Calcium movement and membrane potential changes in the early phase of neutrophil activation by phorbol myristate acetate: a study with ion-selective electrodes.

Abstract

To quantitate Ca movements and membrane potential changes in stimulated [bovine] neutrophils, net fluxes of Ca2+ and the lipophilic cation tetraphenyl phossphonium were measured by a very sensitive ion-selective electrode system. Activation of neutrophils by 3 .times. 10-8 M phorbol 12-myristate, 13-acetate induced a release of .apprx. 20% of total cell Ca, with an initial lag period of < 10 s. The Ca2+ outflux was markedly reduced in ATP-depleted cells and in the presence of a calmodulin inhibitor, thereby suggesting that it occurred by activation of the ATP-drived Ca2+ pump of the neutrophil plasmalemma. Activation of neutrophils also induced a transiently increased exchange of medium 45Ca with cell Ca, which was measurable a few seconds after cell exposure to the stimulant and peaked at .apprx. 40 s. Stimulation of neutrophils after attainment of steady-state accumulation of tetraphenyl phosphonium (resting potential of -67 mV) resulted in a marked depolarization, with a lag period of .apprx. 60 s. The rate and extent of depolarization were reduced by 40 and 65%, respectively, in a low Na+ medium but were not modified by an inhibitor of anion exchange across membranes. A high-K+ medium depolarized neutrophils without either modifying their resting oxidative metaoblism or impairing stimulability by the phorbol ester. Phorbol 12-myristate, which also exhibited no effect on the oxidative metabolism of neutrophils, did not induce Ca2+ extrusion and membrane potential changes. The causal relationship between Ca2+ mobilization, membrane potential changes and activation of neutrophil functions is discussed.