Hormone-controlled CAMP-mediated fluid secretion

in yellow-fever mosquito

DAVID H. PETZEL, MARGARET M. BERG, AND KLAUS W. BEYENBACH

Section of Physiology, Division of Biological Sciences, Cornell University, Ithaca, New York 14853

PETZEL, DAVID H., MARGARET M. BERG, AND KLAUS W. tubules and in intact mosquitos. Our in vitro studies
BEYENBACH. Hormone-controlled CAMP-mediated fluid secre- show that I) CAMP lowers the fractional resistance of

Downloaded from http://ajpregu.physiology.org/ by 10.220.32.247 on September 16, 2016

tion in yellow-fever mosquito. Am. J. Physiol. 253 (Regulatory the basolateral membrane supporting our previous ob-
Integrative Comp. Physiol. 22): R701-R711, 1987.-Evidence servation of an increased Na conductance of that mem-
is presented for hormone-controlled adenosine 3’ ,5’-cyclic brane in the presence of CAMP (24), 2) the secretagogues
monophosphate (CAMP) -mediated NaCl diuresis in Malpigh-
ian tubules of the blood-feeding yellow-fever mosquito Aedes theophylline and forskolin increase the intracellular
aegypti. Studies in isolated Malpighian tubules reveal that CAMP concentrations of isolated Malpighian tubules in
CAMP added to the peritubular bath selectively stimulates parallel with the stimulation of NaCl and fluid secretion,
NaCl secretion and not KC1 secretion by increasing the Na and 3) two diuretic peptides, which we have isolated from
conductance of the basolateral membrane of primary cells. the head of the mosquito (19), also increase intracellular
These effects are duplicated by forskolin and theophylline in CAMP concentrations in parallel with stimulating NaCl
parallel with increased intracellular concentrations of endoge- and fluid secretion in isolated Malpighian tubules.
nous CAMP. Two natriuretic peptides that we have isolated by Turning to the intact animal, we have found that
high-pressure liquid chromatography (HPLC) methods from during the blood meal increased rates of NaCl and fluid
mosquito heads also increase NaCl and fluid secretion in iso- excretion from the animal are associated with elevated
lated Malpighian tubules together with increased intracellular
levels of CAMP. These results are consistent with a mechanism intracellular CAMP concentrations in their Malpighian
of NaCl diuresis in which the natriuretic peptides and CAMP tubules. These experiments demonstrate the central role
are respectively the primary and secondary messengers that of CAMP in mediating NaCl and fluid secretion in vitro
couple the ingestion of a blood meal to the excretion of the and in vivo. Accordingly we propose that the initial
unwanted salt and water fraction of the meal. This hypothesis diuresis that begins while the mosquito is feeding on
is supported by in vivo studies that reveal elevated intracellular blood is mediated via CAMP, which stimulates NaCl and
CAMP levels in Malpighian tubules at the time of maximum fluid secretion in the Malpighian tubule. In this process
NaCl diuresis. CAMP plays the classical role as second messenger. We
believe the diuretic peptides, which we have isolated from
salt and water balance in insects; mechanism of epithelial mosquito heads, to be the primary messenger that links
sodium secretion; intracellular adenosine 3’,5’-cyclic mono- the intake of the blood meal to increased intracellular
phosphate; Aedes aegypti; theophylline; forskolin; mosquito CAMP and to accelerated rates of NaCl and fluid secre-
natriuretic factors
tion in Malpighian tubules.

