adenosine-5--(n-ethylcarboxamide) and 4-nitrobenzylthioinosine

While a great deal is known about stimuli that can induce the release of adenosine from brain tissue, relatively little is known about the regulation of the basal extracellular concentration of adenosine that is present in the absence of stimulation. Under normal conditions, enough adenosine is present to tonically activate a significant portion of the high-affinity adenosine A1 receptors. The present experiments demonstrated that the estimated basal concentration of extracellular adenosine in rat hippocampal slices maintained at 21 degrees C (430 nM) is approximately twice that at 32 degrees C (220 nM). The sensitivity of presynaptic modulatory adenosine A1 receptors was not significantly different at 21 degrees C or at 32 degrees C. Slices maintained at 21 degrees C also showed a reduced ability to inactivate extracellular adenosine, which reflects a reduction in adenosine transport across cell membranes. This effect appears to be primarily due to a reduction in the function of the equilibrative, dipyridamole-sensitive (ei) adenosine transporter; the nitrobenzylthioinosine-sensitive equilibrative transporter (es transporter) appears to be relatively less affected by temperature than is the ei transporter. These experiments demonstrate that extracellular concentrations of adenosine in the brain are sensitive to temperature, and suggest that some of the neurological effects of hypothermia might be mediated via increased concentrations of adenosine in the extracellular space.

Topics: Adenosine; Adenosine Deaminase; Adenosine-5'-(N-ethylcarboxamide); Animals; Carrier Proteins; Dipyridamole; Equilibrative Nucleoside Transporter 1; Equilibrative-Nucleoside Transporter 2; Excitatory Postsynaptic Potentials; Extracellular Space; Hippocampus; In Vitro Techniques; Male; Membrane Proteins; Rats; Rats, Sprague-Dawley; Temperature; Thioinosine

Previous work from our laboratory has demonstrated the presence of high-affinity binding sites for [3H]nitrobenzylthioinosine ([3H]NBTI), a marker of adenosine uptake systems, in the mitochondrial fraction of rat testis. Here, we characterize this system functionally through [3H]adenosine uptake assays. This system (K(m)=2+/-1.3 microM; V(max)=86.2+/-15.5 pmol/mg protein/min) was found to be saturable, non sodium-dependent and sensitive to temperature, pH and osmolarity. [3H]Adenosine incorporation was potently inhibited by hydroxynitrobenzylthioguanosine (HNBTG, IC(50)=3 nM) although NBTI inhibited this uptake weakly (IC(50)=72. 7+/-37.1 microM). Dilazep>dipyridamole>/=hexobendine inhibited [3H]adenosine incorporation at low micromolar concentrations. The nucleosides inosine and uridine were weak inhibitors of this system. The adenosine receptor ligands N(6)-phenylisopropyladenosine (PIA) and 2-chloroadenosine inhibited the uptake only at micromolar concentrations. Neither 5'-(N-ethylcarboxamido)-adenosine (NECA) nor theophylline inhibited adenosine uptake by more than 60% but the mitochodrial benzodiazepine receptor ligands 4'-chloro-diazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl) isoquinoline carboxamide (PK 11195) were able to inhibit it. The lack of inhibition by the blockers of the mitochondrial adenine-nucleotide carrier, atractyloside and alpha, beta-methylene-ATP, indicates that [3H]adenosine uptake occurs via a transporter other than this carrier. All these results support the existence of an equilibrative adenosine transport system, which might mediate the passage of adenosine formed in the mitochondria to the cytoplasm.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Benzodiazepinones; Biological Transport; Dilazep; Dipyridamole; Dose-Response Relationship, Drug; Guanosine; Hexobendine; Hydrogen-Ion Concentration; Inosine; Isoquinolines; Kinetics; Male; Mitochondria; Osmolar Concentration; Phenylisopropyladenosine; Rats; Rats, Sprague-Dawley; Sodium; Subcellular Fractions; Temperature; Testis; Thioinosine; Thionucleosides; Time Factors; Tritium; Uridine

