R-N6-phenylisopropyladenosine Research Articles

Effect of central adenosine on brainstem blood flow in fetal sheep.

The effect of the adenosine analogue R-N6-(phenylisopropyl)adenosine (R-PIA) on blood flow to the medulla and pons was examined in unanaesthetized fetal sheep. Microspheres labelled with isotopes were used to determine blood flow before and after instillation of 0.2 or 0.5 microgram R-PIA into the cerebrospinal fluid of the fourth ventricle. Blood flow to the medulla, which had a mean value (+/- S.E.M.) of 285 +/- 41 ml min-1 (100 g)-1 during the control period, was not changed by the central instillation of R-PIA. Blood flow to the pons was also not affected. These data indicate that central adenosine, which depresses respiratory drive in fetal sheep, acts by mechanisms independent of removal of carbon dioxide from the brainstem.

Co-expression of the adenosine A2A receptor and the stimulatory G-protein alpha subunit in a stable cell line using a novel poly-cistronic expression vector

The effects of different adenosine agonists on cAMP levels in isolated adult guinea pig ventricular myocytes were investigated. 2-Chloro-N6-cyclopentyladenosine (CCPA), R-N6-phenylisopropyl-adenosine (R-PIA), S-N6-phenylisopropyladenosine (S-PIA) and 5′-N-ethylcarboxolamidoadenosine (NECA) reduced isoprenaline-stimulated cAMP levels. Both the nonselective adenosine antagonist 8-{4-[([{(2-aminoethyl)amino}carbonyl]methyl)oxy]-phenyl}-1,3-dipropylxanthine (‘xanthine amine congener’; XAC) and the A1-selective adenosine antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) blocked the inhibitory effect of NECA. Basal cAMP levels were not altered by NECA or the highly A2-selective 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine (CGS 21680), either alone or in the presence of DPCPX or XAC. These results provide evidence against the existence of A2 receptors on the ventricular myocyte but support the presence of A1 receptors.

The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine to adenosine A1 receptors in rat smooth muscle preparations.

1. The binding of 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), an antagonist radioligand selective for adenosine A1 receptors, was studied in rat duodenum, colon muscularis mucosae and longitudinal muscle, urinary bladder and vasa deferentia. 2. [3H]-DPCPX bound with high affinity to a single site in all membrane preparations studied with the exception of the rat urinary bladder in which no specific binding was detected. The affinity (Kd) of the binding site for [3H]-DPCPX was similar in all membrane preparations, the colon longitudinal muscle (1.18 +/- 0.47 nM), colon muscularis mucosae (0.84 +/- 0.15 nM), duodenum (1.59 +/- 0.18 nM) and vasa deferentia (0.93 +/- 0.17 nM). The density of [3H]-DPCPX binding sites was similar in the duodenum (38.8 +/- 4 fmol mg-1 protein), muscularis mucosae (43 +/- 3.5 fmol mg-1 protein) and vasa deferentia (43.3 +/- 12.2 fmol mg-1 protein), but in the longitudinal muscle 6-7 fold more binding sites (295 +/- 70 fmol mg-1 protein) were identified. 3. Inhibition studies using DPCPX (0.1-100 nM), N6-cyclopentyladenosine (CPA) (0.1-100 nM), 5'-N-ethylcarboxamidoadenosine (NECA) (2 nM-10 microM) and (R)-N6-phenylisopropyladenosine (R-PIA) (1 nM-1 microM) to displace the binding of [3H]-DPCPX at a concentration around the Kd value (1 nM), demonstrated an order of potency of displacement in all tissues of DPCPX > or = CPA > R-PIA > NECA. This potency order is characteristic of an A1 receptor, indicating that [3H]-DPCPX binds to adenosine A1 receptors in the rat duodenum, colon and vasa deferentia. Two site analysis revealed that the agonists bind to both a high and low affinity state of the receptor.4. The existence of Al binding sites in the rat vasa deferentia, colon muscularis mucosae and duodenum, and their absence in the urinary bladder, is consistent with previous functional studies.However, in contrast to the findings of the [3H]-DPCPX binding assay, no functional response mediated by adenosine Al receptors could be detected by measuring contractile or relaxant responses to CPA in the colon longitudinal muscle. The functional significance of the binding sites in this tissue has therefore yet to be determined.