MATERIALS AND METHODS

REPRBDUCTION IN THE FEMALE Aedes aegypti
MOSQUITO
includes the taking of a blood meal from which she
derives proteins for oogenesis. With the blood meal, the
mosquito acquires large quantities of NaCl and water
(the plasma fraction of the meal) that the mosquito does
not require and that compromises flight maneuverability
and efficiency. The mosquito possesses renal mecha-
nisms that eliminate a large fraction of this NaCl and
water load before flying away. This is accomplished by a
potent NaCl diuresis that commences even before the
blood meal has been completed.
Previous in vivo studies have shown that the NaCl
diuresis stems from accelerated rates of NaCl and fluid
secretion in the Malpighian tubule (30-32) and that
adenosine 3’,5’-cyclic monophosphate (CAMP) plays a
central role in mediating the stimulation of NaCl and
fluid secretion. In the present study we have further
delineated the role of CAMP in regulating secretion rates.
The studies have been conducted in isolated Malpighian
0363-6119/87 $1.50 Copyright
Mosquitos. Adult female mosquitos of the speciesAedes
aegypti (3-5 days postemergence) were used. The mos-
quitos were raised as described by Shapiro and Hagedorn
(25) and maintained on 3% sucrose solution. Photoperiod
was 16:s light-dark cycle. All experiments took place
during the light period at room temperature (2245°C).
Isolation of Malpighian tubules. After the mosquito was
cold anesthetized, the Malpighian tubules were dissected
and kept in Ringer solutions containing (in mM): 150
NaCl, 25 N-Z-hydroxyethylpiperazine-N’-Z-ethanesul-
fonic acid, 3.4 KCl, 7.5 NaOH, 1.8 NaHC03, 1 MgS04,
1.7 CaClz, and 5 glucose. The osmolality of the Ringer
was 308 t 3 mosmolol/kg Hz0 (n = 10). The pH of the
Ringer was 7.1.
Fluid secretion rates. The rate of fluid secretion in
isolated Malpighian tubules was measured by the method
of Ramsay (19, 30) as described previously. Briefly, the
blind end of the tubule was placed in a Ringer droplet
0 1987 the American Physiological Society R701
R702 CAMP-MEDIATED FLUID SECRETION
(50 ~1) under oil, and the open end of the tubule was
pulled into the surrounding oil so that secreted fluid
flowed from the open end and accumulated as a droplet
separate from the incubating Ringer solution. After an
initial 30-min control period, an aliquot of the incubating
Ringer drop was replaced with an aliquot containing the
secretagogue of interest. The nanoliter fluid volumes of
secreted fluid (V) were calculated using Eq. 1, assuming
a prolate spheriod of the secreted fluid, where s and 1
are, respectively, the short and long axis of the secreted
droplet measured in units of micrometers
rIs21
V -- (0
6 X (lo6 pm3/nl)
Tubule electrophysiology. The transepithelial voltage
across the Malpighian tubule wall was measured by per-
fusing single tubules in vitro by the method of Burg et
al. (5). In these experiments the blind end of the tubule
was removed, and an -8OO-PM-long tubule was cannu-
lated with a double-barrel perfusion pipette. The tubule
was perfused with Ringer at rates ranging from 5 to 10
nl/min so that transepithelial voltages were measured in
the absence of transepithelial ionic concentration differ-
ences. The transepithelial voltage was measured with
respect to ground in the bath. Transepithelial resistance
was measured using cable analysis (2). Intracellular elec-
trical potentials of principal cells of the perfused Mal-
pighian tubule were measured with conventional micro-
electrodes (Omega-Dot borosilicate glass, 1.0 mm OD,
Frederick Haer, Brunswick, ME) filled with 3 M KC1
(electrode resistance = 170 MQ). Fractional membrane
resistances were determined as described previously (2).
The fractional resistance of the basolateral membrane
(fRbl) is defined as the voltage divider ratio; it is the ratio
of the basolateral membrane resistance over the total
transcellular resistance according to &. 2 where AVbl
and A& are, respectively, the basolateral membrane
voltage deflection and the transepithelial voltage deflec-
tion consequent to a transepithelial current pulse and
where &,I and R, are, respectively, the basolateral and
apical membrane resistances
A vbl Rbl
f Rbl
= av, = R, + Rbl
(2)
Blood meaL.A female mosquito was placed in a small
test tube, and the mouth of the tube was capped with a
piece of fine netting. The insect fed readily when the
netted end of the tube was placed against the investiga-
tor’s forearm. The meal was always complete within 5
min, insects engorging between stages 4 and 5 on the
scale established by Pilitt and Jones (22). The average
time of a meal (from time fascicle entered skin until time
it was removed) was 2.6 t 0.2 min (60) (mean t SE, n =
number of insects). After the blood meal, mosquitos were
allowed to move about in the test tube at room temper-
ature for periods ranging from 0 to 120 min. At the end
of the timed period the insects were cold anesthetized at
0°C and their Malpighian tubules were quickly dissected.
Mosquitos that had not been offered a blood meal served
as controls.
Time was noted at several steps throughout the pro-
IN MALPIGHIAN TUBULES
cedure: I) at the beginning of the blood meal time 0, 2)
at the end of the meal, 3) when the homogenizer, con-
taining the dissected tubules, was submerged in liquid
nitrogen, and 4) when ethanol was added to the homog-
enizer (Fig. 1).
As shown in Fig. 1, diuresis begins while the mosquito
is still taking the meal. Due to the time required for 1)
the blood meal, 2) onset of anesthesia, and 3) dissection
of the Malpighian tubules, the CAMP concentrations in
the tubules could not be measured earlier than -4 min
after the onset of meal or -2 min after the diuresis has
begun. Diuretic urine flow rates were measured at times
up to 120 min after the beginning of the meal (32) as
were the intracellular CAMP concentrations in the Mal-
Downloaded from http://ajpregu.physiology.org/ by 10.220.32.247 on September 16, 2016
pighian tubules.
Extraction of CAMP. Intracellular CAMP concentra-
tions in Malpighian tubules were measured using a 1125-
labeled CAMP radioimmunoassay kit (New England Nu-
clear, Boston, MA). In brief, five Malpighian tubules
from one female mosquito were dissected free from the
hindgut and adhering tissues. The tubules were trans-
ferred to a small volume homogenizer (Fisher Scientific,
0%416C, Rochester, NY) containing 10 ~1 of Ringer
solution (control). In some experiments the secretagogue
theophylline or forskolin was added to measure the ef-
fects on intracellular CAMP concentrations. The tubules
were then incubated at room temperature for 5 min in
the absence or for 30-60 min in the presence of secreta-
gogues. The homogenizer was then submerged in liquid
nitrogen, with the intent to inhibit both the adenylate
cyclase and phosphodiesterase activities. The interval of
time when the insect was first anesthetized until the
isolated Malpighian tubules were submerged in liquid
nitrogen was 1.37 t 0.47 min (39) (mean t SE, n =
number of insects). The tubules were then homogenized
with a hand-held pestle, and 100 ~1 ethanol were added
to precipitate protein and to extract CAMP (27). The
pestle was rinsed twice with 100 ~1 ethanol, and the
homogenate was centrifuged for 15 min at 2,500 rpm
(4°C). The supernatant was removed to a 12 x 75-mm
test tube and evaporated to dryness in a 100°C water
bath.
CAMP assay. The dried CAMP extract was redissolved
in 100 ~1 sodium acetate buffer (pH 6.2). The CAMP
sensitivity of the assay was increased to the femtomole
range by adding to each sample 5 ~1 of a 2:l mixture of
triethylamine and acetic anhydride. Following acetyla-
tion, 500 ~1 of the sodium acetate buffer were added to
anesthetrze, dissect
I
tubules submerged in lrquld nitrogen
i
1 1 Ii
0 2 4 6 8 10 12 100 120
Time (minutes)
FIG. 1. Temporal relationship between blood meal, diuresis, and
measurement of CAMP content in Malpighian tubules of female Aedes
mosquito. Time is measured with beginning of blood meal at 0 min.
 CAMP-MEDIATED FLUID SECRETION IN MALPIGHIAN TUBULES R703

each tube, making a 1:6 dilution of each sample. A
standard curve was generated by diluting a 5,000-pmol/
ml CAMP standard to the following CAMP concentra-
tions: 0.1, 0.25, 0.5, 1.0, 2.0, and 4.0 pmol/ml. The CAMP
lz51-labeled tracer and prereacted antibody complex were
then added to each tube. Following a 17-h incubation at
4”C, the standards and samples were centrifuged for 15
min at 2,500 rpm (4°C). The supernatants were dis-
carded, and the pellets were counted in a Beckman
Gamma 3000 radiation counter to determine the amount
of bound tracer. Results were calculated by interpolation
from a semilog plot of the CAMP standard samples.
Normalized percent bound of the 1251-labeledtracer is
plotted against CAMP concentration in picomoles per
until the dissected tubules were submerged in liquid
nitrogen. Because of the variability in the time required
for mosquitos to feed, we have pooled the CAMP data
points into 2-min intervals (Fig. 7). Standard errors for
the pooled time intervals were consistently very small
(CO.9 min), thus the horizontal error bars in Fig. 7 were
omitted. All other averages are reported as means t SE
(n = number of sets of tubules). The significance of
difference was examined using the Student’s t test. Dif-
ferences were considered significant for P < 0.05.
RESULTS
Effects of peritubular DB-CAMP on electrophysiology of
Malpighian tubules in uitro. In studies of in vitro perfused

