RESUMO
Knockout mice lacking the adenosine A(2A) receptor are less sensitive to nociceptive stimuli, and this may be due to the presence of pronociceptive A(2A) receptors on sensory nerves. In support of this hypothesis, we have recently shown that in A(2A) receptor knockout mice there are marked reductions in the changes of two markers of spinal cord neuronal activity, [(3)H]MK801 binding to NMDA receptors and uptake of [(14)C]-2-deoxyglucose, in response to formalin injection. We now report that following a more prolonged inflammatory stimulus, consisting of intraplantar injections of PGE(2) and paw pressure, there was in contrast an increase in [(3)H]MK801 binding and [(14)C]-2-deoxyglucose uptake in the spinal cords of the A(2A) receptor knockout mice which was much greater than in the wild-type mice. This increase suggests that when there is a pronounced inflammatory component to the stimulus, loss of inhibitory A(2A) receptors on inflammatory cells outweighs the loss of pronociceptive A(2A) receptors on peripheral nerves so that overall there is an increase in nociceptive signalling. This implies that although A(2A) antagonists have antinociceptive effects they may have only limited use as analgesics in chronic inflammatory pain.
Assuntos
Dor/metabolismo , Receptor A2A de Adenosina/metabolismo , Medula Espinal/metabolismo , Animais , Antimetabólitos/farmacologia , Autorradiografia , Desoxiglucose/farmacologia , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Inflamação/metabolismo , Camundongos , Camundongos KnockoutRESUMO
G-protein coupled receptors interact to provide additional regulatory mechanisms for neurotransmitter signaling. Adenosine A(2A) receptors are expressed at a high density in striatal neurons, where they closely interact with dopamine D2 receptors and modulate effects of dopamine and responses to psychostimulants. A(2A) receptors are expressed at much lower densities in other forebrain neurons but play a more prominent yet opposing role to striatal receptors in response to psychostimulants in mice. It is, therefore, possible that A(2A) receptors expressed at low levels elsewhere in the brain may also regulate neurotransmitter systems and modulate neuronal functions. Dopamine D2 receptors play an important role in autoinhibition of neuronal firing in dopamine neurons of the ventral tegmental area (VTA) and dopamine release in other brain areas. Here, we examined the effect of A(2A) receptor deletion on D2 receptor-mediated inhibition of neuronal firing in dopamine neurons in the VTA. Spontaneous activity of dopamine neurons was recorded in midbrain slices, and concentration-dependent effects of the dopamine D2 receptor agonist, quinpirole, was compared between wild-type and A(2A) knockout mice. The potency of quinpirole applied in single concentrations and the expression of D2 receptors were not altered in the VTA of the knockout mice. However, quinpirole applied in stepwise escalating concentrations caused significantly reduced maximal inhibition in A(2A) knockout mice, indicating an enhanced agonist-induced desensitization of D2 receptors in the absence of A(2A) receptors. The A(2A) receptor agonist, CGS21680, did not exert any effect on dopamine neuron firing or response to quinpirole, revealing a novel non-pharmacological interaction between adenosine A(2A) receptors and dopaminergic neurotransmission in midbrain dopamine neurons. Altered D2 receptor desensitization may result in changes in dopamine neuron firing rate and pattern and dopamine release in other brain areas in response to persistent dopamine release and administration of psychostimulants.
Assuntos
Neurônios/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D2/metabolismo , Área Tegmentar Ventral/metabolismo , Animais , Agonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Camundongos , Camundongos Knockout , Neurônios/efeitos dos fármacos , Quimpirol/farmacologia , Receptores de Dopamina D2/genética , Área Tegmentar Ventral/efeitos dos fármacosRESUMO
Mice lacking the adenosine A(2A) receptor are less sensitive to nociceptive stimuli, and A(2A) receptor antagonists have antinociceptive effects. We have previously shown a marked reduction in the behavioural responses to formalin injection in A(2A) receptor knockout mice. This may be due to the presence of pronociceptive A(2A) receptors on sensory nerves, and if so spinal cords from A(2A) receptor knockout mice may have altered neurochemical responses to a nociceptive stimulus. We tested this hypothesis by studying two parameters known to change with spinal cord activity, NMDA glutamate receptor binding and [(14)C]-2-deoxyglucose uptake, following intraplantar formalin injection in wild-type and A(2A) receptor knockout mice. In naïve untreated A(2A) knockout mice [(14)C]-2-deoxyglucose uptake in all regions of the spinal cord was significantly lower compared to the wild-type, similar to the reduced NMDA receptor binding that we have previously observed. Following formalin treatment, there was an decrease in [(3)H]-MK801 binding to NMDA receptors and an increase in [(14)C]-2-deoxyglucose uptake in the spinal cords of wild-type mice, and these changes were significantly reduced in the A(2A) knockout mice. In addition to altered behavioural responses, there are therefore corresponding reductions in spinal cord neurochemical changes induced by formalin in mice lacking adenosine A(2A) receptors. These observations support the hypothesis that activation of A(2A) receptors enhances nociceptive input into the spinal cord and suggests a possible role for A(2A) antagonists as analgesics.