Adenosine release from mucosal sources during inflammation and ischemia activates intestinal epithelial Cl- secretion. Previous data suggest that A2b receptor-mediated Cl- secretory responses may be dampened by epithelial cell nucleoside scavenging. The present study utilizes isotopic flux analysis and nucleoside analog binding assays to directly characterize the nucleoside transport system of cultured T84 human intestinal epithelial cells and to explore whether adenosine transport is regulated by secretory agonists, metabolic inhibition, or phorbol ester. Uptake of adenosine across the apical membrane displayed characteristics of simple diffusion. Kinetic analysis of basolateral uptake revealed a Na(+)-independent, nitrobenzylthioinosine (NBTI)-sensitive facilitated-diffusion system with low affinity but high capacity for adenosine. NBTI binding studies indicated a single population of high-affinity binding sites basolaterally. Neither forskolin, 5'-(N-ethylcarboxamido)-adenosine, nor metabolic inhibition significantly altered adenosine transport. However, phorbol 12-myristate 13-acetate significantly reduced both adenosine transport and the number of specific NBTI binding sites, suggesting that transporter number may be decreased through activation of protein kinase C. This basolateral facilitated adenosine transporter may serve a conventional function in nucleoside salvage and a novel function as a regulator of adenosine-dependent Cl- secretory responses and hence diarrheal disorders.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Affinity Labels; Biological Transport; Carrier Proteins; Cells, Cultured; Colforsin; Down-Regulation; Humans; Intestinal Mucosa; Kinetics; Membrane Proteins; Nucleoside Transport Proteins; Sodium; Tetradecanoylphorbol Acetate; Thioinosine; Vasodilator Agents

Dipyridamole (37.5 mg/kg, s.c., b.i.d.), a potent inhibitor of nucleoside transport, was administered to guinea pigs for 14 days in order to investigate the effects of: 1) chronic dipyridamole treatment on [3H]nitrobenzylthioinosine ([3H]NBMPR) binding: 2) chronically released endogenous adenosine on adenosine A1 and A2 receptors. Comparisons of the binding capacities (Bmax) and equilibrium dissociation constants (Kd) in vehicle-treated (VTA) and dipyridamole-treated animals (DTA), revealed a 100 percent increase in Kd of [3H]NBMPR binding in the kidney of DTA but not in heart or brain. There were no changes in adenosine A1 or A2 receptor activities in kidney and brain as measured by [3H]R-phenylisopropyladenosine and [3H]5'-N-ethyl-carboxamidoadenosine binding, respectively. The data suggest that cardiac and central nucleoside transporters may be either less susceptible to chronic dipyridamole administration or have a different adaptive mechanism. Also, endogenous adenosine, which may be chronically released upon dipyridamole treatment, has no effect on adenosine receptors.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Biological Transport; Brain; Cardiovascular System; Cell Membrane; Dipyridamole; Guinea Pigs; Kidney; Male; Myocardium; Nucleosides; Rats; Receptors, Purinergic P1; Thioinosine

