Oscillations of Phospholipase C Activity Triggered by Depolarization and Ca Influx in Insulin-secreting Cells*

Phospholipase C (PLC) is a ubiquitous enzyme involved in the regulation of a variety of cellular processes. Its dependence on Ca is well recognized, but it is not known how PLC activity is affected by physiological variations of the cytoplasmic Ca concentration ([Ca ]i). Here, we applied evanescent wave microscopy to monitor PLC activity in parallel with [Ca ]i in individual insulin-secreting INS-1 cells using the phosphatidylinositol 4,5-bisphosphateand inositol 1,4,5-trisphosphate-binding pleckstrin homology domain from PLC 1 fused to green fluorescent protein (PHPLC 1-GFP) and the Ca indicator fura red. In resting cells, PHPLC 1GFP was located predominantly at the plasma membrane. Activation of PLC by muscarinic or purinergic receptor stimulation resulted in PHPLC 1-GFP translocation from the plasma membrane to the cytoplasm, detected as a decrease in evanescent wave-excited PHPLC 1-GFP fluorescence. Using this translocation as a measure of PLC activity, we found that depolarization by raising extracellular [K ] triggered activation of the enzyme. This effect could be attributed both to a rise of [Ca ]i and to depolarization per se, because some translocation persisted during depolarization in a Ca -deficient medium containing the Ca chelator EGTA. Moreover, oscillations of [Ca ]i resulting from depolarization with Ca influx evoked concentration-dependent periodic activation of PLC. We conclude that PLC activity is under tight dynamic control of [Ca ]i. In insulin-secreting -cells, this mechanism provides a link between Ca influx and release from intracellular stores that may be important in the regulation of insulin secretion.