Assuntos
Glucose/metabolismo , Dor/metabolismo , Receptor A2A de Adenosina/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Medula Espinal/metabolismo , Animais , Desoxiglucose/metabolismo , Maleato de Dizocilpina/metabolismo , Deleção de Genes , Masculino , Camundongos , Camundongos Knockout , Medição da Dor , Ensaio Radioligante , Receptor A2A de Adenosina/genéticaRESUMO
BACKGROUND AND PURPOSE: The purinergic system through the A(2A) adenosine receptor regulates addiction induced by different drugs of abuse. The aim of the present study was to investigate the specific role of A(2A) adenosine receptors (A(2A)Rs) in the behavioural and neurochemical responses to morphine associated with its motivational properties. EXPERIMENTAL APPROACH: Mice lacking A(2A)Rs (A(2A) knockout (KO) mice) and wild-type littermates were used to evaluate behavioural responses induced by morphine. Antinociception was assessed using the tail-immersion and the hot-plate tests. Place-conditioning paradigms were used to evaluate the rewarding effects of morphine and the dysphoric responses of morphine withdrawal. Microdialysis studies were carried out to evaluate changes in the extracellular levels of dopamine in the nucleus accumbens of A(2A) KO mice after morphine administration. KEY RESULTS: The acute administration of morphine induced a similar enhancement of locomotor activity and antinociceptive responses in both genotypes. However, the rewarding effects induced by morphine were completely blocked in A(2A) KO mice. Also, naloxone did not induce place aversion in animals lacking the A(2A)Rs. CONCLUSIONS AND IMPLICATIONS: Our findings demonstrate that the rewarding and aversive effects associated with morphine abstinence were abolished in A(2A) KO mice, supporting a differential role of the A(2A) adenosine receptor in the somatic and motivational effects of morphine addiction. This study provides evidence for the role of A(2A)Rs as general modulators of the motivational properties of drugs of abuse. Pharmacological manipulation of these receptors may represent a new target in the management of drug addiction.
Assuntos
Comportamento Animal/efeitos dos fármacos , Dopamina/metabolismo , Morfina/farmacologia , Motivação , Receptor A2A de Adenosina/fisiologia , Animais , Comportamento Animal/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microdiálise , Atividade Motora/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Núcleo Accumbens/fisiologia , Limiar da Dor/efeitos dos fármacos , Receptor A2A de Adenosina/genéticaRESUMO
Adenosine A2A receptors localized in the dorsal striatum are considered as a new target for the development of antiparkinsonian drugs. Co-administration of A2A receptor antagonists has shown a significant improvement of the effects of l-DOPA. The present review emphasizes the possible application of A2A receptor antagonists in pathological conditions other than parkinsonism, including drug addiction, sleep disorders and pain. In addition to the dorsal striatum, the ventral striatum (nucleus accumbens) contains a high density of A2A receptors, which presynaptically and postsynaptically regulate glutamatergic transmission in the cortical glutamatergic projections to the nucleus accumbens. It is currently believed that molecular adaptations of the cortico-accumbens glutamatergic synapses are involved in compulsive drug seeking and relapse. Here we review recent experimental evidence suggesting that A2A antagonists could become new therapeutic agents for drug addiction. Morphological and functional studies have identified lower levels of A2A receptors in brain areas other than the striatum, such as the ventrolateral preoptic area of the hypothalamus, where adenosine plays an important role in sleep regulation. Although initially believed to be mostly dependent on A1 receptors, here we review recent studies that demonstrate that the somnogenic effects of adenosine are largely mediated by hypothalamic A2A receptors. A2A)receptor antagonists could therefore be considered as a possible treatment for narcolepsy and other sleep-related disorders. Finally, nociception is another adenosine-regulated neural function previously thought to mostly involve A1 receptors. Although there is some conflicting literature on the effects of agonists and antagonists, which may partly be due to the lack of selectivity of available drugs, the studies in A2A receptor knockout mice suggest that A2A receptor antagonists might have some therapeutic potential in pain states, in particular where high intensity stimuli are prevalent.
