified uptake and release

by salvage sites, e.g., the liver; and modified uptake minase (56). Significantly, their glutamine synthesis
by sink tissues, including the kidney (see Ref. 180). was nevertheless increased through enhanced synthe-
Although y-glutamyltransferase generates NH,+ in tase activity when the external Glu- was increased, and
the kidney, it appears to play at most a minor role in the net glutamine production was seen when glutamine was
generation of urinary NH,+ relative to the phosphate- omitted from the environment; hence the difference
dependent glutaminase, a consequence of compartmen- from the response of muscle in vivo appears largely
tation in proximal tubular function (181). It is perhaps quantitative.
unprofitable to try to explain by changes in one organ at One can imagine that modulation of the large dif-
a time the orchestration of the redirection of the gluta- ferences in glutamine flows to and from the liver, on the
mine flows. Poorly understood interorgan communica- one hand, and from the corresponding flows from and to
tion systems appear to be needed to explain the increase skeletal muscle, on the other hand, would be achieved
by 5- or lo-fold of renal glutamine extraction in chronic rather awkwardly by simply giving systems A, AX,
acidosis. Nevertheless, chronic metabolic acidosis pro- and L different regulatory properties in the two organs.
duced in rats by adding HCl to their diet has been re- Pertinent differences do occur among tissues: system A
ported to cause intrinsic adaptations both in the apical of the hepatocyte is not repressed by glutamine as it is
membrane Na+-H+ antiporter and the basolateral by various other system A substrates (73), whereas in
membrane Na+-3HCO; symporter of the proximal con- other tissues, such as the Ehrlich cell and the fibro-
voluted tubule (135), both of which are favorable to H+ blast, glutamine is repressive. The fact that more spe-
excretion. cific agencies serve to cause liver and muscle to release
and take up glutamine each in its own way was already
suggested by the discovery of system N in the rat hepa-
3. Role of muscle in determining glutamine$ows tocyte (98), which is a system that we could expect to be
absent in muscle or at least so different in modulation
Glutamine accounts for 2530% of the amino acid so as not to deserve the same name. System N transport
released from muscle and serves as a vehicle for re- is derepressed with respect to hepatic glutamine uptake
moving excess NH& It serves also as a gluconeogenic by amino acid starvation. A rapid, Na+-dependent,
precursor, as a source of renal NH,+ for carrying out H+ amiloride-sensitive activation of system N by amino
excesses, and as a metabolic fuel for the small intestine. acid substrates both of systems A and N, whether me-
The glutamine release, in contrast to that of alanine, tabolizable or not, has been described by Bode and Kil-
continues even in the postabsorptive state of the rat berg (16b). This effect was cycloheximide insensitive
(146). Although the increased renal uptake of glutamine and was not retained in isolated plasma membrane ves-
in metabolic acidosis is balanced by accelerated muscle icles of the hepatocyte.
release of glutamine (151), the role of muscle transport To have precisely the same system N serve for reg-
processes, if any, in controlling glutamine release has ulation of glutamine transport in both liver and muscle
been unknown. Rennie et al. (141) have developed a hy- seemed likely to prove incompatible with the control of
pothesis implicating particularly a more or less specific their very different glutamine flows. Hence a search for
glutamine transport across the sarcolemma of muscle, a somewhat different glutamine transporter more
discussed next. nearly specific to muscle function became appropriate.
The abnormal overall release of nitrogen by the At the same time the previously seen participation of
muscle of the hindquarters in chronic renal failure as systems A, ASC, and L in glutamine transport might be
simulated in partially nephrectomized rats is attrib- expected to frustrate modulation of glutamine flows by
uted to a glucocorticoid-dependent effect of the meta- more or less tissue-specific glutamine transporters in
bolic acidosis and not to the azotemia (111). Also con- liver and muscle. System A, being inducible and partic-
tributing is a further inhibition of insulin stimulation ularly subject to endocrine action, is known to show a
of the synthesis of muscle protein. The same laboratory very wide range in activity. System L transports gluta-
concluded that the decarboxylation and net transami- mine at least in the fetal hepatocyte (see Ref. 172) and
nation of valine and leucine by muscle were accelerated elsewhere. The partial inhibition of glutamine uptake
by chronic NH&l acidosis in the rat (110). The acidosis by the hepatocyte by 25 mM cysteine (98) indicates less
acted both by enhancing flux through the transaminase glutamine flow by system ASC than by system N in that
and through activation of branched chain a-ketoacid cell. The same questions for muscle deserve further
dehydrogenase. These authors emphasize the impor- evaluation.
tance of therapeutic correction of the acidosis (74). This Toward this goal, Rennie et al. (141) adapted a rat
net amino acid breakdown in muscle has an obvious hindlimb preparation whereby a defined mass of leg
relation to the flow of glutamine and alanine to carry tissue was perfused via the femoral artery to the femo-
off the released amino groups. ral vein just below the inguinal ligament. Unidirec-
Attempts to study control of glutamine release by tional transport was assessed by a paired-tracer isotope
muscle in the cultured L6 myogenic cell line have been dilution method. Conceivably, observation of unidirec-
complicated by the finding that the differentiated tional transport, uncorrected for possible exchange,
fibers incubated in an amino acid mixture mimicking may not serve ideally the present need. The medium,
rat plasma consume glutamine rather than release it, 6% bovine serum albumin in Krebs Ringer bicarbonate
presumably because of their very high content of gluta- medium, was allowed to flow through only once. Al-

though phenylalanine, leucine, isoleucine, and valine apparently noncompetitive, by leucine; and its insensi-
were removed during passage as shown by arteriove- tivity to lowered pH. We had delayed in proposing the
nous differences, their transports half-saturated only occurrence of system N in tissues other than the hepa-
at highly unphysiological perfusate concentrations of tocyte (98) because of the inherent plausibility of organ
-20 mM and were insulin insensitive, with the latter specificity in the handling of glutamine. The designa-
result to be expected if system L transport actually tion system Nm for the muscle system was proposed by
dominated in determining rates. The removal of phe- Rennie to emphasize both the similarities to and the
nylalanine was not sensitive to electrical stimulation or differences from the hepatocyte system, without im-
hypoxia. Regulation of passage of these amino acids by plying a possible generic relation between the two, as
action on the transport process indicated seems on the tends indeed to be suspected for the various occurrences
whole unlikely, considering their high apparent Mi- of systems A or ASC. In approving N, we take the
chaelis constant (K,) values. designation system N, as contrasted with N, still to
The rate of glutamine removal became half-satu- apply, as originally specified, only to the hepatocyte.
rated at a lower level, 3 mM, a level perhaps still rather In a collaboration between the Dundee laboratory
high for optimal regulatory control, with a Vmax low and the London laboratory of Jepson and Millward,
relative to those for BCAA. An increase in the extra- Rennie et al. (140) developed a hypothesis based on the
cellular concentration of either of the three BCAA in- properties of the transport system Nm for glutamine in
hibited glutamine efflux by ~30% over the entire range skeletal muscle. These properties are taken to point to a
of glutamine levels tested, a result possibly pertinent to significant function for the system in the control of
the fall of the intramuscular glutamine level in patho- whole body amino acid metabolism. These properties
logical conditions in which extracellular BCAA concen- include its kinetics and the ion dependence, both for
trations are reduced. This removal of glutamine was muscle glutamine and Glu- transport and also the ef-
associated by these authors with a Na+-dependent sys- fects of endotoxins in infections and of BCAA. Further-
tem they designated as system Nm. more, these authors show the presence of a link between
The concentrations of Glu- also characteristically the size of the glutamine pool in muscle and rate of
showed an arteriovenous difference during perfusion. muscle protein synthesis (Fig. 6), which raises in their
On uptake, the Glu- tended to be converted to gluta- thinking possibilities for therapeutic interventions to
mine. The removal of Glu- half-saturated at 75 PM, was limit protein loss in injury, sepsis, and chronic disease.
Na+ independent and insulin insensitive, and was stim- This interesting hypothesis may place membrane
ulated ~30% by a lowering of pH from 7.4 to 7.1. Hence transport at the sarcolemma in a key position in direct-
an increase in H+ concentration might promote the up- ing glutamine flow from muscle and hence in determin-
take of Glu- and secondarily its conversion to gluta- ing protein synthesis in that tissue. The rather high
mine and thereby the transport of H+ to the kidney, apparent Km values observed, even for glutamine trans-
with its ultimate elimination through substitution of port, will encourage further study as to this regulatory
NH,+ for urea excretion. role.
In the same laboratory, MacLennan et al. (104,105) Hundal et al. (85) have extended the study from the
showed conversely that omitting glutamine at 15 mM same laboratory [Rennie et al. (140)] of the overall
from the perfusion fluid, a change that roughly quar- routes of transport of various other amino acids into
tered the calculated glutamine concentration in the in- the perfused rat hindlimb preparation. This prepara-
tracellular water of muscle, led to a strongly acceler- tion, less the skin, is estimated to be 92-95s skeletal
ated release of phenylalanine from the tissue. This rise muscle by weight. Numerous different muscles are of
in phenylalanine in the perfusate along with the accom- course included, but the transport values should reflect
panying dilution of its [lN]Phe content was used as an the overall physiological role of skeletal muscle in
index of the net balance between protein breakdown amino acid flows. The authors conclude that alanine
and synthesis. The high release of phenylalanine in the entry probably occurs mainly via systems ASC and L,
absence of glutamine in the perfusion fluid was almost with system A playing a minor role. Correspondingly,
completely attenuated by the presence of glutamine at insulin sensitivity of alanine transport was not ob-
15 mM. No change occurred concurrently in the release served. These results apparently leave glutamine as the
of 3=methylhistidine, pointing to the absence of acceler- amino acid that has its uptake by skeletal muscle most
ated breakdown of the myofibrillar apparatus. Concur- conspicuously stimulated by insulin (84), presumably
rently no increase in Glu- or BCAA was seen. The ele- via the new system Nm. That feature may place gluta-
vated glutamine was thus taken to inhibit the net loss of mine in a special position for explaining the known abil-
amino acids, corresponding to a fall to about one-third ity of insulin to cause falls in the amino acid levels of
in net protein breakdown. Effects of glutamine and in- plasma, taking into account the association between
sulin appeared not to be additive, so they may act glutamine supply and protein synthesis and hence on
through a common mechanism. the concurrent flows of amino acids other than gluta-
The muscle carrier for glutamine described in Ren- mine. Glutamine inhibited alanine and serine uptake in
nies laboratory shows superficial similarities to system a noncompetitive manner. This noncompetitive inhibi-
N of liver but also shows distinct differences, especially tion led the authors to conclude that glutamine did not
in the sensitivity of muscle glutamine release to the share ASC transport with alanine or serine. One con-
glucocorticoid analogue, dexamethasone; its inhibition, cedes that the failure of interaction between a pair of

