84 ABSTRACTS FROM PURINES 2000
141
INFLUENCE OF A-RECEPTORS AGONISTS ON GLUTATHIONE
SYSTEM IN THE BRAIN
Kolesnichenko, L.S.; Sotnikova, G.V.; Minakina, L.N.; Kulinsky, V.I.
Departments of Bioorganic Chemistry and Biochemistry, Irkutsk State
Medical University, Irkutsk, Russia
Glutathione system (GS) has great importance for many fundamental cell
functions, but its regulations by A-agonists have not yet been elucidated.
We investigated an influence of A-agonists in saturating doses on GS of the
mouse brain by standard spectrophotometric methods. Highly selective
and very active agonists of A1-receptors cyclopentyladenosine (CPA),
ClCPA, cyclohexyladenosine and adenosine amine congener in doses of
7.2 mcmoles/kg do not influence in 1 hour, but increase the activities of
glutathione S-transferase (GST) in 3-6 hours and glutathione peroxidase
(GPx) in 6 hours. Unselective agonist of A1, A2A and A2B-receptors NECA
of 0.32 mcmoles/kg also raises GST activity in 3 hours. Selective A1-an-
tagonist dipropylcyclopentylxanthine (DPCPX) of 0.13 mcmoles/kg does
not prevent these effects, that is difficult for interpretation. Slow increase
of GST and GPx activities can be a result of these enzymes induction.
Reduced glutathione (GSH) concentration decreased by A2A-agonists CGS
21680 of 1.8 mcmoles/kg, N6-[2-(3,5-dimethoxyphenyl)-2-(2-methyl-
phenyl)ethyl]adenosine 3.8 mcmoles/kg (both in 1 hour) and DPCPX +
NECA, that is probably connected with A2A-receptors involvement. But
A1-agonists and NECA do not change GSH level, that appear to be re-
sulted of glutathione synthesis rise. All A-agonists do not influence on glu-
tathione reductase activity. Thus A-agonists influence on GS, that can be
important for cerebral functions.
142
ADENOSINE-GLUTAMATE RECEPTOR-RECEPTOR INTERAC-
TIONS IN THE CENTRAL NERVOUS SYSTEM
McIlhinney, J.(1); Ciruela, F.(1); Mallol, J.(2); Lluis, C.(2); Franco, R.(2)
(1)MRC Anatomical Neuropharmacology Unit Mansfield Road. Oxford OX1
3TH. United Kingdom. (2)Department of Biochemistry and Molecular
Biology. University of Barcelona. Martí Franquès 1. 08028 Barcelona. Spain.
Recently, evidence has emerged that the seven transmembrane G protein-
coupled receptors (GPCR) may be present as a homo- and heterodimers in
the plasma membrane. Here we describe a new molecular and functional
interaction between two functionally unrelated types of GPCR, namely
the metabotropic glutamate type 1alpha and the adenosine A1 receptors.
We provide biochemical evidence: coimmunoprecipitation and confocal
microscopy for a molecular interaction between mGluR1alpha and adenos-
ine A1 receptors in rat cerebellum, in primary cultures of neurons and in a
heterologous system. Using calcium determinations in transiently trans-
fected HEK cells we show a synergy between mGluR1a and A1R in the
receptor-evoked [Ca2+]i signalling. The distribution (measured by confo-
cal laser microscopy) and function (measured by excitotoxicity experiments)
of mGluR1a/A1R heteromers in primary cortical neurons reveals a close
fine association of both receptors. Apart from providing a molecular basis
for the adenosine/glutamate interactions, these results may lead to the
development of novel agents to treat neuropsychiatric disorders in which
glutamatergic neurotransmission is abnormally regulated.
143
CONTROL OF ENZYME ACTIVITY AND ADENOSINE BIND-
ING BY THE COFACTOR NAD+/NADH OF S-ADENOSYLHOMO-
CYSTEINE HYDROLASE
Kloor, D.; Osswald, H.
Department of Pharmacology, Faculty of Medicine, University of Tübingen,
Tübingen, Germany
The activity of S-adenosylhomocysteine-(SAH) hydrolase regulates intra-
cellular SAH concentration. SAH hydrolase contains tightly bound NAD+
which is involved in the oxidation-reduction mechanism of the catalytic
enzyme activity. Adenosine (ADO) which inhibits SAH hydrolysis binds to
SAH hydrolase with high affinity. In the present investigation we exam-
ined the enzyme activity and ADO binding capacity of SAH hydrolase at
different NAD+/NADH ratios of the enzyme.
Methods: SAH hydrolase was purified to homogeneity from bovine kid-
ney with classical chromatographical methods. The tightly bound NAD+
of the enzyme was removed by dialysis and the apo-enzyme was reconsti-
tuted with NAD+/NADH ratios between 1.0 and 0.1. The enzyme activity
was determined photometrically at 292 nm in 50 mM potassium phos-
phate buffer pH 7.4. For binding assay, the enzyme (10 µg/ml) was incu-
bated with 3H-ADO (5nM) over night in Tris/HEPES pH 7.4 at 4° C to
label the high affinity site. The mixture was filtered and the radioactivity
was counted.
Results: SAH hydrolase, in its maximal oxidized (NAD+) form exhibits
an enzyme activity of 0.12±0.02 U/mg and an 3H-ADO binding of 16.1±1.1
pmol/mg. When the apo-enzyme was reconstituted only with NADH, SAH
hydrolase is enzymatically inactive, whereas the 3H-ADO binding increase
to 112.0±2.1 pmol/mg. At a ratio of 0.1 (NAD+/NADH) the enzyme activ-
ity is still 30% when compared to the oxidized form. However, the 3H-
ADO binding is already maximal (112.0±3.2 pmol/mg).
Conclusion: Our data show that enzyme activity and ADO binding at
SAH hydrolase are inversely proportional. Further studies are necessary
to determine whether the binding sites of ADO are located at the catalytic
center of SAH hydrolase.
144
LIGAND-INDUCED INTERNALIZATION OF A1 ADENOSINE
RECEPTORS IS MEDIATED BY CAVEOLAE IN THE SMOOTH
MUSCLE CELL LINE DDT1MF-2
Mallol, J.(1); Escriche, M.(1); Canela E.I.(1); Enrich C.(2); Lluís, C.(1);
Franco R. (1)
(1) Department of Biochemistry and Molecular Biology. University of
Barcelona, Martí Franquès 1, Barcelona, 08028, Spain. (2)Department of
Cellular Biology and Pathological Anatomy, School of Medicine,University
of Barcelona.
Immunoelectron microscopy techniques have been used to study the traf-
fic of A1 adenosine receptors (A1Rs) and adenosine deaminase in the smooth
muscle cell line DDT1MF-2.
In absence of agonists receptors are homogenously distributed on the
cell membrane. However, N6-(R)-(phenylisopropyl)adenosine (R-PIA)-in-
duced stimulation of receptors for 15 to 40 minutes produces clustering
and sequestration of A1Rs and adenosine deaminase in non-coated vesicles
whose morphological characteristics are identical to those of caveolae. In
fact, similar vesicles were stained with an antibody against caveolin-1 and
the internalization process could be disrupted by pre-incubating cells with
filipin, a sterol-binding agent capable of flatten caveolae. Addition of exog-
enous adenosine deaminase (ADA), a protein that binds to and facilitates
A1R-mediated signaling, produced an acceleration of R-PIA-induced A1R
internalization. After 40 minutes of activation, recycling of A1Rs back to
the cell surface occurred. Treatment of cells with N-ethylmaleymide (NEM),
an inhibitor of vesicular transport, prevented such recycling. Interestingly
adenosine deaminase and A1 adenosine receptors recycle back by a differ-
ent route. In fact the two proteins are segregated in endosomes and follow
a differential sorting pathway. These results are important to know the
mechanisms of receptor downregulation and recycling.
 ABSTRACTS FROM PURINES 2000
145
A1 ADENOSINE RECEPTOR ACTIVATION POTENTLY INHIB-
ITS AXON GROWTH.
Thevananther, S; Schwartz, M; Rivkees, SA.
Department of Pediatrics, Neurobiology Program, Yale University, New
Haven, CT
In contrast to classical neurotransmitters that are discretely released at
synapses, adenosine is produced and released by all cells and diffuses into
interstitial fluid. Adenosine exerts potent biological effects by activating
specific receptors. The receptors include A1 adenosine receptors (A1ARs),
which couple with Gi and Go and inhibit cAMP accumulation within cells.
We have discovered that a unique feature of A1ARs is their high expres-
sion on axons. We have also found that A1ARs are expressed in the brain
during critical periods of development.
To test if A1AR activation influence brain formation, neonatal rats were
injected with A1AR agonists from postnatal days (PD) 3-14. When animals
were examined, we found 23-30% (p < 0.01) reductions in total white matter
volume. Indicating that A1AR activation impairs axon formation, electron
microscopy revealed 30-40% reductions in total axon volumes.
Next, we examined effects of adenosine on isolated hippocampal neurons.
Immunocytochemistry studies revealed A1AR expression on axons and
growth cones. Showing that adenosine can act directly on neurons to inhibit
axon growth, A1AR agonists slowed axonal growth in cultured hippocampal
neurons and induced acute changes in growth cone morphology.
Collectively, these observations identify A1ARs as an important regula-
tor of axon growth during development.
146
ADENOSINE A2A RECEPTOR AGONISTS PROMOTE MORE
RAPID WOUND HEALING THAN RECOMBINANT PLATELET
DERIVED GROWTH FACTOR (PDGF)
Victor-Vega, C.(1); Desai, A.(1); Montesinos, M.C.(1); Leung, E.C.(2); Giefer,
E.E.(2); Cronstein, B.N.(1).
(1) New York University School of Medicine, New York, NY, USA (2) Medco
Pharmaceuticals, Research Triangle Park, NC, USA
We previously demonstrated that topical application of adenosine A2A re-
ceptor agonists promotes wound closure in normal mice and diabetic rats.
We compared the effect of MRE0094 (2-[2-(4-chlorophenyl)ethoxy]adeno-
sine), a selective adenosine A2A receptor agonist, to CGS-21680, the ref-
erence selective adenosine A2A receptor agonist, and platelet derived
growth factor (PDGF), an agent currently marketed for promotion of wound
healing in diabetic ulcers, on wound closure in healthy BALB/c mice.
Wounds (1 (space) cm diameter) were created on the dorsum of mice (2
per mouse) and then treated daily with vehicle, 0.01% PDGF gel or differ-
ent doses of the adenosine receptor agonists. The wound margins were
traced onto plastic sheets, the areas digitized and quantitated and com-
pared. We found that both MRE 0094, 1ug/wound and 10ug/wound, and
CGS-21680, 1ug/wound and 5ug/wound, achieved 50% wound closure sig-
nificantly faster than control (Day 1.9, 1.9, 3.2, 3.2 and 4.0 for the two
concentrations of MRE0094, CGS21680 and control, respectively, p<0.05
ANOVA). Neither higher nor lower concentrations of CGS21680 affected
the rate of wound closure, as compared to control. In contrast, PDGF did
not increase the rate at which wounds closed (50% closure by day 7.2,
p=NS vs control) although there was a qualitative increase in the granula-
tion tissue that accumulated in the wounds. These data confirm our prior
observations that adenosine A2A receptor agonists promote wound clo-
sure and suggest that these agents may be as effective if not more effective
than PDGF for the treatment of poorly healing wounds.
85
147
BINDING THERMODYNAMICS AND INTRINSIC ACTIVITY OF
ADENOSINE A1 RECEPTOR LIGANDS: A MODEL OF THEIR
IN VIVO HEART RATE INHIBITION POWER
Gessi, S. (1); Borea, P.A. (1); Varani, K. (1); Pavan, B. (2); Dalpiaz, A. (3).
(1) Department of Experimental and Clinical Medicine, Pharmacology
section, Ferrara University, Italy. (2) Department of Biology, General Physi-
ology Section, Ferrara University, Italy. (3) Department of Pharmaceutical
Sciences, Ferrara University, Italy
A thermodynamic analysis of the binding to rat cortex adenosine A1 recep-
tors of 5'-deoxyribose-N6-cyclopentyladenosine (full agonist) and several
8-alkylamino (methyl, ethyl, propyl, butyl and cyclopentyl) homologues of
N6-cyclopentyladenosine (partial agonists) was performed. The intrinsic
activity of the compounds was moreover evaluated by measurements of
the inhibition of forskolin stimulated 3'-5'-cyclic adenosine monophosphate
(c-AMP) levels in isolated epididymal rat adipocytes. Standard free energy
(∆G°), enthalpy (∆H°) and entropy (∆S°) of the binding equilibrium were
determined by means of affinity measurements carried out at different tem-
peratures (0, 10, 20, 25, 30° C) and van’ t Hoff plots, lnKA versus 1/T. Affin-
ity constants of drug-receptor interactions were obtained by displacement
experiments in the presence of 1nM [3H]N6cyclohexyladenosine, using at
least twelve different concentrations of cold drug. Levels of c-AMP were
evaluated performing competitive binding assays. The affinity of the ligands
increases with temperature enhancement. As a consequence the binding
of full and partial agonists is totally entropy-driven . Standard entropy val-
ues of a wide series of adenosine derivatives, including the compounds
under examination, are strictly correlated to intrinsic activity ones. Simi-
larly, ∆S° values appear correlated to the in vivo capability of the adenos-
ine derivatives to inhibit rat heart rate. In vitro thermodymanic data of
adenosine A1 receptor ligands are proposed as an indicator of their in vivo
pharmacodynamic on cardiovascular system.
148
HIGH RECEPTOR RESERVE FOR A1-ADENOSINE RECEPTOR
AGONISTS TO DECREASE CYCLIC AMP CONTENT OF RAT
ADIPOCYTES
Liang,H.X.; Shryock,J.C.
Department of Medicine, University of Florida, Gainesville, FL, USA
High receptor reserve (spare receptors) is a potential explanation for the
high potency of adenosine to inhibit cAMP formation and lipolysis in adi-
pose tissue, and the tonic activity of adipocyte A1AdoRs in vivo. We used
the irreversible A1AdoR antagonist FSCPX to determine receptor reserves
for adenosine and A1AdoRs agonists to decrease cAMP in rat isolated epi-
didymal adipocytes. Adipocytes were treated with vehicle (control) or
FSCPX (10 µM) for 60 min at 36oC, washed repeatedly, then incubated
with A1AdoR agonists in the presence of 100 nM isoproterenol for 4 min.
Incubations were terminated by addition of HCl, and cAMP of cell ex-
tracts was determined by radioimmunoassay. FSCPX reduced the response
of adipocytes to A1AdoR agonists but not to nicotinic acid, suggesting that
FSCPX selectively altered the function of A1AdoRs. Values of EC50 for
adenosine, CCPA, R-PIA, and S-PIA to attenuate an isoproterenol-induced
increase of cAMP were 1.5, 0.4, 0.2, and 10.5 nM, respectively. Values of
KA for adenosine, CCPA, R-PIA, and S-PIA, were 4559, 177, 76, and 791
nM, respectively. The percentage of spare receptors at 95% of the maximal
responses for adenosine, CCPA, R-PIA, and S-PIA, was 99, 98, 97, and
91%, respectively. We calculate that adenosine at a concentration reported
to be present in adipose tissue in vivo (128 nM) would cause a 96% inhibi-
tion (expressed as % of maximal response) of catecholamine-stimulated
adenylyl cyclase activity. Thus, a high receptor reserve for adenosine can
explain the tonic activity of A1AdoR.
86 ABSTRACTS FROM PURINES 2000
149
COMPARISON OF THE SPECIFIC BINDING OF THE AD-
ENOSINE A2A RECEPTOR ANTAGONIST 3H-ZM-241385 AND
OF THE AGONIST 3H-CGS-21680 TO BOVINE AND HUMAN
A2A RECEPTORS.
Honrubia, M.(1); Tristán, H.(1); Villazón, M.(1); Cadavid, I.(1); Loza, M.I.(1);
Beleta, J.(2); Díaz, J.L.(2); Palacios, J.M.(2).
(1) Department of Pharmacology, Faculty of Pharmacy, University of
Santiago de Compostela, Spain.
(2) Almirall-Prodesfarma Laboratories, Barcelona, Spain.
The specific binding of the potent A2A adenosine receptor antagonist [3H]-
ZM 241385 to membrane preparations of bovine cerebral caudate and of
HEK 293 cells stably expressing human A2A receptors has been deter-
mined. Studies indicated that the binding was saturable (Bmax = 3.19 and
45.77 pmol/mg for bovine and human receptors respectively) and of high
affinity (Kd = 1.08 and 1.73 nM respectively). The curve fitted to the data
obtained is consistent with the presence of a single binding site in both
preparations. The corresponding saturation analysis using the agonist [3H]-
CGS 21680 as the radioligand resulted in Bmax values of 2.22 and 5.46
pmol/mg and affinities of 10 and 17.8 nM for the bovine and human recep-
tors respectively. In displacement studies there was a good correlation
between affinities calculated for standard A2A receptor agonists and an-
tagonists using either radioligand, and no species differences. The Bmax,
however, was found to be 10-fold greater for the antagonist than for the
agonist in human transfected receptors, but not for bovine tissue. Indicat-
ing a higher expression of the receptor in the transfected cells, and sug-
gesting that the proportion of the high affinity state is higher on membranes
derived from tissue than on those from transfected cells. The main advan-
tages of [3H]-ZM 241385 over [3H]-CGS 21680 as a radioligand for the
A2A receptor are its higher affinity and its higher specific binding (48%
and 72% respectively for the bovine and human preparations, compared
with the 17% and 29% for [3H]-CGS 21680 in the same preparations).
150
PHARMACOLOGICAL AND KINETIC CHARACTERISATION OF
NECA-INDUCED A2B RECEPTOR-MEDIATED RESPONSE IN
ISOLATED GUINEA-PIG AORTA.
Tristán, H.(1); Honrubia, M.(1); Villazón, M.(1); Cadavid, I.(1); Loza, M.I.(1);
Díaz, J.L.(2); Palacios, J.M.(2).
(1) Department of Pharmacology, Faculty of Pharmacy, University of
Santiago de Compostela, Spain.
(2)Almirall-Prodesfarma Laboratories, Barcelona, Spain.
