Oxygen metabolism in cloned macrophage cell lines: glucose dependence of superoxide production, metabolic and spectral analysis.

Abstract The requirements of a cloned macrophage-like cell line, J774.16, for oxygen metabolism, and the nature of the defect in oxidative metabolism in a variant clone derived from it, J774.C3C, were studied. Upon stimulation with phorbol myristate acetate (PMA), the parental clone produced approximately 1 nmol O2-/min/10(6) cells, whereas the variant clone produced no detectable O2- under the same conditions. Sustained O2- production by J774.16 was totally dependent on extracellular glucose; in glucose-free medium, the cells initiated O2- production but could not sustain it. When cells were stimulated with PMA, glucose-C-1 oxidation of J774.16 cells increased 20-fold while that of J774.C3C remained at resting levels. O2- production in J774.16 cells was inhibited by some agents known to block mitochondrial electron transport before coenzyme Q, such as rotenone and tetrathiafulvalene, whereas antimycin A enhanced O2- production. A dissociation between O2- production and glucose-C-1 oxidation was observed when J774.16 was treated with certain metabolic inhibitors. Quinacrine, 2,4-dinitrophenol, chlorpromazine, and trifluoperazine inhibited O2- production completely under conditions in which glucose-C-1 oxidation was reduced only by 30%. Rotenone inhibited O2- production with no effect on glucose-C-1 oxidation whereas antimycin A augmented O2- production 50% but inhibited glucose oxidation by 20%. Glucose transport studies, with 2-deoxy-d-glucose, showed that the Km for glucose transport of both clones was about 1 mM, indicating that cells could effectively transport glucose even at low concentrations. The Vmax for glucose transport in both J774.16 and variant J774.C3C cells doubled after PMA stimulation, indicating that the variant was effectively stimulated by PMA, even though O2- was not produced. Similarly, PMA induced protein phosphorylation in both clones. No differences between clones J774.16 and J774.C3C in hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase, or glutathione peroxidase activities could be found. When dithionite-reduced and -oxidized difference spectra of plasma membranes of these clones were compared, comparable levels of b-type cytochrome were found in both clones. However, CO difference spectra indicated that CO was bound to a b-type cytochrome (presumed to be b-245) in clone J774.16 but not in J774.C3C. From these data we conclude that: 1) O2- production by 5774.16 cells is totally dependent on extracellular glucose: upon stimulation with PMA, 50% of incorporated glucose was consumed in glucose- C-1 oxidation, and 13%w as used for O2- production: ii) because PMA doubles the Vmax of the glucose receptor and induces a protein kinase activity in both clones, both are responsive to the stimulant; and 3) because all the enzyme activities associated with the hexose monophosphate shunt were comparable in the variant and parent cells, and cytochrome b was detected in both cells, but failed to bind CO in the variant clone, the defect in the 5774.C3C cells may be associated with a structural or functional abnormality of a b-type cytochrome involved in the generation of O2-.