Protamine induces elevation of cytosolic free Ca2+ in cultured porcine aortic endothelial cells

To test the hypothesis that protamine influences calcium movement in endothelial cells, we measured the concentration of intracellular free calcium ([Ca2+](i)) in cultured porcine aortic endothelial (PAE) cells in Krebs solution (2.5 mM Ca2+, pH 7.4) at 37 degrees C, by fura-2 fluorimetry. The basal [Ca2+](i) of PAE cells was 113 +/- 18 nM (n = 6). Protamine increased [Ca2+](i) in a concentration-dependent manner (EC50, the concentration having 50% of the maximum effect, 1.4 +/- 0.3 mu g mL(-1), n = 6). The response of PAE cells to 100 mu g mL(-1) protamine (330 +/- 80 nM, n = 6) was blocked by a Ca2+ chelator, 5 mM glycoletherdiaminetetraacetic acid (EGTA; 131 +/- 16 nM, n = 6), and by a non-selective Ca2+ channel blocker, 3 mM Co2+ (134 +/- 14 nM, n = 6). These results suggest that Ca2+ influx through cell-membrane Ca2+ channels is mainly responsible for the protamine-induced Ca2+ elevation.