Submitted on July 15, 2008
Revised on September 9, 2008
Accepted on September 10, 2008
Bradykinin regulates calpain and pro-inflammatory signaling through TRPM7-sensitive pathways in vascular smooth muscle cells
Alvaro Yogi1, Glaucia E Callera2, Rita Tostes, and Rhian M Touyz3*
1 Kidney reserach Centre, OHRI
2 Kidney Research Centre, OHRI
3 University of Ottawa
* To whom correspondence should be addressed. E-mail: rtouyz{at}uottawa.ca.
Transient receptor potential melastatin 7 (TRPM7) channels have recently been identified to be regulated by vasoactive agents acting through G protein-coupled receptors in vascular smooth muscle cells (VSMC). However, downstream targets and functional responses remain unclear. We investigated the subcellular localization of TRPM7 in VSMCs and questioned the role of TRPM7 in pro-inflammatory signaling by bradykinin. VSMCs from WKY rats were studied. Cell fractionation by sucrose gradient and differential centrifugation demonstrated that in bradykinin-stimulated cells, TRPM7 localized in fractions corresponding to caveolae. Immunofluorescence confocal microscopy revealed that TRPM7 distributes along the cell membrane, that it has a reticular-type intracellular distribution and that it co-localizes with flotillin-2, a marker of lipid rafts. Bradykinin increased expression of calpain, a TRPM7 target, and stimulated its cytosol:membrane translocation, an effect blocked by 2-APB (TRPM7 inhibitor) and U-73122 (PLC inhibitor), but not by chelerythrine (PKC inhibitor). Expression of pro-inflammatory mediators VCAM-1 and COX2 was time-dependently increased by bradykinin. This effect was blocked by Hoe-140 (B2 receptor blocker) and 2-APB. Our data demonstrate that in bradykinin-stimulated VSMCs, 1) TRPM7 is upregulated, 2) TRPM7 associates with cholesterol-rich microdomains, and 3) calpain and proinflammatory mediators, VCAM-1 and Cox2, are regulated in part via TRPM7- and PLC-dependent pathways through B2 receptors.. These findings identify a novel signaling pathway for bradykinin, which involves TRPM7. Such phenomena may play a role in bradykinin/B2 receptor-mediated inflammatory responses in vascular cells.