analogues for an observed transport process to meet the lysts, along with the recognized heterogeneity of the
kinetic standards for a competitive interaction still participating tissues, and also by conceivable unstirred
leaves unproved the possible sharing of a common layer effects. The perfusate in the skinned hindlimb
transport route by the two. The overall noncompetitive preparation may, however, establish a relation to the
nature of the interaction does not fully exclude that a muscle cells as close as does the flowing blood in the
portion of the glutamine uptake (e.g., by system ASC) living animal. Elsewhere I (34) have updated the chal-
may be shared and be competitive with alanine. The lenges that I feel the homogeneity of a given mediating
presence of such a sh ared portion could be masked bY agency for membrane transport ought appropriately to
the heterogeneity of the contribu ting tran sport ca ta- receive.
As mentioned, the bulky dipolar amino acids en-
tered perfused skeletal muscle fibers by a Na+-indepen-
A dent route showing competitive interactions, but the K,
0 values were so high (20 mM or higher) that the full
+ endotoxcmic qJoo O range of reactive substrates could not be defined, which
0 Cl
0 0
is not an unusual problem. Aspartate and glutamate
OO 0
were competitors for a Na+-independent route of up-
0 0
O0 take. The rate observed for Glu- was low enough that
its extracellular supply would likely limit glutamine
0 0 00
0 t& synthesis. The synthesis of Glu- within the fibers there-
t 0
t Q fore presumably provides most of the precursor for
0 glutamine. Lysine uptake in contrast was Na+ depen-
0 dent, with kinetics suggesting but not establishing the
# oQ participation of two Na+ for each lysine molecule. Mer-
t salyl inhibition, however, suggested heterogeneity in
% t
00 0 the mediation. The uptake of 3-methylhistidine was at
$ o+ least partially inhibited by histidine, leucine, and cys-
t teine but was not further analyzed (85).
Jepson et al. (91) showed that declines in the free
glutamine content of muscle, whether produced by
treatment with the lipopolysaccharide endotoxin (LPS),
2 6
protein deprivation for 10 to 16 days, low protein intake
muscle glutamine (mM/kg) plus LPS, or a 3-day fast, were closely correlated with
the decline in the protein synthetic rate (as in Fig. 6).
B This synthetic rate was measured as a first-order rate
0 diet constant, KS (%/day), from the plateau of protein-
4 endotoxemic bound radioactivity attained 15 min after injecting in-
0 0 0 traperitoneally into a rat a dose of rH]Phe relative to
0 the simultaneous radioactivity of the intracellular free
%O 0 phenylalanine. The decline in the glutamine content of
0 0
80 0 muscle was similarly correlated with the decline of the
0 0 cp
muscle RNA. Muscle contents of glycine or alanine, or
0 0 O
of any other amino acid studied, were not similarly cor-
Q+ t related with the protein synthetic rate. These investi-
0 gators noted, as it happened for cases of endotoxin
treatment, that the plasma glutamine levels were
maintained during the fall in muscle glutamine; hence
the glutamine concentration gradient fell. Enzymatic
changes failed to account for the correlation; therefore
the authors suggest that the glutamine transporter suf-
fers a decline in its ability to maintain the glutamine
gradient across muscle.
These researchers propose that the regulatory
0 4 8
2 6 characteristics of the glutamine transporter have a con-
muscle glutamine (mM/kg) trolling influence on muscle glutamine and at the same
time on the protein synthetic rate of this tissue. The
FIG. 6. A: relation between muscle glutamine concentration and
protein synthetic rate in skeletal muscle of rats on various diets com-
researchers suggest that therapies might be aimed at
pared with those in endotoxemic animals. B: muscle glutamine con- reversing the loss of glutamine during illness, including
centration related to muscle ribosomal activity in same animals. the current initiatives toward provision of stable gluta-
Overall regression equations for 2 relations were calculated. [From mine dipeptides in solutions for parenteral nutrition
Jepson et al. (91).] (la). The question persists: When does glutamine be-

come conditionally essentia 1 for maintenance of these The role of transport system N in determining the
flows? hepatic output of glutamine becomes more interesting
with the demonstration by Gebhardt and Kleeman (63)
4. Hepatic role in glutamine flows and ureogenesis and by Burger et al. (24a) that this transport activity
responds, altnougn 1 somewnat 1 1 slowly, to stimulation by
1 111 1 1 1 l 1 1 l 1

Despite the central role of the liver in amino acid insulin and glucagon, with dexamethasone showing a
metabolism, this organ has not always received propor- permissive contribution to this stimulation. This effect
tional attention with muscle and kidney in the regula- could readily be differentiated from that on system A,
tion of the excretion of H+ from the organism, perhaps which is stronger and more rapid. The increase was due
because of the complexity of interpreting its amino acid to a rise in Vmax, not Km, and was prevented by cyclo-
flows. Schimmasek and Gurok (149) showed that the heximide. System N does respond to adaptive regula-
perfused liver of the rat established perfusate levels of tion (98), but conditions allowing response to pancreatic
the various amino acids [with the understandable ex- hormones were not encountered in that 1980 study.
ception of the 3 BCAAs that unexpectedly do move The liver can alternatively synthesize urea or glu-
across the hepatocyte membrane by system ASC (99)] tamine from NH:, with the enzymes of urea synthesis
quite similar to those found in the blood plasma of the being primarily periportal, whereas the enzymes of
fed rat. This result suggests that the liver plays a rather glutamine synthesis are perivenous. The functional or-
large role in determining the circulating levels of the ganization of the acinus implies that any flux change
amino acids. The hepatic uptake of glutamine by Na+-
through the urea cycle will determine the substrate
dependent transport system N, a system that as sug-
gested may prove unique to the hepatocyte, has been supply for perivenous glutamine synthesis (66). Hepatic
shown rate limiting to subsequent glutamine metabo- glutaminase, present in the mitochondria of the peri-
lism in the liver (79). This system serves predominantly portal portion of the acinus, is seen as acting as an
for the uptake and release of glutamine both by peripor- amplification system in supplying enough NH,+, despite
tal and perivenous hepatocytes, as shown by a perfusion its usual low concentration in portal blood, for carba-
technique. The rate limitation of glutamine metabolism moylphosphate synthetase, which is rate controlling for
by system N transport was shown to arise from its sa- urea synthesis under physiological conditions. The pro-
turability through the consequences of its specific inhi- vision of HCOi for the same reaction may also be di-
bition by histidine at near physiological concentrations. rectly involved in regulating H+ disposition, thereby
For example, when endogenous synthesis was the only permitting a respiratory contribution (180).
source of glutamine, the inhibition of glutamine release The importance of vectorial considerations apply-
produced by histidine led to a 210% increase in the ing to compartmentation in the choice as to whether
hepatic level of glutamine in perfused liver (79). The NH,+ goes into the glutamine or the urea molecule is
inhibition by histidine -was shown to apply both to the emphasized by the reversal of that choice when the he-
periportal uptake and the peri venous release of gluta- patic acinus undergoes retrograde perfusion (77,78; Fig.
mine, the two fluxes that are implied to be of prime 7). The high affinity of glutamine synthetase in the
importance to hepatic glutamine metabolism by the small population of perivenous hepatocytes normally
demonstrated hepatocyte heterogeneity (for review see scavenges the NH: that escaped urea synthesis in the
Ref. 92a; see Ref. 77). periportal hepatocytes (94). Ammonia-metabolizing
When glutamine synthetase was inhibited by me- enzymes are similarly localized in the human liver, ex-
thionine sulfoximine and simultaneously glutamine up- cept that the zonation is not yet established in the peri-
take and Glu- export were stimulated by ketomethion- natal period (115a). Conceivably a chronic reversal of
ine, only the glutamine uptake and not the Glu- export the direction of flow, as after establishment of a porta-
were inhibited by histidine, pointing to glutamine caval shunt, might gradually lead to a reversal of the
transport as the specific site of histidine action. The anatomic distribution of the two populations of hepato-
Na+-dependent Glu- import, K, of 0.29 mM, which ap- cytes. Methods under development for separating the
pears to be rate limiting to metabolism of portal Glu- subpopulations of hepatocytes should assist in examin-
and apparently corresponds to the ubiquitous system ing such problems in vitro (169).
X;o, is localized in the perivenous hepatocytes, as indi- Amino acid compartmentation between periportal
cated by its major loss on selective necrosis of these and perivenous hepatocytes has been shown in vivo, not
cells by acute Ccl4 treatment (164, 165). The results of only for the distribution of glutamine (79) but also for
Haussinger et al. (79) showed that after stimulation of distribution of Glu- and alanine (46). These authors
glutamine-utilizing enzymes in the presence of a gluta- showed half times of a very few seconds in the rapidity
mine concentration physiological for the portal plasm a, of 13N exchange reactions among [13N-amide]Gln,
glutamine transport across the pl asma membran e, [13N]Ala, and [13N]Glu-, with each of these amino acids
especially in the presence of histidine, is not rapid injected as a small bolus into the portal vein of anesthe-
enough to maintain the initial intracellular glutamine tized rats. Evidence for hepatic zonal differences in the
levels. Thus Haussinger et al. (79) conclude that system uptake of all three was obtained. Despite the slower
N transport of glutamine across the plasma membrane turnover of the [13N-amide]Gln, attributed to the rela-
is a potential regulatory site in hepatic glutamine deg- tively high glutamine concentration, it was possible to
radation and synthesis. confirm that the amide group of glutamine is indeed the

