ABSTRACTS FROM PURINES 2000
R01-01
DRUG DISCOVERY AND PURINES
Williams M.
Neurological and Urological Diseases Research, D-464, Pharmaceutical
Products Division, Abbott Laboratories, Abbott Park, IL 60064-6125, USA
Despite skepticism over the past 30 years that often bordered on the irra-
tional, Burnstock’s seminal concept of purinergic neurotransmission is now
well established. The cloning of the P1, P2X and P2Y receptor families has
formed the molecular basis of efforts to define the physiological and patho-
physiological function(s) of adenosine, ATP, ADP and UTP in human tis-
sues and to identify novel therapeutic agents interacting with these targets.
Although a number of novel compounds (NECA, CI-936, CGS 21680,
SCH 58261, ZM241385, KW-6002) have been identified by by big pharma
since the 1970s, only adenosine is approved for human use. The lack of
clinical success of novel purinergic ligands relates to problems with side
effect liabilities, the choice of therapeutic target (hypertension, schizophre-
nia, stroke) and competing non-purine mechanistic approaches.
More recently, several ‘biotechnology’ companies have identified a num-
ber of promising compounds acting via P1 and P2 receptors including P2Y2
agonists(Inspire/ Genentech) for COPD and chronic bronchitis; the hy-
poxanthine analog, Neotrofin (AIT 082, NeoTherapeutics) for neuro-
degenerative disorders, P2 agonists for bronchial disorders (Duska), the
A1 receptor antagonist, BG 9179 (Biogen) for congestive heart failure and
CPX for cystic fibrosis (SciClone).
The emergence of these novel clinical candidates from outside big pharma
underlines the important role that the biotechnology industry plays for the
future in the discovery and early development of truly novel medications.
Big pharma is thus challenged with balancing internal research activities
with identifying and nurturing early stage opportunities in purinergic thera-
peutics from the biotechnology industry.
R01-02
NOVEL TARGETS IN PURINERGIC THERAPEUTICS
Cattabeni, F. and Abbracchio, M.P.
Institute of Pharmacological Sciences, Via Balzaretti 9, 20133 Milan, Italy
In the past, drugs were introduced in therapy based on serendipitous dem-
onstration of their activity. Similarly, certain drugs have been serendipitously
discovered to owe their effectiveness to entirely unexpected biological
activities. In the purinergic field, a typical example is represented by the
anti-aggregant agent dipyridamol. Recently, its effectiveness in reducing
stroke risk in the second European Stroke Prevention Study has been hy-
pothesized to be independent of its anti-platelet activity and to indeed
represent a serendipitous demonstration of cardio- and neuro-protection
induced by endogenous adenosine accumulation (Picano & Abbracchio,
TiPS 19:14-16, 1998). In recent years, it has become evident that the eluci-
dation of the pathophysiological roles (and underlying mechanisms) of en-
dogenous ligands is the best strategy to identify and validate biological
targets for drug development. Such an approach has proved useful also in
disclosing novel targets that could not have been anticipated For example,
by studying the mechanisms underlying reactive gliosis, we have identi-
fied a novel P2Y receptor linked to COX-2 induction, an information that
may have important implications for therapeutic strategies to neuro-
degenerative diseases characterized by inflammation and excessive glio-
sis. Similarly, by analyzing the mechanisms at the basis of adenosine
analogues-induced cell death, we have demonstrated that, in various cell
types, intracellular adenosine kinases and not extracellular receptors rep-
resent key targets for the pro-apoptotic effects induced by these compounds.
These findings may have implications for the development of novel thera-
peutic approaches to pathologies characterized by either excessive or in-
sufficient cell death (e.g., degenerative pathologies and cancer, respectively).
