adenosine-5--(n-ethylcarboxamide) and 8-cyclopentyl-1-3-dimethylxanthine

Using a splanchnic nerve-spinal cord preparation in vitro, we have previously demonstrated that tonic sympathetic activity is generated from the thoracic spinal cord. Here, we sought to determine if adenosine receptors play a role in modulating this spinally generated sympathetic activity. Various adenosine analogs were applied. N6-Cyclopentyladenosine (CPA, adenosine A1 receptor agonist) and 5'-N-ethylcarboxamidoadenosine (NECA, adenosine A1/A2 receptor agonist) reduced, while N6-[2-(4-aminophenyl)ethyl]adenosine (APNEA, non-selective adenosine A3 receptor agonist) did not alter sympathetic activity. The inhibitory effect of CPA or NECA on sympathetic activity was reversed by 8-cyclopentyltheophylline (CPT, adenosine A1 receptor antagonist) or abolished by CPT pretreatment. In the presence of 3,7-dimethyl-1-propargylxanthine (DMPX, adenosine A2 receptor antagonist), sympathetic activity was still reduced by CPA or NECA. Sympathetic activities were not changed by applications of the more selective adenosine A2 or A3 receptor agonists or antagonists, including 4-[2-[[6-amino-9-(N-ethyl-beta-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS21680), 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385), 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Chloro-IB-MECA), and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191). These findings exclude a possible involvement of A2 or A3 receptors in sympathetic regulation at the spinal levels. Interestingly, CPT alone did not affect sympathetic activity, suggesting that adenosine A1 receptors are endogenously quiescent under our experimental conditions. We conclude that intraspinal adenosine A1 receptors may down-regulate sympathetic outflow and serve as a part of the scheme for neuroprotection.

Topics: Adenosine; Adenosine A1 Receptor Agonists; Adenosine A1 Receptor Antagonists; Adenosine A2 Receptor Agonists; Adenosine A2 Receptor Antagonists; Adenosine A3 Receptor Agonists; Adenosine A3 Receptor Antagonists; Adenosine-5'-(N-ethylcarboxamide); Animals; Dihydropyridines; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A1; Spinal Cord; Splanchnic Nerves; Sympathetic Nervous System; Synaptic Transmission; Theobromine; Theophylline; Triazines; Triazoles

The behavioral effect of the adenosine antagonists CPT, PACPX, DPCPX and PD 115,199 on spontaneous locomotor activity was investigated in mice after parenteral administration. CPT, PACPX and PD 115,199 affected locomotor activity in a biphasic way. Doses in the nanomolar/kg range significantly reduced locomotion (PACPX> or =PD 115,199>>CPT). Higher doses were progressively less active until they became ineffective or slightly stimulated locomotion. NECA, a mixed A1/A2 agonist, and CCPA, a highly selective A1 agonist, also induced a biphasic behavior, with low doses stimulating and high doses inhibiting locomotion. The stimulant effect of 1 nmol/kg NECA was antagonized by depressant doses of antagonists, whereas antagonists-induced hypomotility was potentiated by a depressant dose of NECA (20 nmol/kg). It is suggested that the blockade of A1 receptors by antagonists is probably responsible for reducing locomotor activity, whereas the activation of A2 receptors by agonists is likely responsible for reducing locomotion in mice.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Antineoplastic Agents; Male; Mice; Motor Activity; Purinergic P1 Receptor Agonists; Purines; Sulfonamides; Theophylline; Xanthines

