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A M Sebastião

Publications and source records attributed to A M Sebastião.

At least 37 records · Page 2Linked to original sources
PubMed1996

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.

Adenine Nucleotides↗
PubMed1995

Adenosine A2A receptors stimulate acetylcholine release from nerve terminals of the rat hippocampus.

The nature of the adenosine receptors involved in the enhancement of acetylcholine release in the hippocampus was studied. The A2A agonist, CGS 21680, increased the veratridine-evoked release of [3H]acetylcholine from hippocampal synaptosomes. This presynaptic effect of CGS 21680 was greater at 3-30 nM than at 100 nM. The excitatory effect of CGS 21680 was antagonised by the A2 antagonist, DMPX (10 microM), and by the A2A antagonist, CSC (200 nM), but not by the A1 antagonist, DPCPX (20 nM). We also found co-expression of A2A and choline acetyltransferase mRNAs in the nucleus of the diagonal band and the medial septum, where the cholinergic cell bodies that project into the hippocampus are located. These results indicate that A2A adenosine receptors are present in cholinergic nerve terminals in the hippocampus and that activation of these receptors enhances acetylcholine release.

Acetylcholine↗
PubMed1995

Modification of A1 and A2a adenosine receptor binding in aged striatum, hippocampus and cortex of the rat.

Age-related changes of A1 and A2a adenosine receptor binding characteristics were investigated in three regions of the rat brain using the A1 selective antagonist [3H]DPCPX, and the A2a selective agonist [3H]CGS 21680. The density of A1 binding sites in aged rats (24 months) was decreased by 33% in the hippocampus and by 60% in the cortex and was unchanged in the striatum when compared with young adult rats (6 weeks), with no change in KD. There were also age-related changes in the density of A2a binding sites: in the cortex, there was a 94% increase in the number of [3H]CGS 21680 binding sites in aged rats compared with young rats, and a similar tendency was observed in the hippocampus (32% increase in A2a binding sites in aged rats), with no change in KD; in the striatum there was a tendency for a decrease (22%) in the number of [3H]CGS 21680 binding sites in aged rats, and a decrease in KD. These results suggest that there are age-related changes in the balance between inhibitory A1- and excitatory A2a-adenosine receptor-mediated actions, which vary in different brain areas: in the cortex and hippocampus, the balance might be shifted towards adenosine-mediated excitatory actions, since there is an increase in the number of A2a receptors and a decrease in the number of A1 receptors upon ageing. In contrast, in the striatum, the A1/A2a ratio might be only slightly affected upon ageing.

Adenosine↗
PubMed1995

Inhibition of NMDA receptor-mediated currents in isolated rat hippocampal neurones by adenosine A1 receptor activation.

The effect of the stable adenosine analogue, 2-chloro-adenosine (CADO), on the currents elicited by iontophoretic application of N-methyl-D-aspartate (NMDA) to pyramidal cells acutely dissociated from the CA1 area of the rat hippocampus was studied using the patch-clamp technique in the whole-cell configuration. CADO (3-300 nM) reversibly inhibited NMDA receptor-mediated currents (maximal effect: 54.2 +/- 6.6% decrease, EC50 = 10.3 nM). This effect was prevented by the adenosine A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (50 nM). CADO (100 nM inhibited the conductance induced by iontophoretic application of NMDA, without changing its reversal potential, in both the absence and the presence of Mg2+ (30 microM). Adenosine may contribute to the regulation of the NMDA receptor function, particularly under conditions, like hypoxia and ischaemia, leading to excessive NMDA receptor activation.

2-Chloroadenosine↗
PubMed1995

Ascorbate/Fe(3+)-induced peroxidation and inhibition of the binding of A1 adenosine receptor ligands in rat brain membranes.

The effect of peroxidation induced by the ascorbate/Fe3+ system on the binding properties of the A1 adenosine receptor, was studied in rat brain membranes, using the agonist, [3H]R-N6-phenylisopropyladenosine ([3H]R-PIA), and the antagonist, [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX). For the agonist, as well as for the antagonist, the number of binding sites (Bmax) was significantly (P < 0.05) reduced after pretreatment of the membranes with ascorbate/Fe3+. The affinity of the agonist for the binding sites was not statistically modified (P > 0.05) after ascorbate/Fe3+ pretreatment, whereas the Kd value of the antagonist was increased (P < 0.05) by a factor of 2. Ascorbate/Fe3+ pretreatment affected agonist binding in the presence of GTP in a similar way as that observed in the absence of GTP, suggesting that peroxidation also affects agonist binding to A1 adenosine receptors uncoupled to G-proteins. The results suggest that when brain membranes suffer free radical oxidative damage, the adenosine modulation of neuronal activity through A1 receptors could be less efficient.

