Analysis of oxidation of the membrane potential probe, Di-S-C 3(5), during granulocyte activation

Abstract

Measurement of membrane potential with the fluorescent probe, Di-S-C 3(5), suggests that the chemotactic factor, FMLP causes a rapid depolarization followed by an apparent hyperpolarization of human granulocytes. This apparent hyperpolarization seems to be an artifact due to oxidation of Di-S-C 3(5) by a reactive product of the H 2O 2 and myeloperoxidase secreted from activated granulocytes. The artifactual decrease in fluorescence can be prevented by addition of a H 2O 2 scavenger, catalase, or an antioxidant, ascorbate. The calcium ionophore, A23187, causes depolarization but no hyperpolarization of human granulocytes because of its failure to stimulate the release of H 2O 2. Ouabain prevents the delayed hyperpolarization in response to FMLP. Ouabain acts via the Na-K pump to inhibit FMLP-stimulated release of peroxidase from human granulocytes. We conclude that assays of membrane potential should be conducted in the presence of 1 × 10 −4 M ascorbate to prevent oxidation of the fluorescent probe, Di-S-C 3(5).