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 β-glucuronidase release and superoxide production

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 β-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-(β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H- imidazole hydrochloride (SKandF 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 OmV. NSC channels were substantially more permeable to Na⁺ than to Ca²⁺. SKandF 96365 inhibited fMLP- and ATP-stimulated currents with a half-maximal effect at about 3 μM. 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'-0-[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 β-glucuronidase release and O₂^- production were substantially reduced by replacement of extracellular CaCl₂ or NaCI by ethylenebis(oxyethylenenitrilo)tetra-acetic acid and choline chloride respectively. In the absence of Ca²⁺ and Na⁺, fMLP was ineffective. SKandF 96365 inhibited fMLP-induced β-glucuronidase release and O₂^- production in the presence of both Ca²⁺ and Na⁺, and in the presence of Ca²⁺ or Na⁺ alone. NaCI (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. Our results suggest that in Bt₂cAMP-differentiated HL-60 cells, (i) fMLP and ATP activate NSC channels permeable for Ca²⁺, Mn²⁺ and Na⁺, (ii) activation of NSC channels involves G-proteins and is independent of intracellular Ca²⁺ and protein kinases; (iii) Ca²⁺ and Na⁺ influxes are involved in activation of β-glucuronidase release and O_2- production; (iv) an increase in intracellular free Na⁺ concentration may enhance activation of G-proteins, leading, among other possible mechanisms, to signal amplification.