31
R01-03
INDUSTRY AND UNIVERSITY: A PERSONAL VIEW
Juergen Schrader
University of Düsseldorf, Germany
The author is professor for cardiovascular physiology at a typical German
State University and as such has founded a biotech-company (Cardiogene
Inc.) four years ago using venture capital. Cardiogene initial start was based
on patents for the treatment of restenosis after angioplasty using iNOS
and a non-viral delivery system. To day Cardiogene is dedicated to the
development and delivery of innovative products for the treatment of vas-
cular disease that require local vascular and /or cardiac intervention. The
most advanced product (iNOS and restenosis) will enter a clinical trial
Phase I/II this year in the US.
This introduction is meant to indicate that the author has “hands on”
experience on the transition of ideas primarily generated at a University
into products hopefully helpful in the treatment of human cardiovascular
disorders. What I have learned from all of this is - and this is my hypoth-
esis - that industry and university belong to two completely different cul-
tures which operate best when they are kept separately; little will be gained
when the two cultures are merged into one superstructure. Universities
should maintain to be the place where curiosity is the main motivation and
driving force. The primary measure of success is the understanding of a
biological problem and publication of the results in peer reviewed jour-
nals. Industry on the other hand is primarily looking for the return of in-
vestment with all its consequences. Protection of intellectual property by
a strong patent position is more important than publication in a high rank
journal.
R02-01
INTERACTIONS BETWEEN EXTRACELLULAR PURINES AND
GROWTH FACTORS
Middlemiss, P.J. (1); Rathbone, M.P. (1); Caciagli, F. (2); Ciccarelli, R. (2)
(1) Department of Medicine, McMaster University, Hamilton, Ontario,
Canada. (2) Department of Biomedical Sciences, G. D’Annunzio Univer-
sity, Chieti, Italy.
Extracellular non-adenine based purines stimulate proliferation of many
cell types, including cerebral vascular endothelial cells and astrocytes.
Dying or injured cells release extracellularly micromolar concentrations
of purine nucleosides and nucleotides that likely produce multiple ef-
fects on surrounding cells in vivo, including stimulation of mitosis. Both
adenosine and guanosine are mitogenic for endothelial cells. After hy-
poxia and hypoglycemia the extracellular levels of guanosine are higher
than those of adenosine. Additionally, naturally occurring factors such as
basic fibroblast growth factor (bFGF) and the phosphatase inhibitor so-
dium orthovanadate (SOV) also promote cell growth, particularly of en-
dothelial cells. We questioned whether the purines might act
synergistically with bFGF and SOV in promoting growth of mouse en-
dothelial cells in vitro. Adenosine, guanosine, ATP, GTP, bFGF and SOV
each stimulated endothelial cell proliferation in vitro. But when any of
the purines were combined with either bFGF or SOV, the cell growth
was slower than with the factors singly. Moreover, at most concentra-
tions, combinations of adenosine and guanosine were also mutually in-
hibitory. These data indicate that these factors activate different molecular
pathways, the identities of which will be discussed. Additionally, the ra-
tios of various extracellular growth factors may have an important role in
regulation of cell division after injury.
32 ABSTRACTS FROM PURINES 2000
R02-02
THE NEUROPROTECTIVE ACTIVITY OF GUANOSINE IN-
VOLVES THE PRODUCTION OF TROPHIC FACTORS AND THE
OUTFLOW OF PURINES FROM ASTROCYTES
Caciagli, F. (1); Di Iorio, P. (1); Giuliani, P. (1); Middlemiss, M.P. (2);
Rathbone, M.P. (2)
(1) Department of Biomedical Sciences, G. D’Annunzio University, Chieti,
Italy. (2) Department of Medicine, McMaster University, Hamilton, Ontario,
Canada
Exogenous guanosine (Guoe) dose-dependently increased the production of
nerve growth factor (NGF) and transforming growth factor β1 (TGFβ1) from
rat cultured astrocytes, by involving the mitogen-activated protein kinase
(MAPK) cascade, as shown by the strong inhibition of this effect induced by
cell pretreatment with MAPK inhibitors, wortmannin or PD098,059.
