Receptors for adenosine and adenine nucleotides

1. A summary is provided of current classifications of receptors for the adenine nucleosides and nucleotides. 2. The origin of the division of nucleoside (P1) and nucleotide (P2) receptors is discussed as well as the subdivisions of these into A1 and A2, determined by structure activity considerations, and P2x and P2y. 3. Further suggestions have been made recently for identifying A1a and A1b, A2a and A2b subtypes of the P1 receptor family, and for proposing distinctive nucleotide receptors on platelets (P2t) and mast cells (P2z). 4. In addition, an A3 site may exist with properties intermediate between A1 and A2, and a novel P2s receptor on some smooth muscle systems and a P3 site.     

Effects of adenosine analogs and adenine nucleotides on adenosine 5'-diphosphate-induced rat platelet aggregation

Various adenosine analogs and adenine nucleotides have been tested as inhibitors of ADP-induced aggregation of rat platelets. The potent inhibitors of human platelet aggregation, adenosine, 2-fluoroadenosine, 2-chloroadenosine, carbocyclic adenosine and N⁶-phenyl adenosine, had little effect on rat platelet aggregation (0–30 per cent inhibition). The effects of adenosine or its analogs on ADP-induced aggregation of cross-species platelet-rich plasmas (PRPs) (human platelets suspended in rat plasma or rat platelets in human plasma) were similar to those with the native PRPs, indicating that these species differences were due to intrinsic factors in the platelets and not in the plasma. When these analogs were tested in the presence of the cyclic AMP phosphodiesterase inhibitor papaverine, strong inhibiton of rat platelet ADP-induced aggregation was seen. 2′-Deoxyadenosine and 3′-deoxyadenosine were not inhibitory to ADP-induced aggregation of rat PRP even in the presence of papaverine. Adenosine 5′-tetraphosphate strongly inhibited both human and rat platelet aggregation. AMP, like adenosine, did not inhibit rat platelet aggregation but became strongly inhibitory in the presence of papaverine. This inhibitory effect was abolished by preincubating rat PRP with an adenylate cyclase inhibitor, 2′, 5′-dideoxyadenosine or adenosine deaminase. In the later case, however, if the adenosine deaminase inhibitor 2′-deoxycoformycin was included in the incubation mixture, the inhibition by AMP plus papaverine was similar to adenosine plus papaverine. About 50 per cent of [¹⁴C]AMP was converted to [¹⁴ C]adenosine in rat platelet-free plasma or PRP after a 10-min incubation. α,β-Methylene-ADP and β,γ-methylene-ATP (200 μM) inhibited rat platelet aggregation by 50 and 64 per cent, respectively. Cyclic AMP phosphodiesterase of rat and human platelets gave comparable K_m, and V_max values (K_m 0.53 and 0.21μM and V_max 6.0 and 6.7 pmoles/min/10⁷ platelets, respectively).     

In vitro studies of release of adenine nucleotides and adenosine from rat vascular endothelium in response to alpha 1-adrenoceptor stimulation.

1. Noradrenaline-induced release of endogenous adenine nucleotides (ATP, ADP, AMP) and adenosine from both rat caudal artery and thoracic aorta was characterized, using high-performance liquid chromatography with fluorescence detection. 2. Noradrenaline, in a concentration-dependent manner, increased the overflow of ATP and its metabolites from the caudal artery. The noradrenaline-induced release of adenine nucleotides and adenosine from the caudal artery was abolished by bunazosin, an alpha 1-adrenoceptor antagonist, but not by idazoxan, an alpha 2-adrenoceptor antagonist. Clonidine, an alpha 2-adrenoceptor agonist, contracted caudal artery smooth muscle but did not induce release of adenine nucleotides or adenosine. 3. Noradrenaline also significantly increased the overflow of ATP and its metabolites from the thoracic aorta in the rat; however, the amount of adenine nucleotides and adenosine released from the aorta was considerably less than that released from the caudal artery. 4. Noradrenaline significantly increased the overflow of ATP and its metabolites from cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the thoracic aorta and caudal artery. The amount released from the cultured endothelial cells from the aorta was also much less than that from cultured endothelial cells from the caudal artery. In cultured smooth muscle cells from the caudal artery, a significant release of ATP or its metabolites was not observed. 5. These results suggest that there are vascular endothelial cells that are able to release ATP by an alpha 1-adrenoceptor-mediated mechanism, but that these cells are not homogeneously distributed in the vasculature.     

