Role of nitric oxide on ATP-induced Ca 2+ signaling in outer hair cells of the guinea pig cochlea

Abstract

Recently, a negative feedback effect of nitric oxide (NO) on the adenosine 5′-triphosphate (ATP)-induced Ca 2+ response has been described in cochlear inner hair cells. We here investigated the role of NO on the ATP-induced Ca 2+ response in outer hair cells (OHCs) of the guinea pig cochlea using the NO-sensitive dye DAF-2 and Ca 2+-sensitive dye fura-2. Extracellular ATP induced NO production in OHCs, which was inhibited by l- N G-nitroarginine methyl ester ( l-NAME), a non-specific NO synthase (NOS) inhibitor, and suramin, a P2 receptor antagonist. ATP failed to induce NO production in the Ca 2+-free solution. S-nitroso- N-acetylpenicillamine (SNAP), a NO donor, enhanced the ATP-induced increase of the intracellular Ca 2+ concentrations ([Ca 2+] i), while l-NAME inhibited it. SNAP accelerated ATP-induced Mn 2+ quenching in fura-2 fluorescence, while l-NAME suppressed it. 8-Bromoguanosine-cGMP, a membrane permeable analog of cGMP, mimicked the effects of SNAP. 1H-[1,2,4]oxadiazole[4,3-a] quinoxalin-1-one, an inhibitor of guanylate cyclase and KT5823, an inhibitor of cGMP-dependent protein kinase inhibited the ATP-induced [Ca 2+] i increase. Selective neuronal NOS inhibitors, namely either 7-nitro-indazole or 1-(2-trifluoromethylphenyl) imidazole, mimicked the effects of l-NAME regarding both ATP-induced Ca 2+ response and NO production. Immunofluorescent staining of neuronal nitric oxide synthase (nNOS) in isolated OHCs showed the localization of nNOS in the apical region of OHCs. These results suggest that the ATP-induced Ca 2+ influx via a direct action of P2X receptors may be the principal source for nNOS activity in the apical region of OHCs. Thereafter, NO can be produced while conversely enhancing the Ca 2+ influx via the NO-cGMP-PKG pathway by a feedback mechanism.