title: Effects of recombinant human granulocyte-macrophage colony-stimulating factor on intracellular pH in mature granulocytes.
abstract: We studied the effects of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSFrh) on the internal pH of granulocytes using the fluorescent probe BCECF. GM-CSFrh did not directly alter the resting pH of granulocytes isolated from the peripheral blood; however, when the cells were preincubated for 90 minutes with the growth factor and then activated with the chemotactic peptide N-formyl met leu phe (fMLP), they exhibited both an acceleration in the initial rate of acidification and a marked delay in realkalinization. The kinetic changes both in initial acidification and in subsequent realkalinization induced by GM-CSFrh priming were not prevented by protein synthesis inhibitors and were observed in granulocytes harvested from patients with both sex-linked and autosomal recessive chronic granulomatous disease (CGD). By directly quantitating H+ ion secretion, by monitoring the effects of sodium repletion on intracellular pH, and through use of the sodium channel inhibitors amiloride and dimethyl amiloride and the Na+/K+-ATPase inhibitor ouabain, we showed that the altered kinetics of intracellular acidification and alkalinization following fMLP stimulation of GM-CSFrh-primed granulocytes could not be accounted for by changes in transmembrane proton exportation regulated by the Na+/H+ antiport channel. Although the initial acidification following fMLP was abrogated by 2-deoxy-D-glucose in both GM-CSFrh-pretreated and GM-CSFrh-untreated granulocytes, retardation of the subsequent phase of alkalinization was observed in GM-CSFrh-primed cells even after inhibition of both glycolytic and mitochondrial metabolism. Our data indicate that the increased cytosolic acidification following fMLP stimulation in granulocytes "primed" with GM-CSFrh does not result from disordered proton excretion but instead from increased release of intracellular free acid which is only partially coupled to glucose catabolism or to the generation of superoxide anion (O2-).
authors.affiliation: Department of Medicine, Boston City Hospital, MA.
authors.forename: R
authors.initials: R
authors.lastname: Sullivan
authors.forename: J D
authors.initials: JD
authors.lastname: Griffin
authors.forename: J
authors.initials: J
authors.lastname: Wright
authors.forename: D A
authors.initials: DA
authors.lastname: Melnick
authors.forename: J L
authors.initials: JL
authors.lastname: Leavitt
authors.forename: J P
authors.initials: JP
authors.lastname: Fredette
authors.forename: J H
authors.initials: JH
authors.lastname: Horne
authors.forename: C A
authors.initials: CA
authors.lastname: Lyman
authors.forename: K G
authors.initials: KG
authors.lastname: Lazzari
authors.forename: E R
authors.initials: ER
authors.lastname: Simons
mesh_terms: D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000276:immunology; D002352:Carrier Proteins / Q000378:metabolism; D003115:Colony-Stimulating Factors / Q000494:pharmacology*; D003600:Cytosol / Q000502:physiology; D006019:Glycolysis / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006098:Granulocytes / Q000187:drug effects* / Q000502:physiology; D006133:Growth Substances / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000187:drug effects; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000502:physiology; D010042:Ouabain / Q000494:pharmacology; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000502:physiology; D011994:Recombinant Proteins / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003115:Colony-Stimulating Factors; D006133:Growth Substances; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D011994:Recombinant Proteins; D017923:Sodium-Hydrogen Exchangers; C039465:5-dimethylamiloride; D009240:N-Formylmethionine Leucyl-Phenylalanine; D010042:Ouabain; D000584:Amiloride; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D013755:Tetradecanoylphorbol Acetate