Thrombin Activates Ca2+-permeating Nonselective Cation Channels through Protein Kinase C in Human Umbilical Vein Endothelial Cells

We analyzed Ca2+-permeating nonselective cation channels (NSCs) mediating thrombin-induced contraction of human umbilical vein endothelial cells (HUVECs). A Ca2+ chelater, BAPTA-AM (10 μM), significantly inhibited the thrombin-induced contraction of HUVECs. Thrombin induced inward currents at -60 mV in the presence of intracellular MgATP. Removal of extracellular Ca2+ significantly decreased the currents. A selective phospholipase C inhibitor, U73122 (1 μM) but not its inactive analogue, U73343 (1 μM) almost completely inhibited the currents. Neither a selective inhibitor of Ca2+-ATPase of endoplasmic reticulum, thapsigargin (1 μM) nor a diacylglycerol analogue, 1-oleoyl-2-acetyl-glycerol (30 μM) activated the currents. However, a selective protein kinase C inhibitor, bisindolylmaleimide I (500 nM) significantly inhibited the currents. The thrombin-induced currents were significantly inhibited by SKF96365 (50 μM) but not by La3+ (1 mM), ruthenium red (10 μM) or flufenamic acid (100 μM). As assessed with RT-PCR, HUVECs expressed transient receptor potential (TRP) M4, 7, TRPV1, 2, 4, TRPC1, 4 and 6 subunits of NSCs. These results indicate that thrombin activates Ca2+-permeating NSCs containing TRPC4 through protein kinase C in HUVECs. Thus, drugs specifically inhibiting TRPC4-containing channels might be effective to control fatal diseases such as sepsis where thrombin mediates the vicious cycle between inflammation and coagulation.