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milliliter. Correlation coefficients were typically close to Malpighian tubules, the transepithelial voltage is lumen
-1.0. positive (Table 1, also Refs. 3,19,24, and 32). The voltage
Recovery determinations. It was of interest to deter- hyperpol .arizes to even more lumen-positive values in the
mine whether any CAMP activity was lost during the presence of DB-CAMP (Table 1). In the absence of major
extraction procedure. Recovery of CAMP was measured transepithelial ion concentration differences the hyper-
using a [3H]cAMP recovery marker (New England Nu- polarization of the transepithelial voltage indicates one
clear). One microliter of the marker was added to the of two effects: the stimulation of cation transport in a
Malpighian tubule sample after homogenation and be- secretory direction or the stimulation of anion transport
fore centrifugation. The supernatant was transferred to in a reabsorptive direction or a combination of these two
a 12 x 75mm test tube and evaporated to dryness as effects. However, since DB-CAMP stimulates the secre-
discussed above. After the samples were redissolved in tion of NaCl (Table 1 and Ref. 31), it follows that the
200 ~1 sodium acetate buffer, 100 ~1 were removed and hyperpolarization primarily reflects the stimulation of
counted in a Beckman liquid scintillation counter. Re- cation transport in a secretory direction, in particular
covery was found to be 91 t 3% (5) (mean t SE, n = the active transport of Na from the peritubular bath to
number of sets of tubules). the tubule lumen, as we have shown previously (24, 30,
Protein assay. To normalize the intracellular CAMP 31) .
content to the biological mass of the tubule, we measured The hyperpolarization of the transepithelial voltage by
the protein content of Malpighian tubules using the DB-CAMP is accompanied by a significant reduction of
Lowry protein assay (28). Ten Malpighian tubules from the transepithelial resistance, indicating that the stimu-
two female mosquitos were isolated and homogenized in lation of active Na transport involves a major conduct-
10 ~1 Ringer solution. One milliliter fresh alkaline copper ance change in the epithelium (Table 1). Probing the
reagent (0.1% CuS04*5 H20, 0.2% Na-tartrate in 2% transcellular voltage profile with conventional intracel-
Na2C03 and 0.1 N NaOH) was then added to the homog- lular microelectrodes, we have iden .tified one major con-
enate. After a lo-min incubation period at room temper- ductance change at the basolateral membrane (Table 1).
ature, 100 ~1 of Folin-Ciocalteu reagent (Sigma) were
added and the absorbance read at 750 nm. A standard TABLE 1. Effects of DB-CAMP on transepithelial
curve of the protein concentrations vs. absorbance at 750 transport and electrophysiology of isolated
nm was prepared using bovine serum albumin concentra- Malpighian tubules
tions ranging from 5 to 20 rig/ml.
Control DB-CAMP, 1 mM
Pharmacological agents and peptides. Theophylline and
dibutyryl-CAMP (DB-CAMP) were purchased from Sigma Fluid secretion studies
(St. Louis, MO), and forskolin was purchased from Cal- Fluid secretion rate, nl/min 0.65kO.05 (18) 2.17t0.17 (18)*
biochem (San Diego, CA). Native secretagogue peptides Electrolyte secretion rates,
were isolated from mosquito heads as described previ- pmol/min”f
Na 691t7 (18) 454t43 (7)”
ously (19). K 68t8 (18) 43t6 (7)
Statistical analysis. Whenever possible the experi- Cl 118klO (16) 451k50 (5)*
ments were designed to allow paired t test analysis, where
Perfused tubule studies
each Malpighian tubule is used as its own control (as in
Transepithelial voltage, mV 30.1t7.3 (7) 64.1kl3.3 (7)*
studies of the effects of secretagogues on tubule electro- Basolateral membrane voltage, -77.1t6.3 (7) -23.8k2.0 (7)*
physiology and rates of fluid secretion). When single mV
tubules could not be used for an initial control period, as Apical membrane voltage, mV -107.2t7.2 (7) -87.9H4.8 (7)*
in the studies of the effects of secretagogues on intracel- Transepithelial resistance, 14.9t2.6 (7) 9.9t1.9 (7)*
k&cm
lular CAMP concentration (involving homogenation), the Fractional resistance of 0.63t0.11 (7) 0.33kO.08 (7)*
t test for the significance of the difference of two sample basolateral membrane
means (control and secretagogue treated) was used. Values are means t SE for no. of tubules indicated in parentheses.
We have expressed the in vivo CAMP data as a function DB-CAMP, dibutyryl-cyclic AMP. * Significantly different from control,
of the time interval from the beginning of the blood meal paired t test (P < 0.001). t Data from Ref. 31.
R704 CAMP-MEDIATED FLUID SECRETION IN MALPIGHIAN TUBULES

Under control conditions a fractional resistance of 0.63 12

indicates that the basolateral membrane resistance rep-
resents 63% of the total transcellular resistance. The
fractional resistance drops to 33% in the presence of DB-
CAMP. This drop may have several meanings (see Eq. 8
2): either a large increase in the resistance of the apical
membrane or a large decrease in the resistance of the
basolateral membrane or combination of these two pos- a
sibilities. The first possibility is ruled out because a major z
0 4
increase in the apical membrane resistance would have
led to an increase and not a decrease in the total trans- ;z
epithelial resistance (Table 1). Hence it appears that the z
dominant effect of DB-CAMP on tubule electrophysiology cs
is the decrease of the basolateral membrane resistance. LAJ h 0.2 0.4 0.6 0.8

