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.
Similar content being viewed by others
References
Aswanikumar, S., Schiffmann, E., Corcoran, B.A., Wahl, S.M. 1976. Role of a peptidase in phagocyte chemotaxis.Proc. Nat. Acad. Sci. USA 73:2439
Baker, P.R., Meves, H., Ridgway, E.B. 1973. Effects of manganese and other agents on the calcium uptake that follows depolarization of squid axions.J. Physiol (London) 231:511
Baran, D.N., Ahmed, S.A. 1969. Intracellular concentrations of water and of the principle electrolytes determined by the analysis of isolated leukocytes.Clin. Sci. 37:205
Becker, E.L., Davis, A.T., Estensen, R.D., Quie, P.Q. 1972. Cytochalasin B. IV. Inhibition and stimulation of chemotaxis of rabbit and human polymorphonuclear leukocytes.J. Immunol. 108:396
Becker, E.L., Showell, H.J. 1972. The effect of Ca2 and Mg2+ on the chemotactic responsiveness and spontaneous motility of rabbit polymorphonuclear leukocytes.Z. Immunitaetsforsch. Exp. Klin. Immunol. 143:466
Becker, E.L., Showell, H.J., Henson, P.M., Hsu, L.S. 1974. The ability of chemotactic factors to induce lysosomal enzyme release. I. The characteristics of the release, the importance of surfaces, and the relation of enzyme release to chemotactive responsiveness.J. Immunol. 112:2047
Becker, E.L., Showell, H.J., Naccache, P.H., Sha'afi, R. 1978. Enzymes in granulocyte movement: Preliminary evidence for the involvement of Na+, K+ ATPase.In: Leukocyte Chemotaxis. J.I. Gallin and P.G. Quie, editors, p. 113. Raven Press, New York
Becker, E.L., Sigman, M., Oliver, J.M. 1979. Superoxide production induced in rabbit polymorphonuclear leukocytes by synthetic chemotactic peptides and A23187: The nature of the receptor and the requirement for Ca2+.Am. J. Pathol. 95: 81
Becker, E.L., Talley, J.V., Showell, H.J., Naccache, P.H., Sha'afi, R.I. 1978. Activation of the rabbit polymorphonuclear leukocyte membrane “Na+, K+”-ATPase by chemotactic factor.J. Cell Biol. 77:329
Bergmeyer, H.U., Brent, E., Hess, B. 1963. Lactic dehydrogenase.In: Methods of Enzymatic Analysis. H.U. Bergmeyer, editor. p. 736. Academic Press, New York
Boucek, M.M., Snyderman, R. 1976. Calcium influx requirement for human neutrophil chemotaxis: Inhibition with lanthanum chloride.Science 193:905
Boyum, A. 1968. Isolation of mononuclear cells and granulocytes from human blood.Scand. J. Clin. Lab. Invest. 97(Suppl.29:77
Burckhardt, G. 1977. Non-linear relationship between fluorescence and membrane potential.Biochim. Biophys. Acta 468:227
Cividalli, G., Nathan, D.G. 1974. Sodium and potassium concentration and transmembrane fluxes in leukocytes.Blood 43:861
Cochrane, D.E., Douglas, W.W. 1974. Calcium-induced extrusion of secretory granules (exocytosis) in mast cells expressed to 48/80 or the ionophores A-23187 and X537 A.Proc. Nat. Acad. Sci. USA 71:408
Cramer, E.B., Gallin, J.I. 1979. Localization of submembranous cations to the leading end of human neutrophils during chemotaxis.J. Cell Biol. 82:369
Deutsch, C.J., Holian, A., Holian, S.K., Daniele, R.P., Wilson, D.F. 1979. Transmembrane electrical and pH gradients across human erythrocytes and human peripheral lymphocytes.J. Cell. Physiol. 99:79
Dunham, P.B., Goldstein, I.M., Weissmann, G. 1974. Potassium and amino acid transport in human leukocytes exposed to phagocytic stimuli.J. Cell. Biol. 63:215
Gallin, E.K., Gallin, J.I. 1977. Interaction of chemotactic factors with human macrophages: Induction of transmembrane potential changes.J. Cell Biol. 75:277