The effect of 5'-(N-ethyl) carboxamido adenosine (NECA) on the tone of
the isolated, phenylephrine-contracted, guinea-pig aorta has been stud-
ied. The adenosine analogues NECA, 2-chloroadenosine (2-CADO) and
R(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA) exhibited the following rank
order of potency for relaxation: NECA 2-CADO R-PIA, consistent with
previous reports that relaxation in this tissue is mediated through smooth
muscle A2B receptors. The relaxation induced by NECA was followed by
an incomplete fade that lasted for 30 minutes. Addition of a second
submaximal concentration of NECA on the steady state did not produce
any response, whereas isoproterenol and bradikinin were still able to
achieve their maximal relaxation in the preparation under these circum-
stances. The effect of NECA could be reverted completely by 100 mM
alloxacine (a selective A2B antagonist). We propose that the partial time-
dependent loss of the response to NECA on this preparation is the result
of a desensitisation process of the A2B receptors, which can be accurately
described using a three-compartment kinetic model, which defines three
rate constants: relaxation (k1), desensitisation (k2) and resensitisation (k3)
and one steady-state constant (Rss).
151
REGIONAL DIFFERENCES ON THE ADENOSINE-RECEPTOR
MEDIATED MODULATION OF NORADRENALINE RELEASE
IN THE RAT VAS DEFERENS
Queiroz, Q.; Diniz, C.; Leal, S.; Goncalves, J.
Laboratório de Farmacologia (CEQOFF/FCT) Faculdade de Farmácia,
Universidade do Porto, Porto, Portugal
In the rat vas deferens the relative contribuition of ATP and noradrenaline
to the neurogenic contraction varies, the purinergic component predomi-
nates in the prostatic while the noradrenergic predominates in the epid-
idymal portion.
In this study we compared the modulatory effect of adenosine on norad-
renaline release in the epididymal and prostatic portions of rat vas deferens.
Prostatic and epididymal portions of rat vas deferens preincubated with 3H-
noradrenaline were superfused and stimulated electrically with 100p/8Hz.
The estimated tritium overflow was assumed to reflect action potential evoked
noradrenaline release. Adenosine (10-1000µM)decreased noradrenaline re-
lease in both portions, an effect prevented by DPCPX (20nM). CGS 21680
(10-1000nM) increased noradrenaline release up to 142±12% (n=11,
P<0.05), but only in the epididymal portion. The effect of CGS 21680 was
prevented by DMPX (100nM) but not by ZM 241385 (20nM) and was slihtly
increased by DPCPX (20nM). The effect of CGS 21680 is not likely to be
due to activation of postjuntional receptors since CGS 21680 (100nM) in-
hibited contractions induced by noradrenaline (5µM) by 38±5% (n=10,
P<0.05) but only in the prostatic portion, an effect prevented by DMPX
(100nM). The present results show that in the prostatic portion, adenosine
only inhibits noradrenaline release, while in the epididymal portion adenos-
ine is involved in a dual and opposite modulation of noradrenaline release.
It is concluded that regional differences also occur in the adenosine-recep-
tor mediated modulation of noradrenaline release in the rat vas deferens.
Supported by FCT (unidade I&D 226/94), PRAXIS e FEDER) and ANF.
152
INTERACTION OF ADENOSINE AND GUANINE NUCLE-
OTIDES IN THE RAT HIPPOCAMPUS: EFFECTS ON CYCLIC
AMP LEVELS AND ON ADENOSINE AND GMP BINDING
Tasca, C.I.(2); Souza, D.O.(1)
(1)Departamento de Bioquímica,Instituto de Ciencias Básicas da
Saúde,Universidade Federal do Rio Grande do Sul,Porto Alegre,RS, Brasil.
(2)Departamento de Bioquímica,Centro de Ciencias Biológicas, Universi-
dade Federal de Santa Catarina,Florianópolis,SC,Brasil.
Guanine nucleotides (GN) are implicated in many intracellular mechanisms.
Extracellular actions, as antagonists on glutamate receptors, have also been
recently attributed to these compounds. GN may display a neuroprotective
role by inhibiting excitotoxic events evoked by glutamate. Effects of extra-
cellular GN on adenosine-evoked cellular responses have also been reported.
Although, as the exact mechanism of such interaction is not known, in the
present study, we investigated the interactions of adenosine receptors and
adenosine-induced responses with GN. We showed that GN potentiated
adenosine-induced cyclic AMP accumulation in slices of hippocampus from
young rats. However, neither GMP nor a metabotropic glutamate receptor
agonist, inhibited the binding of the adenosine receptor agonist [3H]NECA
(bound to adenosine A2 receptors), or the binding of the adenosine A2a re-
ceptor agonist [3H]CGS 21680 in hippocampal membrane preparations.
GppNHp, probably by interacting with G-proteins, decreased [3H]CGS 21680
binding. [3H]GMP binding was assayed in order to evaluate the GN sites
which are not G-proteins. The interaction of endogenous adenosine with
GMP-binding sites was determined by incubating membranes in the pres-
ence or absence of adenosine deaminase (ADA). Adenosine analogs (NECA,
CADO, CGS 21680 and CPA) increased GMP binding in the presence of
ADA. However, in the absence of ADA, the control levels of GMP binding
were as high as in the presence of added ADA plus adenosine agonists, indi-
cating that endogenous adenosine modulates the binding of GMP. Whether
these GN sites can display a neuroprotective role, adenosine may be in-
creasing its neuromodulatory and proposed protective action.
 ABSTRACTS FROM PURINES 2000
153
G-PROTEIN CROSS-TALK MEDIATES ADENOSINE’S AMPLI-
FICATION OF MECHANICALLY-ACTIVATED 5-HYDROX-
YTRYPTAMINE RELEASE FROM BON CELLS.
Cooke, H.J. (1); Kim, M. (1); Javed, N.H. (2).
(1) Department of Neuroscience, The Ohio State University, Columbus,
OH, USA (2) Department of Physiology and Health Science, Ball State
University, Muncie, IN, USA
5-Hydroxytryptamine (5-HT) from enterochromaffin cells activates neural
reflexes controlling intestinal fluid secretion and peristalsis. While me-
chanical stimuli are known to release 5-HT, the signal transduction path-
ways and their regulation by adenosine, which is elevated in inflammation,
are unclear. The aim was to determine if mechanically-evoked release of 5-
HT is mediated by G-proteins and modified by adenosine. Rotational shak-
ing (shear stress + pressure) at 80 rpm caused a maximum increase in
5-HT release of 212% above basal (0.84±0.08 pmol/well/20 min) in BON
cells. 5-HT release in streptolysin-O-permeabilized cells was inhibited by
GDP-β-S (p<0.05). A 20 bp antisense phosphorothioated oligonucleotide,
but not the scrambled sequence, abolished shaking-evoked 5-HT release
and significantly reduced Gαq protein levels detected by Western blot-
ting without affecting Gα11. These results suggest that shaking specifi-
cally activates Gαq. BON cells were found to have A1 receptor transcripts
determined by RT-PCR. The adenosine A1 receptor agonist, 2-chloro-N6-
cyclopentyladenosine (100 nM CCPA), enhanced 5-HT release by 1.4 fold
during shaking without an effect on basal release (p<0.05). Bethanechol
and isoproterenol, binding to muscarinic and β-adrenergic receptors to
activate Gαq and Gαs, caused a 1.3 and 5.6 fold increase in 5-HT release,
respectively. CCPA enhanced bethanechol-evoked release of 5-HT by 163%
and inhibited isoproterenol-evoked 5-HT release by 57%. Depending on
the stimulus, CCPA has excitatory or inhibitory effects on 5-HT release.
The results suggest that adenosine A1 receptor activation augments me-
chanically-evoked 5-HT release as a consequence of cross-talk between
Gαi and Gαq. Supported by NIH DK37240.
154
EFFECT OF ZM241385 ON PIAL ARTERIOLAR DILATION DUR-
ING SCIATIC NERVE STIMULATION
Crum, A.V.; Meno, J.R.; Winn, H.R.
Department of Neurological Surgery, University of Washington, Seattle,
Washington, USA
Previous studies have shown that adenosine participates in the cerebral
blood flow response to somatosensory stimulation. However, the adenos-
ine receptor subtype(s) involved in this response is undetermined. In the
present study, we report the effects of ZM241385, a selective antagonist
for the adenosine A2A receptor subtype, on pial arteriolar dilation during
contralateral sciatic nerve stimulation (SNS). The pial circulation was ob-
served through a closed cranial window in chloralose-anesthetized Sprague-
Dawley rats. The contralateral sciatic nerve was exposed, positioned on
silver electrodes and stimulated for 20s (0.20V, 0.5ms and 5Hz). Vessel
diameter was measured on-line utilizing an automated video dimension
analyzer. In mock CSF, stimulation of the contralateral sciatic nerve re-
sulted in a 30.6± 5.35% increase in pial arteriolar diameter, in the hindlimb
somatosensory cortex. Systemic administration of ZM241385 (1 mg/kg, I.V.)
had no effect on resting arteriolar diameter (Control: 27.5± 2.5um;
ZM241385: 25.4± 3.1um), but significantly attenuated pial arteriolar dila-
tion during SNS (control: 30.6± 5.35%; ZM241385: 18.5± 7.38%; n=6,
p<0.05). Pial vasodilatation during topical application of adenosine (1 &
10uM) was also significantly (p<0.01, n=5) attenuated by ZM241385. In
contrast, systemic administration of ZM241385 had no effect on either
hypercarbic vasodilatation or somatosensory evoked potentials (SSEPs).
This study demonstrates that the adenosine A2A receptor subtype plays a
role in the pial arteriolar response to SNS.
87
155
ADENOSINE A3 RECEPTORS ARE INVOLVED IN THE ACTI-
VATION OF AN INHIBITORY NEURAL MOTOR PATHWAY IN
RAT COLON
Christofi, F.L.; Yu, J.-G.; Xue,J.; Wang,Y.-Z.; Kim, M.; Cooke, H.J.
Departments of Anesthesiology & Neuroscience, The Ohio State Univer-
sity, Columbus, OH, USA
Adenosine A3 receptors (R) are widely expressed in the mammalian gut.
However, the physiological role of this receptor in enteric neural reflexes
remains unknown. To clarify the role of A3R, we examined its distribution,
expression and function in neuro-effector transmission in rat colon. Laser
confocal imaging detected intense A3R immunoreactivity (IR) in the cell
somas of myenteric neurons, using 3 different anti-A3R antibodies. Re-
gional differences exist in the numbers of neurons expressing A3R IR in
the colon; the expression profile from highest to lowest numbers of neu-
rons was: descending colon-proximal half (~5000 neurons) / distal half-
descending colon & rectum (4000) transverse colon (1400) proximal colon
(650). Some A3R neurons had multipolar morphology or large smooth cell
somas. A3R IR could be abolished by antibody pre-absorption with con-
trol peptide. Western blotting revealed a prominent 44Kda and a faint 63Kda
band in rat myenteric plexus tissues. Strain-gauge recording of muscle
tension (n=15 rats) revealed a cyclic pattern of large amplitude TTX-resis-
tant contractions (LACs, 0.98±.13 g tension; frequency of 0.7±.1/min,
n=7).The A3R agonist N6-(4-amino-benzyl)-9-[5-(methyl-carbonyl)- (-D-
ribofuranosyl] adenine (AB-MECA) or its analog IB-MECA (1-10uM) in-
hibited or abolished LACs. IB-MECA (10uM) abolished the cyclic response
and subsequent TTX treatment reversed the blockade. Neural blockade
with TTX first, prevented the response to IB-MECA. The response to IB-
MECA was insensitive to the A1 antagonist 8-cyclopentyl-1,3-
dimethylxanthine. No A3R IR was detected in smooth muscles.
Conclusions: Neural A3Rs play a physiological role in activating an in-
hibitory neural pathway leading to suppression of neuromuscular activity
(NIH R01 DK44179-06 - FLC & NIH DK37240 - HJC).
156
ROLE OF A2b RECEPTORS IN THE GASTRIC MUCOSAL CELLS
Vallejo, A.I.; Arin, R.M.; Schijvarger, S.A.; Varela, A.
Department of Physiology, Faculty of Medicine, University of the Basque
Country, Bilbao, Spain
From previous pharmacological studies it has been observed a direct ac-
tion of adenosine on the stimulation of the gastric secretion. The action
mechanism of the A2b receptors on gastric mucosal cells has been investi-
gated in this presentation.
Binding studies of NECA and DPCPX to the ECL and parietal cell
plasma membranes have been carried out. Adenylate cyclase stimulation
has been detected when the plasma membranes were incubated with ad-
enosine analogues. This stimulation was reversed by the membrane treat-
ment with some A2b inhibitors. Also the membrane labelling with an
A2b-antibody has been presented.
A possible interrelation between adenosine and histamine (the main gas-
tric secretion secretagogue) was inferred from the pharmacological stud-
ies. This interrelation has been also investigated on the cAMP pathway.
88 ABSTRACTS FROM PURINES 2000
157
ADENOSINE A3 RECEPTOR GENE EXPRESSION IN THE IN-
TESTINAL TRACT OF HUMANS, RATS AND GUINEA-PIGS
Guzman, J.E.; Yu, J.-G.; Xue, J.; Wang, Y-Z; Kim, M.; Cooke, H.J.; Christofi,
F.L.
Departments of Anesthesiology & Neuroscience, The Ohio State Univer-
sity, Columbus, OH, USA.
Functional and biochemical data suggest that A1, A2 and possibly A3 re-
ceptors (R) influence enteric neural reflexes. To further examine the latter
possibility, a comparative analysis of A3R gene expression was carried out
for human, guinea-pig and rat intestines, as well as human epithelial, glial
and enterochromaffin cell lines. Western blotting with 3 different anti-
A3R antisera on mucosa, submucosa, or whole thickness jejunum from
Roux-en-Y patients (OSU CHTN,Western Division) revealed prominent
63Kda, 50Kda and 44Kda bands. The same result was obtained in human
cell lines (BON, T98GC & U373). Western blotting revealed prominent
44Kda bands and a fainter 63Kda band in guinea-pig and rat colon. In rat
jejunum (and colon), strong A3R immunoreactivity (IR) was expressed in
myenteric neurons. In submucous ganglia, intense A3R IR was detected
in varicose nerve fibers, glial cells and rarely in nerve cell bodies. Laser
scan confocal imaging of BON cells revealed the cell membrane localiza-
tion of A3R IR. A3R IR could be abolished by pre-absorption of anti-A3R
antibodies with their control peptides. RT-PCR analysis indicated expres-
sion of adenosine A3R mRNA (band size of 327 base pairs) in neural and/
or non-neural layers of the human jejunum, ileum, colon and cecum. Sub-
mucosal tissues in all regions (but cecum), mucosa, myenteric plexus-smooth
muscle, and T98GC cells (but not HT-29 or T-84) expressed mRNA for
A3R. Conclusions: A3R is widely distributed in neural and non-neural
tissues of the gut. A3R may be involved in the neurophysiological control
of intestinal reflexes (NIH DK44179 to FLC & DK37240 to HJC).
158
NO ABSTRACT
159
INTERACTIONS OF ADENOSINE AND PROSTAGLANDINS IN
THE DIABETIC KIDNEY.
Pflueger, A.; Knox, F. G.
Department of Medicine and Physiology, Mayo Clinic and Foundation,
Rochester, MN, USA.
Previously we demonstrated that adenosine causes renal vasoconstric-
tion via A1 receptor stimulation in control and type-1 diabetic STZ-rats.
Prostaglandins have been proposed to oppose the effects of renal vaso-
constrictors. We investigated the role of prostaglandins in the renal vas-
cular response to exogenous and endogenous adenosine in control and
diabetic STZ-rats. Exogenous adenosine injected into the abdominal aorta,
decreased renal blood flow (RBF) in a dose-dependent manner to a much
greater extent in STZ-rats than in control rats (P<0.001). Inhibition of
prostaglandin synthesis with indomethacin (INDO, 10 mg/kg i.v.) poten-
tiated the adenosine-induced renal vasoconstriction in control rats but
not in STZ-rats. The renal response to endogenous adenosine was as-
sessed by the postocclusive reduction of RBF (POR), an adenosine-me-
diated phenomenon. POR was greater in STZ rats compared to control
rats (P<0.001). INDO markedly enhanced POR in control rats but not
in STZ-rats. Renal cortical and medullary PGE2 microdialysate concen-
trations and urinary PGE2 excretions were clearly not lower in STZ (cor-
tex: 169±61 pg/ml, medulla: 640±28 pg/ml, urine: 138±25 pg/min) as
compared to control rats (cortex: 99±12 pg/ml, medulla: 489±107 pg/
ml, urine: 82±28 pg/min). INDO significantly decreased renal cortical,
medullary, and urinary excretions of PGE2 in control and STZ-rats. These
findings demonstrate that the adenosine-induced renal vasoconstriction
is increased in the presence of INDO in control but not in STZ-rats. The
observations suggest that the diabetic renal vasculature has a diminished
vasodilatory capacity in response to prostaglandins to counteract adenos-
ine-induced renal vasoconstriction.
160
ADENOSINE INDUCED DETRUSOR MUSCLE RELAXATION
IN CONTROL AND STZ-INDUCED DIABETIC RATS: EFFECT
OF HYPOXIA
Gür,S.
Department of Pharmacology, School of Pharmacy, University of Ankara,
Turkey
Urinary bladder dysfunction is a complication of diabetes mellitus and at-
tributed in part to peripheral neuropathy.Adenosine induces relaxant re-
sponses in the bladder. Hypoxia significantly potentiates responses to
adenosine in the different tissues. We investigated whether adenosine in-
duced relaxant responses in the detrusor muscle from control and diabetic
rats were altered during normoxic and hypoxic conditions. Streptozotocin
diabetic animals were studies at 15 weeks. Carbachol precontracted blad-
ders did not exhibit any alterations between control and diabetic animals
in the responses of adenosine (Con: pD2 : 5.68 ± 0.08 ; max. 67.8 ± 3.52,
Dia: pD2 : 5.56 ± 0.08; max. 50.1 ± 4.29). Hypoxia (10 %O2) potentiated
adenosine induced relaxation in the control detrusor muscle (normoxia, :
pD2 : 5.68 ± 0.08 ; max. 67.8 ± 3.52, hypoxia pD2 :5.91 ± 0.15; max. 90.7
± 1.79, p<0.01). The adenosine antagonist, 8-phenyltheophylline (10 mM,)
reduced hypoxia induced supersensitivity to adenosine from control blad-
der. There was no significant difference seen between normoxia and hy-
poxia in the responses to adenosine of diabetic bladders (normoxia pD2 :
5.56 ± 0.08 ; max. 50.1 ± 4.29, hypoxia pD2 :6.20 ± 0.30; max. 60.0 ±
7.37). In conclusion,we suggested that adenosine induced relaxant re-
sponses potentiated during hypoxia is the important finding and A2 recep-
tors could mediate to this enhanced relaxant effect in the bladder from
control animals. We further suggested that diabetic detrusor muscles is
accompanied by abolished relaxant response to adenosine during hypoxia
and this may be due to decreased A2 receptors in diabetic bladders.