Animals↗
PubMed1994

Evidence for functionally important adenosine A2a receptors in the rat hippocampus.

Adenosine A2a receptors are not confined to dopamine-rich areas of the brain, since thermocycling analysis shows that adenosine A2a receptor mRNA is expressed also in the hippocampus (CA1, CA3 and dentate gyrus) and cerebral cortex. The expression of A2a mRNA in three main areas of the hippocampus was confirmed by in situ hybridization; A2a mRNA expression was mainly localized in the pyramidal and granular cells, the same hippocampal regions that showed adenosine A1 receptor mRNA expression. Receptor autoradiographic studies with [3H]CGS 21680 (30 nM), a selective adenosine A2a receptor agonist, showed specific binding sites in the hippocampus. The density of [3H]CGS 21680 binding was greatest in the stratum radiatum of the CA1 area, followed by the stratum oriens of the cornu Ammonis, stratum radiatum of the CA3 are and supra-granular layer of the dentate gyrus. This anatomical distribution of [3H]CGS 21680 binding was similar to the pattern of [3H]CHA binding in the hippocampus. Electrophysiological studies in the Schaffer fibers/CA1 pyramids showed that upon activation of the A2a receptors with CGS 21680 (10 nM) the ability of the adenosine A1 receptor agonist, CPA, to inhibit neuronal activity was significantly attenuated. These results show functionally important co-expression and co-localization of adenosine A2a and A1 receptors in the hippocampus. The results also suggest that adenosine A2a receptor-mediated neuromodulation is not confined to the basal ganglia, but is more widespread throughout the nervous system.

Adenosine↗
PubMed1994

Purinergic modulation of the evoked release of [3H]acetylcholine from the hippocampus and cerebral cortex of the rat: role of the ectonucleotidases.

Modulation by exogenous and endogenous adenine nucleotides and adenosine of [3H]acetylcholine release evoked by veratridine (10 microM) was compared in synaptosomal fractions from the hippocampus and the cerebral cortex of the rat. In both brain areas, exogenously added ATP or adenosine (10-100 microM) inhibited the evoked tritium release. In the hippocampus, ATP gamma S, an ATP analogue more resistant to catabolism than ATP, was virtually devoid of effect on tritium release, and the effect of ATP was prevented by the ecto-5'-nucleotidase inhibitor alpha,beta-methylene ADP (100 microM), by adenosine deaminase (2 U/ml) and by the A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 20 nM). In contrast, in the cerebral cortex, the effect of ATP on tritium release was not prevented by either alpha,beta-methylene ADP (100 microM) or adenosine deaminase (2 U/ml), and several ATP analogues (30 microM) inhibited release. The order of intensity of the inhibitory effects of the ATP analogues was: ATP gamma S > ATP > beta,gamma-imido ATP > beta,gamma-methylene ATP >> 2-methyl-S-ATP, alpha,beta-methylene ATP. The effect of ATP gamma S in the cerebral cortex was prevented by DPCPX (20 nM) and was not affected by the P2 purinoceptor antagonist suramin (100 microM). In the hippocampus, alpha,beta-methylene ADP (100 microM) increased the evoked release of tritium, and adenosine deaminase (2 U/ml) produced an even greater increase; when adenosine deaminase was added in the presence of alpha,beta-methylene ADP, adenosine deaminase still increased the evoked release of tritium. In the cerebral cortex, DPCPX (20 nM) and adenosine deaminase (2 U/ml) increased the evoked tritium release by a similar magnitude, but the effect of adenosine deaminase was smaller than in the hippocampus. It is concluded that in the cerebral cortex ATP as such presynaptically inhibits acetylcholine release, whereas in the hippocampus the role of adenine nucleotides is as a source of endogenous extracellular adenosine that tonically inhibits acetylcholine release. The results also show that besides formation of adenosine from adenine nucleotides, released adenosine as such contributes in nearly equal amounts to the pool of endogenous adenosine that presynaptically inhibits acetylcholine release in the hippocampus.

