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

Abstract

We analyzed Ca²⁺-permeating nonselective cation channels (NSCs) mediating thrombin-induced contraction of human umbilical vein endothelial cells (HUVECs). A Ca²⁺ chelater, BAPTA-AM (10μM), significantly inhibited the thrombin-induced contraction of HUVECs.Thrombin induced inward currents at -60mV in the presence of intracellular MgATP. Removal of extracellular Ca²⁺ significantly decreased the currents. A selective phospholipase C inhibitor, U73122 (1μM) but not its inactive analogue, U73343 (1μM) almost completely inhibited the currents. Neither a selective inhibitor of Ca²⁺-ATPase of endoplasmic reticulum, thapsigargin (1μM) nor a diacylglycerol analogue, 1-oleoyl-2-acetyl-glycerol (30μM) activated the currents. However, a selective protein kinase C inhibitor, bisindolylmaleimide I (500nM) significantly inhibited the currents. The thrombin-induced currents were significantly inhibited by SKF96365 (50μM) but not by La³⁺ (1mM), ruthenium red (10μM) or flufenamic acid (100μM). 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.