161
EFFECTS OF ADENOSINE ON THE CONCENTRATIONS OF
HISTAMINE AND 5-HT IN THE PLASMA OF ACTIVELY
SENSITISED BROWN NORWAY (BN) RATS CHALLENGED
WITH SALINE OR ALLERGEN.
Mazzoni L., Hannon J. P.,. Tigani B & Fozard J. R.
Research Department. Novartis Pharma AG., Basel, Switzerland
We have shown a marked augmentation of the bronchoconstrictor response
to adenosine following allergen (ovalbumin, OA) challenge in actively
sensitised, BN rats; pharmacological analysis indicated that the response is
mediated by A2B receptors and dependent on 5-HT release from mast cells
(Fozard & Hannon, 2000). We now report changes in the plasma concentra-
tions of histamine and 5-HT associated with the bronchoconstrictor response
to adenosine. BN rats, actively sensitised to OA, were anaesthetised and
cannulae inserted into the jugular vein and carotid artery. Intravenous injec-
tion of vehicle did not change the plasma histamine or 5-HT concentrations.
In animals challenged with saline (0.2 ml i.t., -3h), in which adenosine in-
duced only weak bronchoconstriction, adenosine (1 mg/kg i.v) induced
marked and significant increases in the plasma level of histamine (10-fold,
p<0.001) but 5-HT levels were unaffected. In contrast, in animals challenged
with OA (0.3 mg/kg i.t., -3h), in which adenosine induced marked
bronchoconstriction, adenosine induced both a substantial increase in hista-
mine (10-fold, p<0.001) and a significant increase in 5-HT (3.4-fold, p<0.05).
The increase in plasma histamine and 5-HT levels in response to adenosine
were reduced by pretreatment (-5 min) with 8-SPT (40 mg/kg i.v. [hista-
mine, -52%, p<0.05; 5-HT, -69%, p<0.05]), the mast cell mediator release
inhibitor, disodium cromoglycate (40 mg/kg i.v. [histamine, -83%, p<0.01; 5-
HT, -58%, p<0.05]) but unchanged after methysergide (0.01 mg/kg i.v.); all
interventions reduced the bronchoconstrictor response to adenosine by 70%.
The data provide further evidence that bronchoconstrictor responses to ad-
enosine augmented following OA challenge in actively sensitised BN rats
are largely indirect, a consequence of 5-HT release from mast cells.
Fozard J. R. & Hannon J. P. (2000), Clin. Exp. Allergy.(In press).
 ABSTRACTS FROM PURINES 2000
162
MUTUALLY POTENTIATING EFFECTS OF DRUGS ELEVATING
EXTRACELLULAR ADENOSINE AND GRANULOCYTE COLONY-
STIMULATING FACTOR ON ERYTHROPOIETIC RECOVERY
FOLLOWING 5-FLUOROURACIL-INDUCED HAEMATO-
TOXICITY IN MICE
Weiterová, L.(1); Hofer, M.(2); Pospíšil, M.(2); Znojil, V.(3); Vácha, J.(3);
Vacek, A.(2); Pipalová, I.(2)
(1)Faculty of Science, Masaryk University, Brno, Czech Republic. (2)Insti-
tute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech
Republic.(3) Medical Faculty, Masaryk University, Brno, Czech Republic
The presented data suggest the ability of drugs increasing the level of extra-
cellular adenosine act additively with granulocyte colony-stimulating factor
(G-CSF) to enhance erythropoiesis. To demonstrate these interactions, the
combined effects of the drugs on the recovery from erythropoietic damage
induced in mice by a single dose of 5-fluorouracil (5-FU) were investigated.
Elevation of extracellular adenosine and thus activation of adenosine recep-
tors was induced by joint administration of dipyridamole (DP), a drug inhib-
iting the cellular uptake of adenosine, and adenosine monophosphate (AMP),
an adenosine prodrug. The drugs were injected in a 4-d treatment regimen
starting 2 h after 5-FU injection. Both DP + AMP alone and G-CSF alone
induced only weak effects. However, the combination of the three drugs
produced significant elevation of erythrocytes in the peripheral blood which
pertained in the posttreatment period. Stimulation of proliferation of eryth-
roid progenitor cells (BFU-E) in femoral bone marrow and increased levels
of reticulocytes in the peripheral blood were observed in the course of the 4-
d treatment regimen. In addition, significantly decreased mean cell haemo-
globin accompanying the elevated numbers of erythrocytes in the
combination-treated mice was found. This effect could be interpreted as the
result of a sublethal 5-FU-induced damage to erythroid progenitor and pre-
cursor cells forced to proliferate intensively by the combination therapy. The
observed additivity and synergism of G-CSF with elevated extracellular ad-
enosine in terms of erythropoiesis is an interesting finding with potential
implications in clinical practice.
163
DOSE AND TIME EFFECTS OF CAFFEINE INTAKE ON HU-
MAN PLATELET ADENOSINE A2A RECEPTORS: FUNCTIONAL
AND BIOCHEMICAL ASPECTS
Varani, K. (1); Portaluppi, F. (1); Gessi, S. (1);Merighi, S. (1); Ongini, E. (2);
Belardinelli, L. (3); Borea, PA.(1).
(1) Department of Clinical and Experimental Medicine, Ferrara, Italy. (2)
Schering-Plough Research Institute, San Raffaele Science Park, Milan, Italy.
(3)Department of Medicine, Division of Cardiology, University of Florida,
Gainesville, USA.
We determined whether repeated caffeine administration at different dosages
and for different periods of time (400 or 600mg/d for 1 week or 400mg/d for 2
weeks) up-regulates the human platelet A2A adenosine receptors and is ac-
companied by increases in cAMP accumulation, decreases in aggregation and
calcium levels after stimulation of A2A receptors by the agonist 2-hexynyl-NECA
(HE-NECA). Platelets were obtained from peripheral venous blood of 45 hu-
man healthy volunteers at the end of two weeks of caffeine abstinence and at
12, 60 and 108 hours after the last dose of caffeine. The lowest dose of caffeine,
given for 7 days, had no effect. By giving 400mg/d of caffeine for 14 days re-
sulted, in binding assays performed with the selective A2A adenosine receptor
radioligand [3H]SCH 58261 in the upregulation of A2A receptors. Binding pa-
rameters revealed a control (prior to caffeine) BMAX value of 110±3 fmol/mg
protein and a KD value of 1.21±0.08 nM, respectively. At 12 and 60 hours after
caffeine withdrawal the receptor density, BMAX, increased at both time points
of some 20%, but the KD values were unchanged. The potency of HE-NECA
to produce antiaggregatory effects, a rise in cAMP accumulation and a de-
crease in calcium levels was significantly increased. A similar trend in binding
and functional parameters was observed in subjects treated with 600mg/d for
1 week. These results indicate that chronic caffeine intake leads to upregulation
of A2A adenosine receptors which is accompanied by sensitization, in a time
and dose-dependent manner, to the actions of the agonist HE-NECA.
89
164
ADP POTENTIATES PLATELET DENSE GRANULE SECRETION
INDUCED BY U46619 OR TRAP THROUGH ITS INTERACTION
WITH THE P2cyc RECEPTOR
Cattaneo, M.; Lecchi,A.
A. Bianchi Bonomi Hemophilia and Thrombosis Center, Department of
Internal Medicine, IRCCS Ospedale Maggiore, University of Milano. Italy.
Background. By itself, ADP does not induce secretion of the platelet dense
granule constituents directly, but through the aggregation-mediated synthe-
sis of thromboxane A2 (TxA2), which is greatly enhanced when [Ca2+]o is
decreased by citrate. However, ADP that had been secreted by platelets
potentiates secretion independently of aggregation and TxA2 synthesis
(Cattaneo et al, 1998). Aims. To assess which of the 3 platelet receptors for
ADP (P2X1, P2Y1 and P2cyc) is involved in the potentiation by ADP of
platelet secretion. Subjects. Four normal volunteers and patient VR (con-
genitally deficient in P2cyc). Methods. [14C]5HT secretion was measured 2
min after the addition of U46619 (1 umol/L, a TxA2 analogue) or TRAP (20
umol/L, which stimulates the PAR1 receptor) to aspirin-treated washed plate-
lets suspensions containing 2mmol/L CaCl2, which were not stirred (to pre-
vent platelet aggregation). The effects of epinephrine (10 umol/L) and the
following nucleotide analogues was investigated: AR-C69931MX (P2cyc
antagonist), MRS-2179 (P2Y1 antagonist), α,β-me-ATP (P2X1 agonist). Re-
sults. [14C]5HT secretion induced by U46619 or TRAP from normal plate-
lets was inhibited by AR-C69931MX, but was not affeted by any of the other
2 nucleotide analogues. Epinephrine restored normal secretion in platelets
incubated with ARC-69931MX. VR’s platelets secretion was much lower
than normal, and was not affected by any of the three nucleotide analogues.
In contrast, it was greatly enhanced by epinephrine. Conclusion. P2cyc, which
is negatively coupled to adenylyl-cycalse, mediates the potentiation of platelet
secretion by released ADP. Epinephrine also potentiated secretion. There-
fore, the Gi pathway must be stimulated for full platelet secretion.
165
PHARMACOLOGICAL CHARACTERISATION OF P2X1 RECEP-
TORS ON HUMAN WASHED PLATELETS
Fagura, M.S.(1); Shelton, P.A.(2); Kindon, N.D.(3); Henderson, D.J.(2);
Dougall, I.G(1).
Depts of (1) Discovery BioScience, (2) Molecular Biology & (3) Medicinal
Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough,
U.K., LE11 5RH.
Human platelets have been reported to possess P2X1 receptors (as well as
P2T and P2Y1 receptors; Kunapuli., 1998) but their pharmacological
characterisation has received little attention. In this study, we compared the
pharmacological profile in human washed platelets with that obtained in the
human cloned receptor, stably expressed in rat liver epithelial cells (RLEs).
Platelets were obtained as previously described (Humphries et. al., 1994).
Changes in [Ca2+]i were measured using standard fluorescent techniques.
Apyrase (0.06U.ml-1) was present at all times to degrade endogenous pu-
rines. Antagonists for P2Y1 (A3P5P, Boyer et. al., 1996), P2T (AR-C66096,
Humphries et. al., 1994) and P2X1 receptors (AR-C67648, [5-{6-amino-2-
propylthio-9H-purin-9-yl}pentyl]phosphate, monoanhydride with (di-
bromophosphonylmethyl)phosphonic acid, unpublished data) were added
10 seconds before ATP.
A3P5P and AR-C66096, at concentrations 100-fold greater than their
known P2Y1 and P2T affinities, had no effect on ATP responses. AR-C67648
competitively antagonised ATP responses in RLEs and platelets (pKB =
5.60±0.04 and pA2= 5.23±0.09 respectively; mean±s.e.mean, n=4).
Generation of agonist concentration-effect curves yielded the same po-
tency order in both systems (ATP α,β-methylene-ATP D-β,γ-methylene-
ATP = L-β,γ-methylene-ATP). Maximal activation of P2X1 receptors in
platelets had no effect on P2Y1 receptor mediated ADP responses.
These results confirm that platelets contain P2X1 receptors, which are
pharmacologically distinct from P2Y1 or P2T receptors. The functional sig-
nificance of P2X1 receptors on platelets requires further study.
Boyer, J.L. et. al. (1996). Mol. Pharmacol., 50, 1323-1329.
Humphries, R.G. et. al. (1994). Br. J. Pharmacol., 113, 1057-1063.
Kunapuli, S. P. (1998). Drug. Dev. Res., 45, 135-139.
90 ABSTRACTS FROM PURINES 2000
166
SUPERIOR INHIBITION OF ADP-INDUCED HUMAN PLATE-
LET AGGREGATION BY AR-C69931MX THAN CLOPIDOGREL
Jarvis, G.E.; Nassim, M.A.; Humphries, R.G.; Kirk, I.P.; Tomlinson, W.;
Cusworth, E.A.; Midha, A.; Perrett, J.H.; Mobbs, E.J.; Hammersley, M.D.;
Watts, I.S.
AstraZeneca R&D Charnwood, Loughborough, United Kingdom
Introduction: P2X1, P2Y1 and P2T receptors are present on the platelet.
AR-C69931MX, a potent, selective and competitive P2T antagonist, is cur-
rently in development for the treatment of acute coronary syndromes. In
this study, the anti-aggregatory effect of AR-C69931MX was compared to
that of clopidogrel (Plavix®), a pro-drug which inhibits ADP-induced platelet
aggregation (APA) by irreversibly targeting the P2T pathway.
Methods: Clopidogrel (75mg od) was dosed to 8 healthy male volun-
teers for 11 days. Blood was taken on days 0 (pre-dose control), 1, 2, 3, and
11. APA was measured, with and without addition of AR-C69931MX (500
nM) in vitro, using both whole blood (heparinised) impedance aggregometry
(WBIA) and optical aggregometry in citrated PRP. Three indices were
measured using optical aggregometry1: rate of aggregation (RATE), maxi-
mum extent (MEXT) and final extent (FEXT).
Results: ADP pA50 values (mean±sem: n=8) for each response index were:
Day 0 (control, without/with AR-C69931MX): WBIA, 6.31±0.07/
3.96±0.12; RATE, 5.86±0.05/5.48±0.06; MEXT, 5.60±0.05/5.04±0.12;
FEXT, 5.53±0.06/3.86±0.09.
Day 11 (clopidogrel): WBIA, 5.24±0.13; RATE, 5.72±0.05; MEXT,
5.37±0.07; FEXT, 4.80±0.10.
Conclusions: The minimal effect of AR-C69931MX and clopidogrel on
RATE and MEXT reflects the importance of P2Y1 receptor activation on these
indices1. FEXT and WBIA, which are determined by P2T receptor occupancy,
were inhibited to a greater extent by a clinically relevant concentration of AR-
C69931MX than by the normal therapeutic dose of clopidogrel. This suggests
that direct P2T antagonists will provide significant improvement over the mod-
est clinical benefit demonstrated to date with clopidogrel.
1
Jarvis et al (2000) Br J Pharmacol 129 275-282.
167
THE ATP-DEPENDENT INHIBITION OF THROMBIN-IN-
DUCED CELLULAR SIGNALLING IN HUMAN PLATELETS IS
MEDIATED BY A P2X RECEPTOR
Conde, M.; Chamero, P.; Trueba, M.; Macarulla, J.M.; Gómez, A.; Marino, A.
Department of Biochemistry and Molecular Biology, Faculty of Science,
University of the Basque Country. P. O. Box 644, 48080 Bilbao, Spain.
The classical P2T receptor in human platelets has now been resolved into
three P2 receptor subtypes: P2Y1, P2X1 and P2TAC receptors (Kunapuli., 1998).
Extracellular ATP has been widely accepted as a competitive antagonist of
the ADP-induced platelet activation by competition with ADP for P2T re-
ceptors. Recently, differential responses to extracellular ATP have been de-
scribed to play a physiological role in the modulation of platelet responses
(Soslau., 1997). To understand the ATP mediated inhibition of platelet func-
tions, we have studied the ability of ATP and its analogues to inhibit the
[Ca2+]i and platelet aggregation exerted by 0.025 U/ml of thrombin. ATP
concentrations of 400-1000 µM inhibited the thrombin-stimulated [Ca2+]i
and platelet aggregation. ATP analogues inhibited platelet activation, the
rank order being αβ-methylenATPβγ-methylenATPATPBz-ATP. Relatively
high concentrations of MeSATP inhibited platelet activation whilst low con-
centrations potentiated thrombin-induced platelet activation. The inhibi-
tory effect of ATP and its analogues was enhanced significatively in a Mg2+-free
medium, suggesting that this effect could be dependent of ATP4-. Surpris-
ingly, several known P2X7 blockers showed different effects. Thus, HMA
reverted the ATP-mediated inhibition whereas KN-62 was without effect
and oATP potentiated the ATP-dependent inhibition of thrombin-mediated
responses. The inhibition exerted by ATP was independent of cGMP but a
potentiating effect was found in the presence of ATP and NO. The adenylate
cyclase inhibitor, SQ-22536, slightly reverted the ATP-mediated inhibition.
We conclude that human platelets possess a P2X purinoceptor, different of
P2X7 and ADP-stimulated P2T receptors, which is involved in the physi-
ological inhibition of human platelet activation.
168
PHARMACOLOGICAL CHARACTERIZATION THE A2B ADENOS-
INE RECEPTOR IN TYPE 1 CEREBELLAR ASTROCYTES: IN-
TERACTION WITH P2Y RECEPTORS.
Jiménez, A.I.; Castro, E.; Delicado, E.G.; Miras-Portugal, M.T.
Departamento de Bioquímica, Facultad de Veterinaria, Universidad
Complutense de Madrid, Spain.
ATP and adenosine can be accumulated in the extracellular space and could
interact with specific receptors present in the same cell. We have studied
the interaction between P1 and P2 purinoceptors in type-1 astrocytes cul-
tures from postnatal days 7-8 rat cerebella using single cell microfluorimetry
with fura-2. ATP but not adenosine elicited [Ca2+]i transients (EC50 = 7.9
± 0.3 µM). However, co-stimulation of astrocytes with adenosine and in-
effective ATP concentrations (0,1 or 1 µM) evoked [Ca2+]i transients, which
correspond to 60% of the maximal ATP response. NECA was the only ago-
nist that mimicked the adenosine effect with an EC50 value of 0.17 ± 0.01
µM. The cAMP production stimulation was measured in response to sev-
eral effectors. Adenosine and NECA significantly enhanced the cAMP lev-
els. On the contrary, CPA and CGS-21680 were ineffective. These results
together with immunocytochemistry experiments indicated the presence
of A2B receptors in type-1 astrocytes. The treatment of astrocytes with chol-
era toxin potentiated ATP calcium signals, lowering the (EC50 value for
ATP to 1.5 ± 0.2 µM. However, the pretreatment of cells with forkolin or
a permeable cAMP analogue had no effect on ATP calcium responses. These
results indicated that the potentiation mechanism was elicited before the
adenylate cyclase activation and was parallel but independent on cAMP
accumulation suggesting the involvement of βγ subunits released after Gs
stimulation. The present study shows that individual type-1 astrocytes
coexpress P1 and P2 purinoceptors, which interact. Moreover, the fact that
adenosine potentiates ATP calcium responses suggest relevant functions
of purinergic system in astrocytes.
