Platelet activating factor raises intracellular calcium ion concentration in macrophages

Abstract

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells >95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45° angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+](i)), increased as exogenous calcium ion concentration ([Ca2+](o)) was raised to 1 mM. At [Ca2+](o) ≃ 10 μM, [Ca2+](i) = 72 ± 14 nM (n = 26); at [Ca2+](o) = 1 mM, [Ca2+](i) = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals that can quench quin2. Addition of mouse α + β fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not changes [Ca2+](i). However, addition of plateket activating factor (PAF) (2-20 ng/ml) raised [Ca2+](i) by 480 nM within 1 min if [Ca2+](o) = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+](i) by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 μM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+](i) increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+](i) could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+](i) was 0.1 ng/ml. Response of [Ca2+](i) to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+](i) may be an early event in PAF activation of macrophages.