|Budget Amount *help
¥3,000,000 (Direct Cost : ¥3,000,000)
Fiscal Year 1999 : ¥1,100,000 (Direct Cost : ¥1,100,000)
Fiscal Year 1998 : ¥800,000 (Direct Cost : ¥800,000)
Fiscal Year 1997 : ¥1,100,000 (Direct Cost : ¥1,100,000)
The intracellular mechanisms of slow shortening in isolated guinea pig cochlear outer hair cells were investigated using inhibitors and/or an activator of protein kinases and protein phosphatases. The slow shortening was induced by tetanic electrical field stimulation, and changes in the cell length, volume and intracellular Cl^- concentration were microscopically monitored using, a chloride-sensitive fluorescent dye. The slow shortening was inhibited by a calmodulin inhibitor, W-7, and a calcium calmodulin-dependent protein kinase 11 (CaMKII) inhibitor. KN-62. The inhibition by W-7 or KN-62, was abolished by the supplemented conductance of K^+ with valinomycin, Among the protein phosphatase inhibitors tested, a type 1 and 2A protein phosphatase inhibitor, calyculin A, inhibited the slow shortening. The inhibition by calyculin A was abolished by the increased Cl^- permeability, but neither by the increased K^+ conductance with valinomycin nor by the increased Ca^<2+> conductance with
A23187. A protein serine/threonine phosphatase activator, N-acetylsphingosine, inhibited the shortening, which was abolished by either valinomycin or a type 2A protein phosphatase inhibitor, okadaic acid, but not by calyculin A.These findings suggest the following signaling mechanisms in the slow shortening of outer hair cells : the K^+ channel opening is facilitated through protein phosphorylation by CaMKII and suppressed via okadaic acid-sensitive dephosphorylation, and the Cl^- channel opening depends on calyculin A-sensitive protein phosphatase activity.
Tetanic electrical field stimulation elicited a prominent contraction of isolated guinea pig cochlear inner hair cells. This contraction appeared 30-40 sec after field stimulation and lasted for about 1 min. Using a digital imaging microscope and the Cl^- sensitive fluorescence dye, N-(6-methoxyquinolyl) acetoethyl ester, the decrease in cell volume and intracellular Cl^- concentration were concomitant with the cell concentration. Cytochalasin B inhibited this event, suggesting that the contraction is mediated by actin. Less