Assuntos
Gânglios da Base/metabolismo , Hipotálamo/metabolismo , Dor/metabolismo , Receptor A2A de Adenosina/metabolismo , Transtornos do Sono-Vigília/metabolismo , Transtornos Relacionados ao Uso de Substâncias/metabolismo , Adenosina/metabolismo , Antagonistas do Receptor A1 de Adenosina , Antagonistas do Receptor A2 de Adenosina , Animais , Gânglios da Base/efeitos dos fármacos , Gânglios da Base/fisiopatologia , Humanos , Hipotálamo/efeitos dos fármacos , Hipotálamo/fisiopatologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Vias Neurais/fisiopatologia , Dor/tratamento farmacológico , Dor/fisiopatologia , Receptor A1 de Adenosina/metabolismo , Transtornos do Sono-Vigília/tratamento farmacológico , Transtornos do Sono-Vigília/fisiopatologia , Transtornos Relacionados ao Uso de Substâncias/tratamento farmacológico , Transtornos Relacionados ao Uso de Substâncias/fisiopatologiaRESUMO
The aim of this study was to characterise the receptor(s) mediating responses to adenosine and/or adenosine analogues in mouse isolated aorta and carotid artery. In addition, since mice lacking the A(2A) adenosine receptor are reported to be hypertensive, the possibility that this gene deletion or the altered phenotype results in alteration of responses mediated via adenosine analogues was investigated. This was achieved by comparing results obtained in parallel within single experiments using tissues from A(2A) knock-out animals and their wild-type littermates.In aortic rings, adenosine and 5'- N-ethylcarboxamidoadenosine (NECA) caused relaxations above 10 microM and 30 microM, respectively, which were unaffected by either 8-sulphophenyltheophylline (8-SPT, 100 microM) or A(2A) receptor knockout. 2-[ p-(2-Carbonylethyl)phenylethylamino]-5'- N-ethylcarboxamidoadenosine (CGS 21680) was virtually inactive. R- N(6)-Phenylisopropyladenosine (R-PIA) induced relaxations which were not inhibited by 8-SPT (100 microM) or altered by A(2A) receptor knockout. No A(1)-mediated contractile responses were observed in wild-type or knock-out tissues in contrast with results in mice of the same strain obtained commercially rather than from our breeding programme. In carotid artery rings NECA contracted at low concentrations (0.1-1 microM) and relaxed at higher concentrations. Curves to NECA were not different in tissues from wild-type and A(2A) receptor knock-out mice and both the contractile and relaxant phases were right-shifted by 8-SPT (100 microM) in tissues from animals of both genotype. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX, 3 nM) attenuated contractile NECA responses but did not affect relaxant responses. CGS 21680 was inactive in carotid artery rings from both wild-type and A(2A) receptor knock-out mice. In the presence of DPCPX (30 nM) to abolish contractions, R-PIA induced relaxant curves which were not different in tissues from wild-type and A(2A) knock-out mice and were not inhibited by 8-SPT (100 microM). These results confirm the absence of A(2A) or A(2B) receptors in murine aorta and suggest that relaxations to NECA in carotid artery are A(2B) receptor-mediated whilst contractions are A(1) receptor-mediated. They also indicate the presence of an antagonist-resistant site activated by R-PIA in both vascular preparations. There is no evidence for compensatory changes in responses mediated by adenosine and its analogues due to the gene deletion or the reported resulting hypertensive phenotype in either aortic or carotid arterial rings obtained from A(2A) knock-out mice.
Assuntos
Adenosina/análogos & derivados , Aorta/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Receptores Purinérgicos P1/deficiência , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Animais , Aorta/fisiologia , Artérias Carótidas/fisiologia , Técnicas In Vitro , Modelos Logísticos , Masculino , Camundongos , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Fenilefrina/farmacologia , Receptor A2A de Adenosina , Receptores Purinérgicos P1/genética , Vasodilatação/fisiologiaRESUMO
1 The aim of this study was to examine whether sodium nitroprusside (SNP)-induced relaxation of rat fundus longitudinal smooth muscle involves ryanodine-sensitive Ca2+ release. 2 SNP (300 nM-30 microM) elicited concentration-dependent relaxation of precontracted (1 microM carbachol) rat fundus, an effect almost abolished by the selective guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10 microM). 3 SNP-mediated relaxations were almost abolished by 10 microM ryanodine. 4 SNP-mediated relaxations were also reduced by either 1 microM apamin (a selective small conductance Ca(2+)-sensitive K+ channel, SKCa, inhibitor) or the selective L-type Ca2+ channel inhibitor, nicardipine (3 microM). 5 SNP-induced relaxations were insensitive to 1 mM tetraethylammonium chloride (an inhibitor of large-conductance Ca(2+)-sensitive K+ channels) and 1 microM glibenclamide (an ATP-sensitive K+ channel inhibitor). 6 These data suggest that SNP-mediated fundus relaxation occurs via a cGMP-mediated and ryanodine-sensitive mechanism which requires, at least in part, SKCa and L-type Ca2+ channel activity.