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The decrease of the basolateral membrane resistance
can be attributed to a large extent to an increase of the
membrane Na conductance as previously observed (24).
Consistent with this interpretation is the marked depo-
larization of the basolateral membrane voltage from -77
to -24 mV, which can be expected from the entry of Na
from the bath into the cell (Table 1).
CAMP content in Mulpigkiun tubules of non-blood-fed
mosquitos. The measurement of the [cAMP]i content in
33 different sets of five Malpighian tubules each shows
that [cAMP]i in non-blood-fed mosquitos (control) is
normally distributed (Fig. 2). Mean [cAMP]i is 0.43 pmol
Per tubule with a standard deviation of 0.13 pm01 per
tub ule (n = 33). The large standard deviation may stem
in part from different [cAMP]i in different tubules and/
or differences in the biomass of individual tubules. To
avoid errors stemming from the latter possibility, we
normalized the [CAMP]; to the protein content of Mal-
pighian tubules. The protein content per tubule is also
normally distributed, and mean protein content is 1.28
pg protein per tubule with a standard deviation of 0.14
pg protein per tubule (n = 68, Fig. 2) Since CAMP and
protein content per tubule are both normally distributed,
the normalization to milligrams of protein is justified.
Accordingly, [ cAMP]i in Malpighian tubules from non-
blood-fed mosquitos is 339 pmol/mg protein (Fig. 2).
Effect of theophylline and forskolin on fluid secretion
and intracellular CAMP content in Malpighian tubules in
uitro. Since from past experiments we know that the
addition of exogeneous DB-CAMP to the peritubular bath
of Malpighian tubules has clear and consistent signifi-
cant effects on stimulating NaCl and fluid transport
(Table 1 and Ref. 30), it was of interest to see whether
endogenous CAMP, generated by the epithelial cells of
Malpighian tubules themselves, is able to do the same.
Accordingly, we tested the effects of theophylline (an
inhibitor of phosphodiesterase) on fluid secretion and on
[CAMP];. Under control conditions, in which tubules
were incubated in saline alone, [cAMP]i was 339 t 20
pmol/mg protein (Fig. 3A). On the addition of theoph-
ylline, intracellular CAMP content significantly in-
creased in parallel with the significant increase of fluid
secretion (Fig. 3A).
Next to theophylline, another way to increase intra-
cellular CAMP concentrations is to stimulate the ade-
nylate cyclase system with forskolin. As shown in Fig.
3B, forskolin significantly increased the rate of fluid
co ”
m
pmol cAMP/tubule
0
IA 24
0
u
w 16
m
r
3
21
8
Dg protein/tubule
FIG. 2. Normal distribution of adenosine 3’,5’-cyclic monophos-
phate (CAMP) (A) and protein (B) contents in Aedes Malpighian
tubules. Smooth curves were drawn according to equation for normal
distribution.
secretion in concert with the significant increase of the
intracellular CAMP content. The concentration of for-
skolin used in this series of experiments was 50 PM
which, as later experiments showed (Fig. 4), is a supra-
maximal dose. The forskolin concentration for half-max-
imum effects on fluid secretion is 10 PM (Fig. 4).
Effect of native mosquito peptides on fluid secretion and
intracellular CAMP content of Malpighian tubules in vitro.
In previous studies we have isolated three peptides from
the head of the mosquito that affect the electrophysiology
of Malpighian tubules with or without effects on electro-
lyte and fluid secretion (19). The three peptides have
molecular weights ranging between 1,900 and 2,700 (20).
The peptides are called factors I, II, and III. We believe
factor III to be the mosquito natriuretic factor (MNF)
because it mimics all effects of DB-CAMP: it selectively
stimulates a NaCl-rich and fluid secretion while hyper-
polarizing the transepithelial voltage (19) via its effect
on the Na conductance of the basolateral membrane.
Measuring the effects of factors I, II, and III on the
rates of fluid secretion in one set of tubules and the
effects on [cAMP]i in another set of tubules, we observed
that the native peptides, which stimulate fluid secretion
(factors II and III), also stimulate intracellular CAMP
 CAMP-MEDIATED FLUID SECRETION IN MALPIGHIAN TUBULES R705

pco.00 1
T

30min 6Omin FIG. 3. Theophylline (10 mM CAMP mea-

sured after 30 and 60 min) (A) and forskolin
Control Theophylline Control Theophylline (50 PM measured after 5 min) (B) stimulate
intracellular CAMP content and rates of fluid

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secretion in isolated Malpighian tubules. Val-
ues are means t SE; numbers in parentheses

p<o.oo 1 13000 indicate number of tubules in Ramsay fluid

secretion assay and number of radioimmu-
noassays in CAMP determination. See Fig. 2
for definitions.
p<o.oo 1
. . .a
. . . .
. . . a
. . . .
. . . II
. . . .
. . . . . . . (I
b . . . . . . . .
. . . . . . . a
, . . . , . . . .
. . . . . . 0 a
, . . . . . . .
. . . . . . l 4
, . . . a . . . .
. . . . . . . a
1 . . . ti . . . .
. . . . . . .
1 . . . a . . . .
. . . . . . . a
, . . . , . . . .
. . . . . . . a
. . . , . . . .
‘. . . . . . . a
1. . . , . . . .
. . . . . . . a
1 . . . , . . . .
. . . . . . . a

I
1 . . . a . . . .
. . . . . . .4
1. . . ,
1.
. .
.
. .
. q
l. .o.o.o,
. . .
1
.
.
. .
.
.
. .
.
. .
a . .. . .. . .0. aa
. 11-1
. . .
Control Forskolin Control Forskolin

protein content of the high-pressure liquid chromatog-

0
I I
20
I
10
Forskolin
FIG. 4. Dose-response curve of effect of forskolin on fluid secretion.
Values are means t SE; number in parentheses indicates number of
tubules.
concentrations (Fig. 5, B and C). Factor I, which does
not increase electrolyte and fluid secretion, fails to in-
crease intracellular CAMP concentration (Fig. 5A). Of
the two natural secretagogues, factor III (Fig. 5C) appears
to be the more potent natural diuretic. The concentra-
tions of native peptides used in this series of experiments
were 56 pg/ml for factor I, 108 pg/ml for factor II, and
100 pg/ml for factor III. These values reflect the total
raphy (HPLC) fraction and not necessarily the concen-
T tration of the pure diuretic peptide.
Correlation of intracellular CAMP levels and fluid secre-
tion rates in Malpighian tubules in vitro. Since in the
present study the secretagogues theophylline, forskolin,
and factors II and III all increased fluid secretion rate
together with an increase in [cAMP]i, it is possible to
examine the relationship between [cAMP]i and the rates
of fluid secretion. In Fig. 6 the plot of [cAMP]i vs. fluid
secretion rates reveals a saturation curve with a steep
slope. In the absence of secretagogues (control condi-
tion), [cAMP]i is 339 pmol/mg protein, and fluid secre-
tion rate is 0.56 nl/min. Fluid secretion rates saturate at
-2 nl/min in the neighborhood of 900 pmol cAMP/mg
protein. Hence, CAMP-controlled rates of fluid secretion
30
I
40
I I
50
are mediated over a narrow range of -600 pmol CAMP/
mg protein, and intracellular CAMP levels in excess of
(uM) 1,000 pmol/mg protein do not stimulate fluid secretion
any further.
Forskolin as well as factor III (MNF) increased
[cAMP]i far more than is necessary for maximal stimu-
lation of fluid secretion (Fig. 6), indicating an upper limit
of fluid secretion rate. This limit may be set by the
protein kinase activity, membrane permeabilities, and
rates of transporting ion pumps. In addition, MNF or
any other CAMP-mediated secretagogue may have mul-
tiple cellular effects, some of which could limit the rate
of fluid secretion.
Post-blood meal diuresis and intracellular CAMP con-
 CAMP-MEDIATED FLUID SECRETION IN MALPIGHIAN TUBULES