1. Adenosine-5'-triphosphate (ATP) is known to exert a variety of biological effects via the activation of either ionotropic P2x- or G-protein coupled P2Y-purinoceptor subtypes. In this study the effects induced by ATP and ATP analogues on rat bladder strips were characterized at resting tone and in carbachol-prestimulated tissues. 2. ATP exerted a clear concentration-dependent biphasic response, which was maximal at 1 mM concentration and was characterized by an immediate and transient contraction, followed by a slower sustained relaxation. The receptor mediating contraction was susceptible to desensitization by ATP and by the ATP analogue, alpha,beta-methyleneATP (alpha,beta-meATP) showing the typical features of the P2x-purinoceptor; conversely, ATP-evoked relaxation did not undergo tachyphylaxis following either ATP or alpha,beta-meATP. 3. The slower and sustained relaxant phase seemed to be due to activation of P2Y-purinoceptors, based on responses obtained with the P2Y agonist, 2-methyl-thioATP (2-meSATP) and, more importantly, based on the clear involvement of the G-proteins. In fact, the G-protein activator, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) significantly potentiated and the G-protein blocking agent, guanosine 5'-O-(2-thio-diphosphate) (GDP beta S) completely abolished the ATP-induced relaxation. No effects were exerted by these two G-protein modulators on the ATP-induced contraction. 4. The relaxant component of the ATP response of bladder tissue was not significantly influenced by nitro-benzyl-thioinosine (NBTI) or by 8-phenyltheophylline (8-PT), suggesting that the contribution of the ATP metabolite adenosine to this response was negligible. Moreover, relaxation evoked by ATP and by the adenosine analogue, 5'-N-ethylcarboxamidoadenosine (NECA) was additive.5. Suramin was unable to modify either the relaxant or the contractile responses of bladder strips to ATP. However, when tested on the concentration-response curve to the slowly hydrolysable P2x-agonist alpha,beta-meATP, a rightward shift was detected, suggesting that ATP contractile responses are mediated by suramine-sensitive P2x-purinoceptors.6. Uridine-5'-triphosphate (UTP) only induced a rapid and concentration-dependent contraction of the rat bladder preparation, which was not desensitized by pre-exposure to alpha,beta-meATP, suggesting that UTP responses were not mediated by the 'classical' P2X-purinoceptor.7. It is therefore concluded that both P2x- and P2y-purinocepto

Topics: Adenosine; Adenosine Triphosphate; Adenosine-5'-(N-ethylcarboxamide); Animals; Dose-Response Relationship, Drug; Guanosine Triphosphate; Male; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Thioinosine; Uridine Triphosphate; Urinary Bladder; Vasodilator Agents

In the present work we studied the effect of adenosine and various adenosine analogues on cAMP level in guinea pig coronary endothelial cells of microvascular origin. The tested adenosine agonist mediate a concentration-dependent increase in cAMP level. The rank order of potency was 5'-N-ethylcarboxamidoadenosine (NECA) > CGS 21680 > N6-phenylisopropyladenosine (R-PIA) > 2-chloro-N6-cyclopentyladenosine (CCPA) which is typical for an adenosine A2 receptor. Their respective concentrations for half maximal stimulation of cAMP formation were 0.36 microM, 0.82 microM, 4.7 microM and 9.8 microM. The tested agonists showed differences in efficacy, NECA being the most efficacious. R-PIA, CCPA and adenosine were less efficacious, suggesting partial agonism. The efficacy of adenosine was unchanged by the addition of the nucleoside transport inhibitor S(4-nitrobenzyl)-6-thioinosine (NBTI, 10 microM) suggesting that inhibition of adenylyl cyclase through P-site activation is not responsible for the observed low efficacy of adenosine. We could demonstrate CGS 21680 activation of adenylyl cyclase in a peripheral receptor. We therefore suggest that the endothelial adenosine receptor resembles the striatal adenosine A2a receptor.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Animals; Antihypertensive Agents; Cells, Cultured; Colforsin; Coronary Vessels; Cyclic AMP; Dose-Response Relationship, Drug; Endothelium, Vascular; Guinea Pigs; Isoproterenol; Myocardium; Phenethylamines; Phenylisopropyladenosine; Receptors, Purinergic P1; Thioinosine; Vasodilator Agents

