RESUMO
The influence of alpha2-autoreceptors on the facilitation of [3H]-noradrenaline release mediated by angiotensin II was studied in prostatic portions of rat vas deferens preincubated with [3H]-noradrenaline. Angiotensin II enhanced tritium overflow evoked by trains of 100 pulses at 8 Hz, an effect that was attenuated by the AT1-receptor antagonist losartan (0.3-1 microM), at concentrations suggesting the involvement of the AT1B subtype. The effect of angiotensin II was also attenuated by inhibition of phospholipase C (PLC) and protein kinase C (PKC) indicating that prejunctional AT1-receptors are coupled to the PLC-PKC pathway. Angiotensin II (0.3-100 nM) enhanced tritium overflow more markedly, up to 64%, under conditions that favor alpha2-autoinhibition, observed when stimulation consisted of 100 pulses at 8 Hz, than under poor alpha2-autoinhibition conditions, only up to 14%, observed when alpha2-adrenoceptors were blocked with yohimbine (1 microM) or when stimulation consisted of 20 pulses at 50 Hz. Activation of PKC with 12-myristate 13-acetate (PMA, 0.1-3 microM) also enhanced tritium overflow more markedly under strong alpha2-autoinhibition conditions. Inhibition of Gi/o-proteins with pertussis toxin (8 microg/ml) or blockade of Gbetagamma subunits with the anti-betagamma peptide MPS-Phos (30 microM) attenuated the effects of angiotensin II and PMA. The results indicate that activation of AT1-receptors coupled to the PLC-PKC pathway enhances noradrenaline release, an effect that is markedly favoured by an ongoing activation of alpha2-autoreceptors. Interaction between alpha2-adrenoceptors and AT1-receptors seems to involve the betagamma subunits released from the Gi/o-proteins coupled to alpha2-adrenoceptors and protein kinase C activated by AT1-receptors.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Norepinefrina/metabolismo , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Fibras Simpáticas Pós-Ganglionares/metabolismo , Ducto Deferente/metabolismo , Antagonistas Adrenérgicos alfa/farmacologia , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Autorreceptores/efeitos dos fármacos , Autorreceptores/metabolismo , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Subunidades beta da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades gama da Proteína de Ligação ao GTP/antagonistas & inibidores , Masculino , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Toxina Pertussis/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Receptor Cross-Talk/efeitos dos fármacos , Receptor Cross-Talk/fisiologia , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fibras Simpáticas Pós-Ganglionares/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Trítio , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/inervaçãoRESUMO
In the prostatic portion of rat vas deferens, activation of adenosine A 2B-receptors, beta2-adrenoceptors, adenylyl cyclase or protein kinase A caused a facilitation of noradrenaline release. Blockade of alpha2-adrenoceptors with yohimbine (1 microM) attenuated the facilitation mediated by adenosine A 2B-receptors and by direct activation of adenylyl cyclase with forskolin but not that mediated by beta2-adrenoceptors or by direct activation of protein kinase A with 8-bromoadenosine-3',5'-cyclicAMP. The adenosine A 2B- and the beta2-adrenoceptor-mediated facilitation was prevented by the adenylyl cyclase inhibitors, 2',5'-dideoxy-adenosine (3 microM) and 9-cyclopentyladenine (100 microM), at concentrations that also attenuated the release enhancing effect of forskolin, but were not changed by the phospholipase C inhibitor 1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione (U-73122, 1 microM). Facilitation of noradrenaline release mediated by adenosine A 2B-receptors was also attenuated by activation of protein kinase C with the phorbol ester 12-myristate 13-acetate (1 microM) and by inhibition of Gbetagamma subunits with an anti-betagamma peptide; facilitation mediated by beta2-adrenoceptors was mainly attenuated by the calmodulin inhibitor calmidazolium (10 microM) and by the calmodulin kinase II inhibitor (N-[2-[N-(4-chlorocinnamyl)-N-methylaminomethyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzene-sulfonamide phosphate (KN-93, 5 microM). The results suggest that adenosine A 2B- but not beta2-adrenoceptor-mediated facilitation of noradrenaline release is enhanced by an ongoing activation of alpha2-adrenoceptors. They further suggest that adenosine A 2B-receptors and beta2-adrenoceptors are coupled to distinct adenylyl cyclase isoforms what may explain the different influence of alpha2-adrenoceptor signalling pathway on the facilitatory effects mediated by the two adenylyl cyclase coupled receptors.
