Protamine Induces Elevation of Cytosolic Free Ca2+ in Cultured Porcine Aortic Endothelial Cells

Abstract

Abstract 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 (2-5mM Ca2+, pH7.4) at 37°C, by fura-2 fluorimetry. The basal [Ca2+]i of PAE cells was 113 ± 18 nM (n − 6). Protamine increased [Ca2+]iin a concentration-dependent manner (EC50, the concentration having 50% of the maximum effect, 1.4 ± 0.3 μg mL−1, n = 6). The response of PAE cells to 100 μg 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.