Differential regulation of Ca2+ influx by fMLP and PAF in human neutrophils: possible involvement of store-operated Ca2+ channel.
Calcium (Ca2+) influx into human polymorphonuclear cells (PMNs) in response to N-formyl-Met-Leu-Phe (fMLP) and platelet-activating factor (PAF) stimulation was studied. Whole blood was taken by venous puncture from healthy human volunteers. PMNs were isolated, diluted, and incubated with 2 microM fura-2 AM. The cytosolic free calcium concentration, [Ca2+]i, in human neutrophils was determined by microfluorometry. We found that the net area under the fMLP- or PAF-induced [Ca2+]i rise curve in Ca2+-free medium decreased to 75% or 30% of the area under the curve in Ca2+ medium. Treatment of PMNs with phorbol myristate acetate (PMA), a protein kinase C activator, completely abolished the intracellular Ca2+ level stimulated by PAF, but not the intracellular Ca2+ level stimulated by fMLP. Treatment of PMNs with PAF did not abolish the intracellular Ca2+ level elevation stimulated by fMLP. In addition, treatment of PMNs with fMLP did not abolish intracellular Ca2+ level elevation stimulated by PAF. Loperamide, a positive modulator for store-operated calcium (SOC) channels, elicited an increase in intracellular calcium after the activation of SOC channels stimulated by fMLP or PAF. After the addition of guanosine 3',5'-cyclic monophosphate, N2,2'-O-Dibutyryl-, sodium salt (db-cGMP), the initial increase of PAF- or fMLP-induced PMNs intracellular Ca2+ fluorescences was well preserved, but the slope and the peak height of fluorescence curves declined compared with the curves without db-cGMP. In conclusion, we found that PAF and fMLP regulate the Ca2+ influx of PMNs in different ways. Most of the PAF-induced [Ca2+]i rise resulted from Ca2+ influx, and most of the fMLP-induced [Ca2+]i elevation resulted from intracellular stores release. The initial mobilization of intracellular Ca2+ stores in PAF-stimulated signals is mediated by protein kinase C (PKC) phosphorylation, but not in fMLP-stimulated route. SOC channels are present and important in the fMLP- or PAF-induced PMNs Ca2+ influx. There was no apparent cross-regulation between PAF- and fMLP-stimulated intracellular Ca2+ influx.
Chen
L W
LW
Department of Surgery, Veterans General Hospital-Kaohsiung, Taiwan.
Shen
A Y
AY
Chen
J S
JS
Wu
S N
SN
D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D008139:Loperamide / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000494:pharmacology*; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D007093:Imidazoles; D010972:Platelet Activating Factor; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; D008139:Loperamide; D011493:Protein Kinase C; D006152:Cyclic GMP; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium