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Interaction of chemotactic factors with human polymorphonuclear leukocytes: Studies using a membrane potential-sensitive cyanine dye

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Summary

Changes in the fluorescence intensity of the dye 3-3′ dipentyloxacarbocyanine were measured in suspensions of purified human peripheral blood polymorphonuclear leukocytes (PMNs) during exposure to the chemotactic factors N-formyl-methionylleucyl-phenylalanine (f-met-leu-phe) and partially purified C5a. Incubation of PMNs with dye resulted in a stable fluorescence reflecting the resting membrane potential of the cell. Exposure of PMNs to dye did not affect stimulated chemotaxis or secretion. The mechanism of cell-associated dye fluorescence involved solvent effects from partitioning of the dye between the aqueous incubation medium and the cell and not dye aggregation, Chemotactically active concentrations of f-met-leu-phe (5×10−9 m or greater) produced a biphasic response characterized as a decrease followed by an increase in fluorescence. No fluorescence response was seen in lysed PMNs, and no response was elicited by an inhibitor of f-met-leu-phe binding (carbobenzoxy-phenylalanyl-methionine). The ability of several other synthetic peptides to elicit a fluorescence response corresponded to their effectiveness as chemotactic agents. Although the first component of the response suggested a depolarization, it was not influenced by variation in the external concentration of sodium, potassium, chloride, or calcium, and could not be characterized as a membrane potential change. The second component of the response, which was inhibited by both Mg2+ (10mm)-EGTA (10mm) and high external potassium, was compatible with a membrane hyperpolarization. The data indicate that chemotactic factors produce changes in dye fluorescence which can, at least in part, be attributed to a hyperpolarizing membrane potential change occurring across the plasma membrane.

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  1. John I. Gallin

    Present address: Bacterial Disease Section, Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 20205, Bethesda, Maryland

Authors and Affiliations

  1. Neurobiology and Experimental Hematology Departments, Armed Forces Radiobiology Research Institute, 20014, Bethesda, Maryland

    Bruce E. Seligmann, Elaine K. Gallin, David L. Martin, William Shain & John I. Gallin

  2. Department of Chemistry, University of Maryland, 20742, College Park, Maryland

    Bruce E. Seligmann, Elaine K. Gallin, David L. Martin, William Shain & John I. Gallin

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Seligmann, B.E., Gallin, E.K., Martin, D.L. et al. Interaction of chemotactic factors with human polymorphonuclear leukocytes: Studies using a membrane potential-sensitive cyanine dye. J. Membrain Biol. 52, 257–272 (1980). https://doi.org/10.1007/BF01869194

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