3.0
h Factor III
Forskolin (MNF)
.-:
\ Factor II (8,4) (2797)
r
- 2.0 - (26,7) )---1------ -111-1111- -__
.@
n.s.
.-s +I
*

d
n.s. aI +

n
l’“’ 0
;;
r’
:

: 1.0
Control Factor I Control Factor I . Theophylline
- IE
u.- !’ (18,3)
3
C:“trol

:.- I8
z
(108,331

p<o.oo1 0 1000 2000 3000 4000

; 2.0
T
Intracellular CAMP (pmol/mg protein)

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FIG. 6. Correlation of intracellular CAMP levels and transepithelial
secretory volume flow in Malpighian tubules. Intracellular CAMP levels
are elicited by secretagogues indicated. Numbers in parentheses indi-
cate number tubules used in Ramsay assay followed by number of
I”‘] radioimmunoassays. See text for definitions.

Control Factor II Control Factor II blood meal, intracellular CAMP levels peak at 5 min and
25 min after the onset of the meal. The first peak value
of 549 t 108 pmol cAMP/mg protein is statistically
p<o 001 significant (P < 0.05) and precedes the peak rates of
C T NaCl diuresis by 1 min (Fig. 7). This occurs -5 min after
commencing a blood meal. Thereafter intracellular
CAMP levels and the rates of NaCl and fluid secretion
::::::.
.. .... .... ...... ...-
.:.:::::. . . .. .-
. . .. fall. Intracellular CAMP reaches control levels at 10 min
-::::::.
..*.... ::::::.
.- . .. .. .. ..-
*::::::- (Fig. 7C) and then increases again to the second peak.
p<o.oo1 ::::::. . . . . .. ...
:.::::::-
::::.
::::.:.:. . . ‘..... .. Due to the small number of CAMP measurements during
.:::::.:......
_.*:::::: .. . .
.-::::::.
-::::::.
.....f the postpeak phase of diuresis, the second [CAMP] peak
.. ., .. .. ... .. .
-::::::.
-::::::.
.... .... .... .... ...... .... .. at 25 min does not reach statistical significance.
-.:.:.:.;.:.:.
.... ........ .... .... ...... ..
.. ...... .... .... .... .... ..
..::::...
.. ... .. .. ... -
..:.:.:.:.:.:: DISCUSSION
.::::.
.... .......... ..... ...... ......
.-::;:::.
1 **::::::.
.. .. ... .. .. ..
(7) -::::::.
~::::.‘:.
-..:::::.
. .. .-~ . ... .. . .
CAMP-induced Na conductance in basolateral mem-
.
brane. Transepithelial voltage and resistance profiles are
Control Factor III Control Factor III
(MNF) most accurately evaluated in the absence of transepithe-
(MNF)
FIG. 5. Effects of factor I (A), factor II (B), and factor III (C) lial ionic gradients, i.e., when the epithelium is bathed
isolated by high-pressure liquid chromatography from mosquito heads on both sides with the same Ringer solution. This was
on intracellular CAMP content and rates of fluid secretion in isolated accomplished in the present study by perfusing the lumen
Malpighian tubules. Values are means t SE; number in parentheses of isolated Malpighian tubules with the Ringer solution
indicates number of tubules in Ramsay fluid secretion assay and
number of radioimmunoassays in CAMP determination. See text for
present in the bath. Under these control conditions the
definitions. voltage across the basolateral membrane is on average
-77 mV. The voltage across the apical membrane is
tent of Malpighian tubules in vivo. Urine flow rates as a significantly larger, -107 mV. Hence voltage across the
function of time after the beginning of a blood meal are epithelium is 30 mV, lumen positive (Table 1). In the
plotted in Fig. 7A. The peak phase of diuresis lasts -10 presence of DB-CAMP both membranes significantly de-
min, with excretion rates reaching a maximum of 54 nl/ polarize; the basolateral membrane by 53 mV to -24 mV
min at 6 min. Within 60 min, urine flow has decreased and the apical membrane by only 19 mV to -88 mV.
to a level only 28% of peak diuresis (32). However, the dominant voltage effect of DB-CAMP is on
Rates of postblood meal electrolyte excretion vary the basolateral membrane, where the large depolarization
throughout the duration of the diuresis (Fig. 7B). Na and is apparently responsible for the marked hyperpolariza-
Cl excretion rates during the peak phase are highly tion of the transepithelial voltage from 30 to 64 mV.
elevated and rapidly decrease as diuresis continues. In In previous studies we have shown that 1) the electro-
contrast, excretion rates of K are initially low and rise physiological effects of DB-CAMP are dependent on the
after -20 min to a rate that is maintained well into the presence of Na in the peritubular Ringer (32) and that
later phases of diuresis (32). 2) in the presence of DB-CAMP the basolateral membrane
The changes in intracellular CAMP activities in the voltage is influenced significantly more by bath Na con-
Malpighian tubules following the blood meal are shown centrations than in the absence of DB-CAMP (24). These
in Fig. 7C. Intracellular cyclic AMP concentrations in studies suggested that DB-CAMP increases the basolat-
the Malpighian tubules of non-blood-fed controls mea- era1 membrane Na conductance as one of the steps in
sured 339 t 20 (33) pmollme nrotein (Fig. 70. After the stimulating transepithelial secretion of a NaCl-rich fluid.
 CAMP-MEDIATED FLUID SECRETION IN MALPIGHIAN TUBULES R707
- 80- A
c
\-i
60- URINE FLOW RATE
-C
-aJ
rz 40-
3
0