Assuntos
Canais de Cálcio Tipo L/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Nitroprussiato/farmacologia , Canais de Potássio Cálcio-Ativados/efeitos dos fármacos , Rianodina/farmacologia , Animais , Apamina/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Carbacol/farmacologia , GMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Fundo Gástrico/efeitos dos fármacos , Guanilato Ciclase/antagonistas & inibidores , Técnicas In Vitro , Masculino , Contração Muscular/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Nicardipino/farmacologia , Óxido Nítrico/fisiologia , Oxidiazóis/farmacologia , Parassimpatomiméticos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Wistar , Tetraetilamônio/farmacologiaRESUMO
1 The aim of this study was to characterize the adenosine receptor mediating vasodilation in the microvasculature of the hamster cheek pouch in vivo. A range of adenosine agonists was used including N6-cyclopentyladenosine (CPA) (A1 agonist), 5'-N-ethylcarboxamidoadenosine (NECA) (non-selective), 2-chloroadenosine (2CADO) (non-selective), 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) (A2A agonist), N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IBMECA) (A3 agonist) and adenosine, as well as the adenosine antagonists 8-sulphophenyltheophylline (8-SPT) (A1/A2 antagonist), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (A1 antagonist) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) (A2A antagonist). 2 All the adenosine analogues used induced vasodilation at concentrations between 10 nm and 1 microm, and the potency order was NECA > CGS 21680 > 2CADO > CPA=IBMECA >> adenosine, indicating an action at A2A receptors. 8-SPT (50 microm) antagonized vasodilator responses to NECA with an apparent pKB of 5.4, consistent with an action at A1 or A2 receptors and confirming that A3 receptors are not involved in this response. 3 DPCPX (10 nm) had no effect on vasodilation evoked by NECA, suggesting that this response was not mediated via A1 receptors, while ZM 241385 (10 nm) antagonized dilator responses to NECA with an apparent pKB of 8.9 consistent with an action via A2A receptors. 4 Overall these results suggest that adenosine A2A receptors mediate vasodilation in the hamster cheek pouch in vivo.
Assuntos
Agonistas do Receptor Purinérgico P1 , Receptores Purinérgicos P1/fisiologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Animais , Bochecha , Cricetinae , Relação Dose-Resposta a Droga , Masculino , Mesocricetus , Microcirculação/efeitos dos fármacos , Microcirculação/fisiologiaRESUMO
Although adenosine analogues such as 5'-N-ethylcarboxamidoadenosine (NECA) relax the rat thoracic aorta in a partially endothelium-dependent manner via adenosine A(2A) receptors, others such as N(6)-R-phenylisopropyladenosine (R-PIA) act via an endothelium-independent, antagonist-insensitive mechanism. The role of cyclic nucleotides in these relaxations was investigated in isolated aortic rings using inhibitors of adenylate and guanylate cyclases as well as subtype-selective phosphodiesterase inhibitors. The adenylate cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536; 100 microM) significantly inhibited responses to NECA, but not responses to R-PIA. The type IV (cyclic AMP-selective) phosphodiesterase inhibitor 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 20-1724; 30 microM) significantly enhanced responses to NECA and to a lesser extent those to R-PIA. The guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ; 100 microM) significantly inhibited responses to NECA and acetylcholine but not responses to R-PIA. The selective phosphodiesterase V (cyclic GMP-selective) inhibitors, zaprinast (10 microM) and 4-[[3',4'-(methylenedioxy)benzyl]amino]-6-methoxyquinazoline (MMQ; 1 microM), had no significant effect on responses to either NECA or R-PIA, but enhanced responses to acetylcholine. These results are consistent with the effects of NECA being via activation of endothelial receptors to release NO which stimulates guanylate cyclase, as well as smooth muscle receptors coupled to stimulation of adenylate cyclase. The lack of effect of zaprinast and MMQ on responses to NECA are likely to be due to simultaneous activation of both adenylate and guanylate cyclases in the smooth muscle, as cyclic AMP reduces the sensitivity of phosphodiesterase V to inhibitors. These results also suggest that the effects of R-PIA are via neither of these mechanisms.
