Effects of protein kinase and phosphatase inhibitors on slow shortening of guinea pig cochlear outer hair cells.
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 II (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 Ca2+ 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.
Minamino
M
M
Department of Pharmacology, Kansai Medical University, Moriguchi, Osaka 570, Japan.
Hara
M
M
Ohnishi
S
S
Irie
T
T
Yamashita
T
T
Minato
A
A
Inagaki
C
C
D000818:Animals; D048429:Cell Size / Q000187:drug effects; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006168:Guinea Pigs; D018072:Hair Cells, Auditory, Outer / Q000166:cytology / Q000187:drug effects*; D066298:In Vitro Techniques; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D013997:Time Factors
D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D010749:Phosphoprotein Phosphatases