The actions of adenosine on histamine release of human lung fragments were investigated. Histamine release was stimulated either with the calcium ionophore A23187 or with concanavalin A. Adenosine and its analogue 5'-N-ethylcarboxamidoadenosine alone had no significant effect on basal release or on the release elicited by A 23187 or concanavalin A. However, in the presence of the adenosine receptor antagonist 8-[4-[[[[(2-aminoethyl)amino]-carbonyl]methyloxy]-phenyl]-1, 3-dipropylxanthine (XAC), which itself did not affect the release, adenosine increased the stimulated histamine release. On the other hand, in the presence of the nucleoside transport inhibitor S-(p-nitrobenzyl)-6-thioinosine (NBTI), adenosine caused a reduction in stimulated histamine release. NBTI itself caused a stimulation of release. Thus, a stimulatory effect of adenosine was seen in the presence of XAC, whereas an inhibitory effect was unmasked by NBTI. From these data it is concluded that adenosine exerts two opposing effects on histamine release in the human lung which neutralize each other: it inhibits release via a site antagonized by XAC, which presumably represents an A2 adenosine receptor, and it stimulates release via a mechanism that is blocked by NBTI, suggesting that adenosine needs to reach the interior of cells to exert this effect. The slight stimulatory effect of NBTI alone demonstrates that trapping intracellularly formed adenosine inside mast cells leads to sufficient concentrations of adenosine to stimulate histamine release. These findings suggest an important bimodal role of adenosine in regulating histamine release in the human lung.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Calcimycin; Carrier Proteins; Concanavalin A; Histamine Release; Humans; In Vitro Techniques; Lung; Mast Cells; Membrane Proteins; Nucleoside Transport Proteins; Receptors, Purinergic; Thioinosine

The present study compares the characteristics of radioligand binding to adenosine receptors and adenosine uptake sites in 100- and 50-day-old normal and narcoleptic dogs. Binding to A1 receptors was quantified using a selective A1 agonist ([3H]N6-[(R)-1-methyl-2-phenylethyl] adenosine, [3H]R-PIA) and an antagonist ([3H]dipropyl-8-cyclopentyl-xanthine, [3H]CPX). Differences in the binding of [3H]R-PIA and that of [3H]5'-ethylcarboxamide adenosine ([3H]NECA), which binds to both A1 and A2 receptors with similar affinities, were used to quantify A2 receptors. Nucleoside transport sites were labeled with [3H]nitrobenzylthioinosine ([3H]NBTI), a potent inhibitor of nucleoside transport systems. The present study offered no evidence that either adenosine A1 receptors and adenosine uptake sites in the frontal cortex or adenosine A2 receptors in the putamen were altered in narcoleptic dogs. However, we found that adenosine A1 receptors in the dog exist in different affinity states and that the affinity state in which the receptor is found depends on the brain region examined. A characterization of these low- and high-affinity sites was performed and results indicated that these sites cannot be explained by a single interaction of the A1 receptor with a single G-protein population.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Aging; Animals; Brain; Cataplexy; Dogs; GTP-Binding Proteins; Guanylyl Imidodiphosphate; Kinetics; Membranes; Narcolepsy; Phenylisopropyladenosine; Radioligand Assay; Receptors, Purinergic; Thioinosine; Xanthines

We have examined the effects of some analogues of adenosine upon the circadian rhythm in rate of thymidine incorporation by cultured chick pineal glands. Incorporation in the early period of the photoperiod on day 2 of culture was slightly inhibited by the adenosine analogue N-ethylcarboxamido-adenosine, but this effect was not countered by the antagonist 8-phenyl-theophylline. Thymidine incorporation was inhibited when glands were continuously exposed to the adenosine transport inhibitor nitrobenzyl-thioinosine, but ongoing incorporation was not inhibited by addition of this agent. Removal of adenosine and deoxyadenosine supplements from the medium, with or without further addition of adenosine deaminase, had no appreciable effects upon thymidine incorporation. We conclude that adenosine and analogues probably play no role in regulation of the rhythm in rate of thymidine incorporation.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Affinity Labels; Animals; Cells, Cultured; Chickens; Circadian Rhythm; Pineal Gland; Receptors, Purinergic; Theophylline; Thioinosine; Thymidine; Vasodilator Agents