169
CASPASE ACTIVATION IS AT THE BASIS OF APOPTOSIS OF
HUMAN ASTROCYTOMA CELLS BY 2-CHLORO-2'-DEOXY-AD-
ENOSINE AND 2-CHLORO-ADENOSINE
Ceruti, S. (1); Malorni, W. (2); Cattabeni, F. (1); Abbracchio, M.P. (1).
(1) Institute of Pharmacological Sciences, University of Milan, Milan, Italy.
(2) Istituto Superiore di Sanità, Rome, Italy.
Several adenosine analogues behave as potent inducers of apoptosis and
are often utilized as chemotherapic drugs. We have previously demon-
strated that 2-chloro-2'-deoxy-adenosine (Cladribine, already utilized in
leukemias and lymphomas) and the related compound 2-chloro-adenosine
(2-CA), are potent inducers of apoptosis in a human astrocytoma cell line
(ADF cells; Ceruti et al., Journal of Neuroscience Research, in press). Both
derivatives need to be intracellularly phosphorylated by two different ki-
nases to exert their toxic actions. In this study we have evaluated the bio-
chemical steps leading to ADF cell death, with particular interest in caspase
activation. Cell death by either compound involves cleavage and likely
activation of caspase-3, as demonstrated by western blotting analysis. In
our model, caspase activation is not evident at early time points, but only
after a 7-10 hour exposure to either compound. This confirms that other
events (i.e., the intracellular phosphorylation/activation and, in the case of
Cladribine cell cycle block) are needed to lead to activation of the death
pathway. Moreover, inhibition of adenosine kinase and deoxy-cytidine ki-
nase in the case of 2-CA and Cladribine, respectively completely abol-
ished caspase-3 activation, therefore confirming the link between
intracellular phosphorylation of purine compounds and cell death. To clearly
understand the sequential pathways leading to astrocytoma cell death, we
are currently evaluating the upstream activation of caspase-9 and the re-
lease of cytochrome c from mitochondria.
 ABSTRACTS FROM PURINES 2000
170
EXTRACELLULAR ATP FACILITATES CALCIUM-ACTIVATED
POTASSIUM CURRENTS IN RETINAL GLIAL (MÜLLER) CELLS:
POSSIBLE INVOLVEMENT IN STIMULATION OF DNA SYN-
THESIS
Bringmann, A. (1); Moll, V. (1); Pannicke, T. (1); Faude, F. (2); Illes, P. (3);
Reichenbach, A. (1)
(1) Paul Flechsig Institute of Brain Research, University of Leipzig,
Jahnallee 59, D-04109, Leipzig, (2) Department of Ophthalmology, Uni-
versity of Leipzig, Liebigstrasse 10-14, D-04103 Leipzig, (3) Rudolf Boehm
Department of Pharmacology, University of Leipzig, Haertelstrasse 16-18,
D-04107 Leipzig, Germany
Human Müller glial cells express P2 receptors. Receptor activation in-
creases the intracellular Ca2+ level via both Ca2+ release from internal
stores and Ca2+ entry from the extracellular space. We investigated (i) in
human Müller cells whether the ATP-induced Ca2+ entry stimulates
Ca2+-activated K+ (K(Ca)) channels, and (ii) in primary cultures of guinea-
pig Müller cells whether the activity of K(Ca) channels and of voltage-
gated Ca2+ channels is involved in the stimulatory effect of external ATP
on the DNA synthesis. DNA synthesis was measured by the incorporation
of 5-bromo-2'-deoxyuridine. In human Müller cells, external application
of Na-ATP or dibenzoyl-ATP increased biphasically the amplitude of out-
ward K+ currents. The increase of the sustained outward current was
blocked by applying a calcium-free external solution, while the initial tran-
sient increase was independent of extracellular Ca2+. It is concluded that
both a transient Ca2+ release from intracellular stores and a sustained
Ca2+ entry from the extracellular space elevate K(Ca) currents in Müller
cells. The Na-ATP effect was blocked by iberotoxin, indicating the activa-
tion of big K(Ca) (BK) channels. In Müller cell cultures, exposure to Na-
ATP for 16 h elevates the DNA synthesis rate. This effect was decreased
by simultaneous exposure to suramin or PPADS and was reversed by
blockers of BK (iberotoxin) or SK channels (apamin), as well as by blockers
of voltage-gated Ca2+ channels (flunarizine, nimodipine). It is concluded
that the activity of both K(Ca) channels and voltage-gated Ca2+ channels
is necessary for the proliferation-inducing effect of external ATP.
171
TWO DISTINCT PURINOCEPTORS ARE INVOLVED IN PU-
RINE- AND PYRIMIDINE-EVOKED Ca2+ ELEVATION IN MAM-
MALIAN BRAIN ASTROCYTIC CULTURES
Abbracchio, M.P. (1); Caiani, A. (1); Bolego, C. (1); Centemeri, C. (1); Ceruti,
S. (1); Cattabeni, F. (1); Jacobson, K.A. (2); Puglisi, L. (1); Rovati, G.E. (1);
Burnstock, G. (3); Nicosia, S. (1)
(1) Institute of Pharmacological Sciences, University of Milan, Milan, Italy.
(2) Molecular Recognition Section, Lab. Bioorg. Chem., NIDDK, NIH,
Bethesda, MD, USA. (3) Autonomic Neuroscience Institute, Royal Free
Hospital, School of Medicine, London, UK.
We have previously demonstrated that ATP and 2-methyl-thio-ATP (2-Me-
SATP) increase cytosolic calcium concentrations ([Ca2+]i) in rat striatal
astrocytes. The aim of the present study was to: (i) characterize pyrimi-
dine-induced [Ca2+]i increase in the same model, and (ii) try to identify
the P2Y receptor subtypes mediating Ca2+ mobilization. UDP and UTP
triggered a concentration-dependent [Ca2+]i elevation, solely due to mo-
bilization from intracellular stores. This was indicated by the fact that (a)
removing calcium from extracellular medium (with EGTA) or (b) blocking
its influx with Ni2+ did not modify UTP responses; (c) the store-depleting
agent thapsigargin completely abolished UTP-evoked [Ca2+]i increments.
The G-protein inhibitor guanosine-5'-O-(2-thiodiphosphate), GDPβS, par-
tially inhibited, whereas pertussis toxin did not affect UTP responses. The
phospholipase C inhibitor U-73122 significantly reduced UTP-evoked
[Ca2+]i rise. Computer-assisted analysis of concentration-response curves
indicated that UTP and UDP responses are mediated by a single receptor,
while ATP and 2-Me-SATP interact with two distinct receptors. The se-
lective P2Y1 receptor antagonist MRS2179 abolished the higher-potency
component for ATP. Neither suramin nor pyridoxal phosphate-6-azophenyl-
2'-4'-disulphonic acid affected the UTP response. Sequential challenges
with the same nucleotides resulted in an almost complete homologous
desensitization. Pre-exposure to UTP lowered the subsequent responses
to either ATP or 2-Me-SATP. Maximally active concentrations of UTP and
ATP were not additive. In conclusion, [Ca2+]i elevation in astrocytes by
purines and pyrimidines is related to mobilization from intracellular stores
via two distinct P2Y receptors, most likely the P2Y1 and P2Y6 subtypes.
91
172
P2Y-RECEPTOR MEDIATED ASTROGLIOSIS IN VIVO
Franke, H.(1); Krügel, U.(1); Schmidt, R.(2); Preiß, R.(2); Illes, P.(1)
(1) Rudolf Boehm Department of Pharmacology and Toxicology, Univer-
sity of Leipzig, Leipzig, Germany. (2) Department of Clinical Pharmacol-
ogy, University of Leipzig, Leipzig, Germany.
ATP may represent one of the endogenous factors involved in the initiation
of astrogliosis via activation of P2-receptors, a process repeatedly demon-
strated in cultured astrocytes. We have shown that infusion of 2-methylthio-
ATP into the nucleus accumbens (NAc) of rats under in vivo conditions
induces astrogliosis, which was inhibited by the P2 receptor antagonists re-
active blue 2 and PPADS (Glia 28:190-200, 1999). Investigations with more
specific receptor agonists (α,β-methylene ATP for P2X; adenosine 5'-O-(2-
thiodiphosphate), ADP-β-S for P2Y) suggested that both receptor types are
present on astrocytes in the NAc of rats, referring to a dominance of the P2Y-
receptor. To characterize the P2Y-receptor subtypes which are involved in
the induction of proliferation, the effects of ADP-β-S (P2Y1) and uridine 5'-
triphosphate-γ-S (UTP-γ-S, P2Y2/Y4)(100 µM, each) were compared. For
quantification of proliferating astrocytes, double staining with antibodies
against bromodeoxyuridine (BrdU) and glial fibrillary acidic protein (GFAP)
was performed. All GFAP-positive and all GFAP-/BrdU-double stained cells
were counted in identical areas of the NAc. ADP-β-S showed a stronger
mitogenic effect than UTP-γ-S. Both effects were inhibited by PPADS (30
µM). The ADP-β-S-induced morphogenic reaction involved a significant
up-regulation of the GFAP-immunoreactivity (immunohistochemistry) and
an increase of the GFAP-content (Western blotting). The number of c-Fos
like immunoreactive nuclei under the needle tract was increased compared
to that measured in the aCSF treated control side.
The present data suggest that P2Y1-receptors are involved in astrogliosis
in vivo. This may have consequences for the therapy of gliotic reactions in
neurological disorders.
173
A1 ADENOSINE RECEPTORS AND mGLU3 METABOTROPIC
GLUTAMATE RECEPTORS MODULATE PURINE RELEASE
AND NEUROTROPHIN PRODUCTION FROM RAT CULTURED
ASTROCYTES BY DIFFERENT MOLECULAR PATHWAYS.
Ciccarelli, R. (1); D’Alimonte, I. (1); Florio, T. (1); Kleywegt, S. (1); D’Auro,
M.G. (1); Ballerini, P.(1); Bruno, V. (2); Caciagli, F. (1).
(1) Department of Biomedical Sciences, Section of Pharmacology, “G.
D’Annunzio” University, Chieti, Italy. (2) Neuromed, Pozzilli, Venafro. Italy.
As we previously demonstrated in rat hippocampal slices or synaptosomes
(J. Neurochem. 67: 302, 1996), the selective stimulation of A1 adenosine or
mGlu3 receptors with the respective agonists, 2-chloro-N6-cyclopentyl-
adenosine (CCPA) and aminopyrrolidine-2R-4R-dicarboxylate (APDC),
inhibits, in dose-dependent fashion, the electrically-evoked release of pu-
rines from rat cultured astrocytes and reduce forskolin-induced increase
in intracellular cAMP levels. In contrast, these agonists dose-dependently
stimulate astrocytes to synthesise and to release nerve growth factor and
transforming growth factor-β. Interestingly, these drugs activate mitogen-
activated protein (MAP) kinases ERK1/2 (maximal effect 10 min after drug
addition). Culture pretreatment with pertussis toxin as well as with selec-
tive antagonists of A1 and mGlu3 receptors, such as 8-cyclopentyl-1,3-
dipropylxanthine (DPCPX) or (2S,1’S,2’S,3’R)-2-(2'-carboxy-3'-phenyl-
cyclopropyl)glycine (PCCG-IV), prevents both the inhibition of the evoked
purine release and the enhancement of trophic factors production. The
addition of 8-bromo-cyclicAMP to the culture medium significantly coun-
teracts CCPA- or APDC-induced inhibition of the evoked purine release.
In contrast, cell pretreatment with specific inhibitors of MAP kinase path-
way such as wortmannin or PD098,059 does not affect purine release but
substantially reduces trophic factor production. These findings indicate
that, in cultured astrocytes, the activation of A1 and mGlu3 receptors pro-
duce different effects by different signal transduction pathways. Purine
release modulation results linked to the inhibition of cAMP formation
whereas the production of neurotrophins is related to the activation of
MAPK cascade. These effects are likely coupled to the activity of αi or βγ
subunits of receptor-coupled Gi proteins.
92 ABSTRACTS FROM PURINES 2000
174
EVIDENCE FOR A BASAL AND ELECTRICALLY EVOKED RE-
LEASE OF PURINES AND GLUTAMATE FROM CULTURED
MICROGLIAL CELLS OF RAT BRAIN
Di Iorio, P.(1); Giuliani, P.(1); Virgilio, A.(1); Ballerini, P.(1); Patricelli, P.(1);
Poli, A.(2); Copani, A.(3); Caciagli, F.(1).
(1)Department of Biomedical Sciences, G. D’Annunzio University, Chieti,
Italy. (2) Department of Biology, Bologna University, Bologna, Italy. (3)
Department of Pharmaceutical Sciences, Catania University, Catania, Italy.
Superfused microglial cells cultured from rat brain, labeled with 3H-ad-
enosine or 14C-glutamate, released purines and excitatory amino acids,
measured by HPLC using UV, fluorescence and on-line radiochemical
detection. A field electrical stimulation (30 mA/cm2, 2 mV, 5 msec dura-
tion), applied at different frequencies (2.5 - 20 Hz) for 5 min to cultured
cells, caused a frequency-dependent increase in the purine and glutamate
release, with a maximal increase (at 20 Hz) by about 12- and 3-fold respec-
tively, over basal outflow. In basal condition, the purines assayed in the
superfusion medium were mainly represented by nucleosides (about 55%
of the total released 3H-purines) and their metabolites. The stimulation
caused a 20-fold increase in the extracellular levels of nucleotides that ac-
counted for about 40% of total evoked released of 3H-purines; whereas,
the nucleoside levels were increased by only 5-6-fold (35% of total evoked
release). Moreover, released nucleotides and nucleosides underwent a rapid
extracellular breakdown due to membrane-located specific enzymes. In
contrast, the percent rate of released glutamate was not substantially modi-
fied either in basal condition or under electrical stimulation. The activa-
tion of cultured microglial cells with lipopolysaccharide (LPS; 1 µg/ml) for
4 hours did not affect either the basal or the evoked outflow of purines,
whereas it nearly doubled cell capability to release glutamate. The evoked
release of purines and glutamate was modulated by the activation of sub-
types of purine receptors or metabotropic glutamate receptors whose ex-
pression on microglial cells was evaluated by selective immunostaining,
western blotting, or RT-PCR.
175
PROTEIN KINASE C-MEDIATED NEGATIVE FEEDBACK AL-
TERS FREQUENCY DEPENDENCE OF P2Y1-MEDIATED Ca2+
SIGNALLING IN ASTROCYTES
Fam, S.R. (1,2); Gallagher, C.J. (1,3); Salter, M.W. (1,2,3)
(1) Program in Brain and Behaviour, Hospital for Sick Children; (2) Dept.
Physiology, University of Toronto; (3) Institute of Medical Sciences, Uni-
versity of Toronto, Toronto, Canada.
Astrocyte-to-astrocyte transmission of Ca2+ waves is mediated by release
of ATP and activation of P2Y purinoceptors. For astrocytes from the dorsal
spinal cord, we have determined that fully propagating Ca2+ waves re-
quire the P2Y1 subtype of metabotropic P2 purinoceptor. Under physi-
ological conditions, Ca2+ waves oscillate within astrocyte networks which
produces repetitive activation of astrocyte P2Y1 purinoceptors. We there-
fore characterized Ca2+ responses evoked by repeatedly activating these
receptors. We recorded [Ca2+]i using fura-2AM from individual astrocytes
in primary cultures of dorsal spinal cord. P2Y1 receptors were activated by
trains (40-50 min duration) of repeated applications of 2-MeSADP (5-10 s
at regular intervals between 60 and 90 s). We found that the amplitude of
Ca2+ responses declined progressively over the first 5-8 applications in the
train and then alternated between a plateau level and a lower level. Upon
bath application of the protein kinase C (PKC) inhibitor, Gö 6850, the
amplitude of responses declined progressively to the same plateau level
but there was no subsequent alternation of responses. These results indi-
cate the alternation in the amplitude of P2Y1 receptor-mediated responses
is caused by a negative feedback dependent upon PKC. This PKC-depen-
dent modulation of the [Ca2+]i signals is expected to produce divergence in
downstream signalling cascades engaged by P2Y1 receptor activation. (Sup-
ported by the MRC of Canada).
176
METABOTROPIC PURINOCEPTORS MEDIATE THE PROPAGA-
TION OF INTERCELLULAR Ca2+ WAVES IN SPINAL CORD
ASTROCYTES
Gallagher,C.J. (1,2); Fam, S.R. (1,3); Salter, M.W. (1,2,3)
(1) Program in Brain and Behaviour, Hospital for Sick Children. (2) Insti-
tute of Medical Sciences, University of Toronto. (3) Dept. Physiology, Uni-
versity of Toronto. Toronto, Canada
Introduction: Propagation of intercellular Ca2+ waves is a principal mode
of cell-to-cell communication amongst non-excitable cells. Two subtypes
of metabotropic purinoceptor are present on spinal cord astrocytes, P2Y1
and P2U. The main goal of the present study was to determine whether
metabotropic purinoceptors mediate the propagation of Ca2+ waves in a
spinal cord astrocyte network. Further, we examined which purinoceptor
subtype(s) contributes to Ca2+ wave propagation.
Methods: [Ca2+]i was imaged in cultured spinal cord astrocytes using
Fura-2. Ca2+ waves were evoked by mechanical simulation of a single cell
with a fire-polished glass micropipette.
Results: Mechanical stimulation of an individual astrocyte produced a
rise in [Ca2+]i in the stimulated cell which was followed within 3-20 s by
increases in [Ca2+]i in other astrocytes in the field. To investigate the in-
volvement of purinoceptors in the spread of astrocyte Ca2+ waves we bath
applied the pharmacological antagonists A3P5PS, which selectively blocks
P2Y1, and suramin, which blocks both P2Y1 and P2U. In A3P5PS (100
µM) the rise in [Ca2+]i of the stimulated cell was unaffected however the
spread of the Ca2+ wave to surrounding cells was reduced by 55% relative
to control. In suramin (100 µM) the spread of the Ca2+ wave was reduced
by 87% relative to control.