The P1 purinoceptor subtype mediating the negative inotropic responses of guinea-pig left atria and the negative chronotropic responses of beating right atria were characterized. Guinea-pig isolated paced left atria (2Hz, 5ms, threshold voltage+50%) and spontaneously beating right atria were set up in Krebs-bicarbonate solution and isometric tension and rate of contraction, respectively, were recorded. Concentration-response curves for the reduction of tension and rate, respectively, by adenosine receptor agonists, N6-cyclopentyladenosine (CPA), the R- and S- stereoisomers of N6-(2-phenylisopropyl) adenosine (R-PIA and S-PIA), 5'-(N-carboxamido) adenosine (NECA) and 2-p-((carboxyethyl)-phenethylamino)-5'-(N-carboxamido) adenosine (CGS21680) were obtained. The orders of potency on the left atria (CPA = NECA > R-PIA > S-PIA > CGS21680) and right atria (CPA = R-PIA > S-PIA > CGS21680) were consistent with the responses being mediated via A1 receptors. Antagonism of the responses to CPA or R-PIA by 8-cyclo-1,3-dimethylxanthine (CPT) was examined by a full Schild analysis. Concentration-response curves for CPA or R-PIA were obtained in the absence or presence of five or six concentrations (10(-7)-10(-5) or 3 x 10(-5)M) of CPT. The shift in the concentration-response by CPT was expressed as the concentration-ratio (CR) and plotted as -log(CR-1) against log molar concentration of CPT (Schild plot). pA2 values were calculated from the intercept on the concentration axis and by application of the equation; pA2 = log(antagonist concentration) -log (CR-1). The Schild plots had unity slopes indicating competitive antagonism and the pA2 values derived therefrom indicated that the responses were mediated via A1-receptor. Closer inspection of the Schild plots, however, showed that at the higher concentrations of CPT there was a limit to the displacement of the concentration-response curves of the left and right atria to CPA and of the left atria to R-PIA. There were also significant differences in the apparent pA2 values calculated from the equation, when different concentrations of antagonist were examined. These results indicated that at higher concentrations of agonist there may be a component of the response that is resistant to antagonism by CPT. Whether this is related to the proposal that cardiac responses are mediated via A3 receptors is discussed.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Guinea Pigs; Heart Atria; Heart Rate; In Vitro Techniques; Male; Myocardial Contraction; Phenethylamines; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theophylline

Adenosine analogs have been shown to produce antinociception after intrathecal administration. To determine the adenosine receptor subtype involved in spinal antinociception, the effects of selective agonists and an antagonist on the evoked potentials recorded from a neonatal rat spinal cord were studied. The measured potentials are a slow ventral root potential (slow VRP), which is the C-fiber-evoked excitatory response associated with nociceptive information; a monosynaptic reflex (MSR), which reflects a non-nociceptive transmission related to motor function; and a dorsal root potential (DRP), which reflects a gamma-aminobutyric acidA (GABA(A)) receptor-mediated presynaptic inhibition associated with analgesia.. The evoked potentials were recorded in response to electric stimulation of a lumbar dorsal root. Dose-response curves of agonists for these responses were obtained to determine their relative potency order. The antagonist dissociation constants (K(D) values) were estimated by Schild analysis.. Adenosine agonists dose dependently inhibited the slow VRP and the MSR. However, the slow VRP was five to eight times more sensitive to them than was the MSR. The rank order of agonist potency was N6-cyclohexyladenosine (CHA) = N6-(R)-phenylisopropyladenosine (R-PIA) > 5'-N-ethylcarboxamidoadenosine (NECA) >> CGS 21680 in both responses. 8-Cyclopentyltheophylline produced dose-dependent parallel shifts to the right of NECA dose-response curves for these responses. Schild analysis gave linear plots with slopes near unity. The K(D) values of CPT for the MSR and the slow VRP were estimated to be 5.5 nM and 4.3 nM, respectively. The DRP was also depressed by adenosine agonists with potency order of CHA > NECA >> CGS 21680. 8-Cyclopentyltheophylline antagonized the inhibitory effects of CHA on the DRP.. The results indicate that adenosine agonists inhibit spinal sensory transmission related to nociception by acting at the A1 receptors. The A1 receptor also seems to be involved in transmission related to the spinal motor system. Feedback inhibition mediated by GABA(A) receptors does not contribute to this antinociceptive action.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Analgesics; Animals; Dose-Response Relationship, Drug; Female; Male; Pain; Rats; Rats, Wistar; Receptors, Purinergic P1; Reflex, Monosynaptic; Spinal Cord; Theophylline