A1 adenosine receptors. Two amino acids are responsible for species differences in ligand recognition.

Species differences in ligand binding to A1 adenosine receptors were localized to the seventh transmembrane (TM7) region based on the binding of [8-3H]cyclopentyl-1, 3-dipropylxanthine and three other ligands to wild type and six bovine/canine interspecies receptor chimeras expressed in COS-1 cells. Subsequent site-directed mutagenesis experiments identified amino acid 270 (isoleucine/methionine, bovine/canine) as being primarily responsible for species differences in the binding of N6-adenine-substituted compounds, R-N6-phenylisopropyladenosine (R-PIA) and (S)-N6-endonorbornan-2-yl-9-methyladenine, and the C-8-substituted xanthine, [3H]cyclopentyl-1,3-dipropylxanthine. These data are consistent with the hypothesis that the N6 region of adenines and the C-8-region of xanthines bind to the same region of the receptor. A second TM7 amino acid, 277 (serine/threonine, bovine/canine), selectively influences the binding of the ribose-substituted adenosine analog, 5'-N-ethylcarboxamidoadenosine to a variable extent, depending on the nature of amino acid 270. We hypothesize that amino acid 270 of the A1 receptor interacts with the N6 region of adenosine, while amino acid 277 is important, especially in the absence of an N6 substitution, for interactions with a distinct nucleoside region, possibly on the ribose.

Evidence against KATP channel involvement in adenosine receptor-mediated dilation of epicardial vessels.

The purpose of this study was to determine whether ATP-glyburide-sensitive K+ (KATP-glyburide) channels are involved in the adenosine-induced vasorelaxation of porcine and canine epicardial vessels in vitro. Adenosine and its analogues, 2-chloroadenosine (CAD), 5'-N-ethylcarboxamidoadenosine (NECA), R-N6-(2-phenylisopropyl)adenosine (R-PIA), N6-cyclopentyladenosine (CPA), N6-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)]adenosine (DPMA), 2-phenylaminoadenosine (CV-1808), 2-[m-(carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine (CGS-22988), 2-[(2-cyclohexylethyl)amino]adenosine (CGS-22492), 2-[(p-amino)phenethylamino]adenosine (APE), and 2-(1-octynyl)adenosine (YT-146) (10 nM-100 microM), produced concentration-dependent relaxations in endothelium-intact and -denuded arterial ring segments contracted with 30 mM KCl, 10 nM endothelin-1, or 10 microM prostaglandin F2 alpha. Sodium nitroprusside (SNP; 1 nM-10 microM) and KATP-channel activator, pinacidil (10 nM-10 microM), also produced similar vasodilatory responses. Glyburide, a KATP-channel blocker, caused a rightward shift of the concentration-response curve to pinacidil but did not alter the responses elicited by SNP or adenosine and its analogues. The data suggest that KATP-glyburide channels are not involved in the mechanism whereby adenosine and its analogues elicit their vasorelaxant response in isolated porcine or canine epicardial vessels.

Post-junctional excitatory adenosine A 1 receptors in the rat vas deferens

1. 1. At concentrations between 1 nM and 1 μM, the A 1-selective agonists N 6-cyclopentyladenosine (CPA) and (R)-N 6-phenylisopropyladenosine (R-PIA) each enhanced contractions of the rat vas deferens induced by ATP (10 μM), and this enhancement was blocked by an A 1-selective concentration (1 nM) of the antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). 2. 2. No such enhancement was observed with the non-selective agonists adenosine and 5′- N-ethylcarboxamidoadenosine (NECA) at concentrations between 1 nM and 100 μM, which instead inhibited the contractions. 3. 3. These results show that in addition to the previously demonstrated inhibitory A 1 and A 2 adenosine receptors, the rat vas deferens also possesses post-junctional excitatory A 1 adenosine receptors.

