Two distinct mechanisms have been shown to mediate cytoplasmic p H (pH,) recovery in acid-loaded peritoneal macrophages (M+s): Na+/H+ exchange and H + extrusion by vacuolar-type (V-type) H+ ATPases. The present studies examined the relative roles of these two systems in maintaining pH, and cell function.
Measurements of M+ pH, and superoxide (02-) production in response to stimulation with 12-0-tetradecanoyl phorbol 13-acetate (TPA) were made at physiological or acidic extracellular p H (pH,) levels. The V-type H+ ATPase inhibitor, bafilomycin A,, and the potent Na+/H+ exchange inhibitor, N-ethyl-N-propylamino amiloride (EPA), were used to examine the contributions of these ion transporters to pH, regulation and cell function. At pH, 7.35, the complementary activities of the Na+/H+ antiport and the V-type H' ATPase mediate pH, homeostasis. At pH, 6.7, maintenance of pH, depends primarily on H f ATPase activity: bafilomycin A, reduced pH, from 6.8 2 0.02 in control cells to 6.59 4 0.01 (P < 0.01) while EPA was without effect. The functional importance of V-type H+ ATPase-activity in preserving pH, homeostasis at acidic extracellular p H levels was reflected by the impairment of 0,production at pH,, 6.70 when H+ ATPase activity was inhibited: bafilomycin A, reduced 0,production from 13.9 & 1 .O to 9.3 * 0.6 nmolesilo" cells/40 min, in control and bafilomycin A,-treated cells, respectively (P < 0.05), while EPA had no effect. In subsequent studies, pH, was independently manipulated using the ionophore nigericin. Lowering pH, from 6.80 to 6.60 reduced 0,production from 15.3 2 1.8 to 9.8 * 1.6 nmoles/lO" cells/40 min ( P < 0.05), indicating that the cytoplasmic acidification resulting from inhibition of H+ ATPases at low pH, could account for the associated impairment of 0,production. In a more profoundly acidic environment (pH, 6.35), H + ATPases remained active in regulating pH,, but could not preserve a sufficiently physiological pH, to supprt respiratory burst activity. V-type H+