1. The effects of adenosine receptor agonists were examined on isolated rings of guinea-pig pulmonary artery under normoxic and hypoxic conditions. The rings were denuded of endothelium and tissues were precontracted with phenylephrine (3 x 10(-6) M) before constructing cumulative concentration-response curves to the agonists. 2. 5'-(N-ethylcarboxamido)adenosine (NECA) caused concentration-dependent contractions of the pulmonary artery which were not different between hypoxia and normoxia. The contractions were converted to a relaxation in the presence of the cyclooxygenase inhibitor, indomethacin, and again these were unaffected by hypoxia. 3. Examination of a range of agonists under normoxic conditions in the presence of indomethacin revealed relaxations, except for the A2a receptor-selective agonist, CGS 21680. The vasorelaxation was therefore A2b receptor-mediated. 4. In hypoxia, however, in the presence of indomethacin, vasoconstriction occurred to R(-)-N(6)-(2-phenylisopropyl)adenosine (R-PIA) and, to a greater extent, to Nb-cyclopentyladenosine (CPA). In the absence of indomethacin, the constriction by CPA during hypoxia was significantly greater. 5. The indomethacin-resistant contraction by CPA was abolished by the A1 receptor antagonist, 8-cyclopentyltheophylline (CPT, 3 x 10(-6) M). 6. This study has demonstrated cyclooxygenase-dependent and-independent vasoconstrictions to adenosine agonists in guinea-pig pulmonary artery under hypoxic conditions. The cyclooxygenase-independent contraction is mediated via A1 receptors. 7. These results suggest that endogenous adenosine released in the pulmonary circulation under hypoxic conditions will cause vasoconstriction and may contribute to the pulmonary hypertension associated with acute respiratory failure.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Guinea Pigs; Hypoxia; In Vitro Techniques; Indomethacin; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Phenethylamines; Phenylisopropyladenosine; Purinergic P1 Receptor Agonists; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Theophylline; Vasoconstriction; Vasodilation

The A1 adenosine receptor is a member of the seven-transmembrane G protein-coupled, receptor superfamily. This receptor binds the purine nucleoside adenosine with high affinity and inhibits the activity of adenylate cyclase. We have used site-directed mutagenesis and functional expression studies to examine the role of the threonine residue, located at position 277 in transmembrane domain VII of the human A1 receptor. Mutation of Thr-277 to either serine or alanine resulted in the expression of receptors that had essentially no change in binding affinity for the A1 selective antagonist 8-cyclo-pentyl-1,3-dipropylxanthine. Mutation of Thr-277 to serine resulted in modest (4.4-8.6-fold) but significant increases in the observed Ki values for three adenosine agonists, namely N-(1-methyl-2-phenethyl)adenosine (R-PIA or S-PIA) and 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-L- ribofuranuronamide) (NECA). However, mutation of Thr-277 to alanine resulted in no significant changes in the Ki for R-PIA or S-PIA but did result in a highly significant 437-fold increase in the Ki for NECA. This demonstrates that the hydroxyl moiety of Thr-277 mediates agonist but not antagonist binding and, more specifically, that this residue forms a probable molecular contact site with the 5' substitution found in NECA.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Amino Acid Sequence; Binding Sites; Binding, Competitive; Brain; Cell Membrane; Humans; Kinetics; Mutagenesis, Site-Directed; Point Mutation; Radioligand Assay; Receptors, Purinergic P1; Recombinant Proteins; Stereoisomerism; Theophylline; Threonine; Tritium