Purification and characterization of an adenotin-like adenosine binding protein from human platelets.

A low-affinity adenosine binding protein (adenotin) was purified from human platelet membranes by a four-step procedure. Purification was achieved after extraction from human platelet membranes with 0.3% 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Further purification included Sepharose CL6B gel filtration, DEAE-Sepharose CL6B, and hydroxylapatite chromatography. The protein was purified 884-fold to homogeneity with a 25% yield of binding activity from the membranes. 5'-[8(n)-3H]-N-ethylcarboxamidoadenosine ([3H]NECA) binds to the purified protein with a KD of 155 (144-167) nmol/l and a Bmax of 1.85 +/- 0.10 nmol/mg of protein. Sodium dodecylsulfate polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa. The 2-chloro-substituted adenosine analogs 2-chloro-5'-N-methylcarboxamidoadenosine (CIMECA) and 2-chloro-5'-N-ethylcarboxamidoadenosine (CINECA) were identified as new high affinity ligands of the purified protein showing Ki values of 18 nmol/l and 28 nmol/l, respectively. The low-affinity adenosine binding protein showed a pharmacological profile as follows: CIMECA > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine (CIA) > 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosin e (CGS 21,680) > R-N6-phenylisopropyl-adenosine (R-PIA). Amino-terminal sequence analysis revealed homologies to endoplasmin, glucose regulated protein (GRP94), tumor rejection antigen precursor (GP96), and some stress related proteins.

Characterization of adenosine receptor-mediated generation of cyclic AMP in slices of rat cerebral cortex with chronic epileptic activity.

Cyclic AMP accumulations elicited by adenosine analogues 2-chloroadenosine (2-CADO), R-N6-phenylisopropyladenosine (R-PIA), and N6-cyclohexyladenosine (CHA) were investigated in cortical slices of chronic iron-induced epileptic rats. Cyclic AMP accumulation was elicited 9- to 18-fold by 2-CADO and it was elicited 5- to 7-fold by either R-PIA or CHA; 2-CADO was more potent than R-PIA or CHA in eliciting cyclic AMP accumulation. The adenosine analogues elicited cyclic AMP accumulation in a dose-dependent manner, and the elicitation was inhibited by the adenosine antagonist 8-phenyltheophylline. The 2-CADO-elicited accumulation of cyclic AMP was greatly increased in the cortical region on the primary epileptic side, while the R-PIA- or CHA-elicited accumulation did not change in any cortical region. The deviation detected only in the 2-CADO-elicited accumulation of cyclic AMP may be due to the difference in relative potency for adenosine receptors of the adenosine analogues. The results suggest that adenosine receptor-mediated generation of cyclic AMP is altered in the primary region of iron-induced epileptic cortex, in which heterogeneous alterations in different adenosine receptor subtypes may occur in the epileptic process.

Effects of adenosine and its analogues on isolated intracerebral arterioles. Extraluminal and intraluminal application.