The addition of Guo to the superfusion medium of cultured astrocytes
enhanced the outflow of purines assayed by HPLC using UV, fluorescence
and on-line radiochemical detection. The amount of extracellular adenine-
based nucleotides was increased by about 6-fold over the basal outflow,
whereas that of nucleosides was enhanced by only 3-fold. Guoe caused an
extracellular accumulation of adenosine and inosine, without any corre-
sponding increase in the extracellular levels of hypoxanthine. These find-
ings are compatible with the inhibition of an unknown purine nucleoside
phosphorylase-like ectoenzyme. The Guoe-induced accumulation of ex-
tracellular nucleotides was further enhanced by the pretreatment with the
inhibitor of ecto-5'-nucleotidase, αβ-methyleneADP. The trophic factors
released in the medium of cultured astrocytes, previously exposed to Guoe,
were responsible for a remarkable protective activity against NMDA-in-
duced excitotoxicity in neuronal cultures. The blockade of A1 adenosine
receptors, the stimulation of which also activates the production of NGF
and TGFβ1 from astrocytes, only in part reduced the ability of Guoe to
stimulate the synthesis and release of these trophic factors. Thus, Guoe
produces a neuroprotective effect by directly stimulating astrocytes to pro-
duce trophic factors and by suitably affecting the outflow of adenine pu-
rines from these cells as well as their extracellular breakdown.
R02-03
EVIDENCE FOR A GUANOSINE RECEPTOR FUNCTIONALLY
LINKED TO MITOGEN-ACTIVATED PROTEIN KINASE PATH-
WAY
Traversa, U. (1); Florio, T. (2); Virgilio, A. (2); Middlemiss, P.J. (3); Rathbone,
M.P. (3)
(1) Basic Research and Integrative Neuroscience Centre-Department of
Biomedical Sciences, University of Trieste, Italy. (2) Department of Bio-
medical Sciences, G. D’Annunzio University, Chieti, Italy. (3) Department
of Medicine, McMaster University, Hamilton, Ontario, Canada
It is well known that exogenous guanosine (Guoe) causes multiple trophic
effects in the nervous tissue such as the neurite outgrowth and astrocyte
proliferation. We recently found that the exposure of rat cultured astro-
cytes to Guoe activated the mitogen-activated protein kinase (MAPK) cas-
cade. The maximal phosphorylation of ERK 1/2 MAPKs occurred within
10 min of astrocyte exposure to Guo. This effect was reduced by cell pre-
treatment with pertussis toxin (100 ng/ml) and completely prevented by
inhibitors of MAPK pathway such as wortmannin (100 µM) or PD098,059
(10 µM). Since these actions can be receptor-mediated, we investigated
whether an extracellular receptor for guanosine could exist.
3 vated by growth factors (in PC12 cells), and to be inhibited by the purine
H-Guanosine bound to membranes of rat brain as a linear function of
membranes concentration and the binding was temperature dependent
over a range 4° - 56°C. Both association and dissociation are very rapid.
The plateau of the association was reached at 10 min. The Kobs = 0.2945
± 0.005 min-1 and t1/2 = 2.36 ± 0.036 min. The dissociation of 3H-Guo was
80% within 1 min after addition of 1mM guanosine, and Koff = 1.5122 ±
0.46 min-1. The Kd calculated by kinetic constant was 62.1 nM which was
in agreement with Kd calculated by saturation studies (Kd = 82.5 ± 19.4
nM; Bmax = 0.643 ± 0.07 pmol mg-1 prot). Homologous competition studies
showed a Ki = 142 nM. These data suggest the presence of an high affinity
binding site which could be consistent with an extracellular receptor me-
diating the observed effects on MAPK.
