Inhibition of monocyte-mediated antibody-dependent cellular cytotoxicity by ouabain: relationship between cytotoxic function, glycolysis, and intracellular cation homeostasis.

Abstract

Ouabain, a specific inhibitor of membrane Na+K+-ATPase, was used to study the relationship between monovalent cation, homeostasis and cytotoxic effector function. 5 X 10(-6) M ouabain completely inhibited monocyte monovalent cation active transport within 30 min but caused only 50% inhibition of monocyte-mediated antibody-dependent cellular cytotoxicity (MMADCC) in a subsequent 20-hr assay. Longer (6-hr) preincubation of monocytes in ouabain was required for nearly complete inhibition of cytotoxic function. The previously reported augmentation of monocyte glycolysis caused by the sensitizing antiserum was decreased by 71% in 5 X 10(-6) M ouabain, identifying monovalent cation transport as the cellular function utilizing a major portion of the ATP generated by this augmentation of glycolysis. The lag in inhibition of MMADCC indicated, however, that a delayed effect of ouabain (e.g., changes in intracellular [K+] and [Na+]), and not the rapid ouabain inhibition of cation transport, caused inhibition of cytotoxic function. Four additional findings support this interpretation: i) a 30-min preincubation in ouabain caused little inhibition of MMADCC in a 4-hr assay; ii) monocytes lost cytotoxic activity when incubated in medium (0.2 mEq/cytotoxic activity when incubated in medium (0.2 mEq/liter) that reduces intracellular [K+]; iii) the rapid recovery of cytotoxic activity after readjustment of extracellular [K+] to 6 mEq/liter was blocked by ouabain; iv) increasing membrane K+ permeability by valinomycin while blocking active transport by ouabain caused 50% inhibition under conditions in which neither agent alone significantly affected MMADCC. Thus, it is suggested that homeostasis of monocyte intracellular [K+] and [Na+], but not monovalent cation active transport per se, is required for cytotoxic function.