valinomycin and 3-3--dipentyl-2-2--oxacarbocyanine

Previous studies using membrane potential sensitive probes have provided evidence that chemotactic factors elicit membrane potential changes in normal human neutrophils (PMN). In addition to stimulation of PMN motility, chemotactic factors also stimulate degranulation and superoxide ion (O-2) generation and it has been suggested that alteration of membrane potential activates these events (Korchak, H. M., and G. Weissmann. 1978. Proc, Natl, Acad, Sci. U. S. A. 75: 3818--3822). To further define the inter-relationship of these functions, studies were done with two indirect probes of membrane potential, 3-3'-dipentyloxacarbocyanine and triphenylmethylphosphonium ion (TPMP+) using PMN from normal subjects, from patients with abnormal O-2 production (chronic granulomatous disease [CGD]), and from patients with defective degranulation and/or chemotaxis (Cheddiak-Higashi syndrome and patients with elevated immunoglobulin (Ig)E and recurrent staphylococcal infections). The stimuli used were the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and the secretagogues ionophore A23187 and phorbol myristate acetate (PMA). The results obtained with 3-3'-dipentyloxacarbocyanine and TPMP+ were comparable. The apparent membrane potential changes elicited by f-Met-Leu-Phe and PMA in normal PMN were reduced or entirely absent in PMN obtained from patients with CGD but normal in PMN from other patients. PMN from patients with CGD had normal calculated resting membrane potentials and normal responses elicited by the potassium ionophore valinomycin. The responses to calcium ionophore A23187 were only slightly impaired. The abnormality of the elicited response of CGD cells of f-Met-Leu-Phe and PMA could not be attributed to the absence of O-2, hydroxyl radical, singlet oxygen, or hydrogen peroxide acting on the probes. Instead this abnormality appears to be associated with a dysfunction in the normal molecular mechanism(s) stimulated upon neutrophil activation. The data suggest chemoattractant alteration of membrane potential in normal PMN is related to activation of oxidative metabolism but the relationship to chemotaxis and degranulation remains to be established.

Topics: Adolescent; Adult; Calcimycin; Carbocyanines; Chediak-Higashi Syndrome; Chemotactic Factors; Chemotaxis, Leukocyte; Child; Child, Preschool; Female; Granulomatous Disease, Chronic; Humans; In Vitro Techniques; Male; Membrane Potentials; Neutrophils; Onium Compounds; Superoxides; Tetradecanoylphorbol Acetate; Tetraphenylborate; Trityl Compounds; Valinomycin

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.

Topics: Albumins; Animals; Carbocyanines; Chemotactic Factors; Chemotaxis, Leukocyte; Egtazic Acid; Fluorescent Dyes; Humans; Magnesium; Membrane Potentials; N-Formylmethionine; N-Formylmethionine Leucyl-Phenylalanine; Neuraminidase; Neutrophils; Oligopeptides; Potassium; Quinolines; Rats; Valinomycin