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

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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 Zn2+ , 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 A1 (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.