Responses of the longitudinal muscle and the muscularis mucosae of the rat duodenum to adenine and uracil nucleotides.

1. Previous studies have shown that the rat duodenum contains P1 and P2Y purinoceptors via which it relaxes to adenosine and adenosine 5'-triphosphate (ATP) respectively. It has also been shown to contract to uridine 5'-triphosphate (UTP) and adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), and based on their differential inhibition by the P2 antagonist suramin it has been suggested that they act via two separate receptors. In addition, the rat duodenum has been shown to dephosphorylate ATP rapidly via ectonucleotidases and adenosine deaminase. In this study the responses of two preparations from the rat duodenum, the longitudinal muscle and the muscularis mucosae, were investigated using a series of nucleotides and suramin. 2. 2-Methylthioadenosine 5'-triphosphate (2-MeSATP), ATP, ATP-gamma-S and adenosine 5'-alpha,beta-methylene-triphosphonate (AMPCPP) each relaxed the longitudinal muscle, with an agonist potency order of 2-MeSATP > ATP = ATP-gamma-S > AMPCPP, while UTP and uridine 5'-diphosphate (UDP) were not observed to elicit relaxation. This indicates the presence of a relaxant P2Y-purinoceptor on the longitudinal muscle. The longitudinal muscle did not contract to any of the agonists at concentrations of 300 microM, apart from ATP-gamma-S which caused very weak contractions. 3. ATP-gamma-S, adenosine 5'-methylenediphosphonate (AMPCP), AMPCPP, ATP, UTP, adenosine 5'-diphosphate (ADP), UDP and 2-MeSATP each contracted the muscularis mucosae with an agonist potency order of ATP-gamma-S > or = AMPCP > or = AMPCPP = ATP = UTP = ADP = UDP >> 2-MeSATP, although maximal responses were not obtained at concentrations of 300 microM. The muscularis mucosae did not relax to any of the agonists at concentrations of 300 microM. 4. Suramin (1 mM) inhibited relaxations induced by ATP on the longitudinal muscle, shifting the relaxation concentration-response curve to the right. This further supports the presence of a P2Y-purinoceptor on this muscle layer. Suramin (1 mM) inhibited contractions induced by AMPCPP, but not those induced by ATP, UTP or ATP-gamma-S, in the muscularis mucosae. Desensitization of the muscularis mucosae was seen with AMPCPP, but not with UTP or ATP-gamma-S, and no cross-desensitization between AMPCPP and UTP or ATP-gamma-S was observed. This suggests there are two receptors which mediate contraction on the rat duodenum muscularis mucosae, one suramin-sensitive and the other suramin-insensitive. 5. ATP was rapidly degraded by the muscularis mucosae to ADP, adenosine 5'-monophosphate (AMP) and inosine, with no adenosine being detected. A similar rate of degradation was seen for UTP with UDP, uridine 5'-monophosphate (UMP) and uridine being formed and for 2-MeSATP with 2-methylthioadenosine 5'-diphosphate (2-MeSADP), 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) and 2-methylthioadenosine being formed. AMPCPP and ATP-gamma-S were both degraded more slowly, AMPCPP being degraded to AMPCP, and ATP-gamma-S to ADP, AMP and inosine. Suramin (1 mM), did not significantly affect the rate and pattern of degradation of these nucleotides, apart from AMPCPP which was degraded slightly more slowly in the presence of suramin. 6. These results show that there is a P2Y-purinoceptor which mediates relaxation in the rat duodenum longitudinal muscle. They also show that there is a contraction-mediating suramin-sensitive receptor on the rat duodenum muscularis mucosae which is desensitized by AMPCPP, and thus is probably of the P2X subtype. In addition, there is a contraction-mediating suramin-insensitive receptor on the rat duodenum muscularis mucosae which is not desensitized by UTP or ATP-gamma-S, and at which ATP and UTP show equal potency, and is thus probably of the P2U subtype. In addition, the rat duodenum muscularis mucosae contains ectonucleotidases and adenosine deaminase, which rapidly degrade nucleotides, although the inhibition by suramin of this deg     

Adenosine receptor activation by adenine nucleotides requires conversion of the nucleotides to adenosine

