Development of a H(+)-selective conductance during granulocytic differentiation of HL-60 cells.

The NADPH oxidase is one of the main microbicidal systems of granulocytes. Stimulation of the oxidase during infection leads to a burst of metabolic acid generation. Potentially deleterious cytosolic acidification is prevented by the simultaneous activation of homeostatic H+ extrusion mechanisms, including a recently described H+ conductance. Studies in granulocytes from chronic granulomatous disease patients have suggested a relationship between the oxidase and the H+ conductive pathway. In this report we compared the expression of the H+ conductance and the NADPH oxidase during granulocytic differentiation of dimethyl sulfoxide-induced HL-60 cells. Patch-clamp determinations demonstrated that the H(+)-selective current detectable in differentiated HL-60 cells is virtually absent in uninduced cells. The H+ conductance was also estimated fluorimetrically, measuring changes in the cytosolic pH of suspended cells. Imposition of an inward protonmotive force failed to induce significant cytosolic acidification. In contrast, a sizable conductive H+ extrusion was detected in acid-loaded differentiated cells, consistent with the rectifying properties of the current measured electrophysiologically. By the spectroscopic method, the H+ conductance was not detectable in uninduced cells, developing gradually during granulocytic differentiation. Development of the conductive pathway was found to parallel the biochemical and functional appearance of the NADPH oxidase. These findings suggest that the H+ extrusion mechanisms required for the maintenance of the intracellular pH during granulocyte activation develop pari passu with the acid generating systems and suggest a functional and possibly structural association between the H+ conductance and the NADPH oxidase.

A pH-sensitive and voltage-dependent proton conductance in the plasma membrane of macrophages.

Phagocytes generate large amounts of metabolic acid during activation. Therefore, the presence of a conductive pathway capable of H+ extrusion has been suggested (Henderson, L. M., J. B. Chappell, and O. T. G. Jones. 1987. Biochemical Journal. 246:325-329). In this report, electrophysiological and fluorimetric methods were used to probe the existence of a H+ conductance in murine peritoneal macrophages. In suspended cells, recovery of the cytosolic pH (pHi) from an acid-load in Na+ and HCO3(-)-free medium was detectable in depolarizing but not in hyperpolarizing media. The rate of alkalinization was potentiated by the rheogenic ionophore valinomycin. These findings are consistent with the existence of a conductive H+ (equivalent) pathway. This notion was confirmed by patch-clamping and fluorescence ratio measurements of single adherent cells. When voltage was clamped in the whole-cell configuration, depolarizing pulses induced a sizable outward current which was accompanied by cytosolic alkalinization. Several lines of evidence indicate that H+ (equivalents) carry this current: (a) the conductance was unaffected by substitution of the major ionic constituents of the intra-and/or extracellular media, (b) the reversal potential of the tail currents approached the H+ equilibrium potential; and (c) the voltage-induced currents and pHi changes were both Zn2+ sensitive and had similar time course and potential dependence. The peak whole-cell current displayed marked outward rectification and was exquisitely H+ selective. At constant voltage, the H+ permeability was increased by lowering pHi but was inhibited by extracellular acidification. Together with the voltage dependence of the conductance, these features ensure that H+ extrusion can occur during activation, while potentially deleterious acid uptake is precluded. The properties of the conductance appear ideally suited for pHi regulation during phagocyte activation, because these cells undergo a sustained depolarization and an incipient acidification when stimulated. Comparison of the magnitude of the current with the amount of metabolic acid generated during macrophage activation indicates that the conductance is sufficiently large to contribute to the H+ extrusion required for maintenance of pHi.