Relative roles of Na+/H+ exchange and vacuolar‐type H+ ATPases in regulating cytoplasmic pH and function in murine peritoneal macrophages

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Abstract
Two distinct mechanisms have been shown to mediate cytoplasmic pH (pH i) recovery in acid‐loaded peritoneal macrophages (Mψs): 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 pHi and cell function. Measurements of Mψ pHi and superoxide (O2−) production in response to stimulation with 12‐O‐tetradecanoyl phorbol 13‐acetate (TPA) were made at physiological or acidic extracellular pH (pHo) levels. The V‐type H+ ATPase inhibitor, bafilomycin A1, and potent Na+/H+ exchange inhibitor, N‐ethyl‐N‐propylamino amiloride (EPA), were used to examine the contributions of these ion transporters to pHi regulation and cell function. At pHo 7.35, the complementary activities of the Na+/H+ antiport and the V‐type H+ ATPase mediate pHi homeostasis. At pHo 6.7, maintenance of pHi depends primarily on H+ ATPase activity: bafilomycin A1 reduced pHi from 6.8±0.02 in control cells to 6.59±0.01 (P+ ATPase‐activity in preserving pHi homeostasis at acidic extracellular oH levels was reflected by the impairment of O2− production at pHo 6.70 when H+ ATPase activity was inhibited: bafilomycin A1 reduced O2− production from 13.9±1.0 to 9.3±0.6 nmoles/106 cells/40 min, in control and bafilomycin A1‐treated cells, respectively (P≤0.05), while EPA had no effect. In subsequent studies, pHi was independently manipulated using the ionophore nigericin. Lowering pHi from 6.80 to 6.60 reduced O2− production from 15.3±1.8 to 9.8±1.6 nmoles/106 cells/40 min (P≤0.05), indicating that the cytoplasmic acidification resulting from inhibition of H+ ATPases at low pHo could account for the associated impairment of O2− production. In a more profoundly acidic environment (pHo 6.35), H+ ATPases remained active in regulating pHi, but could not preserve a sufficiently physiological pHi to supprt respiratory burst activity. V‐type H+ ATPases constitute the dominant mechanism by which the pHi of peritoneal Mψs is maintained in an acidic extracellular environment.