Shrinkage-Induced Activation of Na⁺/H⁺ Exchange: Role of Cell Density and Myosin Light Chain Phosphorylation

Previously, we suggested that myosin light chain kinase (MLCK) is involved in shrinkage-induced activation of the Na ⁺ /H ⁺ exchanger in rat astrocytes. Here we have studied the effects of hyperosmotic exposure in C ₆ glioma cells, a common model for astrocytes. Shrinkage-induced activation of the Na ⁺ /H ⁺ exchanger in C ₆ cells is directly proportional to the degree of shrinkage, results in an alkaline shift in the pK' of the exchanger, is dependent on ATP, and is inhibited by ML-7 (an MLCK inhibitor) and by various calmodulin inhibitors. Cell shrinkage also results in increased phosphorylation of myosin light chain (MLC). Interestingly, shrinkage-induced activation of the exchanger does not occur in subconfluent C ₆ cells. However, phosphorylation of MLC still occurs in subconfluent cultures of C ₆ cells on shrinkage, suggesting that the lack of activation in these cells occurs at a point between MLC phosphorylation and Na ⁺ /H ⁺ exchange activation. The lack of activation of Na ⁺ /H ⁺ exchange in subconfluent C ₆ cells can be utilized to further elucidate the shrinkage-induced activation pathway.