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Glucose- and phorbol myristate acetate-stimulated oxygen consumption and superoxide production in rat peritoneal macrophages is inhibited by dexamethasone.

ABSTRACT: 1. Rat peritoneal macrophages stimulated with phorbol 12-myristate 13-acetate (PMA) (40 nM) show an increase in the rate of oxygen consumption (measured with an O2 electrode) and the production of superoxide (measured by cytochrome c reduction), which are both dependent on the presence of exogenous glucose. There is a 1:1 correlation between the oxygen consumed and the superoxide produced over a range of glucose concentrations (0-10 mM). 2. Preincubation of macrophages with dexamethasone (1 microM) for 3 h significantly decreased the Vmax. for PMA-induced glucose-dependent oxygen consumption (P < 0.001) and glucose-dependent superoxide production (P < 0.001). However, dexamethasone did not significantly change the Km for glucose in either PMA-induced oxygen consumption or superoxide production. Dexamethasone is therefore a non-competitive inhibitor of PMA-stimulated glucose-dependent oxygen consumption (Ki = 0.83 +/- 0.09 microM) and superoxide generation (Ki = 0.87 +/- 0.09 microM). 3. The PMA-induced rate of oxygen consumption by macrophages was decreased at oxygen concentrations below approx. 15 microM. The Km of oxygen for PMA-induced oxygen consumption was 1.28 +/- 0.13 microM (n = 12), and this was not significantly different in the presence of dexamethasone; Km = 1.61 +/- 0.31 microM (n = 12). It is therefore concluded that in vivo macrophage superoxide production is not limited by external oxygen or glucose concentrations, even in hypoxic joints.

SUBMITTER: Rist RJ

PROVIDER: S-EPMC1132554 | biostudies-other | 1993 Apr

REPOSITORIES: biostudies-other

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Project description:2-D-Deoxyglucose (2-dGlc) uptake and accumulation into rat peritoneal macrophages was increased by colony-stimulating factor (mCSF) by stimulating the coupling between endofacial hexokinase activity and the sugar transporter. The evidence for this is as follows: (1) mCSF significantly decreased the Km for zero-trans uptake (P less than 0.05), without altering Vmax.; (2) the accumulation of free 2-dGlc was increased by mCSF (P less than 0.05); (3) mCSF retarded the rate of exit of accumulated free 2-dGlc. The mCSF-dependent increase in 2-dGlc uptake by macrophages was enhanced by preincubation of the cells in mCSF-free solution. The activity of the hexose monophosphate shunt (HMPS) measured by the differential uptake of 2-d[1-3H]Glc and 2-d[2,6-3H]Glc was not stimulated by mCSF. Also, in quiescent cells, superoxide production, as determined by cytochrome c reduction, was unaffected by mCSF. Phorbol myristate acetate (PMA; 40 nM) stimulated both the HMPS activity and superoxide production. Both these effects were dependent on the uptake of external sugar (2-dGlc). Incubation of the macrophages with mCSF enhanced the sugar transport and PMA-dependent stimulation of HMPS activity and superoxide production, indicating a role for mCSF in the 'priming' of macrophage functions. Both HMPS activity and superoxide production are entirely dependent on uptake of exogenous sugar, since the potent sugar-transport inhibitor cytochalasin B competitively inhibited 2-dGlc uptake, HMPS activity and superoxide generation in PMA-activated cells (Ki approximately 0.3 microM for all three processes). Over a wide range of 2-dGlc concentrations, 4 mol of superoxide were generated/mol of 2-dGlc metabolized in the HMPS pathway, indicating coupling between these processes. The Km of 2-d[2,6-3H]Glc uptake in PMA-treated cells was 0.45 +/- 0.07 mM, and Vmax. was 1.32 +/- 0.05 mumol.min-1.ml of cell water-1. It is evident that there is a large degree of slippage between HMPS activity and membrane-associated hexokinase activity, since the Km for HMPS activity was 0.06 +/- 0.02 mM and the Vmax. was 0.10 +/- 0.03 mumol.min-1.ml of cell water-1.

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Glucose- and phorbol myristate acetate-stimulated oxygen consumption and superoxide production in rat peritoneal macrophages is inhibited by dexamethasone.

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