source of urea nitrogen in vivo and that the 13N of ala- other. Clearly, parallel discrimination of flows within
nine must pass through that amide pool. an organ must be made in other cases, for example,
Although the uptake of the labeled glutamine into between neurons and glia.
the periportal cells was taken to be unidirectional, only As we examine summary studies on the role of
10% of the portally injected glutamine was taken up transport in amino acid flows involved in regulating the
into that pool. The contribution of that uptake was ultimate net excretion of H+, we see successively for
minimized by bidirectional passage to the perivenous muscle, kidney, and liver the emerging parts of a whole
pool (much of it presumably an exchange of glutamine arrangement for which appropriately regulatable
molecules for glutamine molecules), a pool assumed to transport appears to participate at each organ. How
contain most of the hepatic glutamine. these interdigitate with each other and with the rates of
The results of Cooper et al. (46) agree with those of purely enzymatic processes should gradually become
Taylor and Rennie (164) in suggesting a role for peri- more apparent.
venous hepatocytes in providing Glu- for hepatic gluta-
mine synthesis: [13N]Glu- taken up perivenously by the
liver proved a better source for [13N-amide]Gln synthe- E. Lysosomal Proteolysis and Lysosomal
sis than is [13N]Gl~- formed in the liver from exogenous Amino Acid Transport
Although the studies of Cooper et al. (46) do not One regulatable flow that deserves our attention is
segregate the times required for membrane transport that of amino acids released by proteolysis in the lyso-
from the times required for purely enzymatic reaction some. This net flow must contribute, in variable de-
stages, they show, along with other studies mentioned grees, to the net of flows from and to various cells, e.g.,
in this section, that we need to consider amino acid the flow of glutamine from muscle (see Fig. 5). Methods
flows from one portion of the liver to another along for separately identifying the regulatable component of
with the previously cited flows from one organ to an- lysosomal origin are impressively reviewed by Morti-

A Periportal hepatocytes Perivenous hepatocytes

jlutamine glutamat e
Uf *ea glutamate

.- 0
ammonia C /

I ammonia
-urea E

Par tol vein Hep a tic vein


FIG. 7. A and B: schemes offered Am monia

by Haussinger for cycling of intracellu-

lar glutamine during ureogenesis from
GIU t amine Glutamine
ammonia, showing roles of periportal
and perivenous hepatocytes. B adds
subcellular compartmentation for B Periportal Hepatocyte
Penvenous Heoatocyte
flows. CP, carbamoyl; CPS, carbamoyl
cvtosol cylosol
phosphate synthetase; Glnase, gluta-
minase. [A from Haussinger (77); B
from Hkssinger (78). Reprinted by
permission from Biochemical Society
Transactions, copyright (c)1987 The
Biochemical Society, London.]

NH; Gln



more and P&ii (117) (see also Refs. 116, 117a) in an hibition at low (0.5 X plasma) and high (4 X plasma)
article that provides most of the basis for this sum- concentrations in liver perfusates, but in the vicinity of
mary. Lysosomes may not be the only proteolytic source the plasma levels, inhibitory action is unexpectedly lost,
of amino acids, but for the present they appear to be the although it is retained by the complete amino acid mix-
most important and most explicable source. N-ethyl- ture. The loss was traced to the omission of alanine per
maleimide treatment in vivo modifies actomyosin and se, which at 0.5 mM restored the inhibition to the (1 X
other muscle proteins so that their degradation is accel- plasma) regulatory mixture. More than one regulatory
erated. Increases in such modified proteins led to in- site seems necessary to explain the modulation of lyso-
creases in cathepsin B and other muscle proteins in- somal proteolysis by amino acids. Two modes of coreg-
volved in protein turnover. Nonlysosomal proteolytic ulation by alanine can be recognized by their different
enzymes were concurrently not significantly in- responses to caloric deprivation (118). In muscle, where
creased (63a). leucine is rapidly transaminated, the equally effective
I omit from consideration the release of amino inhibitor, Z-keto-isocaproate, may well be the endoge-
acids from short-lived sources, since they conceivably nous regulator. In liver, in contrast, these two com-
represent cleavage products of unused portions of newly pounds are probably separately recognized. Other nu-
synthesized proteins, e.g., signal peptides. That compo- tritional factors, including caloric deprivation (116a),
nent is not subject to sufficient regulation to be of obvi- strongly affect lysosomal autophagy.
ous relevance to this review, although for experimental Transport became further part of the story of lyso-
purposes it can be blocked by leupeptin. somal proteolysis with the discovery of mediated trans-
If the sequestration of intracellular macromole- port of cystine from secondary lysosomes and its failure
cules occurs within discrete macrovacuoles and involves in cystinosis (61,132-134). Before that discovery, a pas-
uptake of distinct portions of cytoplasm, then the pro- sive role tended to be assumed for pores in the lyso-
cess is designated macroautophagy. To another class somal membrane in letting out the proteolytically gen-
representing instead sequestration of molecules, the erated amino acids. Undoubtedly a full account of the
term microautophagy is applied. Among the agents and transport of inorganic as well as organic ions, not only
conditions that affect the flow of products of lysosomal into lysosomes but probably also into secretory gran-
cleavage of long-lived proteins, the most relevant to this ules (see Ref. 62), and the various elements of the Golgi
review are probably the regulatory effects of circulat- apparatus is about to unfold. Cariappa and Kilberg
ing amino acids. These have been observed especially (25a) provide evidence for Na+-dependent system A
for the perfused liver of the rat. A perfusate containing transport and its hormone-stimulated synthesis in
insulin or the full range of amino acids, each at 10 times Golgi vesicles. I review elsewhere (33) the interesting
its plasma level, terminates macroautophagy and has comparisons and contrasts between eight amino acid
been used to study the regulatory properties of mi- transport systems so far detected in fibroblast lyso-
croautophagy, e.g., in fasted rats (116a). Stringent somes and some of the plasma membrane systems listed
omission of amino acids in perfusion of the liver induces in Table 1 (for review see Ref. 59b). Their separate ge-
a strong macroautophagic response as does glucagon. netic determination, as already implied for the case of
The amino acids reaching this tissue may be regarded cystinosis and supported by differences seen in every
as the primary regulators of autophagy; complete case so far from the systems of plasma membranes,
amino acid mixtures can evoke a full range of modified seems biologically predictable, considering the func-
breakdown in the absence of hormonal agents. Because tional differences in transport across these two bar-
hepatocytes represent 99% of the total cellular protein riers. One might have expected cystine transport out of
of the liver and because their sensitivity after isolation the lysosome to resemble cystine transport into the cell
to inhibitory effects of amino acids is not as great as by system xi, since we deal in both cases with transport
that of the perfused liver, the perfused organ has been from a cystine-rich phase into the cytoplasma kept cys-
preferred for the papers from Mortimores laboratory. tine poor by prompt reduction of entering cystine to
The full inhibitory effect of plasma amino acids can cysteine. Glutamate inhibition of cystine passage from
be obtained with leucine plus proline, each at four times the lysosome has not, however, been seen (61, 133), as
its plasma concentration. A combination that also in- would be expected for system xi. On second thought one
cludes tyrosine, phenylalanine, glutamine, histidine, realizes, however, that it would probably not assist the
tryptophan, and methionine is effective at lower levels lysosome in its need for net elimination of free amino
(117). Leucine is by far the most effective single amino acids to exchange cystine for glutamate. One should not,
acid, suppressing by 60% at 0.8 mM the response to however, exclude a possible role for inward lysosomal
amino acid deprivation. The amino acids branched on transport of one or more amino acids, for example, in
their P-carbon, isoleucine and valine, show no inhibi- receiving possible signals as to the overall amino acid
tory action. In electropermeabilized hepatocytes histi- economy. Transport inward has proved useful in study-
dine proved the strongest inhibitor, whereas leucine ing such lysosomal systems. Indications have already
proved to be much less so (156), supporting a role for cell been described for a regulatory role of circulating levels
entry in determining effectiveness. of various amino acids on the autolytic function of the
Alanine is assigned a permissive role in regulation lysosome (116a).
of hepatic microautophagy by the amino acids. The reg- Cysteamine and analogous thiol compounds have
ulatory mixture of eight amino acids gives identical in- proved therapeutically useful in cystinosis by allowing