2 20-
aI FIG. 7. A: time course of post-blood meal
.-C diuresis in female Aedes aegypti mosquito.
3 0' Time 0 corresponds to commencement of
meal. Peak phase of diuresis, as referred to in
n
C
text, is indicated as period lasting 10 min after
.-

Downloaded from http://ajpregu.physiology.org/ by 10.220.32.247 on September 16, 2016

onset of meal. (From Ref. 33.) B: post-blood
\E B ELECTROLYTE EXCRETION RATE meal excretion rates of Na, Cl, and K in
a 8 0 0 o r *... female A. aegypti mosquito. Time axis is as
“b described in (A). (From Ref. 33.) C: post-blood
4 0 0 0 l ‘\:.....
.
* ... ... ... .. ... . N a
meal intracellular CAMP concentrations in
b Malpighian tubules of A. uegypti. Time is
i
\ -----’ Cl
period until tubules had been isolated from
40 -K blood-fed mosquito and submerged in liquid
nitrogen. Data were pooled into 2-min inter-
vals, and each point represents average time
period from pooled data. Since SE for average
20 time points were consistently very small, her-
izontd error bars are omitted. Concentrations
of adenosine 3’,5’-cyclic monophosphate
(CAMP) are expressed as means t SE (num-
ber of sets of tubules). Control (non-blood
fed) CAMP concentration was 339 t 20 pmol/
8001 c mg protein (33), shaded urea indicates range
of SE of control values. Values significantly
CAMP different from control are indicated by an
+ P < 0.05‘ asterisk; * P < 0.05. (Data points have been
connected for ease of interpretation; this does
not necessarily imply that CAMP activity im-
plicitly follows such a time course.)

1
1
200-

1 I I 1 I
0’ ’ ’

510 20 40 60 80 100 120

Peak
I phase \ Time (min)

Our present measurements of fractional membrane re- They show that the primary cells are the sites for CAMP-
sistances confirm this conclusion. Under control condi- mediated Na transport. A fractional resistance of the
tions 63% of the transcellular resistance resides at the basolateral membrane of 63% indicates that under con-
basolateral membrane. In the presence of DB-CAMP, the trol conditions the conductance of the apical membrane
fractional resistance drops to 33%, which is consistent is greater than that of the basolateral membrane. This
with the induction of a significant membrane conduct- is not very surprising in view of the amplification of the
ance. A marked increase of the Na conductance would apical membrane surface area by way of the luminal
be expected to significantly depolarize the basolateral brush border (12).
membrane, which indeed is observed. Hence the effect Intracellular voltages of Malpighian tubules have been
of DB-CAMP on increasing the basolateral membrane Na measured previously by O’Donnell and Maddrell (17, 18)
conductance is now well documented in Aedes Malpigh- in the blood-feeding insect Rhodnius, by Morgan and
ian tubules and is internally consistent with the effects Mordue (15) in Locusta, and by Weltens and van Ker-
of CAMP on electrophysiology L -d stimulating the secre- khove (29) in the worker ant Formica. In general, our
tion of a NaCl-rich fluid secretion and not KCl-rich fluid measurements in Aedes yield more electronegative values
secretion. than those in Rhodnius, Locusta, or Formica. A critical
Our observations of the electrophysiological effects of comparison is not appropriate, since our studies in Aedes
DB-CAMP in primary cells of Malpighian tubules are the were done in the absence and those in Rhodnius, Locusta,
first to evaluate the fractional membrane resistances. and Formica were done in the presence of transepithelial
R708 CAMP-MEDIATED FLUID SECRETION IN MALPIGHIAN TUBULES

BLOODMEAL fective doses of extracellular DB-CAMP concentration,

which stimulate fluid secretion, where a concentration
of 10v5 M is the half-maximum dose for stimulating fluid
secretion (30).
The CAMP measured in Aedes Malpighian tubules is
1 MIDGUT VOLUME
similar to that measured in Locusta Malpighian tubules
(23). However, wide variations of [cAMP]i are noted in
0I Locusta tubules (13, 14, 23), which we do not observe in
Aedes Malpighian
measurements
tubules. The differences between our
in Aedes and those in Locusta are not
i DISTENSION OF ABDOMINAL
clear. Nevertheless, the comparison shows that the Mal-
STRETCH RECEPTORS pighian tubules of blood feeders (Aedes) and plant feeders
(Locusta) employ CAMP as a mediator of stimulating
fluid secretion.

