The effects of phorbol myristate acetate and chemotactic peptide on transmembrane potentials and cytosolic free calcium in mature granulocytes evolve sequentially as the cells differentiate.
We isolated myeloid precursors from human marrow and studied the effects of phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (fMLP) upon transmembrane potentials and cytosolic calcium ([Ca2+]i) as the cells matured. Using a panel of fluorescent probes, we found that membrane depolarization induced by PMA and fMLP in granulocytes, and elevation in [Ca2+]i stimulated by fMLP, were absent in myeloblasts. When we induced differentiation with granulocyte-macrophage colony-stimulating factors, we found that both ionic responses appeared at approximately the promyelocyte stage. By using di-O-C5(3), we detected an initial phase of fMLP-induced hyperpolarization which appeared ontogenetically earlier than depolarization and which could be evoked in mature granulocytes with lower concentrations of the ligand. Hyperpolarization was partially dependent on extracellular Na+, was abrogated by increasing the external K+ concentration, and was accompanied by mild acidification of the cytoplasm. Bordetella pertussis toxin abolished both hyperpolarization and depolarization. Our findings indicate that shifts in [Ca2+]i and membrane potential changes in response to PMA and fMLP evolve as granulocytes mature. In addition, transmembrane ionic fluxes induced by fMLP appear to be more complex than previously considered, involving at least two separable phases of membrane potential change.
D000634:Aminoquinolines; D052160:Benzothiazoles; D001854:Bone Marrow Cells; D002118:Calcium / Q000378:metabolism*; D002232:Carbocyanines; D002454:Cell Differentiation; D002462:Cell Membrane / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D006098:Granulocytes / Q000166:cytology / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000166:cytology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007377:Interleukin-3 / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D037342:Pertussis Toxin; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000634:Aminoquinolines; D052160:Benzothiazoles; D002232:Carbocyanines; D005456:Fluorescent Dyes; D007377:Interleukin-3; D010566:Virulence Factors, Bordetella; C012944:3,3'-dipropyl-2,2'-thiadicarbocyanine; D009240:N-Formylmethionine Leucyl-Phenylalanine; C018528:3,3'-dipentyl-2,2'-oxacarbocyanine; D012964:Sodium; D037342:Pertussis Toxin; D013755:Tetradecanoylphorbol Acetate; C035739:Quin2; D011188:Potassium; D002118:Calcium