Mechanism of Non-Capacitative Ca²⁺ Influx in Response to Bradykinin in Vascular Endothelial Cells

Abstract

Bradykinin is a potent vasoactive nonapeptide. It elicits a rise in cytosolic Ca²⁺ (Ca²⁺)_i in endothelial cells, resulting in Ca²⁺-dependent synthesis and release of endothelial vasodilators. In the present study, we investigated the mechanism of bradykinin-induced Ca²⁺ influx in primary cultured rat aortic endothelial cells and in a mouse heart microvessel endothelial cell line (H5V). Bradykinin-induced Ca²⁺ influx was resolved into capacitative Ca²⁺ entry (CCE) and non-CCE. The non-CCE component was inhibited by a B2 receptor antagonist (HOE140; 1 µM) and a phospholipase C (PLC) inhibitor (U73122; 10 µM). 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.

Altered Protein Kinase C Regulation of Pulmonary Endothelial Store- and Receptor-Operated Ca2+Entry after Chronic Hypoxia

“…The store-dependent arm of this signaling pathway is activated by IP 3 binding to IP 3 receptors, depleting endoplasmic reticulum Ca 2ϩ and stimulating SOC entry. However, there is considerable evidence that PLC-dependent DAG production mediates store-independent Ca 2ϩ influx Leung et al, 2006). The present study demonstrates that endothelial cells from small pulmonary arteries possess store-independent Ca 2ϩ entry elicited by either OAG or ATP application after CPA-induced store depletion (Fig.…”

Update on vascular endothelial Ca2+signalling: A tale of ion channels, pumps and transporters

“…Mouse pulmonary artery ECs [292] Rat aortic ECs [233,239] -(WB) +(WB) -(WB) +(WB) +(WB) Rat pulmonary artery ECs [311] +(RT-PCR) Rat mesenteric artery ECs [227,312] +(RT-PCR, WB, IHC)…”

“…TRPC3 and TRPC6 are widely expressed both in cultured ECs and in situ endothelium, while TRPC7 is far less abundant (Table 2). DAG stimulates TRPC3-and TRPC6-gated Ca 2+ entry into ECs exposed to a number of agonists, including VEGF [18,230,231] , ATP [232][233][234] , and bradykinin [232,235] . TRPC3-and TRPC6-induced Ca 2+ inflow underpin the stimulatory effect of VEGF on endothelial proliferation, migration and permeability [18,231,236] , while ATP exploits TRPC3 to activate NF-κB and increase vascular cell adhesion molecule-1 expression [237] .…”

CNGA2 Contributes to ATP-Induced Noncapacitative Ca²⁺ Influx in Vascular Endothelial Cells

“…This sequence is specific to CNGA2 only, and it does not cross-react with other CNG isoforms. These 2 strands were annealed and then cloned into a self-constructed siRNA expression vector pcDU6C [5]. pcDU6C contains a U6 RNA polymerase III promoter and a blasticidin resistance gene.…”

“…The ATP-induced [Ca 2+ ] i rise consists of a large initial transient followed by a small sustained phase [5]. The initial [Ca 2+ ] i rise is mostly due to Ca 2+ release from intracellular Ca 2+ stores, whereas the sustained elevation is due to Ca 2+ influx.…”