Extracellular adenosine is transported into chromaffin cells by a high-affinity transport system. The action of adenosine receptor ligands was studied in this cellular model. 5'-(N-Ethylcarboxamido)adenosine (NECA), an agonist of A2 receptors, activated adenosine transport. Km values for adenosine were 4.6 +/- 1.0 (n = 5) and 10.2 +/- 3.0 microM (n = 5) for controls and 100 nM NECA, respectively. The Vmax values were 66.7 +/- 23.5 and 170.2 +/- 30 pmol/10(6) cells/min for controls and 100 nM NECA, respectively. The A1 agonist N6-cyclohexyladenosine, the A1 antagonist 8-cyclopentyl-1, 3-dipropylxanthine, and the A1-A2 antagonist 1,3-dipropyl-8-(4-[(2-aminoethyl)amino]-carbonylmethyloxyphenyl)- xanthine did not significantly modify the adenosine transport in this system. Binding studies done with [3H]dipyridamole, a nucleoside transporter ligand, did not show changes in either the number or affinity of transporter sites after NECA treatment. This ligand can enter cells and quantifies the total number of transporters. The binding studies with [3H]-nitrobenzylthioinosine, which quantifies the plasma membrane transporters, showed a Bmax of 19,200 +/- 800 and 23,200 +/- 700 transporters/cell for controls and 100 nM NECA, respectively. No changes in the KD were obtained. The effects of NECA were not mediated through adenylate cyclase activation, because its action was not imitated by forskolin.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Affinity Labels; Animals; Biological Transport; Cells, Cultured; Chromaffin System; Colforsin; Dipyridamole; Kinetics; Nucleosides; Receptors, Purinergic; Thioinosine

1. Denbufylline has been examined for its ability to inhibit cyclic nucleotide phosphodiesterase isoenzymes from rat cardiac ventricle and cerebrum, as well as for its affinity for adenosine A1 and A2 receptors and the re-uptake site. For comparison, SK&F 94120, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) were examined as phosphodiesterase inhibitors whilst N6-cyclohexyladenosine, R(-)-N6-(2-phenylisopropyl)-adenosine, 5'-N-ethylcarboxamido-adenosine, 2-nitrobenzylthioinosine, theophylline and IBMX were examined for their affinity for adenosine binding sites. 2. This investigation confirmed the presence of four phosphodiesterase activities in rat cardiac ventricle; in rat cerebrum only three were present. 3. Denbufylline selective inhibited one form of Ca2+-independent, low Km cyclic AMP phosphodiesterase. The form inhibited was one of two present in cardiac ventricle and the sole one in cerebrum. This form was not inhibited by cyclic GMP. The inotropic agent SK&F 94120 selectively inhibited the form of cyclic AMP phosphodiesterase which was inhibited by cyclic GMP present in cardiac ventricle. Theophylline and IBMX were relatively non-selective phosphodiesterase inhibitors. 4. Denbufylline was a less potent inhibitor of ligand binding to adenosine receptors than of cyclic AMP phosphodiesterase. This contrasted with theophylline, which had a higher affinity for adenosine receptors, and IBMX which showed no marked selectivity. Denbufylline, theophylline and IBMX all had a low affinity for the adenosine re-uptake site. 5. Denbufylline is being developed as an agent for the therapy of multi-infarct dementia. The selective inhibition of a particular low Km cyclic AMP phosphodiesterase may account for the activity of this compound.

Topics: 1-Methyl-3-isobutylxanthine; 2',3'-Cyclic-Nucleotide Phosphodiesterases; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Cerebral Cortex; In Vitro Techniques; Kinetics; Male; Myocardium; Phenylisopropyladenosine; Rats; Rats, Inbred Strains; Receptors, Purinergic; Theophylline; Thioinosine; Xanthines

1. Adenosine and its metabolically stable analogue N-ethyl-carboxamidoadenosine (NECA) enhance histamine release from rat peritoneal mast cells when these are stimulated by calcium-mobilizing agents. NECA and adenosine shift the concentration-response curve of the calcium ionophore A23187 to lower concentrations. 2. The potencies of NECA or adenosine in enhancing A23187-induced histamine release are dependent on the level of stimulated release in the absence of adenosine analogues. At high levels of release their potencies are up to 20 times higher than at low levels. Consequently, averaged concentration-response curves of adenosine and NECA for enhancing histamine release are shallow. 3. The adenosine transport blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) has no effect by itself at low levels of stimulated histamine release, but abolishes the enhancing effect of adenosine. At high levels of release, however, NBTI alone enhances the release of histamine. 4. It is concluded that adenosine and calcium reciprocally enhance the sensitivity of the secretory processes to the effects of the other agent. The levels of intracellular adenosine obtained by trapping adenosine inside stimulated mast cells are sufficient to enhance histamine release substantially, suggesting that this effect may play a physiological and pathophysiological role.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Calcimycin; Calcium Channel Agonists; Histamine Release; In Vitro Techniques; Male; Mast Cells; Peritoneal Cavity; Rats; Thioinosine

