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

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