escape of lysosomal cystine in the form of a substitute studied after inactivation of y-glutamyl transpeptidase
amino acid, generated by a sulfhydryl-disulfide ex- with a specific affinity-labeling reagent. None of the
change with accumulated lysosomal cystine. The novel three component amino acids nor gly l gly or
amino acid thus formed then passes out of the lysosome gly . gly gly compete with GSH for this transport. Only

by a different, persisting transport system c (33, 133)? the carboxy-terminal ethyl ester of the tripeptide has so
Harper et al. (76) have provided the first demon- far been identified as a competitor with GSH. Its ethyl
stration of a lysosomal transport system responsive to a group is removed from the ester only after its entry into
hormone pertinent to the cells in which it occurs. kidney or liver @a). Such esters have been explored as
FRTL-5 thyroid cells grown at least 48 h with thyrotro- vehicles for increasing cellular GSH levels. Glutathione
pin had sevenfold higher lysosomal tyrosine counter- is present at 5-7 mM in the liver, where it plays a major
transport activity than cells grown without thyrotro- role in various detoxification and regulatory processes.
pin. The carrier exchanging tyrosine also recognizes Its exodus from the liver accounts, however, for most of
phenylalanine and leucine, and countertransport of the hepatic GSH turnover. The exodus process is satu-
these amino acids is three- and sixfold higher, respec- rable and is subject to trans-stimulation and to a re-
tively, after thyrotropin treatment of FRTL-5 cells. versible, competitive inhibition by unconjugated biliru-
The contrasts among the specific amino acid trans- bin, by bromsulfalein, and by various analogous anionic
port systems of various subcellular organelles, includ- compounds (128). The canalicular plasma membrane
ing microsomal, lysosomal, and other Golgi-associated plays a role, along with the rest of the plasma mem-
vesicles, including also the synaptic vesicles held to brane of the hepatocyte, in the hepatocyte transport of
store glutamate, glycine, and y-aminobutyrate for Glu- and Gly. Although the functional significance of
neurotransmission, promise to be highly informative the process is undefined, it may represent the recovery
for reaching an understanding of the full range of of these amino acids from the cleavage of glutathione
the membrane capabilities for the transport of amino excreted into the bile &a, 119). Glutathione replenish-
acids (99a). ment is lower in the isolated perivenous than in the
periportal hepatocytes (96).
Since 1982 the role of amino acid transport in
F. Amino Acid Transport and Protective Function maintaining the protective cellular reservoirs of GSH
of Glutathione has been substantially explained and was recently re-
viewed by Bannai and Tateishi (13), whose laboratory
has contributed heavily to this progress. This tripep-
I. Rate-limiting steps in maintaining cellular
tide, y-L-glutamyl-L-cysteinylglycine, is characterized
glutathione levels by its reactive sulfhydryl group, which gives it the sig-
nificant function of protecting cellular molecules, par-
The interorgan flows of glutathione (GSH) were ticularly the membrane lipids, from various electro-
reviewed briefly by me in 1982 (29) as a significant ex- philic agents. These agents may arise from the physical
ample of membrane-determined flows and again by environment or they may be products of metabolism,
Curthoys in 1986 (48). For GSH, the flow is mainly from for example, from the peroxidative catabolism of pu-
the wide range of cells of its origin, especially the liver, rines. Glutathione serves, to a large extent under the
to the kidney where the peptide is degraded and its catalytic action of the selenium-containing enzyme, glu-
amino acids reused. In renal and intestinal basolateral tathione peroxidase, to reduce the hydroperoxides aris-
membranes a Na+-dependent active transport for GSH ing from oxidized species, such as superoxide and lipo-
has been identified (103). Vincenzini et al. (176) have, peroxides. These species may also arise from the respi-
however, detected a Na+-independent GSH transport in ratory burst of polymorphonuclear leucocytes and
brush-border membrane vesicles of rabbit small intes- macrophages in response to phagocytic stimuli, in the
tine. The transport of the intact tripeptide was best biosynthesis of prostaglandins and leucotrienes from
arachidonate, and by the autooxidation of arachidonate
2The thiol drug may be selected to need another persisting and other fatty acids. Enormously increased attention
transport system to gain access to the lysosome, for example, by has fallen on the maintenance of the protective function
including a structure so hydrophilic as to handicap its permeation of of cellular glutathione levels.
lipid barriers lacking a suitable transport system. This approach is Glutathione is synthesized in cells in general in two
suggested for mercaptoethylgluconamide (MEG) by results of Pisoni steps: the first reaction involves y-glutamylcysteine
et al. (132a). This drug or pro-drug may need to traverse the cyto- synthetase
plasm to form a product, possibly as suggested cysteamine itself, by
deamidation that can pass the lysosomal membrane. This require-
ment would mean also that it must be able to pass the plasma mem-
L-G~u- + L-CySH + ATP s T-L-G~uCYSH + ADP + Pi
brane, perhaps even selectively for preferred cells. A sialic acid trans-
porter newly shown for rat liver lysosomes to be effective for mono- and the second reaction involves glutathione synthetase
carboxylic acid sugars (108a) might help explain the new results of
Pisoni et al. (132a), one presumably a transport and the other an
enzymatic step, could conceivably give a pro-drug, such as MEG, im-
y-GluCySH + Gly + ATP e GSH + ADP + Pi
portant degrees of specificity to selected cells of the cystenotic pa-
tient, at the same time avoiding the stench of free cysteamine. The first reaction is taken to be rate limiting and

