Olson James E, Li Guang-Ze, Wang Ling, Lu Luo
Department of Emergency Medicine, Wright State University School of Medicine, Cox Institute, Kettering, Ohio 45429, USA.
Glia. 2004 May;46(4):391-401. doi: 10.1002/glia.20014.
We examined the calmodulin dependence of anion channel activation during hypo-osmotic swelling in rat cerebral astrocytes. Control cells bathed in iso-osmotic (290 mOsm) phosphate-buffered saline (PBS) and recorded using a patch electrode containing 140 mM KCl increased membrane conductance threefold over basal levels after 12 min in hypo-osmotic (200 mOsm) PBS. Cells injected with monoclonal anticalmodulin antibody demonstrated no increase in membrane conductance during a subsequent exposure to hypo-osmotic PBS. In contrast, cells iontophoretically injected with monoclonal antiglial fibrillary acidic protein antibody or with anticalmodulin antibody absorbed with an excess of free calmodulin demonstrated an increase in conductance during hypo-osmotic exposure similar to that of control cells. Conductance in iso-osmotic conditions was unchanged by antibody injection. Similar results were obtained when using patch electrode and bath solutions containing chloride as the only cell permeant ion, indicating a calmodulin-dependent anion current is activated with this degree of hypo-osmotic treatment. Western blots confirmed the specificity of the anticalmodulin and antiglial fibrillary acidic protein antibodies used in this study for proteins of 17 and 51 kD, respectively. In addition, in vitro studies demonstrated inhibition of the calmodulin-dependent activation of phosphodiesterase by the anticalmodulin antibody. Thus, binding of this antibody to calmodulin causes functional inhibition of calmodulin activity. No change in the intensity or cellular distribution of calmodulin immunostaining was observed during 30 min of hypo-osmotic exposure. However, increased immunostaining for activated calmodulin kinase IIalpha was observed after 10 min of hypo-osmotic exposure, suggesting initiation of calmodulin-dependent processes by cell swelling. The data indicate calmodulin activity is critical for activation of volume-regulated anion channels in rat cerebral astrocytes.
我们研究了大鼠脑星形胶质细胞在低渗肿胀过程中阴离子通道激活对钙调蛋白的依赖性。将对照细胞置于等渗（290 mOsm）磷酸盐缓冲盐水（PBS）中，并用含有140 mM KCl的膜片电极进行记录，在低渗（200 mOsm）PBS中孵育12分钟后，膜电导比基础水平增加了三倍。注射单克隆抗钙调蛋白抗体的细胞在随后暴露于低渗PBS期间，膜电导没有增加。相反，经离子导入注射单克隆抗胶质纤维酸性蛋白抗体或注射用过量游离钙调蛋白吸收的抗钙调蛋白抗体的细胞，在低渗暴露期间电导增加，与对照细胞相似。在等渗条件下，抗体注射对电导没有影响。当使用仅含氯离子作为唯一细胞通透离子的膜片电极和浴液时，也得到了类似的结果，表明在这种程度的低渗处理下，激活了一种钙调蛋白依赖性阴离子电流。蛋白质免疫印迹证实了本研究中使用的抗钙调蛋白和抗胶质纤维酸性蛋白抗体分别对17 kD和51 kD蛋白质的特异性。此外，体外研究表明抗钙调蛋白抗体抑制了钙调蛋白依赖性磷酸二酯酶的激活。因此，该抗体与钙调蛋白的结合导致钙调蛋白活性的功能抑制。在低渗暴露30分钟期间，未观察到钙调蛋白免疫染色强度或细胞分布的变化。然而，在低渗暴露10分钟后，观察到活化的钙调蛋白激酶IIα的免疫染色增加，提示细胞肿胀引发了钙调蛋白依赖性过程。数据表明钙调蛋白活性对于大鼠脑星形胶质细胞中容积调节性阴离子通道的激活至关重要。