Assuntos
Adenina/análogos & derivados , Adenosina/análogos & derivados , Adenosina/farmacologia , Aorta Torácica/efeitos dos fármacos , Nucleotídeos Cíclicos/farmacologia , Vasodilatação/efeitos dos fármacos , 4-(3-Butoxi-4-metoxibenzil)-2-imidazolidinona/farmacologia , Acetilcolina/farmacologia , Adenina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Inibidores de Adenilil Ciclases , Animais , Aorta Torácica/fisiologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Técnicas In Vitro , Masculino , Nitroprussiato/farmacologia , Nucleotídeos Cíclicos/fisiologia , Oxidiazóis/farmacologia , Purinonas/farmacologia , Quinazolinas/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Wistar , Vasodilatadores/farmacologiaRESUMO
Relaxations to adenosine and analogues were investigated in the mouse aorta in the presence of the adenosine A(1) receptor-selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 30 nM), which did not affect relaxations to adenosine or its analogue N(6)-R-phenylisopropyladenosine (R-PIA) but abolished contractile adenosine A(1) receptor-mediated responses to these agonists. Relaxations to adenosine, 5'-N-ethylcarboxamidoadenosine, R-PIA, 2-[p-(2-carbonylethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS 21680), and N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were unaffected by the adenosine A(1)/A(2) receptor antagonist 8-sulphophenyltheophylline (100 microM). IB-MECA relaxations were unaffected by the adenosine A(3) receptor-selective antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS1191, 30 microM) and R-PIA relaxations were unaffected by N(G)-nitro-L-arginine methyl ester (100 microM) and endothelium removal. In conclusion, relaxant responses to adenosine and analogues do not involve adenosine A(1), A(2) or A(3) receptors and are endothelium- and nitric oxide-independent.
Assuntos
Adenosina/análogos & derivados , Adenosina/farmacologia , Aorta/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Aorta/fisiologia , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Masculino , Camundongos , NG-Nitroarginina Metil Éster/farmacologia , Receptor A3 de Adenosina , Receptores Purinérgicos P1/efeitos dos fármacos , Receptores Purinérgicos P1/fisiologia , Vasodilatação/fisiologiaRESUMO
The aim of this study was to characterise the receptor(s) mediating relaxations to adenosine and its analogues in the hamster isolated aorta. Adenosine relaxed the aorta but there was no significant difference between pIC20 values in the absence and presence of 8-sulphophenyltheophylline (8-SPT, 50 microM), although there was a small right-shift (approximately threefold) of the lower portion of the curve in the presence of 8-SPT. However, in the presence of the adenosine uptake inhibitor nitrobenzylthioinosine (NBTI, 1 microM), curves to adenosine were left-shifted by approximately 100-fold and an apparent pK(B) for 8-SPT of 5.79+/-0.05 was obtained. Likewise, 5'-N-ethylcarboxamidoadenosine (NECA) relaxed the aorta but curves were biphasic. The first phase of the curve was blocked by 8-SPT (10-100 microM, pA2 = 5.75+/-0.14) and the A2A-selective antagonist 4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-ylaminolethyl) phenol (ZM 241385, 3 nM-1 microM, pK(B)=9.17+/-0.10). Similarly, the A2A-selective agonist 2-[p)-(2-carbonylethyl)-phenylethylamino]-5'-N-ethylcarboxam idoadenosine (CGS 21680) relaxed the tissues but curves were biphasic and the first phase was again blocked by ZM 241385 (10 nM, apparent pK(B)=9.06+/-0.34). In contrast, relaxations to N6-R-phenylisopropyladenosine (R-PIA), N6-cyclopentyladenosine (CPA), 2-chloroadenosine (2-CADO) and N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) were not blocked by 8-SPT (50 microM). Responses to IB-MECA were also not blocked by the A3 receptor antagonist 3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihyd ropyridine-3,5-dicarboxylate (MRS 1191, 30 microM). The asymptote of the first phase of curves to NECA was markedly reduced (and in some preparations the first phase was completely abolished) both in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME, 0.1 mM), and in the absence of endothelium. Likewise, the first phase of curves to CGS 21680 was abolished both in the presence of L-NAME (0.1 mM) and in the absence of endothelium. In contrast, there were only relatively small shifts to the right of curves to adenosine and the other analogues in the presence of L-NAME or the absence of endothelium (between three- and fivefold). The data suggest the presence of A2A receptors which are located on the endothelium and mediate release of nitric oxide. These receptors are activated by NECA, CGS 21680 and adenosine (in the presence of uptake blockade). The resistance to blockade of relaxations to adenosine (in the absence of uptake inhibitor), CPA, R-PIA, 2-CADO, IB-MECA and high concentrations of NECA and CGS 21680 by 8-SPT or ZM 241385 suggests the presence of an additional mechanism(s). Data obtained with adenosine in the absence and presence of NBTI suggest that the endogenous ligand may cause relaxation via an intracellular mechanism.