We evaluated the responses of brain parenchymal arterioles to intraluminal and extraluminal application of adenosine and its analogues. Intracerebral arterioles (28.4- to 60.3-microns diameter) were isolated from Sprague-Dawley rats, cannulated with micropipettes, and perfused in vitro. Both extraluminal and intraluminal adenosine, 5'-(N-ethylcarboxamido)adenosine (NECA), R-N6-(phenylisopropyl)adenosine (R-PIA), and S-N6-(phenylisopropyl)adenosine (S-PIA) elicited concentration-dependent dilation of these arterioles, but intraluminal application was less potent and efficacious than extraluminal application. Inosine was not vasoactive. A common order of agonist potency (NECA > adenosine > R-PIA > or = S-PIA) was determined for both extraluminal and intraluminal application. Theophylline (10 microM) caused a rightward shift of the adenosine concentration-response curve and a 50-fold reduction in potency. Intraluminal theophylline was one sixth as effective as extraluminal theophylline in antagonizing the extraluminal adenosine response, whereas intraluminal 8-sulfophenyltheophylline, a polar theophylline derivative, was ineffective. Polyadenylic acid (PolyA, 1 microM), an adenosine polymer that does not penetrate the endothelium, induced a dilation of 44.2 +/- 5.3% when applied extraluminally but had no effect when infused intraluminally. The dilator effect of PolyA was antagonized by theophylline. We conclude that: (1) intraluminal adenosine and its analogues are effective dilators of intracerebral arterioles, (2) the dilator effects of both intraluminally and extraluminally applied adenosine are predominantly mediated by A2-type receptors, and (3) adenosine receptors mediating vasodilation are not present on the luminal surface of the endothelium.

Pronounced direct inhibitory action mediated by adenosine A1 receptor and pertussis toxin-sensitive G protein on the ferret ventricular contraction

An adenosine A1 receptor agonist R-N6-phenylisopropyladenosine (R-PIA) elicited a pronounced negative inotropic effect with the EC50 value of 0.69 mumol/l in the presence of a beta-adrenoceptor blocking agent bupranolol (0.3 mumol/l) in the isolated ferret papillary muscle. The negative inotropic effect of R-PIA was not associated with changes in cyclic AMP level. Adenosine and other A1 receptor agonists also elicited a negative inotropic effect. DPCPX (1,3-dipropyl-8-cyclopentyl xanthine) antagonized the negative inotropic effect of R-PIA in a competitive manner (pA2 value = 8.4). The inhibitory action of R-PIA was markedly attenuated in the ventricular muscle preparation isolated from ferrets pretreated with pertussis toxin that caused ADP-ribosylation of 39 kDa proteins in the membrane fraction. In the membrane fraction derived from the ferret ventricle, [3H]-DPCPX bound to a single binding site in a saturable and reversible manner with high affinity (Kd value = 1.21 +/- 0.41 nmol/l; Bmax = 12.8 +/- 3.02 fmol/mg protein; n = 7). The binding characteristics of [3H]-DPCPX in the rat ventricle (Kd value = 1.51 +/- 0.09 nmol/l; Bmax = 12.7 +/- 1.47 fmol/mg protein; n = 5) were similar to those in the ferret. On the other hand, the content of G(o), a major pertussis toxin-sensitive G protein in the ferret heart, was much higher in the ferret than in the rat ventricle. The present results indicate that adenosine receptors may play an important role in the inhibitory regulation of ventricular contractility in the ferret in contrast to other mammalian species. The signal transduction process subsequent to agonist binding to A1 receptors including the pertussis toxin-sensitive G protein and ion channels may be responsible for the unique inhibitory action of adenosine in this species.

Contraction of the rat isolated spleen mediated by adenosine A1 receptor activation.

1. A series of adenosine receptor agonists of varying degrees of selectivity induced concentration-dependent contraction of the rat isolated spleen. With the exception of the response to the selective A2A receptor agonist, 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine (CGS 21680), responses to each ligand were blocked surmountably and to a broadly similar extent by 8-p-sulphophenyltheophylline (10(-5) M). 2. There was a significant correlation between the pEC50 values obtained on the spleen and the binding affinities (pKD; measured with [3H]-NECA) for the A1 receptor of pig striatum (r = 0.98, P < 0.001) but not the A2A receptor (r = 0.14, NS). 3. The antagonist potencies of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and 9-chloro-2-furyl [1,2,3]triazolo[1,5-C]quinazoline-5-amine (CGS 15943) were measured against the prototype selective A1 receptor agonist, R-N6-phenylisopropyladenosine (R-PIA). The resulting pKB values of 8.67 and 7.70, respectively are consistent with the A1 receptor subtype mediating splenic contraction. 4. The response to R-PIA was unaltered in the presence of a concentration (10(-7) M) of CGS 21680 which is 6 fold its KD concentration at the A2A binding site in pig striatum but below the threshold for causing contraction per se; thus, A2A receptors inhibitory to contraction appear to be absent. 5. The response to R-PIA was resistant to blockade by prazosin (10(-7) M) and by nifedipine (10(-6) M) but partially blocked by indomethacin (10(-6) M). 6. The results show that the rat isolated spleen responds to adenosine receptor agonists with contraction. Both the relative potencies of agonists and the effects of antagonists indicate mediation by the A1 receptor subtype. alpha1-Adrenoceptor activation is not involved in contraction but a role for products of cyclo-oxygenase and calcium from a source not dependent on entry through L-channels is implicated.