Assuntos
Adenilil Ciclases/metabolismo , Norepinefrina/metabolismo , Receptor Cross-Talk/fisiologia , Receptor A2B de Adenosina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Ducto Deferente/metabolismo , Inibidores de Adenilil Ciclases , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Calmodulina/antagonistas & inibidores , Calmodulina/metabolismo , Colforsina/farmacologia , Inibidores Enzimáticos/farmacologia , Masculino , Terminações Pré-Sinápticas/metabolismo , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Ratos , Ratos Wistar , Receptor Cross-Talk/efeitos dos fármacos , Receptor A2B de Adenosina/efeitos dos fármacos , Receptores Adrenérgicos alfa 2/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fibras Simpáticas Pós-Ganglionares/metabolismo , Ducto Deferente/inervaçãoRESUMO
In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1-30 microM), but not the A(2A) agonist CGS 21680 (0.001-10 microM), caused a facilitation of electrically evoked noradrenaline release (up to 43 +/- 4%), when inhibitory adenosine A(1) receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A(2B) receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of alpha(1)-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A(2B) receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase-protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A(2B) receptor-mediated facilitation of noradrenaline release.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Norepinefrina/metabolismo , Próstata/metabolismo , Proteína Quinase C/metabolismo , Receptor A2B de Adenosina/metabolismo , Ducto Deferente/metabolismo , Adenosina/metabolismo , Agonistas do Receptor A2 de Adenosina , Animais , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Masculino , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III , Prostaglandinas/metabolismo , Próstata/efeitos dos fármacos , Próstata/fisiologia , Proteína Quinase C/antagonistas & inibidores , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Ducto Deferente/efeitos dos fármacos , Ducto Deferente/fisiologiaRESUMO
The role of angiotensin II receptors, bradykinin receptors and ß-adrenoceptors in the modulation of noradrenaline release and the influence of α(2)-autoinhibition in these effects was investigated in the mesenteric artery and vein. Rings of mesenteric vessels of male Wistar rats were labelled with [(3)H]-noradrenaline and the effects of modulators on tritium overflow evoked by 100 pulses at 2Hz (marked α(2)-autoinhibition) and by 20 pulses at 50Hz or 100 pulses at 2Hz plus yohimbine (1µM; reduced α(2)-autoinhibition) were evaluated. Angiotensin II and bradykinin enhanced noradrenaline release evoked by 100 pulses at 2Hz, in a concentration-dependent manner, in both vessels. These effects were attenuated under conditions of reduced α(2)-autoinhibition. The attenuation was partially reversed by activation of adenosine A(1) receptors in both vessels and by activation of P2Y receptors in the vein. Isoprenaline and the selective ß(2)-adrenoceptor agonist formoterol enhanced tritium overflow independently of α(2)-autoinhibition in the vein. In the artery, the enhancement by formoterol was only observed under reduced α(2)-autoinhibition. Pharmacological characterization of the ß-adrenoceptors indicated that in the artery the effect of isoprenaline was mediated by the ß(1)-subtype under marked α(2)-autoinhibition and by the ß(2)-subtype under reduced α(2)-autoinhibition whereas in the vein the effect was independent of α(2)-autoinhibition. The results indicate that α(2)-autoinhibition is a key determinant of the magnitude of facilitation caused by angiotensin II and bradykinin in both types of mesenteric vessels and regulates the effects mediated by ß(1)-and ß(2)-adrenoceptors which co-exist in the artery.