Brief exposure of primary cultures of hepatocytes to ethanol had a biphasic effect on glucagon receptor-dependent cyclic AMP (cAMP) production: 25-50 mM ethanol decreased cAMP levels, whereas treatment with 100-200 mM ethanol increased cAMP. This biphasic effect was also observed after pretreatment with 10 microM 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. Adenosine A1 and A2 receptors in primary cultures of rat hepatocytes are coupled to inhibition and stimulation of adenylyl cyclase, respectively. Since primary cultures of hepatocytes release adenosine into their extracellular media, we tested whether the acute effects of ethanol on cAMP were mediated by extracellular adenosine. Co-incubation with 2 U/mL adenosine deaminase prevented inhibition of cAMP production by 25-50 mM ethanol, but had no effect on stimulation by 100-200 mM ethanol. Pretreatment of hepatocytes with 110 nM 8-cyclopentyl-1,3-dimethylxanthine, an adenosine A1 receptor antagonist, also completely blocked the inhibitory effects of ethanol on cAMP production. Low concentrations of ethanol enhanced the inhibitory effects of R(-)N6-(2-phenylisopropyl)adenosine, an A1 receptor agonist, on cAMP production in cells pretreated with adenosine deaminase to remove endogenous adenosine. These data suggest that endogenously produced adenosine can be an important modulator of the effects of ethanol on receptor-stimulated cAMP production in primary cultures of rat hepatocytes.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cells, Cultured; Cyclic AMP; Drug Interactions; Ethanol; Glucagon; Liver; Male; Phenylisopropyladenosine; Rats; Rats, Wistar; Receptors, Purinergic P1; Theophylline

The behavioral effects of the adenosine agonists 5'-N-ethylcarboxamidoadenosine (NECA) was investigated in two strains of inbred mice, CD1 and CBA. NECA dose dependently reduced spontaneous locomotor activity with similar potency (ED50 = 36 +/- 1.5 and 36 +/- 1.1 nmol/kg IP for CBA and CD1 mice, respectively) and efficacy (> 90% at 100 nmol/kg) in the two strains. One nmol/kg NECA, an ineffective dose in CBA mice, exerted a significant stimulant action in CD1 mice. In saturation experiments, no differences were found in the density or in the affinity of striatal A2a receptors labeled with [3H]NECA. A strain-related difference was found in the density of striatal A1 receptors labeled with [3H]CCPA. In CBA mice, the Bmax value was 32% less than in CD1 mice (0.646 +/- 0.037 and 0.951 +/- 0.073 pmol bound/mg protein, respectively, p < 0.05). No differences in [3H]CCPA binding parameters were found in cortical and hippocampal membranes obtained from the two strains, whereas a higher density of A1 binding sites was found in the cerebellum of CBA mice. The present results show a close correlation between binding studies and the depressant action of NECA and present evidence for strain-related differences in regional distribution of central adenosine receptors and in behavioral response to purinergic drugs.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Behavior, Animal; Brain; Dose-Response Relationship, Drug; Male; Membranes; Mice; Mice, Inbred CBA; Mice, Inbred Strains; Motor Activity; Platelet Aggregation Inhibitors; Radioligand Assay; Receptors, Purinergic P1; Species Specificity; Theophylline

The effect of the purine agonist N-ethylcarboxamido-adenosine (NECA) on apomorphine-induced rotation was investigated in rats with unilateral 6-hydroxydopamine lesions of the nigrostriatal pathway. Intrastriatal administration of NECA on the denervated side caused a dose-dependent inhibition of contralateral rotation. This inhibition was prevented by prior intrastriatal injection of theophylline. The adenosine A1 receptor antagonist 8-cyclopentyltheophylline was ineffective at concentrations which block this receptor, but effective in preventing the action of NECA at concentrations which block the adenosine A2 receptor. In the absence of apomorphine, NECA had no effect on behaviour. It is concluded that A2 receptor activation counteracts apomorphine effects in the striatum. Since the A2 receptor may be localized to striatal cholinergic neurones, the possible role of these neurones in purine-induced behaviours is discussed.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Apomorphine; Brain Chemistry; Corpus Striatum; Denervation; Dopamine; Dose-Response Relationship, Drug; Hydroxydopamines; Oxidopamine; Rats; Receptors, Purinergic; Stereotyped Behavior; Substantia Nigra; Sympathectomy, Chemical; Theophylline

