Interaction of chemotactic factors with human polymorphonuclear leukocytes: studies using a membrane potential-sensitive cyanine dye.
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-methionyl-leucyl-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 eye between the aqueous incubation medium and the cell and not dye aggregation, Chemotactically active concentrations of f-met-leu-phe (5 x 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+ (10 mM)-EGTA (10 mM) 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.
D000418:Albumins / Q000494:pharmacology; D000818:Animals; D002232:Carbocyanines / Q000378:metabolism*; D002630:Chemotactic Factors / Q000494:pharmacology*; D002634:Chemotaxis, Leukocyte; D004533:Egtazic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D009239:N-Formylmethionine / Q000031:analogs & derivatives / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009439:Neuraminidase / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects* / Q000502:physiology; D009842:Oligopeptides / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011804:Quinolines / Q000378:metabolism*; D051381:Rats; D014634:Valinomycin / Q000494:pharmacology
D000418:Albumins; D002232:Carbocyanines; D002630:Chemotactic Factors; D005456:Fluorescent Dyes; D009842:Oligopeptides; D011804:Quinolines; D014634:Valinomycin; D009239:N-Formylmethionine; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; C018528:3,3'-dipentyl-2,2'-oxacarbocyanine; D009439:Neuraminidase; D008274:Magnesium; D011188:Potassium