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In vitro stimulation of intracellular CAMP levels and
fluid secretion in Malpighian tubules by theophylline and
forskolin. Intracellular CAMP levels are dependent on
the rate of CAMP synthesis by adenylate cyclase and the
rate of CAMP degradation by phosphodiesterases. Hence
both stimulation of synthesis and inhibition of degrada-
tion can be expected to increase intracellular CAMP
levels.
The parallel stimulation of intracellular CAMP levels
and fluid secretion by theophylline, an inhibitor of phos-
phodiesterases (6), supports the second-messenger role
! NaCI-RICH FLUID SECRETION of CAMP in stimulating fluid secretion. Theophylline
BY MALPIGHIAN TUBULES elicits submaximal responses (Fig. 3A) despite the high
FIG. 8. Hypothetical physiological feedback loop of control of di- concentrations used (10 mM). There may be several
uresis by mosquito natriuretic factor (MNF). reasons for the weak, though significant, effects of the-
ophylline on intracellular CAMP levels and fluid secre-
ion gradients. It is presently unknown how transepithe-
tion. The activity of the adenylate cyclase may be low
lial ion gradients affect the membrane and transepithe-
under control conditions so that the inhibition of CAMP
lial electrophysiology of Malpighian tubules from Rhod-
nius, Locusta, and Formica. In Aedes Malpighian tubules, breakdown does not lead to large increases in [cAMP]i
as was suggested previously by Maddrell et al. (11) in the
ion concentrations in the tubule lumen appear to have
case of Rhodnius Malpighian tubules. Alternatively, the
minor effects on the transepithelial electrical parameters
effects of theophylline on Malpighian tubules may be
(24)
Intracellular CAMP levels in Aedes Malpighian tubules more complex than the mere inhibition of phosphodies-
in vitro. The role of CAMP in stimulating fluid secretion terase. For instance, in locust Malpighian tubules, the-
in secretory epithelia in general (7) and in insect Mal- ophylline does not affect intracellular CAMP levels but
pighian tubules in particular (1, 13, 14, 23) is widely it does stimulate fluid secretion (13). Clearly, increased
appreciated. Since in our previous studies the effects of rates of fluid secretion must not always be associated
CAMP were observed on applying DB-CAMP externally, with elevated [CAMP];, and there may be CAMP-inde-
by adding the nucleotide to the bathing Ringer solution, pendent mechanisms of stimulating fluid secretion. Fur-
it was of interest in the present study to find out whether thermore, there are no strong reasons to assume that
endogenous intracellular CAMP, generated by the epithe- CAMP-mediated fluid secretion rates must always be
lial cells of the Malpighian tubules themselves, had sim- associated with proportionate intracellular CAMP levels
ilar effects. The answer to this question necessitated the changing in parallel. In the strictest sense, CAMP may
measurement of intracellular CAMP levels. indeed be a second messenger only, giving a signal that
Under control conditions (in the absence of secreta- is necessary to initiate a response but not to sustain it.
gogues) the [cAMP]i of Aedes Malpighian tubules is Forskolin, which stimulates CAMP synthesis via stim-
normally distributed around a mean of 339 pmol/mg ulating adenylate cyclase, proved to be a potent secret-
protein or 0.43 pmol/tubule. The latter measurement agogue as well as a potent stimulator of [CAMP]; (Figs.
allows a rough estimate of the intracellular CAMP con- 3B and 4). A concentration of only 30 yM is necessary
centration. On the basis of the tubule geometry (length, for maximal stimulation of fluid secretion. The marked
3 mm, OD 75 pm, ID 15 pm), the estimate is 8 PM of increase in the intracellular CAMP levels by forskolin
intracellular CAMP under control conditions. This is supports the idea expressed above that adenylate cyclase
probably an overestimate, since the epithelial cells of activity indeed is low under control conditions when
Malpighian tubules are fusiform (4) and not columnar Aedes Malpighian tubules secrete fluid at subnanoliter
as has been assumed in the above calculation. Neverthe- rates with equimolar concentrations of Na and K (30).
less, a control [cAMP]i of -10m5 M appears to be a Stimulation of intracellular CAMP levels and fluid se-
reasonable estimate (6). It compares favorably with ef- cretion in Malpighian tubules by peptides isolated from
 CAMP-MEDIATED FLUID
BATH PRINCIPAL
< CELL
BASOLATE-RA L APICAL
MEMBRANE
ATP THEOPHYLLINE
0 cretion
FORSKOLIN-
PDE
CAMP
mosquito heads. In previous studies we have isolated
three peptides from the head of Aedes aegypti, which
have major effects on the Malpighian tubules of the same
species (19, 20). The three peptides differ from each
other in their elution from reverse-phase HPLC columns,
in molecular weight, and in the specific effect on Mal-
pighian tubules. In brief, factor I does not stimulate fluid
and electrolyte secretion, but it depolarizes the transep-
ithelial voltage of the perfused Malpighian tubule (Fig.
5A). Factors II and III stimulate NaCl-rich fluid secre-
tion; however, factor II depolarizes and factor III hyper-
polarizes the transepithelial voltage (Fig. 5, B and C).
The ability of these peptides to stimulate fluid secre-
tion depends on their ability to stimulate CAMP levels
in Malpighian tubules. Factor I fails to stimulate fluid
secretion, and it also fails to increase [,cAMP]i. In con-
trast, factors II and III increase fluid secretion in parallel
with increased [cAMP]i. In particular, factor III appears
to be the most potent secretagogue as well as the most
potent sti mulator of in tracellular CAMP levels (Fig. 5).
There are marked similarities between the effects of
external DB-CA .MP and factor III on the transport phys-
iology of Aedes Malpighian tubules. Both stimulate NaCl
secretion and not KC1 secretion, and both hyperpolarize
V, (19, 30, 31). More specifically, both stimulate active
transport of Na. For this reason, we have called factor
III the MNF (19,ZO). Finding that MNF increases intra-
cellular CAMP levels in Aedes Malpighian tubules sug-
SECRETION IN MALPIGHIAN TUBULES R709
LUMEN
FIG. 9. Transport model for Na se-
Downloaded from http://ajpregu.physiology.org/ by 10.220.32.247 on September 16, 2016
in principal cells Malpighian tu-
bules of Aedes aegypti. MNF, mosquito
- 5’-AMP natriuretic factor; CAMP, adenosine
3 ’ ,5’ -cyclic monophosphate; PDE, phos-
phodiesterase.
gests that the specificity of this peptide stems from
stimulating the adenylate cyclase system. If this is true,
then the MNF may be considered to be a hormone whose
cellular mechanism of action is mediated via increased
[cAMP]i. The role of this hormone in triggering the
initial NaCl diuresis during the blood meal is discussed
below.
Post-blood meal diuresis and intracellular CAMP con-
tent of Malpighi .an tubules in vivo. As in other hemato-
phagous insects, the female mosquito undergoes a period
of rapid diuresis immediately following the large blood
meal (16). The initial natriuresis in Aedes aegypti lasts
-10 min. Na and Cl excretion rates decline thereafter
and K excretion rates rise as diuresis continues and as
the K contained in the more slowly digested blood cells
is absorbed into the hemolymph from the midgut (32).
The peak in [cAMP]i in the Malpighian tubules pre-
cedes the peak in excretion rate by 1 min (Fig. 7). Cyclic
AMP activities retu rn to control at 9 min, as 1) diuresis
steadi 1Y declines, 2) N a and Cl excretion decreases, and
3) K excretion increases. Hence changes in intracellular
CAMP con .centrations neatly parallel , if not precede,