Assuntos
Adenosina/análogos & derivados , Aorta/efeitos dos fármacos , Receptores Purinérgicos P1/fisiologia , Teofilina/análogos & derivados , Tioinosina/análogos & derivados , Vasodilatação/efeitos dos fármacos , 2-Cloroadenosina/farmacologia , Adenosina/farmacologia , Animais , Aorta/fisiologia , Cricetinae , Relação Dose-Resposta a Droga , Técnicas In Vitro , Masculino , Mesocricetus , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/fisiologia , Fenetilaminas/farmacologia , Teofilina/farmacologia , Tioinosina/farmacologiaRESUMO
Adenosine 5'-diphosphate (ADP) is a paracrine mediator that activates human blood platelets, causing them to become adhesive and thereby contributing to their role in hemostasis. The actions of ADP were initially thought to be mediated by a unique ADP receptor termed P2(T) found only on platelets and antagonized by ATP, but it appears that at least two P2Y receptor subtypes are involved, a P2Y(1) receptor linked in some way to control of intracellular-free calcium levels and another P2Y receptor linked via an inhibitory G protein to adenylate cyclase. In addition, the presence of excitatory P2X(1) receptors that mediate the influx of monovalent and divalent cations in response to both ADP and ATP has been demonstrated. The precise contribution that each of these P2 receptors make to the overall phenomena associated with platelet aggregation, adhesion and hemostasis is yet to be defined. Antithrombotic agents that interfere with the actions of ADP are marketed, and P2 receptor antagonists are entering clinical trials for acute treatments of thrombosis. This review seeks to summarize the present state of knowledge of platelet P2 receptor pharmacology and therapeutics.
Assuntos
Plaquetas/metabolismo , Receptores Purinérgicos P2/sangue , Difosfato de Adenosina/antagonistas & inibidores , Difosfato de Adenosina/metabolismo , Animais , Humanos , Antagonistas do Receptor Purinérgico P2RESUMO
Adenosine 5'-triphosphate (ATP) has a higher potency at the prostatic than at the epididymal end of the guinea pig vas deferens. We bisected the tissue and measured the breakdown of ATP by each half, and although the half-lives differed, the rate constants per gram of tissue were not significantly different. For a range of tissue portions, a correlation was found between the portion weight and the half-life of ATP. The difference in half-life at the two ends is therefore due to the different weights of the tissues, and the difference in potency of ATP cannot be explained by differences in degradation.
Assuntos
Trifosfato de Adenosina/metabolismo , Epididimo/metabolismo , Próstata/metabolismo , Ducto Deferente/metabolismo , Adenosina/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Animais , Espaço Extracelular/metabolismo , Cobaias , Técnicas In Vitro , Masculino , Fatores de TempoRESUMO
The effects on ATP breakdown of some modulators of adenosine transport or metabolism were studied in the rat colon muscularis mucosae, a tissue which contracts to ATP and is thought to contain P2Y1 receptors. The compounds tested were the xanthine oxidase inhibitor allopurinol, the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and the adenosine uptake blocker S-(4-nitrobenzyl)-6-thioinosine (NBTI). The degradation of adenosine 5'-triphosphate (ATP) (100 microM) and the appearance of metabolites was followed by high pressure liquid chromatography during incubation of isolated tissue preparations alone or in the presence of the drugs, following preincubation with the drugs for 1 h. In the absence of drugs ATP was rapidly degraded by the rat colon muscularis mucosae with a half-life of 6.1 +/- 0.7 min, the major breakdown product being inosine rather than adenosine. Allopurinol (1 microM) and NBTI (10 microM) had no effect on the rate of breakdown of ATP or on the pattern of metabolites produced. EHNA (1 or 10 microM) also had no effect on the half-life of ATP, but in the presence of EHNA (1 microM) the rate of production of inosine was significantly reduced and some adenosine was detected, while in the presence of 10 microm EHNA the production of inosine was abolished and adenosine became the final breakdown product. These results indicate that allopurinol (1 microM) and NBTI (10 microM) have no detectable effect on extracellular purine metabolism in this tissue, and that the build-up of adenosine produced by treatment with EHNA does not have a feedback effect on ATP breakdown.
Assuntos
Adenina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Alopurinol/farmacologia , Colo/metabolismo , Inibidores Enzimáticos/farmacologia , Mucosa Intestinal/metabolismo , Músculo Liso/metabolismo , Tioinosina/análogos & derivados , Adenina/farmacologia , Adenosina/metabolismo , Inibidores de Adenosina Desaminase , Animais , Colo/efeitos dos fármacos , Meia-Vida , Técnicas In Vitro , Mucosa Intestinal/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores Purinérgicos P1/efeitos dos fármacos , Receptores Purinérgicos P2/efeitos dos fármacos , Tioinosina/farmacologiaRESUMO
1. The effects of noradrenaline (NA), the calcium ionophore A23187, forskolin, sodium nitroprusside (SNP) and the K+-channel blocker glibenclamide on the degradation by ectonucleotidases of extracellular adenosine 5'-triphosphate (ATP) were studied in the rat vas deferens. 2. ATP (100 microM) was rapidly broken down by the rat vas deferens with a half-life of 5.83 +/- 0.40 min via adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP), with the final degradation product being inosine and with little adenosine being detected in the samples. 3. Preincubation for 1 h with NA (10 microM), A23187 (10 microM), or glibenclamide (100 microM) had no significant effect on the breakdown of ATP or the production of metabolites. However, both forskolin (10 microM) and SNP (1 microM) significantly increased the concentrations of AMP detected with time. In the case of SNP (1 microM) there was also a significant reduction in the rate of production of inosine, while in the case of forskolin (10 microM) there was a significant increase in the rate of removal of ATP. 4. These results suggest that preincubation with SNP may inhibit 5'-nucleotidase and so reduce the metabolism of AMP, while preincubation with forskolin may increase the activity of the ectonucleotidases responsible for production of AMP from ATP.