Gallin, E.K., Seligmann, B., Gallin, J.I. 1979. Alteration of macrophage and monocyte membrane potential by chemotactic factor.In: The Third International Congress on the Mononuclear Phagocyte. R. Van Furth, editor. Martinus Nijhoff BV, The Hague (in press)
Gallin, E.K., Wiederhold, M.L., Lipsky, P.E., Rosenthal, A.S. 1975. Spontaneous and induced membrane hyperpolarizations in macrophages.J. Cell. Physiol. 86:653
Gallin, J.I., Clark, R.A., Kimball, H.R. 1973. Granulocyte chemotaxis: An improvedin vitro assay employing51Cr labelled granulocytes.J. Immunol. 110:233
Gallin, J.I., Durocher, J.R., Kaplan, A.P. 1975. Interaction of leukocyte chemotactic factors with the cell surface. I. Chemotactic factor-induced changes in human granulocyte surface charge.J. Clin. Invest. 55:967
Gallin, J.I., Gallin, E.K., Malech, H.L., Cramer, E.B. 1978. Structural and ionic events during leukocyte chemotaxis.In: Leukocyte chemotaxis. J.I. Gallin and P.G. Quie, editors, p. 123. Raven Press, New York
Gallin, J.I., Rosenthal, A.S. 1974. The regulatory role of divalent cations in human granulocyte chemotaxis: Evidence for an association between calcium exchanges and microtubule assembly.J. Cell Biol. 62:594
Goldstein, I.M., Brai, M., Osler, A.G., Weissmann, G. 1973. Lysosomal enzyme release from human leucoytes: Mediation by the alternate pathway of complement activation.J. Immunol. 111:33
Goldstein, I.M., Hoffstein, S., Gallin, J.I., Weissmann, G. 1973. Mechanisms of lysosmal enzyme release from human leukocytes: Microtubule assembly and membrane fusion induced by a component of complement.Proc. Nat. Acad. Sci. USA 70:2916
Hank, R.A., Ingraham, L.M., Baehner, R.L. 1979. Membrane fluidity in human and mouse Chediak-Higashi leukocytes.J. Clin. Invest. 64:138
Hladky, S.B., Rink, T.J. 1976. Potential difference and the distribution of ions across the human red blood cell membrane: A study of the mechanism by which the fluorescent cation, di-S−C3(5) reports membrane potential.J. Physiol. (London) 263:287
Hodgkin, A.L., Keynes, R.D. 1955. The potassium permeability of a giant nerve fiber.J. Physiol. (London) 128:61
Jasaitis, A.A., Kuliene, V.V., Skulachev, V.P. 1971. Anilino-naphthalenesulfonate fluorescence changes induced by non-enzymatic generation of membrane potential in mitochondria and submitochondrial particles.Biochim. Biophys. Acta 234:177
Klebanoff, S.J. 1979. Oxygen intermediates and the microbicidal event.In: The Third International Congress on the Mononuclear Phagocyte. R. Van Furth, editor (in press)
Korchak, H.M., Weissmann, G. 1978. Changes in membrane potential of human granulocytes antecede the metabolic responses to surface stimulation.Proc. Nat. Acad. Sci. USA 75:3818
Laris, P.C., Bahr, D.P., Ghaffee, R.R.J. 1975. Membrane potentials in mitochondrial preparations measured by means of a cyanine dye.Biochim. Biophys. Acta 376:415
Lichtman, M.A., Weed, R.I. 1970. Electrophoretic mobility and N-acetylneuraminic acid content of human normal and leukemic lymphocytes and granulocytes.Blood 35:12
Litwack, G. 1955. Photometric determination of lysozyme activity.Proc. Soc. Exp. Biol. Med. 89:401
Martin, P.L., Shain, W. 1979. High affinity transport of taurine and β-alanine and low affinity transport GABA by a single transport system in cultured gliama cells.J. Biol. Chem. (in press)
McCord, J.M., Fridovich, I. 1969. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein).J. Biol. Chem. 244:6049
McKinley, D., Meissner, G. 1978. Evidence for a K+, Na+ permeable channel in sarcoplasmic reticulum.J. Membrane Biol. 44:159