Acetylcholine↗
PubMed1994

Vasoactive intestinal peptide in the hamster seminal vesicle: distribution, binding sites and possible functions.

The presence and functional role of vasoactive intestinal peptide in the hamster seminal vesicle were studied by a combination of structural and functional approaches. The use of an immunofluorescence staining technique in both cryostat sections and whole-mount preparations revealed that vasoactive intestinal peptide-immunoreactive nerve fibres were mainly localized in the lamina propria of the mucosal layer. In double-stained preparations, vasoactive intestinal peptide immunoreactivity was found to be localized in nerves also containing acetylcholinesterase activity. At the ultrastructural level, the use of an immunogold staining method showed that vasoactive intestinal peptide immunoreactivity occurred in large granular vesicles (80-150 nm in diameter) in nerve varicosities which also contained small pleomorphic agranular vesicles. In order to evaluate the anatomical distribution of vasoactive intestinal peptide binding sites in the seminal vesicle, we have utilized an in vitro receptor autoradiographic technique. Vasoactive intestinal peptide binding sites were localized in the basal region of the secretory epithelium, in the muscle layer and in the wall of blood vessels. In vitro incorporation of [3H]L-leucine into protein by tissue slices revealed that vasoactive intestinal peptide (1 microM) significantly increases the amount of released protein. Vasoactive intestinal peptide (0.1-1 microM) did not affect the resting tension of the muscle but significantly inhibited the increase in muscle tension induced by carbachol. Atropine prevented the effect of carbachol, indicating that the latter is mediated by muscarinic receptors. Our results suggest that in the hamster seminal vesicle, vasoactive intestinal peptide is involved in the modulation of muscarinic function and in the control of secretion.

Acetylcholinesterase↗
PubMed1994

Excitatory and inhibitory effects of A1 and A2A adenosine receptor activation on the electrically evoked [3H]acetylcholine release from different areas of the rat hippocampus.

The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [3H]acetylcholine ([3H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A1/A2 agonist 2-chloroadenosine (CADO; 2-10 microM) inhibited, in a concentration-dependent manner, the release of [3H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 microM) on [3H]ACh release was prevented by the A1 antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [3H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 microM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [3H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [3H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Chloroadenosine↗
PubMed1993

Adenosine and adenine nucleotides are independently released from both the nerve terminals and the muscle fibres upon electrical stimulation of the innervated skeletal muscle of the frog.

The independent release of adenosine and adenine nucleotides upon electrical stimulation was studied in the innervated sartorius muscle of the frog after blockade of the extracellular catabolism of adenosine monophosphate (AMP) through exo-AMP deaminase and ecto-5'-nucleotidase. Nerve stimulation (30 min, 0.2Hz) induced the release of both adenosine (19 +/- 3 pmol) and adenine nucleotides (101 +/- 7 pmol). Experiments performed in the presence of tubocurarine (5 microM) to prevent purine release due to nerve-evoked muscle twitching, or under direct stimulation of the muscle in low calcium solutions to prevent pre-synaptic release of purines, showed that there was an evoked release of adenosine and adenine nucleotides both from the nerve endings and from the twitching muscle fibres. Removal of ecto-5'-nucleotidase inhibition shows that the catabolism of adenine nucleotides released during stimulation contributes in about 50% to the amount of endogenous extracellular adenosine. When only one of the enzymes catabolizing AMP (ecto-5'-nucleotidase or exo-AMP deaminase) was inhibited, the evoked release of adenine nucleotides was undetectable, suggesting that each enzyme is able to catabolize all the AMP formed from adenine nucleotides released upon stimulation. It is concluded that the concentration of endogenous extracellular adenosine is under the control of the relative activities of exo-AMP deaminase and ecto-5'-nucleotidase.

AMP Deaminase↗
PubMed1993

On the high affinity binding site for [3H]-1,3-dipropyl-8-cyclopentylxanthine in frog brain membranes.

1. Radioligand binding properties of the adenosine receptor ligands, [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), and [3H]-R-phenylisopropyladenosine ([3H]-R-PIA) were investigated in frog brain membranes. 2. The specific binding of the adenosine antagonist, [3H]-DPCPX to frog brain membranes showed one binding site with Kd and Bmax values of 43.8 nM and 0.238 +/- 0.016 pmol mg-1 protein, respectively. Guanosine 5'-triphosphate (GTP, 100 microM) decreased to 72 +/- 7% and Mg2+ (8 mM) increased to 121 +/- 3% [3H]-DPCPX (40 nM) binding to frog brain membranes. 3. [3H]-DPCPX saturation binding experiments performed in the presence of Mg2+ (8 mM), or in the presence of GTP showed that Mg2+ ions decreased the Kd value of [3H]-DPCPX to 14 nM, and GTP increased this value to 65.6 nM. Bmax values were not significantly (P > 0.05) modified (0.261 +/- 0.018 pmol mg-1 protein, with Mg2+, and 0.266 +/- 0.026 pmol mg-1 protein, in presence of GTP) by the presence of Mg2+ or GTP. 4. The specific binding of [3H]-R-PIA (15 nM) was decreased to 37 +/- 6% by GTP (100 microM) and increased to 123 +/- 4% by Mg2+ (8 mM). [3H]-R-PIA saturation binding experiments performed in the presence of Mg2+ (8 mM) showed one binding site with Kd and Bmax values of 0.9 nM and 0.229 +/- 0.008 pmol mg-1 of protein, respectively. 5. The concentration-inhibition curves of adenosine agonists and antagonists versus [3H]-DPCPX binding showed the following order of potencies: CPA> R-PIA~ NECA> S-PIA> > CGS 21680, for the agonists, and XAC ~-DPCPX> > XCC> PACPX, for the antagonists.6. The present results suggest that the adenosine binding site in the frog brain membranes is G-protein coupled, but that the antagonist affinities and the pharmacological profile is different from the Al or A2 adenosine receptors.

Adenosine↗
PubMed1992

Adenine nucleotide analogues, including gamma-phosphate-substituted analogues, are metabolised extracellularly in innervated frog sartorius muscle.

The metabolism of adenine nucleotides and of their analogues by ecto-enzymes in the innervated frog sartorius muscle was investigated with HPLC. The breakdown of beta, gamma-methylene-ATP was also evaluated by studying the ability of the adenosine uptake inhibitor, dipyridamole, and of the adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), to modify the effect of beta, gamma-methylene-ATP on nerve-evoked twitches. ATP-gamma-S at low (10 microM) but not at high (> or = 100 microM) concentrations was quickly metabolised into a compound with a higher negative charge. L-ATP, homo-ATP and 2-methylthio-ATP were metabolised into compounds with a lower negative charge. Beta-gamma-Imido-ATP and gamma-anilino-ATP were only metabolised slightly. As determined by HPLC, beta, gamma-methylene-ATP was not metabolised. In contrast, this ATP analogue inhibited nerve-evoked twitch responses, an effect which was potentiated by dipyridamole and antagonised by DPCPX. Alpha, beta-Methylene-ATP was dephosphorylated into alpha, beta-methylene-ADP, which was virtually resistant to metabolism in the absence of ATP. In the presence of ATP, alpha, beta-methylene-ADP was transiently phosphorylated into alpha, beta-methylene-ATP. Formation of ATP from ADP was observed even in the absence of an exogenous phosphate donor, and was prevented by the adenylate kinase inhibitor, P1P5-di-(adenosine-5')pentaphosphate (AP5A). AP5A caused only partial inhibition of AMP formation from ADP. The results suggest that some ATP analogues with substitutions in the gamma-phosphate, such as ATP-gamma-S and beta, gamma-methylene-ATP, are metabolised in the innervated frog sartorius muscle. The ADP analogue, alpha, beta-methylene-ADP, might be a substrate for an ecto-nucleoside diphosphate kinase. ADP, besides being dephosphorylated, is also a substrate for an ecto-adenylate kinase in innervated frog sartorius muscle.

Adenine Nucleotides↗
PubMed1992

Evidence for the presence of excitatory A2 adenosine receptors in the rat hippocampus.

