title: Electrogenic H+ pathway contributes to stimulus-induced changes of internal pH and membrane potential in intact neutrophils: role of cytoplasmic phospholipase A2.
abstract: The potential role of cytosolic phospholipase A2 (cPLA2) in the regulation of the electrogenic arachidonic acid (AA)-activatable H+ translocator of neutrophils was investigated. (1) The trifluoromethyl ketone analogue of arachidonate (AACOCF3), a newly developed selective blocker of cPLA2, inhibited both the N-formylmethionyl-leucylphenylalanine (fMLP)- and the phorbol-ester-induced rheogenic H+ efflux (K0.5 approximately 5 microM) and abrogated the stimulus-triggered release of AA from these cells. The drug failed to reduce the fMLP-evoked Ca2+ signal or protein tyrosine phosphorylation and did not affect the activity of protein kinase C. By using the patch-clamp technique we verified that the agent did not interfere with the voltage- and the pH-dependent activation of the H+ conductance of the peritoneal macrophages and therefore is not a direct blocker of the H+ channel itself. AACOCF3, however, slightly decreased the AA-induced stimulation of the H+ currents. We conclude that AA, liberated by the agonist-induced stimulation of cPLA2, is a direct activator of H+ conductance. (2) AACOCF3 did not inhibit superoxide generation, indicating that activation of cPLA2 may not be a prerequisite for turning on NADPH oxidase. (3) Since neither acid generation by the oxidase, nor the basal or stimulated Na+/H+ exchange (the predominant acid-eliminating mechanism) were influenced by the drug, we could use AACOCF3 to address whether the H+ channel in fact opens and plays any physiological role during activation of neutrophils. Stimulus-induced cytosolic alkalinization was smaller, whereas depolarization became larger, in the presence of AACOCF3. Stimulated H+ conductance therefore does contribute to intracellular pH (pHi) homoeostasis and membrane potential changes of intact neutrophils.
authors.affiliation: Department of Physiology and Laboratory of Cellular and Molecular Physiology, Semmelweis University of Medicine, Budapest 8, PO Box 259, H-1444, Budapest, Hungary.
authors.forename: K
authors.initials: K
authors.lastname: Suszták
authors.forename: A
authors.initials: A
authors.lastname: Mócsai
authors.forename: E
authors.initials: E
authors.lastname: Ligeti
authors.forename: A
authors.initials: A
authors.lastname: Kapus
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000097:blood; D001095:Arachidonic Acids / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008815:Mice, Inbred Strains; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019255:NADPH Oxidases / Q000097:blood; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D018408:Patch-Clamp Techniques; D010741:Phospholipases A / Q000097:blood*; D054467:Phospholipases A2; D019000:Phosphotyrosine / Q000032:analysis / Q000276:immunology; D017494:Proton Pumps / Q000097:blood*; D017923:Sodium-Hydrogen Exchangers / Q000097:blood; D013481:Superoxides / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; D017494:Proton Pumps; D017923:Sodium-Hydrogen Exchangers; C081565:arachidonyltrifluoromethane; D013481:Superoxides; D019000:Phosphotyrosine; D016718:Arachidonic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D019255:NADPH Oxidases; D010741:Phospholipases A; D054467:Phospholipases A2; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium