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) stimul ation was studied. Whole blood was taken by venous puncture from healthy hu man volunteers. PMNs were isolated, diluted, and incubated with 2 CIM fura- 2 AM. The cytosolic free calcium concentration, [Ca2+](i), in human neutrop hils was determined by microfluorometry. We found that the net area under t he 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, comple tely 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+ le vel elevation stimulated by PAF. Loperamide, a positive modulator for store -operated calcium (SOC) channels, elicited an increase in intracellular cal cium after the activation of SOC channels stimulated by fMLP or PAF. After the addition of guanosine 3',5'-cyclic monophosphate, N-2,2'-O-Dibutyryl-, sodium salt (db-cGMP), the initial increase of PAF- or fMLP-induced PMNs in tracellular Ca2+ fluorescences was well preserved, but the slope and the pe ak 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 resulte d from intracellular stores release. The initial mobilization of intracellu lar Ca2+ stores in PAF-stimulated signals is mediated by protein kinase C ( PKC) phosphorylation, but not in fMLP-stimulated route. SOC channels are pr esent and important in the fMLP- or PAF-induced PMNs Ca2+ influx. There was no apparent cross-regulation between PAF- and fMLP-stimulated intracellula r Ca2+ influx.