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.
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Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Nanda
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Curnutte
J T
JT
Grinstein
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D000595:Amino Acid Sequence; D000906:Antibodies; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004553:Electric Conductivity / Q000187:drug effects / Q000502:physiology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D015470:Leukemia, Myeloid, Acute; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis* / Q000302:isolation & purification; D019255:NADPH Oxidases; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D013050:Spectrometry, Fluorescence; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010455:Peptides; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate; D004121:Dimethyl Sulfoxide