Intracellular Ca super(2+) Stores Regulate Muscarinic Receptor Stimulation of Phospholipase C in Cerebellar Granule Cells

Muscarinic receptor activation of phosphoinositide phospholipase C (PLC) has been examined in rat cerebellar granule cells under conditions that modify intracellular Ca super(2+) stores. Exposure of cells to medium devoid of Ca super(2+) for various times reduced carbachol stimulation of PLC with a substantial loss (88%) seen at 30 min. A progressive recovery of responses was observed following the reexposure of cells to Ca super(2+)-containing medium (1.3 mM). However, these changes did not appear to result exclusively from changes in the cytosolic Ca super(2+) concentration ([Ca super(2+)] sub(i)), which decreased to a lower steady level ( similar to 25 nM decrease in 1-3 min after extracellular omission) and rapidly returned (within 1 min) to control values when extracellular Ca super(2+) was restored. Only after loading of the intracellular Ca super(2+) stores through a transient 1-min depolarization of cerebellar granule cells with 40 mM KCl, followed by washing in nondepolarizing buffer, was carbachol able to mobilize intracellular Ca super(2+). However, the same treatment resulted in an 80% enhancement of carbachol activation of PLC. In other experiments, partial depletion of the Ca super(2+) stores by pretreatment of cells with thapsigargin and caffeine resulted in an inhibition (18 and 52%, respectively) of the PLC response. Furthermore, chelation of cytosolic Ca super(2+) with BAPTA/AM did not influence muscarinic activation of PLC in either the control or predepolarized cells. These conditions, however, inhibited both the increase in [Ca super(2+)] sub(i) and the PLC activation elicited by 40 mM KCl and abolished carbachol-induced intracellular Ca super(2+) release in predepolarized cells. Overall, these results suggest that muscarinic receptor activation of PLC in cerebellar granule cells can be modulated by changes in the loading state of the Ca super(2+) stores.