The effect of a single electroconvulsive shock (ECS) (30 min and 24 h after treatment) and repeated ECS (10 once-daily) on the adenosine neuromodulatory system was investigated in rat cerebral cortex, cerebellum, hippocampus, and striatum. The present study examined the adenosine A1 receptor using N6-[3H]cyclohexyladenosine ([3H]CHA), the A2 receptor using 5'-N-[3H]ethylcarboxyamidoadenosine ([ 3H]NECA), adenylate cyclase using [3H]forskolin, and the adenosine uptake site using [3H]nitrobenzylthioinosine ([3H]NBI). At 30 min after a single ECS, the Bmax of the [3H]NBI binding in striatum was increased by 20%, which is in good agreement with the well-known postictal adenosine release. The Bmax of [3H]forskolin binding in striatum and cerebellum was increased by 60 and 20%, respectively. In contrast to earlier reported changes following chemically induced seizures, [3H]CHA binding was not altered postictally. At 24 h after a single ECS, there were no changes for any ligand in any brain region. Following repeated ECS, there was a 20% increase of [3H]CHA binding sites in cerebral cortex, which lasted for at least 14 days after the last ECS. [3H]Forskolin binding in hippocampus and striatum was 20% lowered 24 h after 10 once-daily ECS but had already returned to control levels 48 h after the last treatment. Evidence is provided that the upregulated adenosine A1 receptors are coupled to guanine nucleotide binding proteins and, furthermore, that this upregulation is not paralleled by an increase in adenylate cyclase activity as labeled by [3H]forskolin.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Animals; Brain; Cerebellum; Cerebral Cortex; Colforsin; Corpus Striatum; Electroshock; Hippocampus; Kinetics; Male; Rats; Rats, Inbred Strains; Receptors, Purinergic; Thioinosine

Evidence suggests that adenosine modulates neuronal and cerebral vascular functions by interacting with specific receptors on brain cells and blood vessels. Adenosine and other nucleosides are also transported across the blood-brain barrier via a saturable, carrier-mediated mechanism. Using direct ligand binding methods, we studied the two adenosine receptor subtypes, A1 and A2 and the nucleoside transporter moiety in human brain microvessels, pial vessels, choroid plexus, and cerebral cortex membranes. The following specific tritiated ligands were used: cyclohexyladenosine (CHA) for A1 receptors; 5'-N-ethylcarboxamide adenosine (NECA) for A2 receptors; nitrobenzylthioinosine (NBMPR) and dipyridamole (DPY) for nucleoside transporters. We find that cerebral microvessels, pial vessels, and choroid plexus have few, if any, A1 receptors, in contradistinction to cerebral membranes, which have a 10-20-fold higher density of A1 receptor sites. Specific high-affinity NECA binding to A2 receptors in cerebral microvessels, pial vessels, and choroid plexus was saturable and was equivalent to that of cerebral cortical membranes. The Bmax and Kd of the high-affinity NECA binding to vessel preparations were approximately 1.3 pmol/mg protein and approximately 250 nM, respectively, which is similar to our previous findings in the rat and pig. NBMPR and DPY binding were also saturable and were consistent with a single class of high-affinity binding sites. The density of nucleoside transporters was approximately four-fold higher in cerebral microvessels than in cerebral cortex, pial vessels, and choroid plexus. These results suggest that human cerebral microvessels have A2, but not A1, receptors and are particularly enriched with the adenosine transporter moiety.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adult; Blood Proteins; Brain; Carrier Proteins; Cerebral Cortex; Choroid Plexus; Dipyridamole; Female; Humans; Membrane Proteins; Microcirculation; Middle Aged; Nucleoside Transport Proteins; Pia Mater; Receptors, Purinergic; Thioinosine