under feedback inhibition by glutathione. The possibil- reactive with dipolar amino acids of short to medium
ity that GSH synthesis may not always be limited ex- molecular length. Although considerable transmem-
clusively by a single precursor, e.g., the cysteine level, brane exchange occurs with trans-stimulation via sys-
but simultaneously also to a degree say by the glycine tem ASC, steep gradients tend nevertheless to be gen-
supply has come to be appreciated (93). Salter et al. erated. The cysteine flows and gradients are necessarily
(148) have calculated, for example, for the tyrosine influenced by the levels of other ASC substrates com-
economy of the liver the partition of the rate limitation peting for and exchanging by that same system.
between its transport and its enzymatic generation
from phenylalanine. For glutathione synthesis, the lim-
iting mediated transmembrane flow of the precursor 3. A relation between the transport of cystine
amino acids from outside the cell appears generally to and glutamate
concern largely cysteine and only scarcely if at all that
of glutamate or glycine. In contrast to erythrocytes, an as yet undetermined
number of other cells have, beyond the ASC system
transporting cysteine, a transport system for cystine
2. Complexities in the transport steps to glutathione (11, 108), an amino acid that is characteristically at
much higher levels than cysteine in the circulating ex-
Note, however, if a given cell other than an erythro- tracellular fluid. On entry into a cell a prompt subse-
cyte were able to generate an otherwise limiting amino quent reduction of cystine to cysteine may be expected.
acid from an available precursor (e.g., cysteine from This transport system, xi, selects the tripolar form of
methionine), then its cellular supply might be main- cystine, an anion with only one of the two amino groups
tained higher than that afforded by the balance be- protonated, and hence not the tetrapolar, isoionic form,
tween inward and outward transport of cysteine, possi- which is more abundant between pH 5 and 7. Cystine
bly shifting elsewhere the transport limitation for GSH also shares this transport system with the similarly
synthesis (e.g., to the transport of methionine). Note, on charged glutamate ion. The aspartate ion shows in con-
the other hand, that transport of cysteine could be rate trast an inadequate separation of the two carboxylate
limiting despite a high influx, if its outward flux were groups for a corresponding reaction, but a-amino adi-
high enough to cancel out much of the inward flow. I pate and homocystine compete with Glu-. The tripolar
would like to reemphasize that even the ability of a cell cationic form of cystine is apparently selected instead
to synthesize a metabolite, such as cysteine, does not as by the renal brush-border system missing or defective
a general principle guarantee the adequacy of its supply in cystinuria and the tetrapolar form by the system of
or concentration independent of membrane transport. the lysosomal membrane deficient in cystinosis (60,
Supplying the GSH needed for viability and func- 133). The system xi probably would not ordinarily en-
tion appears to be the main biological justification for counter amino acids with which it is reactive, other
retention by the mature mammalian erythrocytes of than cystine and glutamate. These two are exchanged
portions of its abilities at erythroid stages to take up by the system across the membrane of the human fibro-
amino acids selectively. Hemolytic anemias are asso- blast, and, at least in the fetal fibroblast, the two ap-
ciated with GSH deficiency (190). Human erythrocytes pear to be locked into exchange with each other (7).
take up cysteine mainly by a Na+-dependent system of Because the internal supply of one determines the in-
the ASC type (42,189). Extracellular cystine, the disul- flow of the other, it is hard to conceive that the cell
fide, scarcely has access to the human erythrocyte. In could impoverish itself of glutamate to gain the sulfur
other species, e.g., horses and sheep (59), a comparable amino acid.
cysteine-transporting system (designated asc) is Na+ Bannai and Ishii (9) point out that because the in-
independent and appears to be equivalent to the system tracellular pool of cystine is negligibly small and the
of the same designation in the nucleated erythrocytes of pool for glutamate is large, the physiological flows
the pigeon (172a). Phenotypes are known among race through this system are the uptake of cystine coupled to
horses with deficiencies in transport of this category. the exodus of glutamate, with the higher concentration
Inherited GSH deficiency in sheep of the Finnish of the latter providing at least part of the driving force.
Landrace breed is due to defective cysteine transport by These features should make xi an effective scavenger
a similar system. These sheep develop hemolytic ane- for circulating cystine. Cystine is taken up almost ex-
mia due to cysteine sulfoxide supplied by their winter clusively by this system by human fibroblasts; hence
forage. Glutathione levels are normal in reticulocytes, the adaptable system xc activity greatly influences the
liver, and kidney; hence the system concerned in this cellular levels of cysteine and GSH. Other consider-
deficiency appears to be expressed specifically in the ations led Bannai and Ishii (9) to the cycle pictured in
erythrocyte of the sheep. The concurrent accumulation Figure 8. Cysteine is inevitably released into the exter-
of cationic amino acids in these cells, useful for diag- nal medium by cultured fibroblasts and perhaps by
nosis, may point to an aspect of the incompletely un- other as yet unidentified cells in the intact organism.
derstood interaction of cationic and dipolar amino acids Its rapid external oxidation to cystine necessitates an
that might be anticipated for system ASC (167). inward redirection of the sulfur amino acid flow by
In various other cells, cysteine is also transported system xi. Glutamate is also taken up by various cells
by Na+-dependent system ASC, a system especially via other systems, e.g., the Na+-dependent system X;o

(107), energized in dependently of the cystine or cystei ne rophages, since the latter provides cysteine to the lym-
movement (Table 1); hence the glutamate gradient is phoid cells. Such effects point to nutritional flows be-
not likely to be dissipated by the transmembrane re- tween cell lines, which in turn may correspond to signif-
versible oxidation-reduction cycle. icant intertissue flows in the intact organism and
Cystine after uptake into cells is rapidly reduced to should help us to understand how these are established
cysteine, although probably not directly by NADH or and regulated. One wonders to what extent the cys-
NADPH. Instead the reaction appears to proceed by a teine-cystine cycle of Figure 7, A and B, is futile, except
sulfhydryl-disulfide exchange with GSH to form the as a correction for limits to the cellular expression of
mixed disulfide CySSG, followed by regeneration of system x;, i.e., to what extent if any the reducing equiv-
GSH by NADPH-glutathione disulfide (GSSG) reduc- alents represented by cysteine are to biological advan-
tase. tage expended extracellularly, rather than intracellu-
Bannai et al. (10) propose that the reason the activ- larly as GSH, in combating the hazards represented by
ity of system xi of the hepatocyte is low, as seen in O2 excess and environmental electrophiles.
primary culture compared with that in hepatoma cells Furthermore, glutamine uptake occurs in most
and fetal hepatocytes (107), is that an increase by in- cells via system ASC, not including the rat jejunum at
duction or adaptive regulation has not occurred. Cell I in the basolateral pole, according to Wilde and Kilberg
Figure 8 could then represent the unstimulated hepato- (184a). Glutamine shares this route with cysteine and
cyte in vivo, whereas cell II represents a cysteine source, various other amino acids (99), a competition that can
e.g., the fibroblast, and cell III represents the lympho- be added mentally to Figure 8. A significant flow of
cyte. Bannai et al. (10) raise the question as to how glutamine by system L should also be taken into ac-
many tissues in the intact organism engage in the cys- count (172). The ready percursorship of Glu- also pro-
tine-for-Glu- exchange and how many tissues lacking tects the cellular glutamate economy. External Glu- is
the active exchange process need to be fed either with a competitive inhibitor of system XL uptake of cystine.
cysteine by other cells possessing the x; exchange or It has long been known that the glutamate concentra-
with methionine as a precursor, a situation they pro- tion needs to be kept low to avoid nausea-producing
pose for the repressed hepatocyte. Watanabe and Ban- properties of amino acid mixtures infused for intrave-
nai (179) point out that lymphocytes (and lymphoma nous nutrition (106). On the other hand, the high gluta-
cell lines) cannot survive by themselves in culture me- mine content of cell culture media and even the abun-
dium in vitro, presumably because of their lack of sys- dance of glutamine in plasma undoubtedly act competi-
tem XL and a consequent inability to take up cystine; tively to limit the net influx of cysteine via system ASC.
however, these cells survive when cocultured with mac- This action probably accounts for the inhibition of
growth of fibroblasts in culture by 2.5 mM glutamine,
associated with decreases of both intra- and extracellu-
lar levels of cysteine (8). Excess external glutamine
could also stimulate loss of cellular cysteine via system
ASC. The importance of glutamine for the nourishing
interorgan flows it provides and also for H+-eliminat-
ing flows was already considered. In section IIH, I com-
ment on the service of glutamine, accumulated, for ex-
ample, in glial cells, in exchanging for other amino
acids across the BBB or the neurolemma.

4 Protective action of gzutathione against

electrophilic reagents

Stimulation of cystine transport via system ASC in

a dose-dependent manner into mouse spleen lympho-
cytes cultured in the presence of Z-mercaptoethanol,
after adding 25-400 PM cysteine, was found by Ishii et
al. (87) to be greatly enhanced by the presence of the
polyclonal mitogen, lipopolysaccharide W. of Salmo-
neZZa typhosa 0901. Mitogen stimulation of system ASC
of pig lymphocytes had already been recognized (18,19).
As mentioned earlier, system XL is not expressed in the
FIG. 8. Flows of sulfur amino acids between cells 1, 11, and III.
CeU I: a cell deficient in cystine uptake by exchange for glutamate and activated mouse lymphocyte.
hence nourished more largely by methionine. CeU II: a cell such as a Bannai et al. (12) showed an even more interesting
fibroblast receiving cystine by exchange via system xi for glutamate. influence on GSH levels on induction of cystine trans-
CeU III: a lymphocyte also lacking xi but more largely dependent on port by system XL by electrophilic agents. When cul-
uptake of cysteine via system ASC for a cysteine supply as provided tured human fibroblasts were exposed to diethylma-
by fibroblasts. [From Bannai et al. (lo).] leate, the expected depletion of GSH seen initially was

