title: Formyl peptides and ATP stimulate Ca2+ and Na+ inward currents through non-selective cation channels via G-proteins in dibutyryl cyclic AMP-differentiated HL-60 cells. Involvement of Ca2+ and Na+ in the activation of beta-glucuronidase release and superoxide production.
abstract: In human neutrophils, the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) induces increases in the intracellular free Ca2+ concentration ([Ca2+]i) with subsequent activation of beta-glucuronidase release and superoxide (O2-) production. Results from several laboratories suggest that the increase in [Ca2+]i is due to activation of non-selective cation (NSC) channels. We studied the biophysical characteristics, pharmacological modulation and functional role of NSC channels in dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells. fMLP increased [Ca2+]i by release of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular space. fMLP also induced Mn2+ influx. Ca2+ and Mn2+ influxes were inhibited by 1-(beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365). Under whole-cell voltage-clamp conditions, fMLP and ATP (a purinoceptor agonist) activated inward currents characterized by a linear current-voltage relationship and a reversal potential near 0 mV. NSC channels were substantially more permeable to Na+ than to Ca2+. SK&F 96365 inhibited fMLP- and ATP-stimulated currents with a half-maximal effect at about 3 microM. Pertussis toxin prevented stimulation by fMLP of NSC currents and reduced ATP-stimulated currents by about 80%. Intracellular application of the stable GDP analogue, guanosine 5'-O-[2-thio]diphosphate, completely blocked stimulation by agonists of NSC currents. In excised inside-out patches, single channel openings with an amplitude of 0.24 pA were observed in the presence of fMLP and the GTP analogue, guanosine 5'-O-[3-thio]triphosphate. The bath solution contained neither Ca2+ nor ATP. The current/voltage relationship was linear with a conductance of 4-5 pS and reversed at about 0 mV. fMLP-induced beta-glucuronidase release and O2- production were substantially reduced by replacement of extracellular CaCl2 or NaCl by ethylenebis(oxyethylenenitrilo)tetra-acetic acid and choline chloride respectively. In the absence of Ca2+ and Na+, fMLP was ineffective. SK&F 96365 inhibited fMLP-induced beta-glucuronidase release and O2- production in the presence of both Ca2+ and Na+, and in the presence of Ca2+ or Na+ alone. NaCl (25-50 mM) enhanced the basal and absolute extent of fMLP-stimulated GTP hydrolysis of heterotrimeric regulatory G-proteins in HL-60 membranes. The order of effectiveness of salts in enhancing GTP hydrolysis was LiCl > KCl > NaCl > choline chloride.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D003994:Bucladesine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects; D004789:Enzyme Activation; D005453:Fluorescence; D016257:Fura-2; D019204:GTP-Binding Proteins / Q000502:physiology*; D005966:Glucuronidase / Q000378:metabolism*; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D007951:Leukemia, Myeloid / Q000201:enzymology / Q000378:metabolism* / Q000473:pathology; D008345:Manganese / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D012964:Sodium / Q000378:metabolism / Q000502:physiology*; D015222:Sodium Channels / Q000187:drug effects / Q000502:physiology*; D013268:Stimulation, Chemical; D013481:Superoxides / Q000378:metabolism*; D014407:Tumor Cells, Cultured
chemical_list: D015220:Calcium Channels; D007473:Ion Channels; D015222:Sodium Channels; D013481:Superoxides; D008345:Manganese; D009240:N-Formylmethionine Leucyl-Phenylalanine; D003994:Bucladesine; D000255:Adenosine Triphosphate; D012964:Sodium; D005966:Glucuronidase; D019204:GTP-Binding Proteins; D002118:Calcium; D016257:Fura-2