Summary Adenine nucleotides cause adenosine receptor-mediated increases in cyclic AMP in the VA13 human fibroblast line. Levels of adenosine accumulated in the medium are insufficient to account for the responses to adenine nucleotides. Since rapid conversion of the nucleotides to adenosine by 5-nucleotidase in the vicinity of the receptor might account for the responses, six experimental methods were developed to distinguish between local conversion and direct action of the nucleotides. Results of all six methods favored local conversion. (1) 5-Nucleotidase inhibitors blocked the accumulations of cyclic AMP elicited by AMP, ADP, and ATP, but did not affect the response to adenosine. The most potent inhibitor of both conversion of AMP and response to AMP was ,-methylene-ADP (APCP). (2) Adenosine deaminase blocked the responses to AMP, ADP, ATP, and adenosine-containing coenzymes. (3) Theophylline, a specific competitive adenosine antagonist, was an insurmountable inhibitor of the increases in cyclic AMP caused by AMP, ADP, and ATP. The insurmountability was presumably due to substrate sataration of the converting enzyme 5-nucleotidase. (4) Although ADP and ATP had partial agonist-like dose-response curves, they did not inhibit the response to adenosine. (5) Nine cell lines which responded to adenosine were tested for response to AMP. Cell lines with high levels of 5-nucleotidase had large responses to AMP, those with intermediate levels of 5-nucleotidase had large or intermediate responses to AMP, and those with low 5-nucleotidase levels did not respond to AMP. (6) Inhibition of the uptake of labelled adenosine was used as an indicator of unlabelled adenosine concentrations near the cell membrane. Unlabelled AMP inhibited uptake nearly as effectively as unlabelled adenosine. APCP reversed the inhibition by AMP but not the inhibition by adenosine.The adenosine receptor is concluded to be an enity distinct from adenine nucleotide receptors.Submitted in partial fulfillment of the requirements for the degree Doctor of Philosophy in Neurosciences, University of California, San Diego. Supported by NIMH DA-00265 and PHS RR 05665. The author has been a NSF Graduate Fellow. An abstract of this material has been published (Bruns 1977)     

Preferential activation of excitatory adenosine receptors at rat hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides.

1. In the present work, we investigated the action of adenosine originating from extracellular catabolism of adenine nucleotides, in two preparations where synaptic transmission is modulated by both inhibitory A1 and excitatory A(2a)-adenosine receptors, the rat hippocampal Schaffer fibres/CA1 pyramid synapses and the rat innervated hemidiaphragm. 2. Endogenous adenosine tonically inhibited synaptic transmission, since 0.5-2 u ml-1 of adenosine deaminase increased both the population spike amplitude (30 +/- 4%) and field excitatory post-synaptic potential (f.e.p.s.p.) slope (27 +/- 4%) recorded from hippocampal slices and the evoked [3H]-acetylcholine ([3H]-ACh) release from the motor nerve terminals (25 +/- 2%). 3. alpha, beta-Methylene adenosine diphosphate (AOPCP) in concentrations (100-200 microM) that almost completely inhibited the formation of adenosine from the extracellular catabolism of AMP, decreased population spike amplitude by 39 +/- 5% and f.e.p.s.p. slope by 32 +/- 3% in hippocampal slices and [3H]-ACh release from motor nerve terminals by 27 +/- 3%. 4. Addition of exogenous 5'-nucleotidase (5 u ml-1) prevented the inhibitory effect of AOPCP on population spike amplitude and f.e.p.s.p. slope by 43-57%, whereas the P2 antagonist, suramin (100 microM), did not modify the effect of AOPCP. 5. In both preparations, the effect of AOPCP resulted from prevention of adenosine formation since it was no longer evident when accumulation of extracellular adenosine was hindered by adenosine deaminase (0.5-2 u ml-1). The inhibitory effect of AOPCP was still evident when A1 receptors were blocked by 1,3-dipropyl-8-cyclopentylxanthine (2.5-5 nM), but was abolished by the A2 antagonist, 3,7-dimethyl-1-propargylxanthine (10 microM). 6. These results suggest that adenosine originating from catabolism of released adenine nucleotides preferentially activates excitatory A2 receptors in hippocampal CAI pyramid synapses and in phrenic motor nerve endings.     

Inhibitory action of PPADS on relaxant responses to adenine nucleotides or electrical field stimulation in guinea-pig taenia coli and rat duodenum.

1. The effect of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on the relaxant response to adenine nucleotides was examined in the carbachol-contracted guinea-pig taenia coli and rat duodenum, two tissues possessing P2y-purinoceptors. In addition, in the taenia coli PPADS was investigated for its effect on relaxations evoked by adenosine, noradrenaline and electrical field stimulation. In order to assess the selectivity of PPADS between P2-purinoceptor blockade and ectonucleotidase activity, its influence on ATP degradation was studied in guinea-pig taenia coli. 2. The resulting rank order of potency for the adenine nucleotides in guinea-pig taenia coli was: 2-methylthio ATP >> ATP > alpha,beta-methylene ATP with the respective pD2-values 7.96 +/- 0.08 (n = 23), 6.27 +/- 0.12 (n = 21) and 5.88 +/- 0.04 (n = 24). 3. In guinea-pig taenia coli, PPADS (10-100 microM) caused a consistent dextral shift of the concentration-response curve (CRC) of 2-methylthio ATP and ATP resulting in a biphasic Schild plot. A substantial shift was only observed at 100 microM PPADS, the respective pA2-values at this particular concentration were 5.26 +/- 0.16 (n = 5) and 5.15 +/- 0.13 (n = 6). Lower concentrations of PPADS (3-30 microM) antagonized the relaxant effects to alpha,beta-methylene ATP in a surmountable manner. An extensive shift of the CRC was produced only by 30 microM PPADS (pA2 = 5.97 +/- 0.08, n = 6), and the Schild plot was again biphasic.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extracellular metabolism of adenine nucleotides and adenosine in the innervated skeletal muscle of the frog.

The effects of coformycin, alpha,beta-methylene ADP, dipyridamole in the absence and presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), nitrobenzylthioinosine (NBTI), mioflazine and ouabain on the metabolic pathways of exogenously applied ATP and its metabolites in the frog innervated sartorius muscle were investigated. ATP catabolism yielded ADP, AMP, IMP, adenosine and inosine; the ecto-ATPase in situ was shown to be Ca(2+)- or Mg(2+)-activated with a Kmapp for ATP of 767 +/- 48 microM. AMP catabolism yielded IMP, adenosine and inosine; inosine was formed from either exogenous IMP or exogenous adenosine. Catabolism of AMP into IMP was blocked by coformycin, which enhanced adenosine and inosine formation from AMP. alpha,beta-Methylene ADP blocked adenosine formation from AMP and inosine formation from IMP; formation of IMP from AMP was enhanced by alpha,beta-methylene ADP. Complete blockade of AMP degradation was achieved with the simultaneous use of coformycin and alpha,beta-methylene ADP. Dipyridamole attenuated but did not completely block extracellular adenosine removal and inosine appearance in the bath. EHNA, applied in the presence of dipyridamole, did not cause any further attenuation of extracellular adenosine removal. Mioflazine, NBTI and ouabain did not affect adenosine disappearance from the bath. The results suggest that, in the frog innervated sartorius muscle, ATP can be sequentially catabolized into AMP which is then catabolized either into IMP or into adenosine. This extracellular degradation of AMP into IMP might then constitute a shunt-like mechanism to control the levels of adenosine formed from adenine nucleotides.     

Degradation of adenosine by extracellular adenosine deaminase in the rat duodenum

1. Extracellular degradation of adenosine by adenosine deaminase was studied in the rat duodenum using high performance liquid chromatography (HPLC) and pharmacological techniques. 2. Relaxant responses to adenosine (1-10 microM) were potentiated in a concentration-dependent manner by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and deoxycoformycin, both of which are inhibitors of adenosine deaminase. 3. Breakdown of adenosine, determined by HPLC, due to incubation with segments of rat duodenum was inhibited by both EHNA and deoxycoformycin. Cytosolic sources of adenosine deaminase were excluded. 4. Relaxant responses to adenosine were also potentiated by the adenosine transport inhibitor dilazep, which did not inhibit adenosine deaminase activity. 5. The extracellular adenosine deaminase activity (4 units/g tissue) was high compared with activity previously determined in other organs.     

Comparative effects of adenosine and adenine and guanine nucleotides on drug biotransformation in rats

Summary The effect of various nucleotides and adenosine on the hepatic biotransformation of hexobarbital sodium (HB) and p-chloro-N-methylaniline (PCMA) was determined in male rats. Intraperitoneal administration of dibutyryl cyclic adenosine 3,5-monophosphate (DBcAMP), alone and in combination with theophylline, and the cyclic adenosine 3,5-monophosphate (cAMP)-theophylline combination prolonged HB sleeping time by more than 70%. cAMP or dibutyryl cyclic guanosine 3,5-monophosphate (DBcGMP), alone or in combination with theophylline, failed to significantly alter the duration of HB-induced hypnosis. Plasma levels of HB upon awakening suggested that the increase in sleeping time was apparently not due to an altered sensitivity of the brain to the barbiturate. The effect appears to be related to an impairment of HB metabolism since only those compounds that inhibited HB oxidation when added to liver slices prolonged hypnosis. In addition, 5-AMP, adenosine, and cyclic guanosine 3,5-monophosphate (cGMP) failed to alter HB biotransformation by liver slices. A similar pattern of impairment of metabolism by liver slices was observed when PCMA was used as the substrate. The inhibitory effect of DBcAMP was not altered by concurrent addition of either cGMP or DBcGMP. No impairment in the rate of microsomal PCMA biotransformation resulted from addition of adenosine or any of the nucleotides used in this study.     

Comparison of the changes in adenine nucleotides of rat liver mitochondria induced by tamoxifen and 4-hydroxytamoxifen

The antiestrogen tamoxifen (TAM) inhibits the growth of different estrogen receptor (ER)-negative cells. Recently, multiple effects of TAM on mitochondrial bioenergetic functions have been pointed to explain its ER-independent cell death mechanisms. We have shown that TAM and its major active metabolite 4-hydroxytamoxifen (OHTAM) induce depolarization of the mitochondrial membrane potential (ΔΨ) and uncouple the mitochondrial respiration, depressing the oxidative phosphorylation efficiency. To clarify the biochemical mechanisms underlying the changes in the regulation of ATP synthesis and yield, in this work we evaluated the alterations of mitochondrial adenine nucleotides induced by both drugs and ascertained whether such changes could reflect a specific inhibition of either the adenine nucleotide translocase (ANT) or the phosphate carrier, as well as the activation of ATP hydrolysis due to ΔΨ depolarization. We found that both antiestrogens caused a concentration-dependent decrease in mitochondrial ATP levels. Mitochondrial ADP and AMP were concomitantly increased with a subsequent decrease in the ATP/ADP or ATP/AMP ratios. The total concentration of adenine nucleotides also changed. Additionally, both drugs decreased the ANT content of mitochondria, inhibited the phosphate carrier and induced ATP hydrolysis. However, the effects of TAM were more drastic than those induced by OHTAM. Therefore, the depletion of ATP might result from an activation of ATP catabolism, as well as from a decrease in the mitochondrial content of ANT and partial inhibition of the phosphate carrier. Our data may explain the ER-independent effects and cytotoxicity of both drugs and, in agreement with other previous studies, suggest that OHTAM is much less toxic to mitochondria than TAM.     

Inhibition of adrenergic neuroeffector transmission in rabbit pulmonary artery and aorta by adenosine and adenine nucleotides

The effect of adenosine and adenine nucleotides on sympathetic neuroeffector transmission in the rabbit isolated pulmonary artery and aorta was studied. Adenosine (10(-5)-3 x 10(-4)M) decreased the contractile response of pulmonary artery and aorta evoked by electrical-field stimulation. The decrease was reversible. No tachyphylaxis developed. Inhibition of either adenosine deaminase by deoxycoformycin (3.6 x 10(-6)M) or of adenosine transport by dilazep (3 x 10(-6)M) did not alter the inhibitory effect of adenosine on the neurogenic contractions in the pulmonary artery. However, deoxycoformycin plus dilazep markedly enhanced the inhibitory effect of adenosine. The calcium antagonists nifedipine (1.5 x 10(-8)M) and nimodipine (1.3 x 10(-8)M) had no effect on the adenosine-induced inhibition. This was also the case with theophylline (5 x 10(-5)M), atropine (10(-7)M), and the prostaglandin synthetase inhibitors indomethacin (5 x 10(-5)M and suprofen (3 x 10(-5)M). The contractile response of the pulmonary artery elicited by exogenous (-)-noradrenaline (NA; 10(-9)-3 x 10(-4)M) was essentially not altered by adenosine (10(-5)-3 x 10(-4)M). Adenosine (10(-4)M) did not alter the spontaneous 3H-outflow from rabbit aorta preloaded with 3H-(-)-noradrenaline (3H-NA). Adenosine (10(-5)-3 x 10(-4)M), ADP (10(-4)M), ATP (10(-5)M), and inosine (10(-4)M) diminished the overflow of tritium from pulmonary artery and aorta preloaded with 3H-NA. The spontaneous outflow of tritium from aorta preloaded with 3H-NA consisted of 3H-NA (17%), 3H-dihydroxyphenylglycol (3H-DOPEG; 30%), 3H-dihydroxymandelic acid (3H-DOMA, 4%), 3H-O-methylated and deaminated metabolites (3H-OMDA; 42%), and 3H-normethanephrine (3H-NMN; 2%). Adenosine (10(-5) and 10(-4)M) enhanced 3H-DOPEG and 2H-NMN, decreased 3H-NA, and did not alter 3H-DOMA and 3H-OMDA. The stimulation-evoked overflow of tritium for aorta preloaded with 3H-NA consisted of 3H-NA (31%), 3H-DOPEG (18%), 3H-DOMA (2%), 3H-ONDA (46%), and 3H-NMN (3%). Adenosine (10(-5) and 10(-4)M) enhanced 3H-NA and 3H-DOPEG, decreased 3H-OMDA and did not alter 3H-DOMA and 3H-NMN. Adeosine (10(-6)-10(-4)M) did not alter the accumulation of 3H-NA (10(-8)M) by aorta. It is concluded that adenosine inhibits vascular sympathetic neuroeffector transmission by diminishing the release of transmitter from the nerve terminals.     

Ischemic preconditioning protects post-ischemic renal function in anesthetized dogs： role of adenosine and adenine nucleotides

Aim: To investigate the effects of renal ischemic preconditioning (IPC) on both renal hemodynamics and the renal interstitial concentrations of adenosine and adenine nucleotides induced by ischemia-reperfusion injury. Methods: Renal hemodynamics responses to ischemia-reperfusion injury in mongrel dog models were determined with or without multiple brief renal ischemic preconditioning treatments, as well as the adenosine A1 receptor antagonist (KW-3902), respectively. The renal interstitial concentrations of adenosine and adenine nucleotides in response to ischemia-reperfusion injury, either following 1-3 cycles of IPC or not, were measured simultaneously using microdialysis sampling technology. Results: One 10-min IPC, adenosine A1 receptor antagonist (KW-3902) also shortened the recovery time of renal blood flow (RBF) and urine flow (UF), as well as mean blood pressure (BP). Advanced renal IPC attenuated the increment of adenosine and adenine nucleotides, as well as recovery time during the 60-min reperfusion which followed the 60-min renal ischemia. All of these recovery times were dependent on the cycles of 10-min IPC. The renal interstitial concentrations of adenosine and adenine nucleotides increased and decreased during renal ischemia and reperfusion, respectively. Conclusion: A significant relativity in dog models exists between the cycles of 10-min renal IPC and the recovery time of BP, UF, and RBF during the 60-min renal reperfusion following 60-min renal ischemia, respectively. Renal IPC can protect against ischemiareperfusion injury and the predominant effect of endogenous adenosine induced by prolonged renal ischemia; renal adenosine A1 receptor activation during the renal ischemia-reperfusion injury is detrimental to renal function.     

Relaxations of guinea-pig fundic strip by adenosine, adenine nucleotides and electrical stimulation: antagonsism by theophylline and desensitization to adenosine and its derivatives