On the usefulness of Fura‐2 measurements of intrasynaptosomal calcium levels in rat cortical synaptosomes to study mechanisms of presynaptic function

Levels of [Ca2+]i in rat cortex synaptosomes were measured using the Ca2+ indicator Fura-2. Ca2+ influx was induced by veratridine in a concentration-dependent manner (1-10 microM). The resulting increase in [Ca2+]i was inhibited by tetrodotoxin (TTX). K+ (18 mM) increased the [Ca2+]i which was not influenced by TTX. K(+)-channel blockers such as 4-aminopyridine, alpha- and delta-dendrotoxin pre se were ineffective. The veratridine-induced Ca2+ influx in synaptosomes was reduced by L-type Ca(2+)-channel blockers, such as felodipine, nifedipine and PN-200-110, verapamil and diltiazem. omega-Conotoxin, and N-type Ca(2+)-channel blocker, did not inhibit the veratridine-stimulated [Ca2+]i increase. Bay K 8644, and L-channel agonist, stimulated an increase of [Ca2+]i in synaptosomes which was not sensitive to TTX. R-N6-Phenyl-isopropyl-adenosine (R-PIA) and clonidine, agonists at adenosine A1-receptors and alpha 2-adrenoceptors, respectively, did not influence the veratridine-stimulated [Ca2+]i increase. R-PIA did not interact with Bay K 8644-stimulated [Ca2+]i increase in synaptosomes. The results for all the substances used show major differences between the effects on Ca2+ influx in synaptosomes and on the electrically evoked neurotransmitter release in slice preparations. Thus, the synaptosome preparation is not a generally applicable experimental model for the study of Ca2+ mechanisms of presynaptic neuromodulation.

Reversible Contractile Dysfunction in Reversible Experimental Cardiac Allograft Rejection: Alterations in the β-Receptor-Stimulated Adenylylcyclase Pathway

Alterations in the β-adrenergic receptor adenylylcyclase (AC) pathway are well known in heart failure. Previous studies by our group have demonstrated impaired function of the AC pathway and contractile reserve when stimulated with isoproterenol (ISO) or forskolin (F) in cardiac allografts with moderate or severe rejection. To determine if recovery of the AC pathway occurs when rejection is reversed, we used a rat heterotopic heart transplant model. Lewis (L) rats received either isografts or Lewis-Brown Norway (LBN) allografts (ALLO). The hearts were explanted on Day 4 and reimplanted into a recipient syngeneic with the original donor (L to L, LBN to LBN). Grafts were harvested 2 days later and analyzed. Receptor-mediated modulation of AC activity was investigated using ISO, F, and the metabolic inhibitor of AC, R-N6 (2-phenylisopropyl)-adenosine (R-PIA). ISO- and F-mediated stimulation of AC were significantly impaired in ALLO. CARB and R-PIA remained unchanged. Reimplanted hearts had no histologic evidence of rejection and had normal cAMP production in response to ISO and F. In conclusion, AC alterations are concordant with histologic changes in this reversible model of heart failure. Such alterations may be a component in the contractile dysfunction associated with rejection.

A2-purinoceptor-mediated relaxation in the guinea-pig coronary vasculature: a role for nitric oxide.