Conclusions: We conclude that the propagation of Ca2+ waves in spinal
cord astrocytes is mediated by metabotropic purinoceptors. Furthermore, we
have determined that both subtypes of purinoceptor are required for full Ca2+
wave propagation in spinal astrocytes.(Supported by the MRC of Canada).
177
ATP-ACTIVATED GLUTAMATE RELEASE THROUGH NON-SE-
LECTIVE P2Z/P2X7 LIKE CHANNELS IN CULTURED MOUSE
ASTROCYTES
Anderson, C. (1); Duan, S. (1); Chen, Y. (1); Keung, E (2); Swanson, R. (1)
(1) Department of Neurology and (2) Department of Medicine, University
of California, San Francisco and Department of Veterans Affairs, San Fran-
cisco, CA, USA (2)
Glutamate release from astrocytes can be acheived by reversal of high-
affinity uptake systems, by regulatory volume decrease through volume-
sensitive organic anion channels, and by a Ca2+-dependent mechanism
resembling neuronal vesicular release. ATP has been implicated as a pri-
mary extracellular messenger in astrocytes (Guthrie et al., J Neurosci
1999,19: 520). Using whole cell patch clamp recording we showed that
ATP (0.3-3 mM) and the P2Z/P2X7 receptor specific agonist BzATP (0.03-
0.3 mM) dose dependently induced an inward current that was antago-
nized by PPADS and DIDS. Both ATP- and BzATP-induced inward current
were enhanced in Ca2+ and Mg2+ deficient solution, characteristic of P2X7
receptor channels. The permeability ratio for Na+ : Cl- : glutamate, esti-
mated by ion substitution and measuring the shift in the reversal potential
of BzATP-induced current, was 1 : 0.35 : 0.14. We further showed that ATP
(1 mM) and BzATP (0.1 mM) induced release of preloaded 14C-glutamate
and 3H-D-aspartate in astrocytes. The release was enhanced in Ca2+ and
Mg2+ deficient solution and inhibited by PPADS, DIDS, and the irrevers-
ible P2Z/P2X7 receptor antagonist oxidized ATP. Kainic acid (0.1 mM),
which induced larger depolarization than ATP and BzATP did, did not
induce 3H-D-aspartate release. We suggest that ATP induces glutamate
release in astrocytes through P2Z/P2X7 like non-selective channels. Sup-
ported by NIH RO1 NS31914, Deptartment of Veterans Affairs and the
Medical Research Council and Heart and Stroke Foundation of Canada.
 ABSTRACTS FROM PURINES 2000
178
A1 ADENOSINE RECEPTOR ACTIVATION INCREASES LEPTIN
PRODUCTION BY RAT WHITE ADIPOSE TISSUE
Ozeck, M.J. (1); Liang, H.X. (1); Belardinelli, L. (2); Shryock, J.C. (1).
(1) Department of Medicine, University of Florida, Gainesville, FL, USA.
(2) CV Therapeutics, Inc., Palo Alto, CA, USA.
The effect of A1 adenosine receptor (A1AdoR) activation on leptin production
by adipocytes from rat white adipose tissue and on serum leptin concentration
in Sprague-Dawley rats was determined. The A 1AdoR agonist R-
phenylisopropyladenosine (R-PIA) increased leptin production of isolated
epididymal adipocytes by 38 ± 3% (EC50 value = 5 nM). This effect of R-PIA
was abolished by the A1AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine
(CPX, 10 nM and 1 µM), the phosphatidylinositol 3-kinase inhibitor
wortmannin (30 nM), and the type 3 phosphodiesterase inhibitor cilostamide
(10 nM – 1 µM). Endogenous adenosine released by adipocytes also increased
leptin production. R-PIA (1 nM) augmented the effect of insulin to stimulate
leptin production by adipocytes. Insulin increased leptin release by 29 ± 4
and 104 ± 7% in the absence and presence of R-PIA respectively. R-PIA (19
µg/kg) also increased serum leptin levels in anesthetized Sprague Dawley rats.
These results suggest that activation of A1AdoRs in white adipocytes increases
leptin release and potentiates the effect of insulin to increase leptin release.
An increase of the activity of phosphatidylinositol 3-kinase and a reduction of
cAMP content in adipocytes are associated with and may mediate the action
of an A1AdoR agonist to increase leptin production.
179
THE STABLE PYRIMIDINES UDPbS AND UTPgS DISCRIMI-
NATE BETWEEN THE P2 RECEPTORS THAT MEDIATE VAS-
CULAR CONTRACTION AND RELAXATION OF THE RAT
MESENTERIC ARTERY
Malmsjoe, M. (1); Adner, M. (2); Harden, T.K. (3); Pendergast, W. (4);
Edvinsson, L. (5); Erlinge, D. (6)
(1),(2),(5),(6)Division of Vascular Research, Department of Medicine, Lund
University Hospital, Lund, Sweden. (3)Department of Pharmacology, Uni-
versity of North Carolina, School of Medicine, Chapel Hill, North Caro-
lina, USA. (4)Inspire Pharmaceuticals Inc., Durham, North Carolina, USA.
The contractile and relaxant effects of the different P2-receptors were
characterised in the rat isolated mesenteric artery by use of extracellular
nucleotides, including the stable pyrimidines uridine 5’-O-thiodiphosphate
(UDPβS) and uridine 5’-O-3-thiotriphosphate (UTPγS).
The selective P2X-receptor agonist, ab-methylene-adenosine triphos-
phate αβ-MeATP) stimulated a potent (pEC50=6.0) but relatively weak
contraction (Emax=57% of 60mM K+). The contractile concentration-
response curve of adenosine triphosphate (ATP) was biphasic when added
in single concentrations. The first part of the response could be desensitised
by αβ-MeATP, indicating involvement of P2X-receptors, while the second
part might be mediated by P2Y-receptors. The contractile P2Y-receptors
were further characterised after P2X-receptor desensitisation with 10uM
αβ-MeATP. Uridine diphosphate (UDP), uridine triphosphate (UTP) and
ATP stimulated contraction only in high concentrations (1-10mM). The
selective P2Y6-agonist, UDPβS, and the P2Y2/P2Y4–receptor agonists
UTPγS and adenosine 5’-O-3-thiotriphosphate (ATPγS) were considerably
more potent and efficacious (Emax=250%). Adenosine 5’-O-thiodiphos-
phate (ADPβS) was inactive, excluding contractile P2Y1 receptors.
After precontraction with 1uM noradrenaline, UTP, ADP and ATP in-
duced relaxations with similar potencies (pEC50=5). UTPγS, ADPβS and
ATPγS were approximately one log unit more potent indicating the pres-
ence of endothelial P2Y1- and P2Y2/P2Y4-receptors. The P2Y6-receptor
agonist, UDPβS, had no effect.
In conclusion, UDPβS and UTPγS are useful tools when studying P2-
receptors in tissue preparations with ectonucleotidase activity. Contractile
responses can be elicited by stimulation of P2Y6- and, slightly less po-
tently, P2Y2/P2Y4-receptors. The P2X response was relatively weak, and
there was no P2Y1 response. Stimulation of P2Y1- and P2Y2/P2Y4-recep-
tors elicited relaxation, while P2Y6 did not contribute.
93
180
CHARACTERIZATION OF CONTRACTILE P2 RECEPTORS IN
HUMAN CORONARY ARTERIES BY USE OF THE STABLE PY-
RIMIDINES UDPbS AND UTPgS
Malmsjoe, M. (1); Hou, M. (2); Harden, T.K. (3); Pendergast, W. (4); Pantev,
E. (5); Edvinsson, L. (6); Erlinge, D. (7)
(1),(2),(5),(6),(7)Division of Vascular Research, Department of Medicine, Lund
University Hospital, Lund, Sweden. (3)Department of Pharmacology, Uni-
versity of North Carolina, School of Medicine, Chapel Hill, North Carolina,
USA. (4)Inspire Pharmaceuticals Inc., Durham, North Carolina, USA.
This study was designed to evaluate the relative contribution of the different
contractile P2-receptors in endothelium denuded human coronary arteries
by use of extracellular nucleotides, including the stable pyrimidines uridine-
5’-O-thiodiphosphate (UDPβS) and uridine-5’-O-3-thiotriphosphate
(UTPγS). The isometric tension of isolated vessel segments was recorded in
vitro and P2-receptor mRNA expression was examined by RT-PCR.
α,β-MeATP elicited contractions at a low concentration (pEC50=5.2) indi-
cating the presence of contractile P2X-receptors. The P2Y-responses were
analyzed after P2X-receptor desensitization with 10uM ab-methylene-adenos-
ine-triphosphate (α,β-MeATP). The stable nucleotides UTPγS and adenos-
ine-5’-O-3-thiotriphosphate (ATPγS), which are agonists of P2Y2- or
P2Y4-receptors were approximately 100-fold more potent than the endogenous
uridine-triphosphate (UTP) and adenosine-triphosphate (ATP) (pEC50=4.6
and 3.8 for UTPγS and ATPγS). The efficacy of these responses were approxi-
mately double that of the P2X-agonist α,β-MeATP (Emax=125% for UTPγS,
126% for ATPγS and 68% for α,β-MeATP), suggesting a primary role for con-
tractile P2Y2/4-receptors. The P2Y2-receptor agonist Ap4A also stimulated
contraction, while the selective P2Y1-agonist adenosine-5’-O-thiodiphosphate
(ADPβS) and the selective P2Y6-agonist UDPβS had no effect. RT-PCR analysis
of mRNA from endothelium denuded human coronary arteries demonstrated
strong bands for P2Y2 and P2X1, although bands for P2Y1, P2Y4 and P2Y6
receptor mRNA could be detected.
In conclusion, UDPβS and UTPγS are important tools for P2Y-receptor
subtype characterization in intact tissues with ectonucleotidase activity.
Extracellular nucleotides elicit contraction of human coronary arteries pri-
marily by activation of P2Y2 and P2X-receptors, while a role for P2Y1- and
P2Y6-receptors can be excluded. Antagonists of P2Y2- and P2X-receptors
may be useful in the treatment of coronary vasospastic disorders.
181
PHARMACOLOGICAL CHARACTERIZATION OF NOVEL A2A
ADENOSINE RECEPTOR (A2AADOR) AGONISTS
Gao,Z.(1); Otero,D.H.(2); Zablocki, J.A.(1); Palle,V.(1); Elzein, E.(1); Baker,
S.P.(2); Belardinelli, L.(1)
(1)CV Therapeutics, 3172 Porter Drive, Palo Alto, CA94304. (2) Department
of Pharmacology and Therapeutics, University of Florida, Gainesville, FL32610
Previous research efforts have led to the development of several potent
A2AAdoR-selective agonists. These agonists generally have high affinities
for A2AAdoRs and produce robust and long-lasting physiological responses.
The clinical application of these compounds to situations whereby organ
selectivity and/or short duration of action is desirable (e.g. myocardial im-
aging with radionuclide) can be limited because of their high affinity for
the receptor. In the present study, we have characterized several newly
synthesized compounds [2-(N or C-pyrazolyl)adenosine derivatives] using
radioligand binding and cAMP assays. The aim was to develop short-act-
ing A2AAdoR agonists and compare them to the high affinity agonists,
CGS21680 (CGS) and WRC0470 (WRC). The affinities (Ki) of these com-
pounds for human A2A and A1 adenosine receptors were determined by
competition binding studies and the results (Ki values at hA2A/hA1 recep-
tors in nM) are as follows: CVT2995 (305/866), CVT3033 (2895/5836),
CVT3032 (13651/6350), CVT3146 (1269/16460), CGS (609/3540), WRC
(272/7278), NECA (360/328), and R-PIA (1656/477). Functionally, these
compounds activate A2AAdoRs and cause cAMP accumulation in PC12 cells,
but not in HEK-293 expressing endogenous A2BAdoRs. Furthermore, upon
addition of A2AAdoR antagonist, the rate of decline of cAMP accumulation
elicited by various A2AAdoR agonists in PC 12 cells was found to be in-
versely related (R2=0.85) to the affinity of these agonists for the receptor.
We anticipate that these novel low affinity A2A agonists may have greater
potential for clinical and therapeutic applications.
94 ABSTRACTS FROM PURINES 2000
182
P2Y6 RECEPTOR MEDIATED MITOGENIC EFFECTS ON VAS-
CULAR SMOOTH MUSCLE CELLS
Erlinge, D.(1); Harden, TK.(2); Pendergast, W(3); Baldetorp, B.(4); Möller,
S.(1); Edvinsson, L.(1); Hou, M.(1)
(1)Dept. of Medicine, Lund University, Lund, Sweden. (2)Dept. of Pharma-
cology, North Carolina School of Medicine, Chapel Hill, USA, (3)Inspire
Pharmaceuticals Inc., Durham USA, (4)Dept. Oncology, Lund University,
Lund, Sweden
The mitogenic effects of the P2Y6 receptor have not been possible to exam
due to lack of stable agonists. UDP and UTP stimulated 3H-thymidine in-
corporation in vascular smooth muscle cells (VSMC) with similar pEC50
values. Pretreatment of UDP with hexokinase did not reduce the effect. The
stable pyrimidine agonists UDP-beta-S and UTP-gamma-S did not differ in
potency, and had equal maximum effects as their natural analogues. Similar
results were obtained on cell number and protein synthesis. UDP was syn-
ergistic with PDGF, bFGF and EGF. Flow cytometry demonstrated cell cycle
progression to both the S phase and the G2 phase. The U73122 but not
pertussis toxin or indomethacin, inhibited UDP mediated mitogenesis. None
of the protein kinase C inhibitors K252a, Gö6976, Ro-31-7549 and NGIC or
the protein kinase A inhibitor H-89 had any effects. Tyrphostin 25 inhibited
UDP mitogenesis, but wortmannin, PD98059 or SB203588 had no specific
effects. The half-life of P2Y6 receptor mRNA was less than 1 hour. PD098059
suppressed the basal expression of P2Y6 receptor mRNA and the expression
of P2Y6 receptors was upregulated by ATP and interleukin-1beta.
Our results indicate that UDP stimulate growth of VSMC synthesis by
activation P2Y6 receptors. The intracellular signal pathways are depen-
dent on phospholipase C and a tyrosine kinase pathway independent of
PI3, MAPKK or P38, and independent of Gi-proteins, epoxyeicosanoids,
protein kinase C and protein kinase A. The P2Y6 receptor mRNA has a
high turnover rate and the expression is upregulated by factors known to
important in the development of vascular disease.
183
GREATER ATTENUATION BY ADENOSINE OF THE ARRHYTHM-
OGENIC THAN OF THE POSITIVE INOTROPIC ACTION OF ISO-
PROTERENOL ON GUINEA PIG VENTRICULAR MYOCYTES
Song, Y. (1); Shryock, J.C. (1); Belardinelli, L. (2).
(1)University of Florida, Gainesville, Florida, USA. (2)CV Therapeutics,
Palo Alto, California, USA.
Background-Previous reports suggest that adenosine inhibits isoproterenol-
facilitated ventricular tachycardia but not isoproterenol-stimulated ventricu-
lar contractility. Thus, adenosine might not equally antagonize the various
actions of an β-adrenoceptor agonist. This hypothesis was examined by quan-
titatively comparing the effects of adenosine on isoproterenol-stimulated trig-
gered activity and cell twitch shortening of guinea pig ventricular myocytes.
Methods and Results-Transmembrane voltages and currents were re-
corded with the whole-cell patch electrode technique. Cell twitch shorten-
ing was measured using a video detector. Isoproterenol (25 nmol/L) increased
the amplitude of L-type Ca2+ current (ICa(L)), and the β1-adrenoceptor an-
tagonist CGP-20712A (250 nmol/L) attenuated the action of isoproterenol.
Adenosine (0.1-100 µmol/L) decreased the amplitude of isoproterenol-stimu-
lated delayed afterdepolarizations by 30±6% to 92±5%, but attenuated iso-
proterenol-induced prolongation of the action potential duration by only
14±4% to 59±4%, and had no significant effect on the voltage of action
potential plateau. Likewise, adenosine (30 µmol/L) inhibited isoproterenol-
induced transient inward current by 89±7%, but reduced isoproterenol-
stimulated ICa(L) by only 43±6%. Isoproterenol-induced aftercontractions
were abolished by adenosine (10 µmol/L), whereas the amplitude of cell
twitch shortening either was not significantly affected or was increased. The
actions of adenosine on cell twitch shortening and aftercontractions were
similar to the actions of ryanodine, suggesting that adenosine may reduce
the oscillatory releases of Ca2+ from the sarcoplasmic reticulum. The effects
of adenosine were mimicked and reversed by N6-cyclopentyladenosine and
8-cyclopentyl-1,3-dipropylxanthine, respectively.
Conclusion-Activation of A1 adenosine receptors by adenosine can an-
tagonize the pro-arrhythmic effect of β-adrenergic stimulation on ventricu-
lar myocytes without reducing cell twitch shortening.