Assuntos
Antagonistas de Receptores Adrenérgicos alfa 2/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Veias Mesentéricas/efeitos dos fármacos , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa 2/metabolismo , Angiotensina II/farmacologia , Animais , Bradicinina/farmacologia , Relação Dose-Resposta a Droga , Masculino , Artérias Mesentéricas/metabolismo , Veias Mesentéricas/metabolismo , Ratos , Ratos WistarRESUMO
The role of ATP on the modulation of noradrenaline release elicited by electrical stimulation (100 pulses/8 Hz) was studied in the prostatic portion of rat vas deferens preincubated with [3H]noradrenaline. In the presence of P1 antagonists, the nucleotides 2-methylthioadenosine-5'-triphosphate (2-MeSATP), 2-methylthioadenosine 5'-diphosphate (2-MeSADP), ADP, and ATP decreased electrically evoked tritium overflow up to 44%, with the following order of potency: 2-MeSATP > 2-MeSADP > ADP > or = ATP. The P2Y antagonists reactive blue 2 (RB2) and 2-methylthioadenosine 5'-monophosphate (2-MeSAMP) increased, whereas the P2X antagonist pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) decreased evoked tritium overflow. The inhibitory effect of 2-MeSATP was antagonized by RB2 (10 microM) and by 2-MeSAMP (10 microM) but not by the selective P2Y1 receptor antagonist 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate (MRS 2179; 10 microM). When, besides P1 receptors, inhibitory P2Y receptors were blocked with RB2, alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-meATP), beta,gamma-imidoadenosine 5'-triphosphate (beta,gamma-imidoATP), beta,gamma-methyleneadenosine 5'-triphosphate (beta,gamma-meATP), 2-MeSATP, and ATP enhanced tritium overflow up to 140%, with the following order of potency: alpha,beta-meATP > 2-MeSATP = ATP = beta,gamma-meATP > or = beta,gamma-imidoATP. The facilitatory effects of alpha,beta-MeATP and beta,gamma-imidoATP were prevented by PPNDS. Under the same conditions, apyrase attenuated, whereas the ectonucleotidase inhibitor 6-N,N-diethyl-D-beta,gamma-dibromomethylene 5'-triphosphate enhanced tritium overflow, an effect that was prevented by PPNDS. In the prostatic portion of the rat vas deferens, endogenous ATP exerts a dual and opposite modulation of noradrenaline release: an inhibition through activation of P2Y receptors with a pharmacological profile similar to that of the P2Y12 and P2Y13 receptors and a facilitation through activation of P2X receptors with a pharmacological profile similar to that of P2X1 and P2X3, or PX2/P2X3 receptors.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Norepinefrina/metabolismo , Receptores Purinérgicos P2/metabolismo , Ducto Deferente/efeitos dos fármacos , Animais , Apirase/farmacologia , Masculino , Ratos , Ratos Wistar , Receptores Purinérgicos P2X , Receptores Purinérgicos P2Y1 , Trítio , Ducto Deferente/metabolismoRESUMO
In the epididymal portion of rat vas deferens, facilitation of noradrenaline release mediated by adenosine A2A receptors, but not that mediated by beta2-adrenoceptors or by direct activation of adenylyl cyclase, was attenuated by blockade of alpha2-adrenoceptors and abolished by simultaneous blockade of alpha2-adrenoceptors, adenosine A1 and P2Y receptors. The adenosine A2A receptor-mediated facilitation was not changed by inhibitors of protein kinase A, protein kinase G or calmodulin kinase II but was prevented by inhibition of protein kinase C with chelerythrine or bisindolylmaleimide XI. Activation of protein kinase C with phorbol 12-myristate 13-acetate caused a facilitation of noradrenaline release that was abolished by bisindolylmaleimide XI and reduced by antagonists of alpha2-adrenoceptors, adenosine A1 and P2Y receptors. Activation of adenosine A2A receptors attenuated the inhibition of noradrenaline release mediated by the presynaptic inhibitory receptors. This effect was mimicked by phorbol 12-myristate 13-acetate and prevented by bisindolylmaleimide XI. It is concluded that adenosine A2A receptors facilitate noradrenaline release by a mechanism that involves a protein kinase C-mediated attenuation of effects mediated by presynaptic inhibitory receptors, namely alpha2-adrenoceptors, adenosine A1 and P2Y receptors.