followed by a doubling of the cellular GSH level. The G. A Redw Cycle is Somewhat Anai!qpusly
powerful associated induction of system xi was inter- Dependent on Membrane Transport
preted as a protective action of the cells against an
electrophilic attack (13). Bromosulfophthalein was The interconversions of proline and A-pyrroline-5-
added to the list of electrophilic reagents that cause the carboxylate (P5C) in the human erythrocyte allows the
induction of cystine transport activity, again via system transfer of reducing equivalents across the plasma
XL and in the rat hepatocyte (12). membrane of the human erythrocyte, if proline is
When macrophages that had been elicited during 4 transported inward and P5C is transported outward,
days by the intraperitoneal injection of thioglycollate necessarily by different transport systems. The human
broth into the mouse peritoneum were cultured for 16 h, erythrocyte has a high P5C reductase activity as its sole
their rate of cystine uptake increased 400fold, whereas enzyme of the proline-ornithine-glutamate metabolic
the uptake of dipolar (neutral) amino acids did not system. The glucose-6-phosphate dehydrogenase of the
change (179). The properties of this uptake corre- erythrocyte plays a central role in this proposed shuttle
sponded in detail to those of system XL. As a conse- of reducing equivalents. Four Sardinian subjects whose
quence the GSH levels in the macrophages doubled, and erythrocytes were deficient in glucose-6-phosphate de-
a third compound, taken to be cysteine, was released hydrogenase failed to show the responses seen in eryth-
and accumulated in the culture medium within the time rocytes of control subjects, namely large increases of
frame of the study. Culture under hypoxic conditions pentose shunt activity, the formation of P-P-ribopyra-
depressed the induction of the transport activity; hence nose, and the incorporation of hypoxanthine into ino-
oxygen increase was probably the major factor contrib- sine monophosphate on treatment with P5C (188). A
uting to the induction of system xi. participation of this shuttle in the transfer of oxidizing
In summary, membrane transport appears to play potential from hepatocytes to erythrocytes was pro-
a dominant role in the maintenance, regulation, and posed (69); a parallel shuttle across the inner membrane
actual function of glutathione metabolism in its defense of the hepatic mitochondrion was also proposed (68). An
of cells against oxidative stresses. Transport system XL important component in the evaluation of the various
appears to be primarily influenced by these regulatory proposed transmembrane redox shuttles is the demon-
processes. Meister (112a) has newly reviewed glutathi- stration as to whether the enabling metabolite trans-
one metabolism and its selective modification. port systems are indeed coupled and under regulation.
Understandably the membrane transport of P5C
does not occur by an amino acid transport system [over
5. Cysteamine and other sufiydryl corrqwunds 20 amino acids have been shown not to inhibit P5C pas-
may redirect trawpurt of cystine sulfur sage into CHO cells (115)]. The unfortunate use of the
name oxoproline for pyrrolidone-5-carboxylic acid may
At least two situations have now been recognized in obscure the detail that P5C is not an amino acid. The
which a sulfhydryl compound, often cysteamine, reacts transport of P5C is saturable, temperature dependent,
with cystine by sulfhydryl-disulfide exchange to yield and sensitive to metabolic inhibitors (33a). It is Na+
the mixed disulfide of cysteine and cysteamine, the en- independent and has a pH optimum at 6.4, Furthermore
hanced membrane transport of which then occurs by a the uptake mechanism is linked to the transfer of re-
system different from that ordinarily serving for cys- ducing equivalents, i.e., to the conversion of PX to pro-
tine. In these cases the sulfhydryl compound may be line and the concomitant oxidation of reduced pyridine
said to function as a transmembrane delivery system nucleotide. Hence free P5C could not be detected in the
for cystine sulfur by means of the sulfhydryl-disulfide cells. The participation of proline transport does, how-
exchange stimulated by it. In the most striking case, ever, leave this exchange activity pertinent to this re-
cysteamine treatment serves to remove destructive ac- view.
cumulations of cystine from lysosomes in cystinosis,
with the mixed sulfide moving across the membrane by
a system designated c, which is specific to cationic H. Amino Acid Flows To and From the Brain
amino acids (133,134). In an earlier case the enhanced
movement of the sulfur across the plasma membrane The extracellular environment beyond the BBB is
into L-1210 mouse lymphoma cells treated instead with one we may expect to be relatively impoverished in
mercaptoethanol occurs as the predictable hydroxy-ter- amino acids, as indicated by results of analysis of cere-
minal mixed disulfide via system L (86). For a third brospinal fluid. This poverty is presumably a condition
related case (88), with the use of Wacetylcysteine necessary for special neuronal sensitivity to the release
treatment, the new route of entry made available into of certain of the amino acids into the synaptic junction
CHO cells has not been securely identified but is proba- in neurotransmission. The consequent necessity that a
bly also system L. The amino acid analogue produced by phase unusually dilute in amino acids is to be crossed
deacetylation and an sulfhydryl-disulfide exchange, in from BBB to neuron must inevitably figure in the main-
this case thialysine, is known to be transported into tenance and regulation of the nutritional flows to the
the Ehrlich cell via system L (28). Predictably, this de- brain.
livery of cystine sulfur into the CHO cell should favor We see evidence of the vulnerability of central ner-
the synthesis of GSH. vous tissue to overenrichment of its environment, espe-

cially in glutamate or aspartate, in the excitotoxic ac- monolayers then formed in lo-14 days. Each of these
tion of excesses of these amino acids to produce lesions disks was then placed so as to divide a diffusion cell into
in specific brain areas. Certain circumventricular tis- two side-by-side chambers. One of these, which received
sues lie outside the protection of the BBB. Cellular con- a pulse of [3H]leucine and [14C]sucrose, plus perhaps an
centrations of the dicarboxylic amino acids as high as amino acid analogue to be tested for a possible trans-
0.1 M have been produced in such tissues on feeding a port-competitive action, was called the donor compart-
single dose to 4-day-old mice (136). The excitotoxic ac- ment. The other compartment, the sample compart-
tion of certain pharmacological glutamate analogues is ment, received only 0.6 ml of the same assay buffer
believed to arise on a similar basis. The important ques- serving as solvent in the donor chamber. This assay
tion has been raised as to whether fetal transplants into buffer resembled Krebs Ringer bicarbonate buffer. The
the brain may fail to develop a BBB for the uptake of cellular monolayer was generally placed on the donor
macromolecules (23). The parallel question needs to be side of the cellulose disk. Aliquots of 0.2 ml were taken
asked as to whether the microvessels nourishing such from the sample chamber at selected time intervals
implants ultimately express the BBB properties for after the radioactive pulse, with this volume then being
transport of small molecules. replaced with 0.2 ml of fresh assay buffer. The total
The transport of amino acids and other metabolites volume in the diffusion cell was maintained at 3.0 ml.
and their analogues across the BBB is attributed to the The rate of passage of sucrose through such mono-
layer of endothelial cells that line the brain microves- layer-bearing disks decreased to a minimum after lo-14
sels. Early in vivo studies of this function introduced by days of prior growth of cells to form a monolayer. Even
Oldendorf (123) showed large differences in the mode of then the permeability to sucrose was 170 times that
mediation of passage of amino acids. The initial charac- observed for the BBB in vivo. Leucine passage was at
terization of a transport system, one closely resembling least five times faster when no monolayer was present
system L, was made in the intact animal by Wade and on the disk. The rate of passage of leucine across the
Katzmann (178). Although the passage of threonine monolayer was the same whether the cell monolayer lay
(note, not exactly a large neutral amino acid) across on the donor side or on the sampling side of the cellulose
the rat BBB occurs as elsewhere by system L, a minor disk. On this basis, Audus and Borchardt (4) conclude
part may occur by a suspected Na+-dependent ASC that the system L transport being observed (unconven-
component (168). Hargreaves and Pardridge (74a) also tionally called by them the leucine system) is in vivo a
attribute to system ASC the Na+-dependent, MeAIB-in- bidirectional, equilibrative transport. This conclusion is
dependent movement of dipolar amino acids from iso- one that should perhaps be challenged. I have already
lated human brain capillaries, including part of the discussed evidence for asymmetry in the operation of
movement of leucine but none of that of phenylalanine. system L in other occurrences, and biological advantage
. The microvascular endothelial barrier of the CNS seems inherently plausible for its being able to operate
differs from endothelia in other organs by having asymmetrically at the BBB.
highly resistant tight junctions with almost no fenes- As far as I have inquired, these authors may not
trations and few pinocytotic vesicles (21, 22). Besides have established that their conditions for the develop-
the mediated amino acid transport systems mentioned ment of the monolayer of microvascular endothelial
here, transporters of monosaccharides, monocarboxylic cells actually led to the physiologically polarized distri-
acids, choline and amines, purines, and nucleosides have bution of any transport mediation or indeed of any
been reported. The relation of such systems to the deliv- membrane function between the poles of the cells. Fail-
ery of a number of drugs across the BBB is under vigor- ure of expression of a transport system at either the
ous study. blood side or the brain side of the endothelium could
In vitro studies proceeded with suspensions of en- distort the polarity. Furthermore, the unphysiological
dothelial cells, but these preparations served only to leakiness of the monolayer obtained may have masked
measure cellular uptake of amino acids and not transfer degrees of directional asymmetry. Although experi-
across the endothelium. Because MeAIB uptake was not ments with epithelial monolayers may indeed favor
observed, it was reasoned that if an equivalent of sys- their expectation that any directional asymmetry ought
tem A served for amino acid transport across the BBB, to appear as the monolayer forms tight junctions, the
then that transport must be polarized so as to serve conclusion is too important to be left untested. The
only in the opposed direction toward the brain and meeting of such an expectation should be signaled by
therefore escape observation (16; see Ref. 80). Clearly a the asymmetric appearance of other transendothelial
proper in vitro study calls for a functionally polarized transport activity, involving, perhaps, the participation
preparation, as had earlier been obtained with the sup- of systems A, ASC, or Xio for which operational asym-
ported MDCK monolayers discussed in section IIA. Only metry would be hard to overlook. Furthermore, one
when the isolated endothelial cells were cultured on a would do well to look for transmembrane asymmetry by
porous support, first of nylon (20) and then more prom- comparing the action of a wide range of analogous sub-
isingly on regenerated cellulose disks coated with colla- strates, particularly under conditions producing truns-
gen (4, 5), could one expect to study transendothelial stimulation between contrasting analogues or coopera-
passage. tive effects between different transport systems, each
Endothelial cells isolated from bovine gray matter of which might contribute differentially at the two
were seeded onto the 15-mm cellulose disks on which faces of the endothelium. Even if asymmetry in system