184
A2A ADENOSINE RECEPTOR-MEDIATED CORONARY VASODI-
LATION BY NOVEL, SHORT ACTING 2-(N OR C-PYRAZOLYL)
ADENOSINE DERIVATIVES
Li, Z.(1); Lasley, RD.(2); Wu, L.(3); Zablocki, J.(4); Blackburn, B.(1,4); Elzein,
E.(4); Palle, V.(4); Gao, Z.(1); Belardinelli, L.(1)
(1) Pharmacological Science, CV Therapeutics, Palo Alto, USA. (2) De-
partment of Surgery, University of Kentucky, Lexington, USA. (3) Depart-
ment of Medicine, University of Florida, Gainesville, USA. (4) Bioorganic
Chemistry, CV Therapeutics, Palo Alto, USA
The objective of this study was to compare the coronary vasodilation caused
by two novel low affinity A2A adenosine receptor (A2AAdoR) agonists, CVT3033
(Ki=2.90µM) and CVT3146 (Ki=1.27µM) with adenosine and high affinity
agonists, CGS21680 (Ki=0.61µM) and WRC0470 (Ki=0.27µM). Experi-
ments were carried out in (1) rat and guinea pig isolated perfused hearts and
(2) open-chest anesthetized pigs instrumented for measurements of coro-
nary artery conductance, atrioventricular conduction time (AVNCT) and
coronary blood flow (CBF), respectively. The magnitude (potency) and time
to 50% reversal (t 0.5) of the increase in coronary artery conductance and
CBF were measured for each agonists. The potency (EC50, nM) of agonists
in rat and guinea pig isolaed perfused hearts (rat/guinea pig) were 67.9±16.7/
203±22.0 (CVT3033), 6.4±1.2/18.6±6.0 (CVT3146), 59.2±6.4/86.0±0.5
(adenosine), 0.5±0.1/1.7±0.4 (CGS21680) and 0.6±0.2/2.4±1.1 (WRC0470),
respectively. In rat isolated hearts and open-chest anesthetized pigs, the
duration of action (t0.5, min, rat/pig) of these agonists were 3.4±0.5/3.1±0.9
(CVT3033), 5.2±0.2/2.6±0.4 (CVT3146), 1.6±0.1/1.2±0.2 (adenosine),
14.5±0.9/9.7±0.8 (CGS21680) and 21.9±0.9/9.5±0.8 (WRC0470), respec-
tively. CVT3033 and CVT3146 were equieffective in causing coronary va-
sodilation as compared to CGS21680 and WRC0470. The A2AAdoR-mediated
increase in coronary artery conductance caused by CVT3033 and CVT3146
were abolished by the A2AAdoR antagonist, ZM241385. Both agonists had
no effect on A1AdoR-mediated porlongation of AVNCT. In open-chest anes-
thetized pigs, CVT3033 and CVT3146 increased CBF by 3.33±0.58 and
3.83±0.39-fold respectively. In summary, these low affinity agonists, CVT3033
and CVT3146 are selective and potent A2AAdoR full agonists for coronary
vasodilation. These short-acting A2AAdoR agonists may prove useful for phar-
macological vasodilation during myocardial imaging with radionuclide agents.
185
LOSARTAN PREVENTS 1,3-DIPROPYL-8-SULFOPHENYLXAN-
THINE-INDUCED HYPERTENSION
Albino-Teixeira, A. (1); Sousa, T. (2); Morato, M. (2); Moura, D. (1);
Guimarães, S. (1)
(1) Institute of Pharmacology and Therapeutics, Faculty of Medicine of
Porto, Portugal. (2) IBMC, University of Porto, Portugal.
The prolonged infusion through Alzet minipumps of 1,3-dipropyl-8-
sulfophenylxanthine (DPSPX), a non selective antagonist of adenosine re-
ceptors, causes hypertension and an increase in plasma renin activity,
suggesting activation of the renin-angiotensin system. DPSPX treatment
also induces marked hypertrophic and hyperplastic changes in the cardio-
vascular system. This study aimed at evaluating the effects of losartan, an
AT1 antagonist, in DPSPX-induced hypertension.
Four groups of rats were treated from day 0 to day 7: group A (n=4) -
saline i.p.; group B (n=4) - DPSPX (90 µg/kg/h) i.p.; group C (n=4) - losartan
(15 mg/kg/day) p.o.; group D (n=6) - DPSPX (90 µg/kg/h) i.p. + losartan
(15 mg/kg/day) p.o.. Body weight, water intake and arterial blood pressure
were daily measured since day -7 until the end of the study. On day 7
fragments of the left ventricle and of the mesenteric, renal and caudal ar-
teries were processed for morphological study.
DPSPX increased systolic (SBP) and diastolic (DBP) blood pressure (day
7: group B - SBP: 150.0±5.3 mmHg; DBP: 109.5±2.7 mmHg and group A
-SBP: 110.5±5.0 mmHg; DBP: 75.2±7.9 mmHg; p<0.05). Losartan pre-
vented DPSPX-induced blood pressure raise (day 7: group D - SBP:
109.2±4.7 mmHg; DBP: 77.2±4.4 mmHg). Group C didn’t reveal signifi-
cant differences in blood pressure values compared with group A.
Losartan prevented the hypertrophic and hyperplastic effects of DPSPX
but had no effects by itself.
In conclusion, losartan has no influence in arterial blood pressure and
cardiovascular morphology of normotensive rats but prevents the hyper-
tension and morphological alterations induced by DPSPX.
 ABSTRACTS FROM PURINES 2000
186
CONSISTENCY ETHANOL FEEDING ENHANCE THE VASO-
RELAXING EFFECT OF ADENOSINE RECEPTORS IN ISO-
LATED AORTA FROM SPONTANEOUSLY HYPERTENSIVE RATS
Rekik, M.; El-Mas, M.M.; Abdel-Rahman, A.A.; Mustafa, S.J.
Department of Pharmacology, School of Medicine, East Carolina Univer-
sity, Greenville, NC, USA
The effects of chronic ethanol feeding on blood pressure (BP) and on the
vasorelaxant activity of the adenosine-receptor agonist, 5'-N-ethylcarbox-
amidoadenosine (NECA) were investigated in spontaneously hyperten-
sive rats (SHR). Compared with controls, ethanol (2.5% or 5%, w/v) lowered
BP in a dose-dependent manner. The hypotensive response to ethanol
started after week 1, reached a maximum at week 5 and remained so till
the end of the study. A concentration-dependent relaxation of phenyleph-
rine (1 µM) precontracted thoracic aortic rings caused by NECA was ob-
served in all groups. In endothelium-denuded aortic rings, dose-response
curves for NECA were similar in both control and treated SHR (maximal
relaxation was 26±4, 32±3 and 37±7 % for control, 2.5 and 5% ethanol,
respectively). The relaxation caused byonsist NECA was abolished by 10
µM 8-SPT (adenosine receptor antagonist), but not by 30 µM L-NMMA
(NOS inhibitor). In endothelium-intact rings, acetylcholine caused a dose-
dependent relaxation but no difference was observed between groups.
However, the responsiveness to NECA was significantly increased in all
groups and NECA relaxation was significantly (p<0.05) higher in ethanol-
treated groups compared with control (maximal relaxation was 45±1,
69±10 and 76±3 % for control, 2.5 and 5% ethanol, respectively). L-NMMA
(30 µM) abolished the relaxation caused by NECA whereas 10 µM 8-SPT
inhibited only the responses caused by the lower doses of NECA. These
results suggest that chronic ethanol treatment in the SHR potentiates the
vasorelaxant effect of adenosine receptors in the aorta. This effect is medi-
ated through the endothelium most likely by increasing NO release.
187
NITRIC OXIDE MEDIATES THE AGE-RELATED DESENSITI-
ZATION OF MYOCARDIAL A1 ADENOSINE RECEPTORS
Friedman,E.;Yacoub,H.;Ma,X.L.;Johnson,G.P.;Cai,G.
Department of Pharmacology and Physiology,MCP Hahnemann
University,Philadelphia,PA,USA.
We prevoiously found that A1 adenosine receptor (A1AdoR) function is com-
promised in the senescent rat myocardium; this change is accompanied by
increased A1AdoR phosphorylation. Presently, our aim was to investigate
the potential role of nitric oxide (NO) in A1AdoR desensitization. We found
a 62% increase in NO production in coronary effluent collected from iso-
lated perfused hearts of 24- vs. 6-month-old rats. We also observed age-
dependent increase in myocardial inducible NO synthase expression. In
ventricular membranes from 6- month old rats, NO donors or the cyclic
GMP analog, 8-bromo-cGMP, elicited A1AdoR desensitization. In an at-
tempt to define the role of NO in regulating A1AdoR function, we adopted
CHO cells expressing human A1AdoR as a model to test the effect of the
NO-cyclic GMP cascade on receptor function. Expression of A1AdoR in
these cells was confirmed by [3H]DPCPX binding and by A1AdoR agonist
(SPA)-mediated inhibition of adenylyl cyclase. Treatment with NO donors,
SIN-1 (100 µM) or GSNO (200 µM), reduced A1AdoR-mediated maximal
inhibition of forskolin-stimulated adenylyl cyclase (46.8% for vehicle vs.
39.4% for SIN-1 and 37.9% for GSNO) and shifted the agonist dose-re-
sponse curve to the right (IC50: 0.17 µM for vehicle vs. 0.48 µM for SIN-1
and 0.32 µM for GSNO), indicating functional desensitization of A1AdoR.
The effects of the NO donors were mimicked by 8-bromo-cGMP (100 µM).
The results suggest that desensitization of A1AdoRs is elicited via NO-
cyclic GMP phosphorylation and that this cascade may underlie the age-
associated attenuation of A1AdoR function and of the cardioprotective action
of adenosine.
95
188
NITRIC OXIDE MEDIATES THE ADENOSINE-INDUCED AT-
TENUATION OF THE POSITIVE DROMOTROPIC EFFECT OF
ISOPROTERENOL
Martynyuk, A.E.(1,2); Zima, A.(1); Seubert, C.N.(1); Morey, T.E.(1);
Sumners, C.(2,3); Dennis, D.M.(1,4).
Departments of (1) Anesthesiology, (2) Neuroscience, (3) Physiology, and
(4) Pharmacology & Experimental Therapeutics, University of Florida,
Gainesville, FL, USA.
We hypothesized that nitric oxide (NO) plays an important role in mediat-
ing the antiadrenergic effect of adenosine on atrioventricular (AV) nodal
conduction.
In guinea pig hearts instrumented for measurement of AV nodal con-
duction time (atrium-to-His bundle, A-H, interval), the NO synthase (NOS)
inhibitor, L-NMMA (100 µM), reversibly inhibited 80% (P=0.009, n=6)
of adenosine’s antiadrenergic action on the positive dromotropic effect of
isoproterenol (0.01 µM).
In parallel studies carried out in rabbit AV nodal myocytes, intracellular
mechanisms whereby NO mediates the inhibitory effect of adenosine on
isoproterenol-induced A-H interval shortening were studied. Adenosine
(3 µM) inhibited isoproterenol-stimulated (0.1 µM) ICa,L (ß-ICa,L) by 46±6%
(P<0.001, n=17). Consistent with isolated heart data, the NOS inhibitors,
L-NMMA (100 µM) and L-NNA (500 µM) attenuated the effect of adenos-
ine on ß-ICa,L by 69±8% (P<0.001, n=16) and 69±7% (P<0.001, n=10),
respectively. An inhibitor of NO-stimulated guanylyl cyclase LY83538 (40
µM) reduced the inhibitory effect of adenosine on ß-ICa,L by 97±6%
(P=0.004, n=15). Similarly, the nonspecific inhibitor of cAMP-phosphodi-
esterases IBMX (50 µM) decreased the antiadrenergic effect of adenosine
by 60% (P=0.02, n=6), whereas the extracellular application of the non-
hydrolyzeable cAMP analog 8-Br-cAMP (500 µM) prevented this action of
adenosine. Activation of cGMP-dependent protein kinase (PKG) by CPT-
cGMP (300 µM) diminished ß-ICa,L, but to a significantly smaller degree
(16±4%, P=0.025, n=12) than that caused by adenosine.
We conclude that NO mediates the antiadrenergic effect of adenosine on
AV nodal conduction by a mechanism predominately involving activation of
cGMP-dependent cAMP-phosphodiesterase and to a lesser extent activation
of PKG.
189
CONTRIBUTION OF IK,ADO TO THE NEGATIVE DROMOTRO-
PIC EFFECT OF ADENOSINE
Martynyuk, A.E.(1,2); Morey, T.E.(1); Zima, A.(1); Seubert, C.N.(1);
Belardinelli, L.(4); Dennis, D.M.(1,3)
Departments of (1) Anesthesiology, (2) Neuroscience, and (3) Pharmacol-
ogy & Experimental Therapeutics, University of Florida, Gainesville,
Florida and (4) CV Therapeutics, Inc., Palo Alto, California, USA.
Although IK,ADO has been widely implicated in the negative dromotropic
effect of adenosine, experimental evidence directly linking IK,ADO to the
atrioventricular nodal effects of adenosine is lacking. Therefore, we deter-
mined if and to what extent IK,ADO contributes to the negative dromotropic
effect of adenosine.
In rabbit atrioventricular nodal myocytes studied using the whole-cell
patch clamp technique, Ba2+ (100 µmol/L) selectively and completely blocked
IK,ADO at membrane potentials from -70 to 0 mV and abolished the adenos-
ine-induced leftward shift of the reversal membrane potential, but did not
change the attenuation by adenosine of ICa,L. In rabbit and guinea pig iso-
lated hearts instrumented for His-bundle electrogram recordings, Ba2+ (100
µmol/L) alone did not significantly prolong the atrium-to-His bundle (A-H)
interval, but markedly attenuated A-H interval prolongation caused by ad-
enosine. In guinea pig heart, EC50 values for adenosine-induced A-H inter-
val prolongation increased from 3.3 to 13.2 µmol/L (P<0.001) in the absence
and presence of Ba2+, respectively. Consistent with complete block of IK,ADO,
elevated [K+]o did not augment the negative dromotropic effect of adenosine
in the presence of Ba2+. The Ba2+-sensitive component of the AV nodal ef-
fects of adenosine was concentration-dependent (P<0.001). Despite spe-
cies-dependent differences in sensitivities to adenosine (guinea pigrabbit),
the relative contribution of IK,ADO to atrioventricular nodal conduction slow-
ing was similar. The relative contribution of IK,ADO to A-H interval prolonga-
tion ranged from 37.8% (P=0.013) to 72.5% (P<0.001) in guinea pig hearts
at 2 and 6 µmol/L adenosine, respectively.
IK,ADO significantly contributes to adenosine-induced negative dromotropy,
especially at relatively high concentrations of adenosine.
96 ABSTRACTS FROM PURINES 2000
190
THE ROLE OF PURINERGIC TRANSMISSION IN NEURO-
GENIC VASOCONSTRICTION OF DIFFERENT SIZED RAT
MESENTERIC ARTERIES
Gitterman, D. P.; Evans, R. J.
Department of Cell Physiology and Pharmacology, University of Leices-
ter, Great Britain
P2X receptors are ligand-gated cation channels activated by extracellular
ATP. In arterial smooth muscle their activation leads to membrane
depolarisation, calcium influx and contraction. ATP and noradrenaline are
co-released from sympathetic nerves, both contributing to neuronal con-
trol of vascular tone. The aim of this study was to determine the relative
roles of P2X receptors versus α-adrenoceptors in mediating neurogenic
contraction in different diameter rat mesenteric arteries. Constrictions were
evoked by electric field stimulation using 10 pulses of 0.2ms duration at a
frequency of 10Hz; responses were abolished by tetrodotoxin (0.3µM).
Suramin (100µM) reduced contractions in small (diameter below 125µm)
and medium-sized (diameter 125-250 µm) vessels by 65.3 ± 7.4% and 82.7
± 3.3% respectively but only by 3.1 ± 6.1 % in large arteries (diameter
above 450µm). Prazosin (0.1µM) virtually abolished constrictions in large
arteries and reduced them by 32.6 ± 2.6% and 12.7 ± 6.1% in small and
medium vessels respectively. Co-application of suramin and prazosin abol-
ished responses in all vessels. These results demonstrate that the adrener-
gic component of contraction dominates in large arteries while the
purinergic component dominates in smaller vessels. These results are in
agreement with our data showing substantially lower potency of α,β-
methyleneATP in large arteries. The importance of L-type calcium chan-
nels in sympathetic vasoconstriction was tested using nifedipine (1µM).
Responses were only slightly reduced in the three types of artery indicat-
ing that calcium influx through L-type calcium channels plays only a mi-
nor role in mediating calcium entry for neurogenic responses.
191
VASORELAXATION TO ATP IN THE RAT MESENTERIC ARTE-
RIAL BED IS ATTENUATED BY a,b-METHYLENE ATP BUT
DOES NOT INVOLVE CAPSAICIN-SENSITIVE SENSORY
NERVES
Ralevic, V.
School of Biomedical Sciences, Queen’s Medical Centre, University of
Nottingham, Nottingham NG7 2UH, England
Primary afferent nerves can be activated by a variety of stimuli to release
calcitonin gene-related peptide, which mediates slow vasorelaxation. ATP
can excite primary afferents, including those in rat mesenteric arteries, via
actions at ionotropic P2X receptors, but it is not known whether this elic-
its an efferent function. The present study investigated the possible in-
volvement of P2X receptors and primary afferent nerves in vasorelaxation
to ATP in the rat mesenteric arterial bed.
ATP elicited dose-dependent vasorelaxation in methoxamine-pre-con-
stricted preparations that was biphasic at doses of 50nmol and greater, with
a pD2 value for the initial, rapid, phase of relaxation of 9.2±0.2 and second-
ary, prolonged, relaxation of 56±3% at 50nmol ATP (n=8). Suramin (100µM)
attenuated both rapid (pD2 7.8±0.1) and prolonged relaxation (15±3%;
50nmol ATP) (n=5; P<0.001). Pre-treatment with the sensory neurotoxin
capsaicin (10µM) had no effect on vasorelaxation to ATP. Relaxation was also
unaffected by indomethacin (10µM) and 8-sulfophenyltheophylline (1µM).
α,β-methyleneATP (10µM) had no effect on rapid relaxation to ATP, but
attenuated the second phase (25±8%; 50nmol ATP) (n=8; P<0.01).
These data show that both rapid and prolonged vasodilatation to ATP of
the rat mesenteric arterial bed are mediated by P2 receptors, but neither
phase involves capsaicin-sensitive afferents. This suggests that P2X recep-
tors expressed on sensory nerves in rat mesenteric arteries do not mediate
an efferent function. Adenosine P1 receptors and prostanoids do not con-
tribute to either phase of ATP-mediated vasorelaxation. Attenuation by α,β-
methyleneATP of prolonged vasorelaxation to ATP indicates modulation
by smooth muscle P2X receptors of this response.
VR is a Royal Society University Research Fellow.
192
CHARACTERIZATION OF P2 RECEPTORS IN THE PORCINE
ISOLATED CORONARY ARTERY
Alexander, S.P.H.; Bera, R.; Otterburn, P.M.; Ralevic, V.
School of Biomedical Sciences, Queen’s Medical Centre, University of
Nottingham, Nottingham NG7 2UH, England
P2 receptors in the vascular system are differentially distributed such that
P2X receptors evoke contractions and are expressed primarily on the smooth
muscle, whilst P2Y receptors are relaxatory, associated typically with the
endothelium. Here, we report an investigation of P2 receptor distribution
in the porcine isolated coronary artery.