Assuntos
5'-Nucleotidase/fisiologia , Trifosfato de Adenosina/metabolismo , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/metabolismo , Difosfato de Adenosina/análise , Monofosfato de Adenosina/análise , Animais , Calcimicina/farmacologia , Colforsina/farmacologia , Glibureto/farmacologia , Inosina/análise , Masculino , Nitroprussiato/farmacologia , Norepinefrina/farmacologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Adenosine 5'-diphosphate (ADP) induces human blood platelets to aggregate and change shape, and it has been suggested that these two responses are mediated by more than one subtype of ADP receptor. The structure-activity relationships for several analogues of adenine nucleotides in causing aggregation and shape change were measured and compared in washed platelets using an aggregometer. ADP and its analogues 2-methylthioadenosine 5'-diphosphate (2-methylthio-ADP), adenosine 5'(alpha,beta-methylene)diphosphonate (AMPCP), S(P)-adenosine 5'-O-(1-thiodiphosphate) (AD-P alphaS) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) were used as agonists. Adenosine 5'-triphosphate (ATP) and its analogues, P1, P5-diadenosine pentaphosphate (ApsA), adenosine (5'-(alpha,beta-methylene)triphosphonate (AMPCPP), 2-methylthioadenosine 5'-triphosphate (2-methylthio-ATP) and uridine 5'-triphosphate (UTP), as well as the trypanocidal drug suramin, were used as antagonists. In general, the structure-activity relationships for both responses were similar, but for some analogues differences were observed. ADPalphaS and ADPbetaS were much more potent agonists relative to ADP for shape change than for aggregation and indeed ADPalphaS antagonized ADP-induced aggregation with an apparent pK(B) value of 5.5+/-0.1. 2-Methylthio-ATP also had different effects in aggregation and shape change, being a much higher affinity antagonist of aggregation than of shape change with an apparent pK(B) value of 7.0+/-0.2 for aggregation and 5.2+/-0.2 for shape change. These results support the suggestion that these two responses are mediated by multiple ADP receptors on human platelets, and are consistent with shape change being mediated via one receptor (the P2Y1 receptor) with aggregation requiring the activation of two receptors (the P2Y1 and another P2Y receptor).
Assuntos
Nucleotídeos de Adenina/farmacologia , Difosfato de Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Nucleotídeos de Adenina/química , Difosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Plaquetas/citologia , Tamanho Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Suramina/farmacologia , Tionucleotídeos/farmacologia , Tripanossomicidas/farmacologia , Uridina Trifosfato/farmacologiaRESUMO
The development of adenosine A1 and A2B receptors on the longitudinal muscle and muscularis mucosae of the neonatal rat distal colon has been investigated using homogenate binding, quantitative autoradiography and functional studies. In homogenate binding studies 1,3-[3H]-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) bound with high affinity to A1 receptors in the muscularis mucosae and intact colon from rats aged 10, 15, 20, 25 and 30 days. The affinity of [3H]DPCPX was similar to that in the adult at all ages, but the density of binding sites was higher in the neonatal tissues. Quantitative autoradiography showed a higher density of [3H]DPCPX binding sites in the longitudinal muscle than in the muscularis mucosae at all ages studied (day 10 to adult), and this binding was concentration-dependently displaced by N6-cyclopentyladenosine (CPA). In functional studies the longitudinal muscle relaxed in response to 5'-N-ethylcarboxamidoadenosine (NECA) and CPA at all ages studied (15-30 days), NECA being more potent than CPA. The potency of NECA remained constant and it was antagonised by 1 microM DPCPX at all ages with pA2-values consistent with activation of A2 receptors. The inactivity of 2-[p-(carboxyethyl)-phenylethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) indicated that the A2 receptors were of the A2B subtype. The muscularis mucosae contracted in response to CPA at all ages studied (day 15 to adult) and the antagonism by DPCPX (10 nM) were consistent with activation of A1 receptors. In conclusion, binding, autoradiographic and functional studies identified A1 receptors on the rat colon muscularis mucosae at all ages studied. However, while binding and autoradiographic localisation showed the presence of A1 receptors in the longitudinal muscle at all ages studied, functional data only revealed the presence of A2B receptors.
