title: Role of nitric oxide on ATP-induced Ca2+ 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 Ca2+ response has been described in cochlear inner hair cells. We here investigated the role of NO on the ATP-induced Ca2+ response in outer hair cells (OHCs) of the guinea pig cochlea using the NO-sensitive dye DAF-2 and Ca2+ -sensitive dye fura-2. Extracellular ATP induced NO production in OHCs, which was inhibited by L-NG-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 Ca2+ -free solution. S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, enhanced the ATP-induced increase of the intracellular Ca2+ concentrations ([Ca2+]i), while L-NAME inhibited it. SNAP accelerated ATP-induced Mn2+ 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 [Ca2+]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 Ca2+ 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 Ca2+ 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 Ca2+ influx via the NO-cGMP-PKG pathway by a feedback mechanism.
authors.affiliation: Hearing Research Laboratory, Department of Otolaryngology, Kansai Medical University, Fumizonocho 10-15, Moriguchi, Osaka 570-8507, Japan.
authors.forename: Jing
authors.initials: J
authors.lastname: Shen
authors.forename: Narinobu
authors.initials: N
authors.lastname: Harada
authors.forename: Hiroko
authors.initials: H
authors.lastname: Nakazawa
authors.forename: Toshishiko
authors.initials: T
authors.lastname: Kaneko
authors.forename: Masahiko
authors.initials: M
authors.lastname: Izumikawa
authors.forename: Toshio
authors.initials: T
authors.lastname: Yamashita
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D003051:Cochlea / Q000166:cytology*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D003952:Diagnostic Imaging / Q000379:methods; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005455:Fluorescent Antibody Technique / Q000379:methods; D006168:Guinea Pigs; D018072:Hair Cells, Auditory, Outer / Q000378:metabolism*; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors
chemical_list: D004791:Enzyme Inhibitors; D020030:Nitric Oxide Donors; C110881:S-nitro-N-acetylpenicillamine; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D000255:Adenosine Triphosphate; D010396:Penicillamine; D006152:Cyclic GMP; D002118:Calcium