Impairment of Store-Operated Ca 2+ Entry in TRPC4 −/− Mice Interferes With Increase in Lung Microvascular Permeability

Abstract

We investigated the possibility that the TRPC gene family of putative store-operated Ca 2+ entry channels contributes to the increase in microvascular endothelial permeability by prolonging the rise in intracellular Ca 2+ 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-1 agonist peptide (TFLLRNPNDK-NH 2 ) resulted in a prolonged Ca 2+ transient secondary to influx of Ca 2+ . Ca 2+ influx activated by thrombin was blocked by La 3+ (1 μmol/L). In TRPC4 −/− LECs, thrombin or TFLLRNPNDK-NH 2 produced a similar initial increase of intracellular Ca 2+ secondary to Ca 2+ store depletion, but Ca 2+ influx induced by these agonists was drastically reduced. The defect in Ca 2+ 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,c ), a measure of vascular permeability, by a factor of 2.8 in wt and 1.4 in TRPC4 −/− ; La 3+ (1 μmol/L) addition to wt lung perfusate reduced the agonist effect to that observed in TRPC4 −/− . These results show that TRPC4-dependent Ca 2+ entry in mouse LECs is a key determinant of increased microvascular permeability.