In endothelially-intact segements at resting tension, nucleotides evoked
concentration-dependent contractions with the rank order of potency α,β-
meATP UTP ATP, without achieving a maximal response. In U46619-pre-
constricted arteries, ATP (up to 100µM) and ADP (up to 1mM) evoked
concentration-dependent relaxations (pD2 values of 5.37 and 5.41, respec-
tively), the potency of which was unaltered after endothelial removal (5.46
and 5.10, respectively. 1mM ATP evoked contractions in both intact and
denuded pre-constricted segments. In contrast, responses to UTP (up to
1mM) were converted from a concentration-dependent relaxation in en-
dothelium-intact to a concentration-dependent contraction in denuded
preparations, failing to achieve a maximal response in either situation.
These data indicate that multiple P2 receptors are expressed in porcine
coronary arteries. The likely presence of constrictor P2X and UTP-prefer-
ring receptors on the smooth muscle contrasts with a vasorelaxatory UTP-
preferring receptor present on the endothelium and vasorelaxatory ADP/
ATP-preferring receptors present on the smooth muscle. The porcine coro-
nary artery, therefore, exhibits a novel spectrum of P2 receptors. Whether
these responses are mediated through novel P2 receptors or through “clas-
sical” receptors coupled in a novel fashion remains to be determined.
VR is a Royal Society University Research Fellow.
193
EFFECT OF A DECREASE IN pH ON CONTRACTILE RE-
SPONSE MEDIATED BY P2X RECEPTORS IN THE PORCINE
ISOLATED CORONARY ARTERY
Ralevic,V.; Otterburn,P.M.; Bera,R.; Alexander,S.P.H.
School of Biomedical Sciences, University of Nottingham Medical School,
Nottingham NG7 2UH, England
Recombinant P2X subtypes have been described to display differential
sensitivity to reduced ambient pH. Little is known, however, about the
effect of reduced pH on responses at endogenous P2X receptors in cardio-
vascular tissues. In the present study, the effects of reducing ambient pH
on contractile responses mediated by α,β-meATP, a selective P2X recep-
tor agonist, in the porcine isolated coronary artery have been investigated.
At pH 7.4, responses to contractile agents showed the rank order of re-
sponse: KCl (60mM, 7.80 g) histamine (10µM, 7.01 g) α,β-meATP (10µM,
2.43 g). Replacement of the medium with Krebs’ adjusted to pH 6.9 had
no effect on the magnitude of the KCl-induced contraction, whilst signifi-
cantly reducing responses to histamine and α,β-meATP (45 and 55 % con-
trol, respectively). In time-matched control segments maintained at pH
7.4, responses to the contractile agents were unchanged.
These data indicate that a decrease from physiological pH to pH 6.9
causes a pronounced reduction in the vasocontractile response mediated
by P2X receptors in porcine coronary arteries. This effect of pH is not
selective for P2 signalling as responses to histamine were similarly reduced.
However, contractions to KCl were unaffected by a decrease in pH, which
indicates the decrease in contractions to α,β-meATP and histamine is not
due to a non-specific effect of pH on the vascular smooth muscle. Possible
sites of modulation include pH modulation of the ligands, their receptors
or intracellular signalling mechanisms.
VR is a Royal Society University Research Fellow.
 ABSTRACTS FROM PURINES 2000
194
INFLUENCES OF HYPOXIA ON THE VASOCONSTRICTOR RE-
SPONSES TO SYMPATHETIC ACTIVITY AND EXOGENOUS
NORADRENALINE: POSSIBLE ROLES OF ADENOSINE.
Coney, A.M.; Marshall, J.M.
Department of Physiology, The Medical School, University of Birming-
ham
Muscle vasodilatation evoked in the rat by systemic hypoxia is largely
mediated by adenosine acting on A1 receptors, even though A2a receptors
are also present (Bryan & Marshall, J Physiol 541,151-162, 1999). Further,
the muscle vasoconstrictor response evoked by continuous stimulation of
the sympathetic chain at the low frequency of 2Hz is reduced by systemic
hypoxia and this effect is partially alleviated by the A1 receptor antagonist
DPCPX (Coney & Marshall, J Physiol 1999). We have now tested whether
muscle vasoconstriction evoked by the sympathetic transmitter noradrena-
line (NA) is affected by hypoxia or by adenosine released in hypoxia.
In anaesthetised rats, femoral blood flow and arterial pressure were re-
corded allowing calculation of femoral vascular resistance (FVR). NA was
infused at 10µg.kg-1min-1 i.a. for 3min in normoxia and in hypoxia (breath-
ing 8% O2) before and after DPCPX. NA infusion increased integrated
FVR by 3.3 ± 0.6 Resistance Units (RU) in normoxia, but by only 1.1 ±
0.2 RU in hypoxia (P
Thus, muscle vasoconstriction evoked by exogenous NA is depressed
by systemic hypoxia but this is not attributable to adenosine acting on A1
or A2a receptors. We propose that the ATP released as a co-transmitter
with NA, is more rapidly dephosphorylated to adenosine in hypoxia thus
attenuating the constrictor response to sympathetic stimulation.
195
CHARACTERIZATION OF TRANSGENIC MICE CARDIAC-SPE-
CIFICALLY OVEREXPRESSING THE A3 ADENOSINE RECEP-
TOR
Auchampach, J.A.; Hobson, L.; Jones, W.K.; Bolli, R.; Black, R.G.
Division of Cardiology, University of Louisville, Louisville, KY, USA.
Overexpression of A1 adenosine receptors in the hearts of mice increases
resistance to myocardial ischemia. Conversely, chronic overexpression of
other G protein-coupled receptors in transgenic mice leads to cardiac hy-
pertrophy and heart failure. The goal of these studies was to characterize
the effect of overexpressing A3 receptors in the hearts of transgenic mice.
A3 receptor transgenic mice were created using the α-myosin heavy chain
gene promoter and the A3 receptor cDNA. Two separate lines of mice were
characterized, a low copy number line (A3tg.3; transgene copy number =
2) and a high copy number line (A3tg.10; transgene copy number = 10).
Hearts from both lines of mice displayed marked overexpression of A3 re-
ceptors based on radioligand binding analysis with the agonist radioligand
[125I]-AB-MECA and enhanced agonist-dependent inhibition of isoproter-
enol-stimulated adenylyl cyclase activity. Compared to wild-type mice, heart
rate and blood pressure were not changed in A3tg.3 mice. However, heart
rate was reduced approximately 25% in A3tg.10 mice. Further analysis re-
vealed no changes in A3tg.3 mice, while A3tg.10 mice had increased heart-
to-body weight ratios and altered mRNA expression of markers of
hypertrophy at 17 weeks of age. Thus, A3 receptor transgenic mice are a
useful model to study A3 adenosine receptors in cardiac physiology. We
predict that low-level overexpression of the A3 receptor affords protection
against myocardial ischemia, whereas higher levels of overexpression re-
sults in a compensatory hypertrophic response.
97
196
INVOLVEMENT OF ADENOSINE IN MUSCLE VASODILATA-
TION EVOKED BY SYSTEMIC HYPOXIA AFTER NITRIC OXIDE
SYNTHASE INHIBITION.
Edmunds, N.J.; Marshall, J.M.
Depatment of Physiology, The Medical School, University of Birmingham
Systemic hypoxia results in vasodilatation within rat hindlimb muscle which
can be severely attenuated by the NO synthase (NOS) inhibitor, L-NAME,
but can be restored by infusion of the NO-donor SNP (Edmunds & Marshall,
2000, J. Physiol, in press). Since hypoxia-evoked increases in FVC are partly
mediated by adenosine A1-receptors (Bryan & Marshall, J. Physiol. 514, 151-
162, 1999), we have investigated whether adenosine contributes to the hy-
poxia-evoked vasodilatation during infusion of SNP after NOS inhibition.
In anaesthetised rats, femoral vascular conductance (FVC: femoral blood
flow/mean arterial pressure) increased during systemic hypoxia (12% and
8% inspired O2). L-NAME increased baseline FVC, and severely attenu-
ated the vasodilatation to each hypoxic challenge. Infusion of SNP returned
baseline FVC to levels observed prior to L-NAME, and completely re-
stored hypoxia-evoked changes in FVC. Further, after L-NAME and dur-
ing SNP infusion both the adenosine A1/A2 receptor antagonist, 8-SPT,
and the selective A1-receptor antagonist, DPCPX, attenuated the increase
in FVC evoked by both 12% and 8% O2.
Thus, the vasodilatation evoked by hypoxia after NOS inhibition and dur-
ing SNP infusion, is similar to that seen under control conditions and is, in
part, mediated by adenosine, acting via A1-receptors. It appears that this
action of adenosine A1-receptors may not be mediated by the release of NO,
but merely dependent on basal levels of NO, which could act to facilitate the
dilator actions of adenosine (De Wit et al, Circ. Res. 28, 1513-1518, 1994).
197
THE CONTRIBUTION OF ADENOSINE AND A1 RECEPTORS
TO TONIC VASODILATATION IN RAT HINDLIMB MUSCLE
DURING EARLY CHRONIC SYSTEMIC HYPOXIA.
Walsh,M.;Marshall,J.M.
Physiology Department, The Medical School, Birmingham University,
Birmingham,UK.
In rat hindlimb muscle, acute systemic hypoxia evokes an NO-dependent
vasodilatation mediated by the action of adenosine on A1 receptors, even
though stimulation of A2A receptors can also evoke NO-dependent muscle
vasodilatation (Bryan & Marshall, J.Physiol. 514, 151-166). When rats were
exposed to chronic systemic hypoxia (breathing 12%O2), an NO-dependent
tonic muscle vasodilatation was present at 1 and 3 days, but waned by 7 days
(Walsh & Marshall, J. Physiol, 515P, 144P, 1999). We have now tested whether
this tonic dilatation is mediated by adenosine acting on A1 or A2A receptors.
In anaesthetised, normoxic (N) rats breathing air and in 1 and 7 day chroni-
cally hypoxic (1 and 7 CH) rats breathing 12% O2, femoral blood flow and
arterial pressure were recorded; femoral vascular conductance (FVC) was
computed. In N and 7CH rats, baseline values of FVC were similar (0.009
± 0.001 and 0.009± 0.001 conductance units, CU: mean ± S.E.M) and
were unaffected by the A1 receptor antagonist DPCPX (0.1 mg.kg-1) or by
subsequent administration of the non-selective adenosine receptor antago-
nist 8-SPT (20 mg.kg-1). In contrast, 1CH rats had higher baseline FVC
(0.0144 ± 0.001*) than N and 7CH rats and this was reduced by DPCPX
(to 0.0110 ± 0.001*, *: p < 0.05 ANOVA and Bonferroni post hoc test), 8-
SPT producing no further affect
These results indicate that in 1CH rats ~50 % of the tonic NO-depen-
dent hindlimb vasodilatation is mediated by adenosine acting on A1, rather
than A2A receptors and that by 7 days of chronic hypoxia, this adenosine–
dependent dilatation has waned.
98 ABSTRACTS FROM PURINES 2000
198
A2A RECEPTORS ARE INVOLVED IN ABNORMAL RESPONSES
TO ADENOSINE IN THE MESENTERIC ARTERY IN PORTAL
HYPERTENSIVE RABBITS
Villa-de-Brito, M.T.(1);Canto, A.(2);Cunha, R.A.(2); Duarte Correia, J.H.(1);
Marques, M.C.(3)
(1)CIISA, Fac. Veterinary Medicine, Lisbon Tech. Univ., Portugal. (2)Lab.
Neurosciences, Fac. of Medicine, Univ. Lisbon, Portugal. (3)Lab. Physio-
pathology, Fac. of Pharmacy, Univ. Lisbon,Portugal
Exougenously added adenosine-induced vasodilation is diminished in portal
hypertensive rabbits(PHR) in the cranial mesenteric artery (Villa de Brito
et al., 1998, NS Arch. Pharmacol., 358, Suppl.1:R258). We now investi-
gated if this modification could result from desensitisation of vasodilatory
adenosine A2A receptors. Thus, we evaluated adenosine plasma levels
(Cunha et al., 1989, Chromatographia 28: 610) and A2A receptor binding
density; using the A2A receptor agonist [3H]CGS 21680 (20 nM) and
[3H]ZM 241385 (2 nM)(Cunha et al.,1999, NS Arch. Pharmacol.,359:295),
in cranial mesenteric arteries of normal male New Zealand rabbits (NR,
n=6) and portal hypertensive rabbits (PHR, n=6), six weeks after partial
portal vein ligation upon ketamine (25mg/Kg I.M.) and medetomidine
(0.5mg/Kg I.M.) anaesthesia.
Adenosine plasma levels were lower (p<0.001) in PHR (78±5 nM) when
compared with NR (244±13 nM).The density of A2A receptors estimated
as [3H]ZM 241385 binding was lower (p<0.05) in PHR (355±45 fmol/mg
protein) than in NR (491±8 fmol/mg protein) the same occurring when
evaluating [3H]CGS 21680 binding (233±29 fmol/mg protein in PHR ver-
sus 334±12 fmol/mg protein in NR).
The observed decrease in adenosine plasma levels suggests that adenos-
ine metabolism is altered in PHR. Furthermore, the decreased density of
A2A receptors in the mesenteric artery of PHR might contribute for the
diminished adenosine-induced vasodilation observed in PHR.
199
MECHANISM OF POTENTIATION OF THE NEGATIVE DRO-
MOTROPIC EFFECT OF ADENOSINE BY THE ALLOSTERIC
ENHANCER PD 81,723
Martynyuk, A.E.(1,2); Seubert, C.N.(1); Zima, A.(1); Morey, T.E.(1);
Belardinelli, L.(4); Dennis, D.M.(1,3)
Departments of (1) Anesthesiology, (2) Neuroscience, and (3) Pharmacol-
ogy & Experimental Therapeutics, University of Florida, Gainesville,
Florida and (4) CV Therapeutics, Inc., Palo Alto, California, USA.
The 2-amino-3-benzoylthiophene PD 81,723, an allosteric enhancer of A1-
adenosine receptor binding, potentiates the negative dromotropic effect of
adenosine, despite blocking IK,ADO. Therefore, the mechanism(s) whereby
PD 81,723 potentiates the AV nodal effects of adenosine were studied in
guinea pig and rabbit isolated hearts instrumented for measurement of AV
nodal conduction (atrium-to-His bundle, A-H interval) and in guinea pig
and rabbit atrial myocytes using conventional patch-clamp techniques. PD
81,723 (5 µM) markedly potentiated prolongations of the A-H interval caused
by adenosine. This negative dromotropic effect of adenosine was still poten-
tiated by PD 81,723, even after IK,ADO was blocked by Ba2+ (100 µM) and
after the magnitude of ICa,L was fixed at a constant level by 8-Br-cAMP (1.5
mM). In atrial myocytes, PD 81,723 did not significantly change the inhibi-
tion of basal ICa,L caused by adenosine. However, PD 81,723 blocked IK,ADO
recorded at negative Vms and increased the adenosine-augmented outward
K+ current (IK) at positive Vms. Similar to IK,ADO, adenosine-augmented IK
was attenuated by the A1-adenosine receptor antagonist CPX (0.1 µM), but
in contrast to IK,ADO, adenosine-augmented IK was insensitive to Ba2+ (100
µM) and to removal of K+ from the extracellular solution. IK was maximally
augmented at 1 µM adenosine, whereas IK,ADO increased exponentially over
the concentration range of adenosine studied (0-10 µM). We conclude that
adenosine, via the A1-adenosine receptor, activates IK,ADO and augments IK.
Adenosine-sensitive IK is augmented by PD 81,723 and may mediate the
potentiation by PD 81,723 of the negative dromotropic effect of adenosine.
200
DIADENOSINEPOLYPHOSPHATES (APnA) EXERT CONTRAC-
TILE AND DILATOR EFFECTS ON HUMAN MESENTERIC AND
RENAL SMALL ARTERIES
Steinmetz, M.(1); Janssen, A.K. (1); Pelster, F. (2); Piechota, H.J. (2); Rahn,
K.H. (1); Schlatter, E. (1)
(1) Dept. of Internal Medicine, (2)Center of Surgery, University of Münster,
Münster, Germany
Objective: A role of APnA (n=3-6) as neurotransmitters has been suggested
for several tissues of different species. Heterogenous vasoactive qualities
by APnA were demonstrated in many different animal vascular beds. Here
we determined the arterial effects of APnA in human mesenteric (hMA,
diameter: 350 µm) and renal arteries (hRA: 500 µm)
Method: In tissue preparations obtained from the local center of sur-
gery small resistance arteries were isolated under the microscope. Arter-
ies were mounted in a myograph for measurement of isometric wall tension.
Results: In hMA and hRA all agents (0.1–500µM) induced concentra-
tion-dependent contractions, (rank order of potency in hMA: AP6AAP5AA-
P4AAP3A; hRA: AP5AAP6AAP4AAP3A). In both arteries responses were
transient within 1-2 min. During precontraction induced by endothelin
(0.02 µM) APnA induced biphasic responses in hMA: An increase in tone,
exceeding the level of precontraction was followed by a sustained net
vasorelaxation. This relaxation was maximal 74±6% of the precontraction
with AP6A, 70±11% with AP4A, 65±8% with AP5A and 45±17% with
AP3A. Comparable biphasic vascular responses induced by APnA were
observed in phenylephrine (10 µM) precontracted hRA.
Conclusions: APnA induce contraction and vasorelaxation in both of the
human arteries, hMA and hRA, depending on the degree of precontraction.
The contractile potency of APnA in hMA is dependent on the length of the
phosphate chain, this rule does not seem to be equally stringent for the vasodi-
lator potency. The rank order of potency of the APnA induced effects does not
seem to be totally uniform in human arteries from different vascular beds.
201
A2A-ADENOSINE RECEPTORS MEDIATE CORONARY VASODI-
LATION BY ATP IN GUINEA PIG ISOLATED HEARTS
Erga, K.S.(1); Seubert, C.N.(2); Liang, H.X.(3); Wu, L.(3); Shryock, J.C.(3);
Belardinelli, L.(4)
(1) Department of Physiology, University of Bergen, Bergen, Norway. (2)
Department of Anesthesiology, University of Florida, Gainesville, FL , USA.
(3) Department of Medicine, University of Florida, Gainesville, FL , USA.
(4) CV Therapeutics Inc., Palo Alto, CA, USA.
Adenosine-5'-triphosphate (ATP) and adenosine are potent coronary va-
sodilators. ATP is rapidly converted to adenosine by ectonucleotidases.