1. The Langendorff heart preparation was used to investigate the mechanism of action of the endothelium-dependent vasodilatation evoked by adenosine and its analogues in the guinea-pig coronary vasculature. 2. The relative order of potency of adenosine and its analogues in causing a reduction in perfusion pressure was D-5'-(N-ethylcarboxamide)adenosine (NECA) = 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N- ethylcarboxamidoadenosine (CGS 21680)> R-N6-(2-phenylisopropyl)adenosine (R-PIA) = adenosine = 2-chloroadenosine (2-CA) > S-N6-(2-phenylisopropyl)adenosine (S-PIA) = N6-cyclopentyl-adenosine (CPA); thus suggesting the presence of A2-purinoceptors in this preparation. 3. 8-(p-Sulphophenyl)theophylline (8-PSPT; 3 x 10(-5) M) significantly reduced both the maximum amplitude and area of the vasodilatation produced in response to adenosine (5 x 10(-10) -5 x 10(-8) mol) without having any effect on the response to the P2-purinoceptor agonist, 2-methylthioATP. The relaxation induced by adenosine (5 x 10(-12) -5 x 10(-8) mol) was unaffected by the selective A1-purinoceptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10(-8) M). This antagonist profile suggests that only A2-purinoceptors are present in the guinea-pig coronary vasculature. 4. The areas of the vasodilator response to adenosine (5 x 10(-10) -5 x 10(-7 mol), NECA (5 x 10(-12) -5 x 10(-7) mol) and CGS 21680 (5 x 10(-12) -5 x 10(-10) mol) were significantly reduced by NG-nitro-L-arginine methyl ester (L-NAME; 3 x 10(-5) M). The amplitude of the responses to low concentrations of adenosine (5 x 10-10-5 x 10-9mol), NECA (5 x 1011 mol) and CGS 21680 (5 x 1011-5 x 10-9mol)were significantly reduced by L-NAME (3 x 10-5 M).5. L-Arginine (1.5 x 10-3 M) significantly reversed the inhibition, by L-NAME (3 x 10-5 M), of the relaxant response to adenosine (5 x 10-8 mol), NECA (5 x I0- mol) and CGS 21680 (5 x 10-11 mol).6. Indomethacin (10-6 M) did not inhibit the response to adenosine, except at low doses (5 x 10-11-5 x 10-10 mol).7. It is concluded that in the guinea-pig coronary vasculature, while a major part of the vasodilator action of adenosine is probably directly via A2-receptors on the smooth muscle, activation of a subpopulation of A2-purinoceptors on endothelial cells by adenosine and its analogues induces relaxation via production of nitric oxide; prostanoids appear to play a minimal role in the relaxation induced by adenosine as in most other preparations.

Cardiovascular effects of adenosine and the adenosine A1 Receptor antagonist NPC 205 are altered with age in guinea pigs

AbstractResponsiveness of the cardiovascular system to the effects of vasoactive agents can vary with age. The endogenous metabolite adenosine has negative chronotropic, inotropic, and hypotensive effects. The purpose of this study was to determine if the effects of exogenously administered adenosine and the adenosine A1 receptor antagonist 1,3‐di‐n‐propyl‐8‐(hydroxyphenyl) xanthine (NPC 205) vary with age. The effects of adenosine on mean arterial blood pressure (MABP), heart rate, and cardiac contractility (LV dP/dt) were investigated in two groups of guinea pigs: a young group (3–4 weeks) and an older group (72–75 weeks). In vivo, the older animals were shown to be significantly more sensitive to the negative inotropic, chronotropic, and hypotensive effects of adenosine, whereas the responses to R‐N6‐phenylisopropyladenosine (R‐PIA), a metabolically stable analog of adenosine, were similar in both age groups. In vitro, right atria from young (3–4 weeks) and old (72–75 weeks) guinea pigs exhibited no age‐related differences in the sensitivity to the negative chronotropic effects of adenosine or R‐PIA. Younger animals were more sensitive to the positive inotropic effects of the adenosine A1 receptor antagonist, NPC 205. In addition, basal heart rates were significantly lower in the older group of animals. It may therefore be concluded that there age‐related effects of adenosine. Furthermore, comparison of the effects of adenosine to the effects of the stable analog R‐PIA and the adenosine antagonist NPC 205 suggests that these differences may be due to changes in adenosine metabolism with age. © Wiley‐Liss, Inc.

