RESUMO
Nitric oxide (NO) production during hyposmotic stimulation in outer hair cells (OHCs) of the guinea pig cochlea was investigated using the NO sensitive dye DAF-2. Simultaneous measurement of the cell length and NO production showed rapid hyposmotic-induced cell swelling to precede NO production in OHCs. Hyposmotic stimulation failed to induce NO production in the Ca2+-free solution. L-NG-nitroarginine methyl ester (L-NAME), a non-specific NO synthase inhibitor and gadolinium, a stretch-activated channel blocker inhibited the hyposmotic stimulation-induced NO production whereas suramin, a P2 receptor antagonist did not. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor inhibited the hyposmotic stimulation-induced increase in the intracellular Ca2+ concentrations ([Ca2+]i) while L-NAME enhanced it. 1H-[1,2,4]oxadiazole[4,3a]quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase (PKG) mimicked effects of L-NAME on the Ca2+ response. Transient receptor potential vanilloid 4 (TRPV4), an osmo- and mechanosensitive channel was expressed in the OHCs by means of immunohistochemistry. 4alpha-phorbol 12,13-didecanoate, a TRPV4 synthetic activator, induced NO production in OHCs. These results suggest that hyposmotic stimulation can induce NO production by the [Ca2+]i increase, which is presumably mediated by the activation of TRPV4 in OHCs. NO conversely inhibits the Ca2+ response via the NO-cGMP-PKG pathway by a feedback mechanism.
Assuntos
Tamanho Celular , Células Ciliadas Auditivas Externas/metabolismo , Óxido Nítrico/metabolismo , Órgão Espiral/metabolismo , Transdução de Sinais , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Cálcio/metabolismo , Carbazóis/farmacologia , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , GMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Inibidores Enzimáticos/farmacologia , Gadolínio/farmacologia , Guanilato Ciclase/antagonistas & inibidores , Guanilato Ciclase/metabolismo , Cobaias , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Soluções Hipotônicas , Indóis/farmacologia , Cinética , Potenciais da Membrana , NG-Nitroarginina Metil Éster/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Órgão Espiral/citologia , Órgão Espiral/efeitos dos fármacos , Pressão Osmótica , Ésteres de Forbol/farmacologia , Potássio/metabolismo , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/metabolismo , S-Nitroso-N-Acetilpenicilamina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Cloreto de Sódio/metabolismo , Suramina/farmacologia , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/metabolismoRESUMO
Nitric oxide (NO) production during hyposmotic stimulation in outer hair cells (OHCs) of the guinea pig cochlea was investigated using the NO sensitive dye DAF-2. Simultaneous measurement of the cell length and NO production showed rapid hyposmotic-induced cell swelling to precede NO production in OHCs. Hyposmotic stimulation failed to induce NO production in the Ca(2+)-free solution. L-N(G)-nitroarginine methyl ester (L-NAME), a non-specific NO synthase inhibitor and gadolinium, a stretch-activated channel blocker inhibited the hyposmotic stimulation-induced NO production whereas suramin, a P2 receptor antagonist did not. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor inhibited the hyposmotic stimulation-induced increase in the intracellular Ca(2+) concentrations ([Ca(2+)](i)) while L-NAME enhanced it. 1H-[1,2,4]oxadiazole[4,3a]quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase (PKG) mimicked effects of L-NAME on the Ca(2+) response. Transient receptor potential vanilloid 4 (TRPV4), an osmo- and mechanosensitive channel was expressed in the OHCs by means of immunohistochemistry. 4alpha-phorbol 12,13-didecanoate, a TRPV4 synthetic activator, induced NO production in OHCs. These results suggest that hyposmotic stimulation can induce NO production by the [Ca(2+)](i) increase, which is presumably mediated by the activation of TRPV4 in OHCs. NO conversely inhibits the Ca(2+) response via the NO-cGMP-PKG pathway by a feedback mechanism.