L transport were not found, the search for the contrast tem A speaks for the choice bulky, whereas the trans-
of asymmetry in transport functions where it should be port selection of histidine in its dipolar rather than its
inevitable seems too interesting to delay. Conceivably tripolar form deserves a precision scarcely provided by
the necessary inward asymmetry in the flow of amino applying the adjective neutral for this basic amino acid.
acids to the brain, for example, for tyrosine or trypto- Assuming that we cannot expect at this time to escape
phan, arises not from the inherent asymmetry in the the habitual abbreviation in neurochemistry, LNAA, let
pertinent transport system but from the rate limitation us at least ignore the original and inappropriate evoca-
imposed by these low K, transports in conjunction with tions of the letters L and N. At least let L stand for
the sustained gradients from blood to brain. If so, regu- system L, an L that does not of course stand for leucine.
latability of the transport would appear to be biologi- The metabolism-resisting substrate, l-aminocyclohex-
cally important even if the mediation is inherently sym- ane-[llC]carboxylic acid, has been chosen in Agranoffs
metrical. laboratory for positron emission tomography of the
Audus and Borchardt (4) also note that the rate of brain (3).
leucine transport was increased by 148% when the sup- In a 1983 review, Christensen and Makowske (43)
ported monolayers had been pretreated with cyclohexi- urged a search for procedures that might allow presum-
mide. They propose that this effect was due to the pre- ably stoichiometric binding of Glu- as a neurotransmit-
vention by cycloheximide of diversion of a major part of ter to be distinguished from its transport into a neuron
the labeled leucine to protein synthesis during migra- or, if the two events should indeed prove parts of the
tion within the monolayer. This explanation could eas- same phenomenon, procedures that would demonstrate
ily be tested by substituting for L-leucine a labeled the association of these two. A demonstration by
amino acid less likely to be incorporated into protein. Kessler et al. (97) that a portion of the total fixation of
Among the observed potent inhibitors of L-leucine Glu- to brain membranes could be reversed by adding
transport, L-dopa or D-leucine might serve this purpose, external cystine promises to achieve one such distinc-
as might the model system L substrate BCH, which is tion. The chloride-dependent fixation of Glu-, pre-
built on the norbornane nucleus. DeFeudis et al. (53) viously regarded as not more than a binding but pre-
have emphasized the possibility of intermediate meta- sumably an information-carrying binding, proved by
bolic modification of amino acids within the BBB. that test to be a sequestration, which is to say a catal-
Cangiano et al. (25) have proposed that brain mi- ysis of transport to a compartment beyond the external
crovessels, serving as an in vitro model of the BBB, take membrane. The exchange of external cystine for inter-
up bulky dipolar amino acids in exchange for glutamine nalized Glu- was logically taken to identify the trans-
previously accumulated into these cells by a MeAIB-in- port with system XT for which, incidentally, chloride
hibitable route. Both aspects of the exchange, gluta- dependence has not to my knowledge been identified as
mine for leucine and leucine for glutamine, were dem- a universal feature. An analogue, e.g., homocystine,
onstrated, leading the authors to the suggestion that that might on binding stimulate Glu- exodus by ex-
this effect is due to cooperative interactions between change, e.g., from astrocytes, could conceivably occasion
systems A and L. Tests for such exchanges ought, if excitotoxic effects.3
possible, to be made in endothelial cells demonstrably After we consider passage across the BBB, impor-
functioning as an endothelium between compartments. tance falls next on the transport into nerve cells and
A somewhat similar exchange is believed to serve glia of the amino acids that have passed the BBB. This
in the function of glutamine exodus in removing the transport has often been studied in brain slices and
ammonium ion from the brain and hence in the defense vesicles (sometimes in vesical preparations that include
against hepatic encephalopathy. Intracellular gluta- free mitochondria). Uptake appears to occur, as usual,
mine tracer has, however, been observed not to ex- by a variety of transport systems. For example, Rhoads
change in mouse astrocytes with extracellular gluta- et al. (142) have shown the operation of a system analo-
mine, and astrocytes in culture have been shown to gous to that known as the iminoglycine system in epi-
consume glutamine at physiological levels as rapidly as thelial tissues. Separate, single high-affinity transport
they synthesize it (150, 196). Before these results are systems for 4-aminobutyrate, for taurine (these two
taken to exclude the proposed service of glutamine for with some overlap in affinity), for glycine, and by a
other amino acids within the brain, other external system common for Glu- and Asp- have been observed
amino acids might be tested, since the exchange might for the cortical synaptosomal tissue of mouse brain by
be obligatorily asymmetric. Also the behavior of the Debler and Lajtha (51). As methods are further im-
astrocytes in culture should be tested for its relevance proved for studying transport across the isolated endo-
in vivo. thelial cells, the need grows for distinguishing their
Without rejecting the established abbreviation, contribution to transport across the BBB from the ap-
LNAA, intended to refer to large neutral amino acids, I parently similar transport within compartments of the
would urge that it really should mean the bulky dipolar
amino acids, the original terms for system L substrates 3A further case of erroneous discrimination was uncovered by
(130), rather than shifting the system L terminology for Hollmann and Seifert (82a). The sensitivity to temperature, to high
neurochemistry only to the inappropriate adjectives, osmolarity, to ultrasonification, and also to detergent treatment and
large neutral. The selectivity for leucine, valine, and receptor solubilization together provided the new evidence for trans-
threonine, relative to norleucine, for system L over sys- port and not just binding.

brain beyond the BBB. To what degree is the amino acid flow of amino acids is a subject of controversy (see Ref.
uptake seen with brain slices due to the endothelial cells 187). For example, Striver and Clow (152) pointed out
of the microvessels? How may the stages by which nu- that PKU is a disease of a phenylalanine-rich environ-
trients reach the glia, the neuron, and organelles be ment, although an ideal way to decrease that richness
conveniently discriminated? and yet obtain a diet fully acceptable to school children
Although MeAIB inhibition of glutamine uptake by and pregnant phenylketonuric women seems not yet to
the microvessels of the BBB has already been men- have been achieved. The vulnerability of the CNS to
tioned, tests with MeAIB have failed to provide evi- dietary components added for effects on tastes (e.g.,
dence for an analogue of system A operating in a direc- monosodium glutamate and aspartame) is also of con-
tion across the BBB inward to the brain (see Refs. 16, cern, but the risk of their being detrimental is rather
64). Such results do not mean that only bulky dipolar more equivocal than the risks inherent in the strong
amino acids cross the BBB. In our emphasis of the accumulation of certain amino acids in congenital dis-
LNAA we could well overlook important mediated eases in which the catabolism of an amino acid is ob-
transport of less bulky amino acids by one or more structed. Phenylketonuria will serve for this discussion
transport systems with affinities too low to interrupt of resultant transport competition. The reality of this
the apparent linearity of the curve relating rate to con- risk, previously proposed for transport to brain, was
centration. Even among the high-affinity substrates, as first demonstrated for untreated PKU by Neame (X21),
much as 40% of the uptake of an amino acid has ap- although interestingly for brain slices rather than spe-
peared to be of the nonsaturable category (see Refs. 113, cifically for the BBB. McKean et al. (112) showed that
131). Note, however, that this categorization by no high circulating phenyalanine levels cause depletions of
means establishes movement exclusively by diffusion, the brain methionine, isoleucine, leucine, histidine,
as is so often erroneously assumed. This fallacy was tryptophan, and tyrosine. Progress toward the under-
demonstrated long ago by Wilbrandt and Rosenberg standing of this special vulnerability of the developing
(184). Rather this term usually means that we do not brain to competition by the elevated phenylalanine
yet understand why the movement fails to saturate concentration (for review see Ref. 95) reached a high
within the reasonable range of our tests. To the extent point with the demonstration by Oldendorf et al. (126)
that this component contributes other than artifac- that this amino acid is an inhibitor more effective than
tually, it surely must figure in the nutrition of the methionine for the brain uptake of [75Se]selenomethi-
brain. One may doubt that it really escapes all biologi- onine in the intact rat. Oldendorf et al. (127) proceeded
cal regulation. Even the component of transmembrane to show in 9 untreated PKU patients that brain uptake
migration identified by analogues as largely lacking in of this gamma-emitting amino acid analogue, measured
structural specificity (see Ref. 38) or significant tem- by external counting, was reduced sharply (P < 0.001)
perature sensitivity (41) and presumably all regulation compared with 12 matched mentally defective children.
may be appreciable. In a subsequent paper, Oldendorf (124) showed that in
As in other tissues, histidine becomes less and less rats the phenylalanine in serum of PKU patients signif-
a substrate of system L at the BBB and more and more icantly diminished the brain uptake of 14C-labeled tyro-
an inhibitor of cationic amino acid transport as histi- sine, leucine, isoleucine, methionine, tryptophan, histi-
dine is protonated. Its cationic form appears, how- dine, valine, 3,4=dihydroxyphenylalanine (DOPA), and
ever, not to be transported by the cationic system it in- threonine but not that of arginine, ornithine, or lysine.
hibits (125). Elsewhere, I (32) emphasized that when phenylala-
Lest we suppose that leucinemia disturbs BBB nine is acutely or chronically accumulated, it tends to
transport only by amino acid transport systems, note compete with the exodus of various amino acids from
that Banks and Kastin (6) have concluded that the cir- muscle, liver, and probably other tissues, leading to se-
culating leucine level modulates a proposed specific, questration of the affected amino acids in such tissues
saturable blood-to-brain transport of the tetrapeptide and presumably to consequent acceleration of their ca-
with opiate activity, Tyr . Pro Leu Gly amide desig-
l l l
tabolism. Experimental hyperphenylalaninemia in rats
nated Tyr MIF-1, across the BBB. At plasma levels leads not only to severe depression of the brain levels of
found in maple syrup urine disease, leucine inhibits this alanine, valine, methionine, isoleucine, and leucine but
transport but apparently not by a competitive sharing also to more than a doubling of brain glycine (54), a
of a common route. Tyrosine, isoleucine, or valine did result suggesting that inhibitory effects on amino acid
not share this inhibition. Regulation of the passage of exodus may occur also for this tissue. In this connection
this and related peptides in the blood-brain direction is it is interesting to note a tendency of free amino acids of
indicated by the results summarized by Banks and muscle to be increased in maternal PKU of the fetus
Kastin (6), although the nature of its mediation re- (65). These effects of phenylalanine excesses seem likely
mained undemonstrated. to contribute a second component to the amino acid
deprivation of the CNS, with disastrous consequences in
I. Dietary and Other Environmental Influences infantile PKU and presumably also in maternal PKU of
on Brain Nutrition by Amino Acids the fetus beyond those arising from phenylalanine
competition with amino acid transport at the BBB. The
Just how vulnerable the brain may be to dietary two components might prove additive for an amino acid,
variations and their competitive effects on the balanced such as leucine, responsive to both of these influences.