changes in NaCl and u rine excretion.
Is MNF diuretic hormone in vivo? Our studies in iso-
lated Malpighian tubules strongly suggest that the pep-
tide MNF, which we have isolated from the head of the
mosquito, activates the adenylate cyclase system in Mal-
pighian tubules, thereby increasing intracellular CAMP
R710 CAMP-MEDIATED FLUID SECRETION
activity and stimulating the secretion of a NaCl-rich
fluid (Fig. 5). In vivo the blood meal stimulus also in-
creases CAMP concentrations in the Malpighian tubules
(Fig. 7), followed by the excretion of a NaCl-rich urine
in the intact mosquito. Hence, the data support the
hypothesis that MNF is indeed a hormone.
Several criteria must be satisfied before the hormone
status of MNF is established: to prove an endocrine
feedback loop, Johnson (9) uses the following criteria: 1)
a physiological event must be demonstrated to stimulate
one part of the insect that subsequently alters activity
in another part. We have demonstrated in the present
study that the blood meal stimulus does indeed alter
activity in the Malpighian tubules by increasing intra-
cellular CAMP concentrations and by stimulating the
rates of NaCl and fluid secretion.
2) The effect must persist after all nervous connec-
tions between the two parts have been severed. Maddrell
(10) has established the presence of neurosecretory axon
endings lying close to the basement membrane of Rhod-
nius Malpighian tubules and has determined that they
do not contain the diuretic hormone. Their function in
the neural control of Malpighian tubules is unknown.
Such axons terminating at the Malpighian tubules have
not been found in Aedes aegypti. In addition we have
demonstrated, at any rate, that MNF directly stimulates
CAMP production in isolated tubules, where it is certain
that the nervous system plays no role.
3) Consequent to the stimulus, a substance must be
isolated in the hemolymph that mimics the effects of the
stimulus. We have found enhanced diuretic activity in
the hemolymph collected from blood-fed mosquitos (3).
Hemolymph from mosquitos on a sucrose diet has little
diuretic potency, and hemolymph taken from blood-fed
and then decapitated mosquitos loses diuretic potency
with time. These observations strongly support the hy-
pothesis of a hemolymph-borne primary messenger.
4) The substance must be identified chemically and
its structure confirmed by synthesis.
Factors II and III have recently been characterized as
thermostable peptides having molecular weights of 1,900
and 2,700, respectively (20). Their structures have not
yet been confirmed by synthesis.
Based on data from this and other studies supporting
the role of a natriuretic factor as the diuretic hormone
in Aedes aegypti, we may propose a hypothetical model
for the control of postblood meal diuresis (Fig. 8).
We speculate that, as in the case of Rhodnius (17), the
activation of abdominal stretch receptors following en-
gorgement is the first step in the control of post-blood
meal diuresis. Gwadz @a) has established the presence
of such receptors in Aedes and has determined that they
are responsible for the proper termination of feeding. We
speculate that they may also play a role in the initiation
of diuresis by causing the release of the MNF.
Stobbart (26) has found out that the head as well as
an intact ventral nerve cord from the abdomen to the
head are necessary for the initiation and maintenance of
diuresis in Aedes aegypti. This demonstrates that the
brain is included in the nervous relay controlling diuresis.
Though our studies have demonstrated that the head is
IN MALPIGHIAN TUBULES
one site of storage of natriuretic factors (20), diuretic
activity has also been found in the abdomen and thorax
as well (unpublished observations). This. may indicate
additional storage sites for the diuretic peptides or sites
of their release. Specific neurohemal areas for the release
of natriuretic factors have not been determined, but the
speed of the diuretic response (~2 min) might suggest
that MNF is released into the hemolymph at a sight near
the Malpighian tubules, as has been found in other blood-
feeding insects (16).
Gee (8) has postulated that, to most efficiently control
post-blood meal diuresis in Glossina, the diuretic hor-
mone must be destroyed almost as quickly as it is released
into the hemolymph, lest its concentrations accumulate
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and make lengthy and difficult the cessation of the initial
diuresis. The Malpighian tubules themselves are known
to destroy the diuretic hormone in both Glossina (8) and
in Rhodnius (17), effectively reducing its activity in the
hemolymph. If this is the case in Aedes as well, the
feedback mechanism for the cessation of diuresis is
rather straightforward. As copious amounts of water are
excreted in the initial natriuresis, distension of the mid-
gut is reduced, and the stretch receptors become quies-
cent. Release of the natriuretic factors is no longer stim-
ulated, their concentration in the hemolymph is reduced,
and the Malpighian tubules are no longer stimulated to
produce CAMP.
Cellular mechanism of MNF action in Aedes Malpigh-
ian tubule. Fluid secretion by insect Malpighian is driven
by tfie active transport of cations. Though in general K
is the ion involved in this process, Na is sometimes the
preferred ion in blood-feeding insects (21).
Figure 9 represents our current understanding of the
cellular mechanism of MNF action and the mechanism
of Na secretion in Aedes aegypti. Previous studies have
determined that the basolateral membrane is permeable
to both Na and K to approximately the same degree
under control conditions (24), which is consistent with
similar rates of Na and K secretion in unstimulated
tubules (30). The dominant effect of DB-CAMP was found
to be the increase of the basolateral membrane conduct-
ance to Na (24), consistent with the specific stimulation
of Na and Cl secretion by DB-CAMP in vitro. We propose
that MNF, by directly activating the adenylate cyclase
system in the Malpighian tubules and by increasing
intracellular CAMP activity, increases the basolateral
membrane conductance to Na. This increase of the ba-
solateral membrane Na conductance allows for increased
rates of Na entry from the hemolymph into the primary
epithelial cells of the Malpighian tubule. Since transep-
ithelial Na movement in the secretory direction is against
the electrochemical potential (31), the active transport
step must be at the apical membrane, presumably in-
volving an electrogenic Na pump, as is generally assumed
for all Malpighian tubules. The transepithelial pathway
for Cl secretion is unknown at this time.
We thank Guillermo Pierluisi, Charles Prosper, and Jan Veenstra
for technical assistance and Sue Hawk for secretarial assistance.
This study was supported by National Science Foundation Grant
PCM-8403305.
Preliminary reports of this study were presented in abstract form at
the Fall 1985 and Spring 1986 Meetings of The American Physiological
 CAMP-MEDIATED FLUID SECRETION IN MALPIGHIAN TUBULES R711
Society and the XXXth Congress of the International Union of 16. NIJHOUT, H. F., AND G. M. CARROW. Diuresis after a bloodmeal
Physiological Sciences. in female Anopheles freeborni. J. Insect Physiol. 24: 293-298, 1978.
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Received 15 September 1986; accepted in final form 1.2 June 1987. B. HARRISON. Elaborations of the basal surface of the cells of the
Malpighian tubules of an insect. Tissue Cell 17: 865-881, 1985.
18. O’DONNELL, J. M., AND S. H. P. MADDRELL. Secretion by the
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