R02-04
METABOLISM OF GUANINE AND GUANINE NUCLEOTIDES
IN PRIMARY RAT NEURONAL CULTURES
Sperling, O. (1,2); Zoref-Shani, E. (2)
(1) Department of Clinical Biochemistry, Rabin Medical Center, Petah-
Tikva, and (2) Department of Clinical Biochemistry, Sackler Faculty of
Medicine, Tel-Aviv University, Tel-Aviv, Israel
The aim of this study was to characterize the metabolism of guanine and
guanine nucleotides in neurons. Rat primary neuronal cultures, matura-
tion in vitro, resulted in a marked increase in the specific activity of gua-
nase, creating advantage for guanine degradation to xanthine over its
reutilization for synthesis of GMP. In accordance, in intact mature neu-
rons, the rate of guanine degradation to xanthine was 10-fold the rate of its
conversion to nucleotides. The turnover rate of guanine nucleotides was
found to be fast, reflecting mainly synthesis of nucleic acids and degrada-
tion to xanthine and other purine bases and nucleosides. Small propor-
tions of guanine nucleotides were converted to adenine nucleotides. The
accumulation of label in xanthine indicates (in the absence of xanthine
oxidase) that the main degradative pathway of GMP is that through gua-
nosine and guanine to xanthine, rather than that through IMP to inosine
and hypoxanthine. Most of the IMP produced from hypoxanthine was con-
verted to adenine nucleotide (91.5%), but a significant proportion (6%)
was found in guanine nucleotides. These results indicate that the two arms
of the guanine nucleotide cycle (IMP to GMP and GMP to IMP) are ac-
tive, but its rate of activity and its metabolic importance are yet uncertain.
A hypoxanthine-guanine phosphoribosyltransferase-deficient neuroma cell
line was found to excrete into the culture media normal amounts of xan-
thine, but 15-fold the normal amount of hypoxanthine. In conclusion, in
the neurons, guanine nucleotides are produced mainly from IMP (not from
guanine) and degraded mainly through guanosine and guanine to xanthine.
R02-05
INOSINE ACTS THROUGH AN INTRACELLULAR MECHANISM
TO ALTER NEURONAL GENE EXPRESSION AND INDUCE
AXON REGENERATION IN VITRO AND IN VIVO
Benowitz, L.I.
Children’s Hospital and Harvard Medical School, Boston, MA, USA
Although CNS neurons normally fail to regenerate injured connections,
they are able to do so if extracellular conditions are altered. In contrast,
lower vertebrates spontaneously regenerate their axons in vivo, and in a
cell culture model of this phenomenon, we discovered that the purine
nucleoside inosine stimulates axogenesis from goldfish retinal ganglion
cells. Guanosine was somewhat less active, whereas adenosine was inac-
tive unless hydrolyzed to inosine (via ADA). Pyrimidine nucleosides,
purine nucleotides, and hypoxanthine were inactive. Inosine acts via an
intracellular mechanism, and stimulates expression of GAP-43 and other
genes associated with axon growth. Its likely target is N-kinase, a par-
tially characterized, 47kDa S/T kinase previously reported to be acti-
analog, 6-thioguanine. Inosine acts as a competitor to 6-thioguanine, and
is probably an agonist of N-kinase. In vivo, inosine induced extensive
axon growth in the injured rat spinal cord. Following unilateral transec-
tion of the corticospinal tract, inosine administered to the non-axotomized
motor cortex (via minipump, 2 weeks) stimulated hundreds, and in some
cases thousands, of axons to cross over to the denervated side of the spi-
nal cord and replace the severed axons. Using stem cells in the lesion
site, we are now studying whether inosine can also stimulate the dam-
aged axons to grow across the lesion site. In summary, inosine activates a
central program of gene expression for axon growth, and may have clini-
cal value in the treatment of CNS injury.
 ABSTRACTS FROM PURINES 2000
R02-06
INOSINE AND GUANOSINE PROTECT NEURONAL AND GLIAL
CELLS DURING INHIBITION OF MITOCHONDRIAL ELEC-
TRON TRANSPORT
Jurkowitz, M. S. (1); Hohl, C.M. (1); Lucas, J. H. (2)
(1) Department of Molecular and Cellular Biochemistry and (2) Depart-
ment of Physiology, The Ohio State University Medical School, Colum-
bus, Ohio, 43210, U.S.A.