Although our biological interests bring our special emphasized the possible intermediate modifications of
attention to the abnormal passage of tyrosine and amino acids within the placental barrier. Obviously the
tryptophan and their close analogues across the BBB in amino acid flow to the fetus represents other than a
PKU or in leucinemia, especially since high levels of simple sharing in the flow from the maternal alimen-
phenylalanine and leucine interfere competitively with tary tract. In an earlier study in the pregnant guinea
these flows, we should remember that this transport pig (44), we proposed that a second sequential site
system is also highly sensitive to the BCAA and other where amino acids appeared to be concentrated on their
bulky amino acids and is at least as important to their way from maternal organism to fetal muscle was in the
flow to the brain. Brosnan et al. (24) have shown that final stage of their entries into the fetal tissues per se
rats experimentally made diabetic show slowed entry of from the fetal plasma. This proposal deserves a more
tyrosine and decreased brain levels of tryptophan as a detailed study with analysis of a wider range both of
result of transport competition by the elevated circu- amino acids and tissues, with consideration of the role
lating levels of branched amino acids, a competition played by various transport systems, e.g., system Nm of
that presumably applies also in the opposite direction. muscle. After delivery the fetal tissues are exposed to
Rigotti et al. (144) have proposed the use of methionine an abrupt impoverishment of the amino acid concen-
to modify therapeutically the mix of amino acids pass- trations of their extracellular environment. An exten-
ing the BBB. A carefully selected artificially bulky ana- sion of this perinatal change is seen in a decline from
logue might perhaps prove to serve quite as safely for childhood to maturity in 2aminoisobutyrate accumu-
this purpose, avoiding possible troublesome metabolic lation into muscle as seen in biopsy samples taken from
effects of excess methionine or other indispensable human subjects at elective surgical procedures. The
amino acids. phenomenon has been referred to as tissue amino acid
The older, less quantitative thinking led investiga- hunger decreasing with age (45). A recent paper by
tors for many years to be reluctant in accepting the Kudo et al. (102) showed, in extension of earlier find-
possibility of toxic action by excesses of an essential ings, in purified vesicles of the syncytiotrophoblast
nutrient. By now, however, we are so familiar with the brush border a wide-scope Na+-dependent mediation
modulating action of metabolites on metabolic reac- resembling system A. The weakness of its inhibition by
tions in general and the incompletely specific recogni- BCH, lysine, and leucine argues against an alternative
tion of each molecular species, especially that for mem- similarity to the wide-scope Na+-dependent system(s)
brane transport, so that accumulations of amino acids, of renal and intestinal brush borders and of the mouse
especially likely for essential amino acids, are more and blastocyst (175). A second Na+-dependent component
more readily understood as potentially injurious, par- contributes also to the uptake of MeAIB and proline,
ticularly when a large quantity of one is ingested alone. although a mutual inhibitory interaction with methio-
It has been proposed to avoid toxic effects of cys- nine uptake led to an unexpected assignment of methio-
teine fed or otherwise administered as the thiol per se nine transport also to this route. Note that the tropho-
by supplying this amino acid, where conditionally es- blast presents two membranes with probably different
sential, as L-Z-oxothiazolidine-4-carboxylate in which transport mediators at each surface, as for other epi-
the thiol group is masked (3). This anionic compound is thelia. Comparisons between the transport character-
transported to the brain and presumably to other tis- istics of membrane vesicles derived from the two sur-
sues perhaps by the same route as the analogous pyro- faces are needed to supplement direct tests for the two
glutamate. Within the cells of the mouse or rat brain it surfaces (181b). The cationic amino acids, as repre-
is enzymatically decyclized to form cysteine. Supplying sented by lysine, appear to be transported by system y+
the cysteine moieties as the thiazolidine derivative pro- in the artificially perfused guinea pig placenta (181b).
vides a vehicle for its transport to the brain, thus mini- It appears from studies of both surfaces of the tro-
mizing the toxic effects that may be seen when the phoblast that Glu- and Asp- are accumulated into the
amino acid is supplied as the thiol per se. Other thiols human placenta from both sides (81). This result em-
might usefully be supplied therapeutically as their phasizes the role trophoblast metabolism may play in
thiazolidine derivatives. net placental flows.
In concluding consideration of amino acid nutrition
for the fetal stage of our lives, note that more pertinent
J. Flows of Amino Acids Across Placental Barrier to this review would be further evidence, beyond that in
Between Maternal and Fetal Organism the reviews cited in the opening paragraph of this sec-
tion, as to the degree to which transport processes, rela-
An early set of observations suggesting that amino tive to enzymatic differences, contribute to the faster
acids are at higher levels in human fetal than in mater- growth of the fetus and the unequivocal flow of amino
nal plasma (e.g., see Ref. 137) fell so far from later acids to the fetus at maternal expense (44). Although
values, so the results were obviously vitiated by analyti- the phenylketonuric pregnant female clearly tolerates
cal problems, understandable for 1914. Subsequent the chronically elevated levels of circulating phenylala-
values not only demonstrated gradients but showed nine, the fetus is undoubtedly rendered much more
amino acid movements against gradients (44). A sym- sensitive than it otherwise would be to the perturba-
posium on this subject (192) was followed by a valuable tions of amino acid transport because of the trans-
review by Yudilevich and Sweiry (194). Young (191) has membrane enrichment of its internal environment.


TRANSPORT ACROSS BIOMEMBRANES I now return to my opening question (sect. IB) in a
unified form: Can we understand yet how a set of trans-
port systems, including several rather weak in specific-
Molecular explanations of regulation of amino acid ity, with overlapping affinities for as many as a dozen
transport are at best at provisional stages. For the so- amino acids can contribute along with enzymatic reac-
dium-independent systems for dipolar amino acids, reg- tions to the biological ne eds for va rious regu lated
ulation tends to be relatively inconspicuous but may amino acid flows? This remains a highly challenging
nevertheless prove more widely explanatory. In Esche- question. Information for one or another flow shows
richia coZ& transport by system LIV-J is regulated by that regulatable membrane transport participates in
the concentration of L-leucine in the growth medium modulating these various flows. One is encouraged to
(136b). Transport by this system is derepressed when a believe that explanations of that regulation when
leuine auxotroph is grown at rather low levels of leu- reached will include modulation of membrane trans-
tine, an effect reversed by adding leucine but no other port.4
amino acid. In section IG, I stressed evidence as to how
widely leucine, levels influence amino acid metabolism We need to imbed the knowledge of enzymes (and transport
in the higher animal. By use of temperature-sensitive systems) in a broader panoply of their functional relation-
leucyl-tRNA synthetase mutants of E. coZi, it could be ships in the cell (and the organism).
shown that leucine is effective to the degree that it Joshua Lederberg
permits the charging of leucyl-tRNA, with that product Foreword. In: Kornberg, A. For Love of Enzymes.
rather than free leucine being the effector. Its action is Harvard Univ. Press, 1989
held to arise from an attenuation of transcription in-
volving the termination factor rho (130a). It is the close I gratefully acknowlege critical readings by and advice from
coupling between transcription and translation in pro- Drs. Stanley Schultz and Lon Van Winkle. I am also grateful to Jac-
caryotes that allows regulation by attenuation to be queline Benson for detailed, repeated typings of the manuscript.
demonstrated, although attenuation does not in all in- Experiments in my laboratory were supported by National In-
stitutes of Health Grants DK-32281 and HD-01233.
stances depend on rho. Derepression of LIV-J transport
was found in strains mutated in rho or 1euS in support
of the proposed attenuation mechanism (136~). REFERENCES
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