We used video-microscopic techniques to study responses of rat cremaster muscle arterioles to adenosine (ADO) placed in a bathing solution in an effort to determine 1) the sensitivity of these vessels to local interstitial ADO concentration and 2) the parameters of interstitial adenosine handling. Two vessels, located at different depths (approximately 40 and 115 microns) below the surface of the tissue, were studied simultaneously. Invariably, a higher bath ADO concentration was required to induce vasodilation in the deeper vessel; the concentration required for 50% dilation response (EC50) for ADO dilation increased at an average of 1.8 +/- 0.2 log10 U/100 microns of depth into the tissue. This result was shown to be due to a standing gradient in interstitial ADO concentration. By extrapolating results to the tissue surface, we estimate that the EC50 for arteriolar dilation to local interstitial ADO is approximately 0.1 microM. The steepness of the tissue ADO gradient indicates that the rate constant for interstitial ADO loss is near 0.24/s. The gradients for nonmetabolizable adenosine analogues were less than 1/10th as steep as that for ADO itself. Qualitatively similar results were obtained from experiments on hamster cremaster muscle preparations.

Topics: 2-Chloroadenosine; Adenine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Arterioles; Biological Assay; Cricetinae; Diffusion; Dipyridamole; Extracellular Space; Kinetics; Methods; Muscles; Phenylisopropyladenosine; Rats; Rats, Inbred Strains; Thioinosine; Vasodilation

The activities of an endogenous nucleoside, 5'-deoxy-5'-methylthioadenosine (MTA), on adenosine sensitive sites such as adenosine A1 and A2 receptors and the P-site, as well as on purine nucleoside transport, have been studied. This nucleoside competitively antagonized the A2 receptor-mediated stimulation of neuroblastoma adenylate cyclase, produced a GTP-dependent and 8-p-sulfophenyltheophylline-sensitive inhibition of adenylate cyclase activity in rat cerebellar membranes, and decreased the spontaneous contractile activity of isolated segments of rabbit jejunum. MTA was neither active at the P-site nor did it diminish the binding of [3H]nitrobenzylthioinosine, a nucleoside transport inhibitor. We conclude that (a) MTA is an agonist at the adenosine A1 receptor but an antagonist at the A2 receptor, and (b) the adenosine receptor which causes relaxation of rabbit jejunum is not a neuroblastoma-type A2 receptor which activates adenylate cyclase.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclase Inhibitors; Animals; Cerebellum; Deoxyadenosines; In Vitro Techniques; Jejunum; Mice; Muscle Contraction; Neuroblastoma; Rabbits; Rats; Receptors, Purinergic; Thioinosine; Thionucleosides

5'-N-ethylcarboxamideadenosine (NECA) greater than 2-chloroadenosine greater than adenosine greater than (-)-N6-(R-phenyl-isopropyl)-adenosine greater than (+)-N6-(S-phenylisopropyl)-adenosine, in that order of potency, inhibited in vitro antigen-induced histamine release from human basophils in a dose-dependent fashion. Inhibition occurred only during the first stage of antigen-induced histamine release and the nucleosides failed to inhibit the release caused by the Ca2+ ionophore, A23187. 6-nitrobenzylthioinosine and dipyridamole, which inhibit adenosine uptake, and erythro-9-(2-hydroxy-3-nonyl)adenine, which blocks adenosine metabolism, did not impair the inhibition caused by NECA and adenosine. 8-phenyltheophylline and theophylline, two competitive antagonists of adenosine receptors, blocked the inhibition caused by NECA and adenosine. These data suggest that NECA and other adenosine analogs activate a specific cell surface adenosine receptor which possesses properties similar to those of an adenosine A2/Ra receptor.

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Adenylyl Cyclases; Basophils; Biological Transport; Calcimycin; Histamine Release; Humans; Receptors, Cell Surface; Receptors, Purinergic; Structure-Activity Relationship; Theophylline; Thioinosine