Bradykinin is a potent vasoactive nonapeptide. It elicits a rise in cytosolic Ca<sup>2+</sup> (Ca<sup>2+</sup>)<sub>i</sub> in endothelial cells, resulting in Ca<sup>2+</sup>-dependent synthesis and release of endothelial vasodilators. In the present study, we investigated the mechanism of bradykinin-induced Ca<sup>2+</sup> influx in primary cultured rat aortic endothelial cells and in a mouse heart microvessel endothelial cell line (H5V). Bradykinin-induced Ca<sup>2+</sup> influx was resolved into capacitative Ca<sup>2+</sup> entry (CCE) and non-CCE. The non-CCE component was inhibited by a B2 receptor antagonist (HOE140; 1 µ M) and a phospholipase C (PLC) inhibitor (U73122; 10 µ M). The action of bradykinin could be mimicked by 1-oleoyl-2-acetyl-glycerol, an analogue of diacylglycerol (DAG), and by RHC80267, a DAG-lipase inhibitor. Immunoblots showed that TRPC6 was one of the main TRPC channels expressed in endothelial cells. Transfection of H5V cells with two siRNA constructs against TRPC6 both markedly reduced the TRPC6 protein level and, at the same time, decreased the percentage of cells displaying bradykinin-induced non-CCE. siRNA transfection also reduced the magnitude of non-CCE among the cells responding to bradykinin. Taken together, our data suggest that bradykinin-induced non-CCE is mediated via the B2-PLC pathway, and that DAG may be involved in this process. Further, TRPC6 is one of the important channels participating in bradykinin-induced non-CCE in endothelial cells.
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