Ca2+-dependent protein kinase-A modulation of the plasma membrane Ca2+-ATPase in parotid acinar cells

Cross-talk between cAMP and [Ca2+](i) signaling pathways represents a general feature that defines the specificity of stimulus-response coupling in a variety of cell types including parotid acinar cells. We have reported recently that cAMP potentiates Ca2+ release from intracellular stores, primarily because of a protein kinase A-mediated phosphorylation of type H inositol 1,4,5-trisphosphate receptors (Bruce, J. I. E., Shuttleworth, T. J. S., Giovannucci, D. R., and Yule, D. I. (2002) J. Biol. Chem. 277, 1340-1348). The aim of the present study was to evaluate the functional and molecular mechanism whereby cAMP regulates Ca2+ clearance pathways in parotid acinar cells. Following an agonist-induced increase in [Ca2+](i) the rate of Ca2+ clearance, after the removal of the stimulus, was potentiated substantially (similar to2-fold). by treatment with forskolin. This effect was prevented completely by inhibition of the plasma membrane Ca2+-ATPase (PMCA) with La. PMCA activity, when isolated pharmacologically, was also potentiated (similar to2-fold) by forskolin. Ca2+ uptake into the endoplasmic reticulum of streptolysin-O-permeabilized cells by sarco/endoplasmic reticulum Ca2+-ATPase was largely unaffected by treatment with dibutyryl cAMP. Finally, in situ phosphorylation assays demonstrated that PMCA was phosphorylated by treatment with forskolin but only in the presence of carbamylcholine (carbachol). This effect of forskolin was Ca2+-dependent, and protein kinase C-independent, as potentiation of PMCA activity and phosphorylation of PMCA by forskolin also occurred when [Ca2+](i) was elevated by the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid and was attenuated by pre-incubation with the Ca2+ chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The present study demonstrates that elevated cAMP enhances the rate of Ca2+ clearance because of a complex modulation of PMCA activity that involves a Ca2+-dependent step. Tight regulation of both Ca2+ release and Ca2+ efflux may represent a general feature of the mechanism whereby cAMP improves the fidelity and specificity of Ca2+ signaling.