Montecucco, L., Pozzan, T., Rink, T. 1979. Dicarbocyanine fluorescent probes of membrane potential block lymphocyte capping, deplete cellular ATP and inhibit respiration of isolated mitochondria.Biochim. Biophys. Acta 552:552
Naccache, P.H., Showell, H.J., Becker, E.L., Sha'afi, R.I. 1977. Sodium, potassium and calcium transport across rabbit polymorphonuclear leukocyte membranes: Effect of chemotactic factor.J. Cell Biol. 73:428
Padday, J.F. 1968. Metachromasy of a thiocarbocyanine dye in aqueous solution: The formation of dimers and trimers.J. Physical Chem. 72:1259
Philo, R.D., Eddy, A.A. 1978. The membrane potential of mouse ascites-tumour cells studied with the fluorescent probe 3,3-dipropyloxadicarbocyanine.Biochem. J. 174:801
Schiffmann, E., Corcoran, B.A., Aswanikumar, S. 1978. Molecular events in the response of neutrophils to synthetic N-fMET chemotactic peptides: Demonstration of a specific receptor.In: Leukocyte Chemotaxis. J.I. Gallin and P.G. Quie, editors. p. 97. Raven Press, New York
Schuldiner, S., Kaback, H.R. 1976. Membrane potential and active transport in membrane vesicles fromEscherichia coli.Biochemistry 14:5451
Seligmann, B., Gallin, J.I. 1980. Secretagogue modulation of the response of human neutrophils to chemoattractants: Studies with a membrane potential sensitive cyanine dye.Mol. Immunol. (in press)
Seligmann, B., Gallin, E.K., Martin, D.L., Shain, W., Gallin, J.I. 1977. Evidence for membrane potential changes in human polymorphonuclear leukocytes during exposure to the chemotactic factor f-met-leu-phe as measured with the fluorescence dye dipentyloxacarbocyanine.J. Cell Biol. 75:103a
Showell, H.J., Becker, E.L. 1976. The effect of external K+ and Na+ on the chemotaxis of rabbit peritoneal neutrophils.J. Immunol. 116:99
Showell, J.J., Freer, R.J., Zigmond, S.H., Schiffmann, E., Asuanikumar, S., Corcoran, B., Becker, E.L. 1976. The structure-activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal enzyme secretion for neutrophils.J. Exp. Med. 143:1154
Showell, H.J., Naccache, P.H., Sha'afi, R.I., Becker, E.L. 1977. The effects of extracellular K+, Na+ and Ca++ on lysosomal enzyme secretion from polymorphonuclear leukocytes.J. Immunol. 119:804
Simons, T.J.B. 1979. Actions of a carbocyanine dye on calcium-dependent potassium transport in human red cell ghosts.J. Physiol. (London) 288:481
Sims, J., Waggoner, A.S., Wang, C.-H., Hoffman, J.R. 1974. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles.Biochemistry 3:3315
Talalay, P., Fishman, W.H., Huggins, C. 1946. Chromagenic substrates. II. Phenolphthalein glucuronic acid as substrate for the assay of glucuronidase activity.J. Biol. Chem. 166:757
Utsumi, K., Sugiyama, K., Miyahara, M., Naito, M., Auai, M., Inoue, M. 1977. Effect of concanavalin A on membrane potential of polymorphonuclear leukocyte monitored by fluorescent dye.Cell Struct. Funct. 2:203
Waggoner, A. 1976. Optical probes of membrane potential.J. Membrane Biol. 27:317
Ward, P.A., Becker, E.L. 1970. Potassium reversible inhibition of chemotaxis by ouabain.Life Sci. 9:355
Wilkinson, P.C., Allan, R.B. 1978. Assay systems for measuring leukocyte locomotion: An overview.In: Leukocyte Chemotaxis. J.I. Gallin and P.G. Quie, editors. p. 1. Raven Press, New York
Zigmond, S.H. 1978. Chemotaxis by polymorphonuclear leukocytes.J. Cell Biol. 77:269
Zilberstein, D., Schuldiner, S., Padan, E. 1979. Proton electrochemical gradient inEscherichia coli cells and its relation to active transport of lactose.Biochemistry 18:669
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01869194