Adenosine and related compounds produced a concentration-dependent relaxation of the guinea-pig fundic strip in non-treated preparations and in preparations treated with atropine, tetrodotoxin (TTX), guanethidine, reserpine or 6-hydroxydopamine (6-OH-DA). The relative potencies in descending order were ATP ? ADP + adenosine. Theophylline (THEO) at low concentrations (25–75 μM) selectively antagonized the inhibitions produced by ATP, ADP and adenosine without antagonizing those due to noradrenaline (NA), isoprenaline (ISO) and papaverine (PAPAV). Schild plots yielded apparent pA₂ values for THEO of1.4 × 10^?5M, 1.8 × 10^?5 M and 2.8 × 10^?5 M for ATP, ADP and adenosine respectively, with slopes which were not significantly different from 1 (p + 0.05). These results suggest competitive antagonism. Theophylline was a more specific antagonist of the relaxations induced by the purines than were compounds such as quinidine, phentolamine or 2,2′-pyridylisatogen (PIT). In atropine (1 × 10^?7to 5 × 10^?7 M) pretreated fundic strips, transmural or periarterial nerve stimulation produced relaxations which were frequency-dependent. The effects of periarterial nerve stimulation were abolished by phentolamine plus propranolol, by guanethidine, by bretylium, by TTX and by pretreatment with reserpine or 6-OH-DA. The responses to transmural nerve stimulation were abolished only by TTX. The relaxations induced by transmural nerve stimulation were reduced or abolished by THEO, whereas those produced by periarterial nerve stimulation were not affected. Theophylline was more selective than quinidine or phentolamine in blocking such responses. Exposure of the tissues to high concentrations of ATP, ADP or adenosine caused tachyphylaxis to the relaxations induced by the purines as well as those to transmural nerve stimulation, while the responses to NA, ISO, PAPAV or periarterial nerve stimulation were either augmented or unaffected. The effects of desensitization were reversible. This results suggests that the response to transmural nerve stimulation may be due to neural release of adenosine or a related compound. These results are discussed in relation to the release of ATP or related nucleotides from purinergic nerves since the responses to the purines and those to transmural nerve stimulation were reduced or abolished by either THEO or by desensitization with ATP, ADP or adenosine. The possibility is raised that an adenine nucleotide or nucleoside released from nerves might have a physiological function in the “receptive relaxation” of the stomach following stimulation of the pharynx and upper oesophagus by passage of a bolus of food.     

Effect of acute lung injury on metabolism of adenine nucleotides in rat perfused lung.

1. The hydrolysis of adenosine di- and monophosphate (ADP, AMP) was studied in perfused lungs isolated from rats treated with alpha-naphthylthiourea (ANTU) to induce acute lung injury. This injury is associated with damage to the endothelium, the locus of the ADP and AMP hydrolysing enzymes. 2. Treatment with ANTU did not change the proportion of [3H]-ADP surviving a single passage through the pulmonary circulation, at any time up to 50 h after ANTU. Less than 8% and 2% respectively of 1 or 0.1 mumol ADP, given as a bolus, appeared in lung effluent. 3. The metabolites of ADP, AMP and adenosine in lung effluent were increased fro 2 h after ANTU. 4. Metabolism of [3H]-AMP as substrate was always low but, following ANTU treatment, the adenosine content of lung effluent increased four fold. 5. It appears that, in spite of considerable endothelial cell damage, as demonstrated by pulmonary oedema, the ectoenzymes catalysing ADP and AMP hydrolysis were relatively little affected by ANTU.     

Inhibitory effect of adenosine and adenine nucleotides on potassium-evoked efflux of [3H]-noradrenaline from the rat isolated heart: lack of relationship to prostaglandins.

1 In the isolated heart of the rat prelabelled with [3H]-noradrenaline (NA) and perfused with Krebs solution, administration of potassium (K+ 60 mumol) increased the efflux of total radioactivity and of [3H]-NA in the perfusate. 2 Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine, but not inosine, dibutyryl cyclic adenosine 3',5'-monophosphate (db cyclic AMP) or db cyclic GMP reduced the K+/-evoked overflow of total radioactivity and of intact [3H]-NA, in concentrations too low to cause release of prostaglandins. ATP, ADP and adenosine did not affect tyramine-evoked overflow of tritium. 3 Blockade of prostaglandin synthesis with indomethacin did not alter the inhibitory effect of either ATP, ADP or adenosine on K+/-induced overflow of tritium, thereby indicating that these nucleotides inhibit adrenergic transmission by a mechanism unrelated to stimulation of prostaglandin synthesis. 4 Theophylline which increases entry of calcium (Ca2+) across the cell membrane and reduces its binding in the cell, enhanced K+/-evoked overflow of tritium and diminished the inhibitory effect of ATP, ADP and adenosine on K+/-evoked overflow of tritium from the heart, presumably by interfering with transneuronal Ca2+ metabolism.