Our goal was to determine whether exogenous ATP causes vasodilation by
P2Y- or by A2A-adenosine receptor activation.
The effects of ATP and adenosine on coronary conductance in guinea pig
isolated hearts perfused at a constant flow of 10 ml min-1 were compared.
ATP and adenosine both caused sustained, concentration-dependent in-
creases of coronary conductance. Maximal responses were equivalent. Ad-
enosine was significantly more potent than ATP (pD2±SEM, 6.68±0.04 for
ATP and 7.06±0.05 for adenosine, p<0.0001, n = 10). The values of pIC50
for the selective A2A-adenosine receptor antagonist SCH58261 to antago-
nize equivalent responses to ATP and adenosine were 8.28±0.08 and
8.28±0.06 (p=0.99, n=6), respectively. The non-selective adenosine recep-
tor antagonists xanthine amine congener (XAC) and CGS15943 antagonized
similarly the equivalent vasodilations caused by ATP (pIC50 values 7.48±0.04
and 7.45±0.06, respectively) and adenosine (pIC50 values 7.37±0.13 and
7.56±0.11). In contrast to ATP and adenosine, the two P2Y agonists 2-
methylthio-ATP and uridine-5'-triphosphate failed to cause stable increases
of coronary conductance, caused desensitization of vasodilator responses,
and were not antagonized by SCH 58261, 8-parasulfophenyltheophylline,
or XAC. Adenosine deaminase significantly attenuated ATP-induced increases
in coronary conductance. Glibenclamide attenuated coronary vasodilations
caused by ATP and adenosine by 88 and 89%, respectively, but failed to
attenuate those caused by 2-methylthio-ATP, whereas L-NAME attenuated
all three agonists equivalently.
These results strongly suggest that sustained, submaximal coronary va-
sodilation caused by exogenous ATP is entirely mediated by adenosine
acting upon A2A-adenosine receptors.
 ABSTRACTS FROM PURINES 2000
202
PURINERGIC MEDIATED MODULATION OF COCHLEAR
BLOOD FLOW
Thorne, PR. (1); Muñoz,DJB. (1); McFie, C. (2); DeVal, D. (1); Nikolic, P.
(1) and Housley, GD. (1).
(1) Department of Physiology, University of Auckland. Auckland, NZ (2)
Department of Audiology, Green Lane Hospital, Auckland, NZ
Sensory, neural and secretory tissues of the cochlea have a variety of P2
receptors. Given the vasoactive role of extracellular ATP in other tissues,
experiments were undertaken to investigate its potential role in regulating
cochlear blood flow. The perilymphatic compartment of anaesthetized
guinea-pigs’ cochleae was perfused with ATP, adenosine, UTP and ATPgS
while blood flow was monitored by laser Doppler flowmetry in the lateral
wall. ATP and adenosine significantly increased the cochlear blood flow
(28.8 – 229.0 % and 35.8 - 258.1 % respectively) while UTP did not pro-
duce any change. The effect of ATP was reduced by RB2 and PPADS,
whereas the effect of adenosine was reduced by theophylline, but not with
either DMPX or DPCPX. The effect of ATP was unaffected by the inclu-
sion of theophylline to the perfusate while ATPgammaS produced an in-
crease in cochlear blood flow similar to the one observed with ATP
indicating that the ATP effect is not mediated by adenosine derived from
ectonucleotidase activity. These findings indicate the presence of both P1
and P2 receptors in cochlear blood vessels. Preliminary immunocytochemi-
cal studies undertaken to investigate the nature and location of P2 recep-
tors involved in regulating cochlear blood flow revealed staining with P2X
receptor antisera localised to the smooth muscle layer of the spiral modiolar
artery. These studies demonstrate that extracellular ATP and adenosine
may exert a modulatory role in cochlear blood flow.
Supported by Deafness Research Foundation of New Zealand, Auckland
Medical Research Foundation and Health Research Council of New Zealand.
203
APOPTOSIS OF CARDIOCYTES IS INDUCED BY A3 ADENOS-
INE RECEPTOR AGONIST AND IT IS SUPPREESSED BY ISO-
PROTERENOL
Shainberg, A.(1); Jacobson, KA,(2); Nawrath, H.(3); Zinman, T.(1); Isaac,
A,(1); Shneyvays, V.(1)
(1) Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel. (2)
Mol. Recogn. Section, Lab. Bioorg. Chem., NIH, Bethesda, Maryland, USA.
(3) Institute for Pharmacology, University of Mainz, Germany
The purpose of the present study was to investigate the mechanisms in-
volved in the induction of apoptosis in newborn cultured cardiomyocytes by
activation of A3 adenosine receptors and to examine the protective effects of
b-adrenoceptors. The selective agonist for A3 adenosine receptors Cl-IB-
MECA (2-chloro-N6-iodobenzyl-5-N- methylcarboxamidoadenosine) and the
antagonist MRS1523 (5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-
phenylpyridine-5- carboxylate) were used. High concentrations of the Cl-
IB-MECA (10 µM) induced cardiomyocytes apoptosis. In addition,
Cl-IB-MECA caused a sustained and reversible increase in [Ca2+]i, which
was prevented by the selective antagonist MRS1523. Furthermore, MRS1523
protected the cardiocytes if briefly exposed to Cl-IB-MECA, and partially
protected from prolonged (48 h) agonist exposure. Apoptosis induced by Cl-
IB-MECA was not redox-dependent, since the mitochondrial membrane
potential remained constant until the terminal stage of cell death. Cl-IB-
MECA activated caspase-3 (CPP32) protease after 7 h of treatment. Bcl-2
protein was readily detected in control cells, and its expression was signifi-
cantly decreased after 24 and 48 h of treatment with Cl-IB-MECA. Beta-
adrenergic stimulation antagonized the pro-apoptotic effects of Cl-IB-MECA,
probably through a cAMP/protein kinase A-independent mechanism, since
addition of dibutyryl-cAMP did not abolish the apoptosis induced by Cl-IB-
MECA. Incubation of cultured myocytes with isoproterenol (5 µM) for 3 or
24 h almost completely abolished the increase in [Ca2+]i. Prolonged incu-
bation of cardiomyocytes with isoproterenol and Cl-IB-MECA did not in-
duce apoptosis. Our data suggest that the apoptosis-inducing signal from
activation of adenosine A3 receptors (or contracting beta-adrenergic signal)
leads to the activation of the G-protein-coupled enzymes and downstream
pathways to a self-amplifying cascade.
99
204
A COMPETITIVE RT-PCR ASSAY FOR THE QUANTIFICATION
OF P2 RECEPTOR TRANSCRIPTS FROM HUMAN VASCULAR
SMOOTH MUSCLE.
Webb, T.E.;Boarder, M.R.
The Cell Signalling Laboratory, Dept of Biological Sciences, De Montfort
University, Leicester, LE1 9BH.
Vascular smooth muscle cells (VSMC) exhibit two states known as contrac-
tile and synthetic. A reversion to a synthetic phenotype is prerequisite for
the involvement of VSMC in the formation of restenosis and atheroslerosis.
We are interested in the role of nucleotides, primarily acting via P2Y recep-
tors, in the cellular control of this process. Using internal mammary artery
and saphenous vein fragments from heart bypass operations, we have shown
that nucleotides are able to modulate the incorporation of [3H]-thymidine
into DNA in cultured VSMC from both vessels. Furthermore, RT-PCR analy-
sis revealed the expression of multiple P2 receptor transcripts in VSMC.
In order to quantify the level of expression, and changes in expression
levels, of these receptor mRNAs in VSMC a competitive RT-PCR assay
was developed. A synthetic RNA was generated as an internal standard for
each receptor sequence to be studied (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11
and P2X1). Each standard contained a 100 base pair deletion and con-
tained the relevant primer sequences for the targeted receptor transcript.
A known amount of the synthetic RNA fragment was added to each RT-
PCR reaction and the ratio of the resulting amplicons allowed a calcula-
tion of the number of mRNA molecules in the sample. Data generated by
this procedure will be presented and will include transcript expression
levels from saphenous vein fragments and cultured VSMC taken through
several passages and comparing cultures of internal mammary artery and
saphenous vein VSMC derived from the same patient.
Funded by the Wellcome Trust.
205
ANTIPROLIFERATIVE EFFECTS OF UTP ON HUMAN VASCU-
LAR SMOOTH MUSCLE CELLS.
Kumari, R.; White, P.J.; Boarder, M.R.
The Cell Signalling Laboratory, Deptartment Of Biological Sciences, De
Montfort University, Leicester, Uk.
Proliferation of vascular smooth muscle causes narrowing of blood vessels
leading to atherosclerosis and hypertension. Nucleotides such as atp and
utp are released from activated platelets. In normal blood vessels these act
on p2y receptors on endothelial cells, stimulating the release prostacyclin
and nitric oxide, which have relaxant and anti-proliferative effects on vas-
cular smooth muscle cells (vsmc). However, in diseased state damaged
endothelium allows nucleotides to act directly on vsmc. Both atp and utp
stimulate proliferation of animal vsmc in culture. This study focuses on
p2y receptor control of proliferation in human saphenous vein (sv) and
internal mammary artery (ima) vsmc cultures.
Thymidine incorporation was used to measure dna synthesis as an index
of proliferation. Utp significantly attenuated platelet derived growth fac-
tor (pdgf) induced dna synthesis in both ima and sv cells. Atp significantly
enhanced pdgf responses in ima vsmc. Atp and utp were able to generate
similar increases in cytosolic calcium levels in both ima and sv vsmc. Phos-
phorylation of shc and mitogen activated protein kinase (mapk) was also
increased above basal levels when cells were stimulated with atp. The utp
inhibitory effect on the pdgf response was not seen in mapk phosphoryla-
tion as determined by western blotting using anti-phospho mapk specific
antibodies. In conclusion atp acts synergistically with pdgf as a pro-prolif-
erative agent in imas. However, utp acts as an anti-proliferative regulator
in both human arterial and venous vsmc.
Funded by Medical Research Council and the Wellcome Trust.
100 ABSTRACTS FROM PURINES 2000
206
DIFFERENTIAL ROLE OF ATP-SENSITIVE POTASSIUM CHAN-
NELS IN ADENOSINE-INDUCED VASODILATION IN RAT
CEREBRAL VESSELS: LARGE CONDUIT ARTERIES VERSUS
mICROVASCULAR ARTERIOLES.
West,G. A.,Oh, B., Nguyen, T-S., Meno, J.,Winn, H.R.
Department of Neurological Surgery, University of Washington, Seattle,
WA, USA.
Adenosine (ADO) is a potent cerebrovasodilator and potential metabolic
mediator of cerebral blood flow. However, the mechanism of action by
which adenosine causes vasodilatation in the cerebral circulation is cur-
rently unknown. In this study we sought to define the role of ATP-sensi-
tive potassium (KATP) channels in ADO-induced vasodilation in the arterial
network using the KATP blocker glibenclamide (GLIB) on ADO-induced
vasodilation in rat secondary branches of basilar artery (20BA) and
intraparenchymal arterioles (IPA). Vessels were isolated, cannulated using
a micropipette system, and luminally pressurized to 60 mm Hg. In 20BA
(146±5 µm, n=7) and IPA (97±9 µm, n=15) vessels contracted spontane-
ously to 75% and 64% of passive diameter, respectively. In both groups,
ADO induced comparable dose-dependent dilations (-log EC50 0.8 µM
and 3.4 µM, respectively). In the 20BA, 1 µM GLIB partially antagonized
ADO-induced dilation; however, 1 µM GLIB had no effect on ADO dose-
response in IPA. GLIB had no effect on baseline tone nor dilation induced
by acidic (pH 6.8) buffer in either groups. Pinacidil, KATP channel opener,
induced dilation in both sets of vessels that was nearly completely blocked
by GLIB, n=6. We conclude that opening KATP channels leads to
vasorelaxation in both vascular beds. However, while ADO-induced va-
sodilation is partially mediated by KATP channels in 20BA, there is no
role of KATP channels in IPA. Thus, there appears to be a differential
segmental response in role of KATP channels in ADO vasodilation in the
cerebrovasculature.
207
ADENOSINE-INDUCED DILATION OF RAT INTRACEREBRAL
ARTERIOLES INVOLVES THE A2A RECEPTOR SUBTYPE.
Coyne, E.F.; Ngai, A.C.; Winn, H.R.
Department of Neurological Surgery, University of Washington, Seattle,
WA, USA.
We have previously shown that adenosine (ADO)-induced dilation of ce-
rebral arterioles is mediated by A2 adenosine receptors (Circ. Res. 73: 448,
1993). The purpose of the present study is to further characterize the re-
ceptor subtypes involved. Intracerebral arterioles were isolated from adult
rats, cannulated using a micropipette system, and luminally pressurized to
60 mmHg. The vessels developed spontaneous tone to approximately
63±0.8% of passive diameter (82±6um). Both ADO and the A2A receptor
agonist CGS21680 induced dose-dependent dilation of the cerebral arte-
rioles. The more potent vasodilator was CGS21680 (-logEC50:
CGS21680=8.7, n=37 vs. ADO=6.4, n=41), but ADO was more effica-
cious (maximal dilation: ADO=133±1.3% vs. CGS21680=120±2.1%). The
selective A2A receptor antagonist ZM241385 (0.1uM) completely inhibited
CGS21680-induced vasodilation, and significantly attenuated ADO-in-
duced vasodilation. However, ZM241385 did not affect dilation to acetyl-
choline (10uM) or to acidic (pH 6.8) buffer. The non-selective ADO receptor
antagonist CGS15943 (0.1uM), in combination with ZM241385 (0.1uM),
did not further attenuate ADO-induce vasodilation. We also evaluated the
effects of CPX (0.1uM), a selective A1 receptor antagonist, and MRS1191
(0.1uM), a selective antagonist for the A3 receptor subtype. Neither of these
antagonists had an effect on the ADO dose response. We conclude that the
A2A adenosine receptor subtype plays a significant role in adenosine-in-
duced dilation of cerebral arterioles. We question whether the A2B recep-
tor subtype may also be involved in dilation of intracerebral arterioles at
higher concentrations of adenosine. However, selective A2B receptor an-
tagonists are currently unavailable to determine this unequivocably.
208
ROLE OF CALCIUM-DEPENDENT POTASSIUM CHANNELS IN
ADENOSINE-INDUCED VASODILATION IN RAT CEREBRAL
INTRAPARENCHYMAL ARTERIOLES.
West, G. A., Nguyen, T-S, Oh B., Meno, J., Winn, H.R.
Department of Neurological Surgery, University of Washington, Seattle,
WA, USA
Adenosine (ADO) is a potent cerebrovasodilator and important metabolic
regulator of cerebral blood flow. The ion channels mediating ADO vasodi-
lation is well characterized. We investigated the role of calcium-activated
potassium channels (KCa) in ADO (n=7), sodium nitroprusside (SNP, n=5),
and 8-bromo-cGMP (8-Br-cGMP, n=5) induced vasodilation using
Iberiotoxin (IBTX, 1 nM), a potent blocker of KCa channels, in rat mi-
crovascular intraparenchymal arterioles. Vessels were isolated, cannulated
using a micropipette system, and luminally pressurized to 60 mm Hg. In
all three experimental groups, arterioles had similar resting diameters (95±3
µm, 97±3 µm, and 90±5 µm, respectively) and developed comparable
baseline tone (66%, 69%, and 68%, respectively). IBTX blocked vasodila-
tion induced by 0.5 µM and 10 µM ADO by 10% and 29%, respectively.
IBTX blocked vasodilation induced by 1 µM SNP by 65%. IBTX blocked
vasodilation induced by 100 µM 8-Br-cGMP by 73%. IBTX had no effect
on either baseline tone nor dilation induced by acidic (pH 6.8) buffer in all
groups. Glibenclamide (GLIB), a blocker of ATP-sensitive potassium
(KATP) channels, had no effect on ADO, SNP nor 8-Br-cGMP dilations.
NS-1619, an opener of KCa, induced IBTX-sensitive dilations in these
vessels and was completely blocked by IBTX. These data suggest a partial
role of KCa but not KATP channels in ADO-induced vasodilation in VSM
from rat intraparenchymal arterioles, likely involving ADO A2a receptor
activation, stimulation of guanylate cyclase, activation of PKG, and subse-
quent opening of KCa channels.
209
PKC BETA MEDIATES THE INHIBITION OF P2Y RECEPTOR-
INDUCED INOSITOL PHOSPHATE FORMATION IN ENDOT-
HELIAL CELLS
Lin, W.W.; Chen, B.C.
Department of Pharmacology, College of Medicine, National Taiwan Uni-
versity, Taipei, Taiwan.
Bovine pulmonary artery endothelium (CPAE) expresses phospholipase C
(PLC)-linked P2Y1 and P2Y2 receptors, for them 2MeSATP and UTP are
respective agonists. Here, we have investigated the particular protein ki-
nase C (PKC) isoform(s) responsible for the inhibition of P2Y1 and P2Y2
receptor-evoked inositol phosphate (IP) formation by phorbol 12-myristate
13-acetate (PMA). Although short-term (20 min) pretreatment of cells with
PMA attenuated 2MeSATP- and UTP-induced phosphoinositide (PI) break-
down, this inhibition was lost after 15 h. Preincubation with PMA for 24 h,
on the contrary, potentiated 2MeSATP and UTP responses. The IP forma-
tion stimulated by NaF was unaltered by PMA pretreatment. Western blot
analysis showed that treatment of CPAE with PMA resulted in a rapid
translocation of PKC isoform β, ε and µ, but not λ, from the cytosol to the
membrane fraction. Pretreatment of the selective PKC inhibitor Ro 31-
8220 attenuated the inhibitory effect of PMA on IP formation. Go 6976 (an
inhibitor of conventional PKCα, β and γ) and LY 379196 (a selective PKCβ
inhibitor) also dose-dependently inhibited the PMA-mediated desensiti-
zation. Transfection of PKCβ-specific antisense oligonucleotide reduced
PKCβ protein level and inhibited PMA-mediated PI reduction. RT-PCR
analysis showed that PMA treatment for 4-24 h up-regulated P2Y1 and
P2Y2 receptors at the mRNA levels. These results suggest that PKCβ may
exert a negative feedback regulation on endothelial P2Y1 and P2Y2 recep-
tor-mediated PI turnover. The down-regulation of PKCβ and enhanced
P2Y receptor expression together might contribute to the late PI enhanc-
ing effect of PMA.