ABSTRACT
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.
Subject(s)
Cell Size , Hair Cells, Auditory, Outer/metabolism , Nitric Oxide/metabolism , Organ of Corti/metabolism , Signal Transduction , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Animals , Calcium/metabolism , Carbazoles/pharmacology , Cell Size/drug effects , Cells, Cultured , Cyclic GMP/metabolism , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic GMP-Dependent Protein Kinases/metabolism , Enzyme Inhibitors/pharmacology , Gadolinium/pharmacology , Guanylate Cyclase/antagonists & inhibitors , Guanylate Cyclase/metabolism , Guinea Pigs , Hair Cells, Auditory, Outer/drug effects , Hypotonic Solutions , Indoles/pharmacology , Kinetics , Membrane Potentials , NG-Nitroarginine Methyl Ester/pharmacology , Nitric Oxide Donors/pharmacology , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Organ of Corti/cytology , Organ of Corti/drug effects , Osmotic Pressure , Phorbol Esters/pharmacology , Potassium/metabolism , Purinergic P2 Receptor Antagonists , Receptors, Purinergic P2/metabolism , S-Nitroso-N-Acetylpenicillamine/pharmacology , Signal Transduction/drug effects , Sodium Chloride/metabolism , Suramin/pharmacology , TRPV Cation Channels/agonists , TRPV Cation Channels/metabolismABSTRACT
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.
