Protein kinase C activation accelerates proton extrusion by vacuolar-type H +-ATPases in murine peritoneal macrophages

Abstract

The role of protein kinase C in the regulation of vacuolar-type H +-ATPase (V-ATPase) activity was studied in thioglycolate-elicited mouse peritoneal macrophages. Acid-loaded macrophages suspended in a Na +- and HCO − 3-free K +-medium containing Zn 2+ , a H +-conductance blocker, exhibited an initial intracellular pH recovery rate of 0.33 ± 0.04 pH/min ( n = 9). Pretreatment with 12- O-tetradecanoyl phorbol 13-acetate (TPA) or mezerein for as little as 3 min induced a marked (82%) increase in the initial pH recovery rate. Stimulation was prevented by the V-ATPase inhibitor, bafilomycin A 1 (200 nM) indicating that the effect of the protein kinase C agonists was via augmentation of proton pump activity. The protein kinase C inhibitor, staurosporine (100 nM) completely blocked the stimulatory effects of TPA and mezerein, suggesting involvement of protein kinase C. In keeping with this notion, the inactive analogue of TPA, 4-phorbol didecanoate did not stimulate recovery from an acid load. Extracellular pH determinations revealed that the observed increase in cytosolic pH recovery rate by the protein kinase C agonists was due to increased extrusion of protons from the cells, likely through V-ATPases located in the plasma membrane. Considered together, these data demonstrate regulation of plasmalemmal V-ATPase-mediated proton extrusion by protein kinase C.