Facilitation of [ 3H]-ACh release by forskolin depends on A 2-adenosine receptor activation

The effect of forskolin (FSK) on [ 3H]-acetylcholine release ([ 3H]-ACh) from the phrenic motor nerve terminals, and its modification by adenosine deaminase (ADA), by the A 2-adenosine receptor agonist 2-[ p-(2-carboxyethyl)phenethylamino]-5′- N-ethylcarboxamide adenosine (CGS 21680C), by the A 1-adenosine receptor agonist R- N 6-phenylisopropyl adenosine (R-PIA), by the A 2-antagonist N-(2-(dimethylamino)-ethyl)- N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1 H-purine-8-yl)-benzene sulphonamide (PD 115,199), and by the A 1-antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were studied on the rat phrenic-hemidiaphragm preparation. It is concluded that the excitatory effect of FSK on evoked [ 3H]-ACh release depends on tonic A 2-adenosine receptor activation.

G-protein-coupled A 1 adenosine receptors in coated vesicles of mammalian brain: Characterization by radioligand binding and photoaffinity labelling

A 1 adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaffinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1,3-[ 3H]dipropyl-xanthine ([ 3H]DPCPX) gave a K d value of 0.7 nM and a B max value of 82 ± 13 fmol/mg protein. For the highly A 1-selective agonist 2- chloro-N 6-[ 3 H]cyclopentyladenosine ([ 3H]CCPA) a K d value of 1.7 nM and a B max value of 72 ± 29 fmol/mg protein was estimated. Competition of agonists for [ 3H]DPCPX binding gave a pharmacological profile with R- N 6-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5′- N-ethylcarboxamido-adenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCPA to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido- N 6- 125I-p-hydroxyphenylisopropyladenosine ([ 125I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A 1 receptors labelled in brain membranes. Therefore, coated vesicles contain A 1 adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A 1 receptors in these vesicles are not a processed receptor form. These results confirm that A 1 receptors in coated vesicles are coupled to a G-protein, and it appears that the A 1 receptor systems in coated vesicles and in plasma membranes are identical.

Relative agonist potencies of C 2-substituted analogues of adenosine: Evidence for adenosine A 2B receptors in the guinea pig aorta

Nine C 2-substituted adenosine analogues that are potent and selective for the A 2-adenosine receptor were tested for their ability to induce relaxations of the guinea pig aorta. Compounds tested were 2-phenylethoxyadenosine (PEA), 2-phenylethoxy-5′- N-ethylcarboxamidoadenosine (PENECA), 2-cyclohexylethoxyadenosine (CEA), 2-fluorophenylethoxyadenosine (FPEA), 2-methoxyphenylethoxyadenosine (MPEA), 2-naphthylethoxyadenosine (NEA), 2-phenylaminoadenosine (CV-1808), 2-phenylethylaminoadenosine (PEAA) and 2-carboxyethylphenethylamino-5′-N-ethylcarboxamidoadenosine (CGS21680). The responses to these agents were compared to those of three standard adenosine receptor agonists, 5′-N-ethylcarboxamidoadenosine (NECA), N 6-cyclohexyladenosine (CHA) and R-N 6-phenylisopropyladenosine (R-PIA). The C 2-ethoxyadenosine analogues were 30- to 140-fold less potent than NECA and the C 2-amino-substituted analogues were 250 to 1000-fold less potent than NECA at inducing relaxations of the guinea pig aorta. All of the analogues were also less potent than the A 1-selective agonist R-PIA. However, only responses to NECA were competitively antagonized by the non-selective adenosine receptor antagonist 8-phenyltheophylline (8-PT), pK B=6.83±0.05. The results suggest that the C 2-substituted analogues produce relaxations of the guinea pig aorta through a combination of actions at A 2-adenosine receptors and at xanthine resistant sites. The lack of potency of these analogues at activating the xanthine sensitive A 2-receptors in the guinea pig aorta suggests that these adenosine receptors may be of the A 2b-subtype.

