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Comparison of PAF- and fMLP-induced [Ca2+]i transients in human polymorphonuclear leukocytes
D Nowak1, P Bialasiewicz, A Antczak
1Department of Pneumology and Allergology, Medical University of Lodz, Poland.
Abstract:
Changes of [Ca2+]i in human polymorphonuclear leukocytes (PMNL) were studied. PMNL suspension was activated three times every 5 min with 10(-7) M PAF and fMLP. Both PAF and fMLP, induced three consecutive [Ca2+]i transients in PMNL suspended in medium with 1 mM Ca2+. The first Ca2+ response was a result of Ca2+ release from internal stores and the extracellular Ca2+ influx, while the second and third responses were completely dependent on Ca2+ influx from extracellular space. The contribution of Ca2+ from intracellular stores to the first PAF-induced Ca2+ response was about 1.4-fold lower in comparison with the first fMLP induced Ca2+ response (27 +/- 1 vs 37 +/- 6% (p < 0.05). Previous addition of PAF enhanced 3-fold (p < 0.001) the PMNL response to fMLP while cells pretreated with fMLP failed to increase their [Ca2+]i after challenge with PAF. PMNL from 40% of donors did not respond to PAF in the presence of 100 nM Ca2+. However, the cells responding to PAF as the cells treated with fMLP or cyclopiazonic acid released almost the entire Ca2+ from intracellular stores after challenge. Subtraction of mean [Ca2+]i transients in the presence of 100 nM Ca2+ from that obtained in medium with 1 mM Ca2+ showed that, in PMNL stimulated with PAF in contrast to the cells treated with fMLP, the onset of Ca2+ influx from extracellular space precedes Ca2+ release from intracellular stores. These results suggest that PAF-induced Ca2+ influx from extracellular space is at least partly independent of Ca2+ release from intracellular stores.
Insights
Platelet-activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (fMLP) both trigger calcium transients in human polymorphonuclear leukocytes (PMNL). PAF-induced calcium influx is partly independent of intracellular calcium release, unlike fMLP.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Human polymorphonuclear leukocytes (PMNL) play a critical role in immune responses.
- Intracellular calcium ([Ca2+]i) signaling is a key mechanism regulating PMNL functions.
- Platelet-activating factor (PAF) and formyl-methionyl-leucyl-phenylalanine (fMLP) are potent activators of PMNL.
Purpose of the Study:
- To investigate and compare the dynamics of intracellular calcium ([Ca2+]i) changes in human PMNL induced by PAF and fMLP.
- To elucidate the distinct mechanisms of calcium mobilization by PAF and fMLP in PMNL.
Main Methods:
- Human PMNL suspensions were stimulated sequentially with PAF and fMLP at defined concentrations and time intervals.
- Intracellular calcium ([Ca2+]i) transients were measured using calcium indicator dyes in PMNL suspended in media with varying calcium concentrations (1 mM Ca2+ and 100 nM Ca2+).
- The contribution of intracellular calcium release versus extracellular calcium influx was analyzed for both stimuli.
Main Results:
- Both PAF and fMLP induced three consecutive [Ca2+]i transients, with initial responses involving both intracellular release and extracellular influx, while subsequent responses depended solely on extracellular influx.
- The contribution of intracellular calcium stores to the first PAF-induced response was significantly lower than that of fMLP.
- PAF pretreatment potentiated the fMLP response, but fMLP pretreatment did not enhance the PAF response.
- Crucially, PAF-stimulated calcium influx in PMNL preceded intracellular calcium release, suggesting a degree of independence, unlike fMLP.
Conclusions:
- PAF and fMLP activate distinct calcium signaling pathways in human PMNL.
- PAF-induced calcium influx is at least partly independent of intracellular calcium release, a mechanism differing from fMLP.
- These findings highlight the differential regulation of calcium homeostasis by inflammatory mediators in PMNL, impacting immune cell function.