Assuntos
Colo/crescimento & desenvolvimento , Colo/fisiologia , Desenvolvimento Muscular , Músculo Liso/crescimento & desenvolvimento , Músculo Liso/fisiologia , Receptores Purinérgicos P1/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/farmacologia , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Envelhecimento/metabolismo , Animais , Autorradiografia , Sítios de Ligação , Ligação Competitiva , Colo/metabolismo , Masculino , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Ensaio Radioligante , Ratos , Ratos Wistar , Frações Subcelulares/metabolismo , Vasodilatadores/farmacologia , Xantinas/metabolismo , Xantinas/farmacologiaRESUMO
1. Adenosine and ATP have well-established functions as neuromodulator and neurotransmitter, respectively, in smooth muscle preparations, and purinergic control may be an early form of autonomic control in both evolution and ontogenesis. 2. This review describes the postnatal development of responses mediated by the various receptors for adenosine and for nucleotides in the rat duodenum, colon, urinary bladder and vas deferens and considers the implications that this development may have for the importance of purinergic control in neonates and adults.
Assuntos
Músculo Liso/fisiologia , Receptores Purinérgicos/fisiologia , Fatores Etários , Animais , Intestinos/fisiologia , Masculino , Ratos , Bexiga Urinária/fisiologia , Ducto Deferente/fisiologiaRESUMO
The functional effects of adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), adenosine 5'-tetraphosphate (AP4) and the diadenosine polyphosphates P1,P3-diadenosine triphosphate (Ap3A), P1,P4-diadenosine tetraphosphate (Ap4A) and P1,P5-diadenosine pentaphosphate (Ap5A) were studied in two isolated smooth muscle preparations thought to contain P2Y (P2Y1) receptors, the guinea-pig taenia caeci (which relaxes to ATP) and the rat colon muscularis mucosae (which contracts to ATP). In addition, the breakdown of these compounds by the rat colon muscularis mucosae was investigated by high pressure liquid chromatography. In the guinea-pig taenia caeci all the purine nucleotides caused relaxation with a potency order of Ap3A=Ap4A> ATP>AP4=Ap5A, and these relaxations were antagonised by suramin with apparent pA2 values in the region of 5, consistent with activation of a P2Y1 receptor. In the rat colon muscularis mucosae the nucleotides caused contraction with a potency order of Ap3A = Ap4A>ATP=AP4 =Ap5A >UTP. However, while suramin (100 microM) inhibited responses to ATP and UTP at all concentrations of agonist, it only inhibited contractions induced by the higher concentrations of AP4, Ap3A and Ap4A and had little effect on contractions induced by Ap5A. A higher concentration of suramin (1 mM) enhanced contractions induced by ATP but greatly inhibited those induced by UTP and had no effect on responses to the other agonists. The A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 10 nM) had no effect on responses to ATP or UTP but inhibited responses to Ap3A, Ap4A, Ap5A and AP4. A combination of suramin (1 mM) and DPCPX (10 nM) almost abolished responses to all the agonists. ATP and UTP were rapidly degraded by the rat colon muscularis mucosae while AP4, Ap3A, Ap4A and Ap5A were degraded more slowly, and the major product detected after breakdown of the purine nucleotides was inosine rather than adenosine. The breakdown of all the nucleotides was inhibited by suramin (1 mM), although this inhibition did not achieve statistical significance in the case of ATP. These results show that while the diadenosine polyphosphates appear to act as P2 agonists in the taenia caeci, in the rat colon muscularis mucosae their major action is via adenosine A1 receptors rather than via P2 receptors. In addition, although they are more stable than ATP or UTP, their action in this tissue is clearly affected by their degradation which complicates the effects of suramin.
Assuntos
Nucleotídeos de Adenina/farmacologia , Trifosfato de Adenosina/farmacologia , Colo/efeitos dos fármacos , Fosfatos de Dinucleosídeos/farmacologia , Uridina Trifosfato/farmacologia , Nucleotídeos de Adenina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Colo/fisiologia , Fosfatos de Dinucleosídeos/metabolismo , Cobaias , Masculino , Ratos , Ratos Wistar , Suramina/farmacologia , Uridina Trifosfato/metabolismoRESUMO
Although the effects of nucleotides in the cardiovascular system have been known for almost 70 years, it is only in the past few years that some of the P2 receptors at which they act have been cloned and characterized. It is now clear that the control of cardiovascular function by nucleotides is complex, involving multiple receptors and multiple effects in the different cell types of importance. In this review Mike Boarder and Susanna Hourani summarize the P2 receptors that are present in endothelial cells, platelets, smooth muscle and nerves, the signalling pathways that they activate and the responses that are produced. They also discuss the important role of nucleotides in the interactions between the different cell types, and the implications of this in vascular disease.