The A2 adenosine receptor agonist, CGS 21680 in nanomolar concentrations, reversibly increased in a concentration-dependent manner the amplitude of orthodromically-evoked population spikes recorded from the CA1 pyramidal cell layer of rat hippocampal slices. The adenosine receptor antagonist, 3,7-dimethyl-l-propargylxanthine (DMPX, 10 microM), which has selectivity for A2 adenosine receptors, prevented this excitatory effect of CGS 21680. These results suggest that A2 adenosine receptors are present in the rat hippocampus and that activation of these receptors enhance hippocampal excitability.

Adenosine↗
PubMed1992

Ecto-5'-nucleotidase is associated with cholinergic nerve terminals in the hippocampus but not in the cerebral cortex of the rat.

The extracellular catabolism of exogenously added AMP was studied in immunopurified cholinergic nerve terminals and in slices of the hippocampus and cerebral cortex of the rat. AMP (10 microM) was catabolized into adenosine and inosine in hippocampal cholinergic nerve terminals and in hippocampal slices, as well as in cortical slices. IMP formation from extracellular AMP was not detected. alpha, beta-Methylene ADP (100 microM) inhibited almost completely the extracellular catabolism of AMP in these preparations. The relative rate of catabolism of AMP was greater in hippocampal slices than in cortical slices. AMP was virtually not catabolized when added to immunopurified cortical cholinergic nerve terminals, although ATP could be catabolized extracellularly under identical conditions. The comparison of the relative rates of catabolism of exogenously added AMP, calculated from the amount of AMP catabolized after 5 min, in hippocampal cholinergic nerve terminals and in hippocampal slices revealed a nearly 50-fold enrichment in the specific activity of ecto-5'-nucleotidase upon immunopurification of the cholinergic nerve terminals from the hippocampus. The results suggest that there is a regional variation in the subcellular distribution of ecto-5'-nucleotidase activity in the rat brain, the ecto-5'-nucleotidase in the hippocampus being closely associated with the cholinergic nerve terminals, whereas in the cerebral cortex ecto-5'-nucleotidase activity seems to be located preferentially outside the cholinergic nerve terminals.

5'-Nucleotidase↗
PubMed1991

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.

Adenine↗
PubMed1991

Solubilized rat brain adenosine receptors have two high-affinity binding sites for 1,3-dipropyl-8-cyclopentylxanthine.

The specific binding of L-N6-[3H]phenylisopropyladenosine (L-[3H]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[3H]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (KD) values of 0.24 and 3.56 nM and respective receptor number (Bmax) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[3H]PIA also showed two sites with KD values of 24.7 and 811.5 nM and Bmax values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[3H]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with Ki values of 0.29 and 13.5 nM, respectively. [3H]DPCPX saturation binding experiments also showed two binding sites with respective KD values of 0.81 and 10.7 nM and respective Bmax values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.

Animals↗
PubMed1991

Inhibitory and excitatory effects of adenosine receptor agonists on evoked transmitter release from phrenic nerve ending of the rat.

1. The effects of the adenosine analogues, 5'-N-ethyl-carboxamide adenosine (NECA), R-N6-phenylisopropyladenosine (R-PIA), 2-chloroadenosine (CADO), and CGS 21680C on electrically evoked tritium outflow from preparations loaded with [3H]-choline and on evoked endplate potentials (e.p.ps), as well as the ability of the xanthines, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and PD 115,199 to antagonize the effects of the adenosine analogues, were investigated in phrenic nerve-diaphragm preparations. 2. NECA, R-PIA and CADO decreased, in a concentration-dependent manner, the evoked tritium outflow from preparations loaded with [3H]-choline. NECA and R-PIA were about equipotent and more potent than CADO. 3. DPCPX shifted to the right in a near parallel fashion the concentration-response curve for the inhibitory effect of R-PIA on evoked tritium outflow. 4. In the presence of DPCPX, NECA increased, rather than decreased, evoked tritium outflow. PD 115,119 antagonized, in a concentration-dependent manner, this excitatory effect of NECA. 5. CGS 21680C, in low nanomolar concentrations, increased evoked tritium outflow, an effect also antagonized by PD 115,119. 6. CGS 21680C increased, and R-PIA decreased, the amplitude of e.p.ps recorded from preparations paralysed with tubocurarine. Both effects could be observed in the same endplate. 7. It is concluded that both inhibitory (probably A1) and excitatory (probably A2) adenosine receptors coexist at the rat neuromuscular junction, modulating the evoked release of acetylcholine.

Adenosine↗