Puschner B, Schacht J
Kresge Hearing Research Institute, University of Michigan, Ann Arbor 48109-0506, USA.
Hear Res. 1997 Aug;110(1-2):251-8. doi: 10.1016/s0378-5955(97)00086-5.
Cochlear outer hair cells in vitro respond to elevation of intracellular calcium with slow shape changes over seconds to minutes ('slow motility'). This process is blocked by general calmodulin antagonists suggesting the participation of calcium/calmodulin-dependent enzymatic reactions. The present study proposes a mechanism for these reactions. Length changes of outer hair cells isolated from the guinea pig cochlea were induced by exposure to the calcium ionophore ionomycin. ATP levels remained unaffected by this treatment ruling out depletion of ATP (by activation of calcium-dependent ATPases) as a cause of the observed shape changes. Involvement of protein kinases was suggested by the inhibition of shape changes by K252a, a broad-spectrum inhibitor of protein kinase activity. Furthermore, the inhibitors ML-7 and ML-9 blocked the shape changes at concentrations compatible with inhibition of myosin light chain kinase (MLCK). KN-62, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), also attenuated the length changes. Inhibitors with selectivity for cyclic nucleotide-dependent protein kinases (H-89, staurosporine) were tested to assess potential additional contributions by such enzymes. The dose dependence of their action supported the notion that the most likely mechanism of slow motility involves phosphorylation reactions catalyzed by MLCK or CaMKII or both.
体外培养的耳蜗外毛细胞对细胞内钙升高的反应是在数秒到数分钟内发生缓慢的形态变化（“慢速运动性”）。这一过程被通用的钙调蛋白拮抗剂所阻断，提示钙/钙调蛋白依赖性酶促反应的参与。本研究提出了这些反应的一种机制。通过暴露于钙离子载体离子霉素来诱导从豚鼠耳蜗分离的外毛细胞的长度变化。这种处理对ATP水平没有影响，排除了ATP消耗（通过激活钙依赖性ATP酶）是观察到的形态变化的原因。广谱蛋白激酶活性抑制剂K252a对形态变化的抑制提示了蛋白激酶的参与。此外，抑制剂ML-7和ML-9在与抑制肌球蛋白轻链激酶（MLCK）相容的浓度下阻断了形态变化。Ca2+/钙调蛋白依赖性蛋白激酶II（CaMKII）的抑制剂KN-62也减弱了长度变化。测试了对环核苷酸依赖性蛋白激酶具有选择性的抑制剂（H-89、星形孢菌素），以评估此类酶可能的额外作用。它们作用的剂量依赖性支持了这样一种观点，即慢速运动性最可能的机制涉及由MLCK或CaMKII或两者催化的磷酸化反应。
