Anti-TRPC4 polyclonal antibody

We investigated the role of tumor necrosis factor-alpha (TNF-alpha) in activating the store-operated Ca2+ channels in endothelial cells via the expression of transient receptor potential channel (TRPC) isoforms. We observed that TNF-alpha exposure of human umbilical vein endothelial cells resulted in TRPC1 mRNA and protein expression, whereas it had no effect on TRPC3, TRPC4, or TRPC5 expression. The TRPC1 expression was associated with increased Ca2+ influx after intracellular Ca2+ store depletion with either thrombin or thapsigargin. We cloned the 5'-regulatory region of the human TRPC1 (hTRPC1) gene which contained a TATA box and CCAAT sequence close to the transcription initiation site. We also identified four nuclear factor-kappaB (NF-kappaB)-binding sites in the 5'-regulatory region. To address the contribution of NF-kappaB in the mechanism of TRPC1 expression, we determined the effects of TNF-alpha on expression of the reporter luciferase after transfection of hTRPC1 promoter-luciferase (hTRPC1-Pro-Luc) construct in the human dermal microvascular endothelial cell line. Reporter activity increased >4-fold at 4 h after TNF-alpha challenge. TNF-alpha-induced increase in reporter activity was markedly reduced by co-expression of either kinase-defective IKKbeta kinase mutant or non-phosphorylatable IkappaB mutant. Treatment with NEMO-binding domain peptide, which prevents NF-kappaB activation by selectively inhibiting IKKgamma interaction with IKK complex, also blocked the TNF-alpha-induced TRPC1 expression. Thus, TNF-alpha induces TRPC1 expression through an NF-kappaB-dependent pathway in endothelial cells, which can trigger augmented Ca2+ entry following Ca2+ store depletion. The augmented Ca2+ entry secondary to TRPC1 expression may be an important mechanism of endothelial injury induced by TNF-alpha.

Sub-acute hypoxia causes pulmonary vasoconstriction (HPV) is associated with increased intracellular Ca2+ concentration ([Ca2+](i)) and contraction of pulmonary arterial smooth muscle cells (PASMCs) We previous have demonstrated that 15-hydroxyeicosatetraenoic acid (15-HETE). a metabolite of arachidonic acid by 15-lipoxygenase (15-LO), causes elevated [Ca2+](i) in PASMCs partly through Ca2+ entry via other than L-type Ca2+ channels In this study, we used SKF96365/La3+ (SOCC antagonists) and Nordihydroguiairetic acid (NDGA, a blockage of 15-LO) to examine the effect of 15-HETE on capacitative Ca2+ entry and activity/expression of store-operated Ca2+ channels (SOCCs) during sub-acute hypoxic procedure and the contribution of SOCCs on the maintenance of vascular tones The results showed that the 15-HETE induced constriction of PA rings from normoxic and sub-acute hypoxic rats can be abolished by SKI:96365 and La3+. Capacitative Ca2+ entry (CCE) was also enhanced in PASMCs cultured with 15-HETE under subacute hypoxic condition (3% O-2, 48 h) and incubation with NDGA in PASMCs can greatly suppress this enhancement. Moreover, TRPC1, not TRPC4 and TRPC6, m RNA and protein expression were increased in PASMCs during these procedures Meanwhile, the effect of 15-HETE on CCE and TRPC1 expression under sub-acute hypoxic cultivation were greatly suppressed in 15-LO knockdown PASMCs and PAs These results suggest that 15-HETE mediated HPV through increased TRPC1 expression, leading to enhanced CCE, contributing to the maintenance of vascular tone. (C) 2010 Elsevier Inc All rights reserved