The locomotor effects in mice of selective A1 and A2 adenosine agonists, antagonists and combinations of agonists were investigated using a computerized activity monitor. The A2-selective agonist 2-[(2-aminoethylamino)carbonylethylphenylethylamino[-5'-N- ethylcarboxamidoadenosine (APEC), an amine derivative of 2-(carboxyethylphenylethylamino)adenosine-5'-carboxamide, was a more potent locomotor depressant than its amide conjugates. The rank order of potency after i.p. injection for adenosine agonists was 5'-N-ethylcarboxamidoadenosine (NECA) (ED50, 5.8 nmol/kg) greater than APEC (ED50, 25 nmol/kg) greater than N6-cyclohexyladenosine (CHA) (ED50, 270 nmol/kg). An A1-selective, centrally acting, adenosine antagonist, 8-cyclopentyltheophylline (10 mg/kg), completely reversed the locomotor depressant effects of CHA (A1-selective) and NECA (nonselective) at doses of agonists as high as twice the ED50, and shifted the dose-response curves to the right, suggesting a primary involvement of A1 receptors. 8-cyclopentyltheophylline did not affect the depressant effects of APEC at the ED50, consistent with the A2-selectivity of APEC. The locomotor effects of APEC and CHA were completely reversed by theophylline, but not by the peripherally active 8-p-sulfophenyltheophylline, indicating central action of the adenosine agonists. The depressant effects of APEC, but not of NECA or CHA, were reversed significantly by an A2-selective adenosine receptor antagonist, 4-amino-8-chloro-1-phenyl-[1,2,4]triazol[4,3-a]quinoxaline. Low or subthreshold doses of CHA potentiated the depressant effects of APEC. A subthreshold dose of CHA did not alter the depressant effect of NECA, whereas a subthreshold dose of APEC increased the depressant effects of low doses of NECA. Thus, it appears that A1- and A2-selective adenosine agonists have separate central depressant effects, which can be potentiative.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Injections, Intraperitoneal; Locomotion; Male; Mice; Phenethylamines; Receptors, Purinergic; Theophylline; Vasodilator Agents

(-)-N6-(R-phenylisopropyl)-adenosine (R-PIA) depressed tritium overflow and vasoconstriction evoked by electrical stimulation to a similar extent in isolated tail arteries of Wistar rats (WR) preincubated with [3H]noradrenaline. The inhibitory effects of adenosine, 5'-N-ethylcarboxamidoadenosine (NECA) and R-PIA were determined on the constrictor responses of tail arteries obtained from WR, as well as spontaneously hypertensive (SHR) and Wistar Kyoto rats (WKY). In WR and WKY, the rank order of agonist potency (R-PIA greater than NECA greater than adenosine) was compatible with the presence of adenosine A1-receptors. Whereas adenosine, NECA and R-PIA were equiactive in WR and WKY, they produced no or only slight changes in SHR. The left renal arteries of some WR were partially occluded to induce hypertension. R-PIA had the same effect in the tail arteries of these animals as in preparations obtained from sham-operated WR. The above results suggest that the subsensitivity of presynaptic A1-receptors in the blood vessels of SHR is genetically determined. This could contribute in vivo to enhanced transmitter release from terminals of perivascular nerves and subsequent increases in vascular resistance.

Topics: Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Blood Pressure; Electric Stimulation; Hypertension; In Vitro Techniques; Muscle, Smooth, Vascular; Norepinephrine; Phenylisopropyladenosine; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Rats, Inbred WKY; Receptors, Purinergic; Species Specificity; Theophylline; Vasoconstriction