Hypoxic/ischemic injury is accompanied by abnormalities in mitochon-
drial function, a decline in ATP, and altered ion-homeostasis. We induced
alterations in mitochondrial function and ATP levels in ROC-1 glia using
inhibitors of mitochondrial electron transport chain, rotenone or amobar-
bital. Using varied inhibitor concentrations, ATP levels were decreased in
a stepwise manner. Morphological changes were detected after ATP levels
decreased below threshold, and included a period of increasing osmotic
imbalance and cytoskeletal perturbation followed by loss of viability. Strik-
ing protective effects on viability were obtained with inosine and guanosine.
The degree of protection depended on nucleoside concentration (50 - 1500
micromolar) and required transport of nucleoside into the cell. In pro-
tected cells ATP and GTP remained at higher levels, and lactate produc-
tion was increased, both in proportion with nucleoside concentration.
Treatment of murine spinal cord mixed neuronal and astrocyte cultures
with rotenone produced 100% neuronal death within 260 min; astrocytes
died more slowly (50% viable at 565 min). The viabilities of neurons and
astrocytes were also preserved by nucleoside. In all cells the protective
effects of inosine and guanosine did not occur in presence of BCX-34, in-
hibitor of purine nucleoside phosphorylase. Our evidence supports a pro-
tective mechanism in which the ribose moiety of inosine and guanosine is
converted (via pentose phosphate pathway) to phosphorylated glycolytic
intermediates that can be catabolized in glycolysis, yielding ATPs. We sug-
gest that mitochondrial dysfunction plays an important role in cell injury,
and that nucleosides may be used as potential drugs for protecting neural
cells from hypoxic/ischemic injury.
33
R02-07
PURINE-INDUCED APOPTOSIS OF ASTROCYTES IS MEDI-
ATED BY ADENOSINE
Rathbone, M.P. (1); Andrew, C.M. (1); Crocker, C.E. (1); Werstiuk, E.S. (1);
Caciagli, F. (2); Ballerini, P. (2); Ciccarelli, R. (2)
(1) Department of Medicine, McMaster University, Hamilton, Ontario,
Canada. (2) Department of Biomedical Sciences, G. D’Annunzio Univer-
sity, Chieti, Italy
In brain, spinal cord and other tissues, dying cells release micromolar con-
centrations of purine nucleosides and nucleotides that produce multiple
effects on surrounding cells. Many such effects are ‘trophic’, but as well
adenosine agonists induce apoptosis. After hypoxia and hypoglycemia, the
extracellular levels of guanosine are higher than those of adenosine. There-
fore, we investigated whether extracellular guanosine, like adenosine, in-
duced apoptosis in primary cultures of neonatal rat astrocytes. Initial
experiments with primary rat astrocyte cultures showed that while 100µM
adenosine induced a significant percentage of apoptotic cells measured by
both TUNEL and Annexin V labeling quantified by flow cytometry; the
same concentration of guanosine did not affect the number of apoptotic
cells. However, co-treatment with guanosine and the adenosine deami-
nase inhibitor, EHNA, increased adenosine-induced apoptosis and caused
guanosine to induce a significant proportion of apoptotic cells. These ef-
fects were blocked by the A3 receptor antagonist, MRS-1220, indicating
that adenosine is the molecule responsible for purine-induced apoptosis
and that it transduces its signal through the A3 adenosine receptor. We
have shown that guanosine stimulates the release of purines from astro-
cytes, particularly ATP and adenosine. If adenosine were the apoptotic
signal, then inhibition of purine nucleoside transport through exposure of
the astrocytes to an inhibitor of that process, such as NBTI, would reduce
accumulation of extracellular adenosine induced by guanosine and, con-
comitantly, apoptosis. Treatment with NBTI completely inhibited apoptosis
induced by guanosine, but not that induced by adenosine. This provides
further evidence that adenosine, rather than guanosine, is the mediator of
apoptosis.