Role of adenosine receptor activation in myocardial infarct size limitation by ischaemic preconditioning.

The aims were to examine the role of adenosine receptors in the mechanism of preconditioning in a chronic rabbit model of myocardial infarction; to assess whether the preconditioning effect is blocked by an adenosine receptor antagonist, 8-phenyltheophylline; and to determine whether an adenosine A1 receptor agonist, R(-)N6-2-phenylisopropyl adenosine (R-PIA), mimics infarct size limitation by preconditioning. Myocardial infarction was induced in male rabbits by occlusion of the left coronary artery for 30 min, which was followed by 72 h reperfusion. Before the 30 min ischaemia, rabbits were subjected to one of the following six protocols: (1) untreated control; (2) intravenous injection of 8-phenyltheophylline; (3) preconditioning with 5 min ischaemia; (4) pretreatment with 8-phenyltheophylline plus preconditioning; (5) intravenous injection of R-PIA; or (6) R-PIA plus atrial pacing (240.min-1). Infarct size and area at risk were determined by histology and fluorescent particles, respectively. Preconditioning significantly limited infarct size, normalised as a percent of area at risk (%IS/AR), to 19.2 (SEM 2.3)% v control value of 46.5(2.8)%. 8-Phenyltheophylline alone did not modify the %IS/AR, but its injection before preconditioning attenuated the preconditioning effect such that IS/AR = 34.4(2.5)%. While R-PIA did not achieve statistically significant myocardial salvage, R-PIA plus atrial pacing limited infarct size to 33.7(3.0)% (p<0.05 v control). The R-PIA group had severe hypotension and their infarct sizes were inversely correlated with diastolic blood pressure at reperfusion. There was no such correlation in the R-PIA plus pacing group in which bradycardia and hypotension induced by R-PIA were attenuated by atrial pacing. The infarct size limiting effect of preconditioning was attenuated by 8-phenyltheophylline, and pretreatment with R-PIA was able to limit myocardial infarct size when severe hypotension was avoided by atrial pacing. These findings suggest that adenosine receptor activation plays a crucial role in the mechanism of preconditioning.

C13 The role of adenosine in the regulation of energy balance in heart and brain

In vitro contractility studies were conducted in canine gastric muscularis mucosae strips with the adenosine analogues 2-chloroadenosine (CIAD), 5′-N-ethylcarboxamidoadenosine (NECA), 5′-(N-cyclopropyl)-carboxamidoadenosine (CPCA), R-N6-(2-phenylisopropyl)adenosine (R-PIA), S-PIA, N6-cyclohexyladenosine (CHA) and (2-p-carboxyethyl)phenylamino-5′-N-carboxamidoadenosine (CGS21680) as well as the A1-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), Adenosine analogues contracted the muscle strips with the following rank order of potency: CPCA > NECA > CIAD > R-PIA . CHA . S-PIA. CGS21680. CPCA, R-PIA, and CHA were partial agonists. At a concentration selective for adenosine A1 receptors (50 nM), DPCPX did not alter the concentration-response curves to CIAD or CHA. However, at higher concentrations (1–10 μM), DPCPX antagonized CIAD-mediated contractions in a competitive manner (pA2 = 6.96; slope = 0.93). CIAD-meadited contraction was not altered by treatment of the muscle strips with tetrodotoxin (1 μg/ml) or mepyramine (1 μM). Our results indicate that adenosine A1 receptors, nerves or mast cells are not involved in contraction of canine gastric muscularis mucosae by adenosine analogues.