Journal
CIRCULATION RESEARCH
Volume 91, Issue 1, Pages 70-76Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/01.RES.0000023391.40106.A8
Keywords
mouse lung endothelial cells; thrombin-induced Ca2+ influx; TRPC4 knockout; lung microvascular permeability
Funding
- NHLBI NIH HHS [HL-45638, T32-HL-077829] Funding Source: Medline
- NIGMS NIH HHS [GM-58531] Funding Source: Medline
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We investigated the possibility that the TRPC gene family of putative store-operated Ca2+ entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca2+ signaling. Studies were made in wild-type (wt) and TRPC4 knockout (TRPC4(-/-)) mice and lung vascular endothelial cells (LECs) isolated from these animals. RT-PCR showed expression of TRPC1, TRPC3, TRPC4, and TRPC6 mRNA in wt LECs, but TRPC4 mRNA expression was not detected in TRPC4(-/-) LECs. We studied the response to thrombin because it is known to increase endothelial permeability by the activation of G protein-coupled proteinase-activated receptor-1 (PAR-1). In wt LECs, thrombin or PAR-I agonist peptide (TFLLRNPNDK-NH2) resulted in a prolonged Ca2+ transient secondary to influx of Ca2+. Ca2+ influx activated by thrombin was blocked by La3+ (1 mumol/L). In TRPC4(-/-) LECs, thrombin or TFLLRNPNDK-NH2, produced a similar initial increase of intracellular Ca2+ secondary to Ca2+ store depletion, but Ca2+ influx induced by these agonists was drastically reduced. The defect in Ca2+ influx in TRPC4(-/-) endothelial cells was associated with lack of thrombin-induced actin-stress fiber formation and a reduced endothelial cell retraction response. In isolated-perfused mouse lungs, the PAR-1 agonist peptide increased microvessel filtration coefficient (K-f,K-c), a measure of vascular permeability, by a factor of 2.8 in wt and 1.4 in TRPC4(-/-); La3+ (I mumol/L) addition to wt lung perfusate reduced the agonist effect to that observed in TRPC4(-/-). These results show that TRPC4-dependent Ca2+ entry in mouse LECs is a key determinant of increased microvascular permeability.
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