DEVELOPMENT OF A H-SELECTIVE CONDUCTANCE DURING GRANULOCYTIC DIFFERENTIATION OF HL-60 CELLS()

The NADPH oxidase is one of the main microbicidal systems of granulocy tes. Stimulation of the oxidase during infection leads to a burst of m etabolic acid generation. Potentially deleterious cytosolic acidificat ion is prevented by the simultaneous activation of homeostatic H+ extr usion mechanisms, including a recently described H+ conductance. Studi es in granulocytes from chronic granulomatous disease patients have su ggested a relationship between the oxidase and the H+ conductive pathw ay. In this report we compared the expression of the H+ conductance an d the NADPH oxidase during granulocytic differentiation of dimethyl su lfoxide-induced HL-60 cells. Patch-clamp determinations demonstrated t hat the H+-selective current detectable in differentiated HL-60 cells is virtually absent in uninduced cells. The H+ conductance was also es timated fluorimetrically, measuring changes in the cytosolic pH of sus pended cells. Imposition of an inward protonmotive force failed to ind uce significant cytosolic acidification. In contrast, a sizable conduc tive H+ extrusion was detected in acid-loaded differentiated cells, co nsistent with the rectifying properties of the current measured electr ophysiologically. By the spectroscopic method, the H+ conductance was not detectable in uninduced cells, developing gradually during granulo cytic differentiation. Development of the conductive pathway was found to parallel the biochemical and functional appearance of the NADPH ox idase. These findings suggest that the H+ extrusion mechanisms require d for the maintenance of the intracellular pH during granulocyte activ ation develop pari passu with the acid generating systems and suggest a functional and possibly structural association between the H+ conduc tance and the NADPH oxidase.