Biochemical and Biophysical Research Communications
Na+ entry via TRPC6 causes Ca2+ entry via NCX reversal in ATP stimulated smooth muscle cells
Section snippets
Materials and methods
Cell culture and transfection. Rat aortic smooth muscle cells (RASMC) were cultured as previously described [16]. Cells were stored in 90% DMEM 10% DMSO in liquid nitrogen, used between passages 8–13, and cultured at 37 °C in 95% O2/5% CO2. Cells were seeded on 12 mm culture-coated glass coverslips (VWR Scientific) treated with Matrigel (30× dilution, BD Sciences). Empty pcDNA3 vector or dominant-negative TRPC6 (dnTRPC6; C-myc epitope tagged) were transiently transfected into cells 24 h after
Ca2+-influx through receptor-operated channels and reverse-mode NCX
In these RASMC ATP activates metabotropic P2Y G-protein coupled receptors [2], [16]. Stimulation of RASMC with ATP (1 mM) induced a biphasic increase in [Ca2+]i consisting of a large transient increase followed by a plateau phase typical of activation of PLC coupled receptors (Fig. 1A). KB-R7943 (10 μM), a concentration that selectively inhibits reverse-mode NCX [1], [2], abolished the tonic phase of the [Ca2+]i increase (Figs. 1A and B) indicating that a major component of Ca2+ entry stimulated
Acknowledgments
This research was supported by a Grant (20R90431) to C. van Breemen from the Canadian Institutes of Health Research. Virginia S. Lemos thanks CNPq/Brazil (Conselho Nacional de Desenvolvimento Cientifico e Tecnológico) for financial support. Financial support for Damon Poburko was provided by the Michael Smith Foundation for Health Research and the Natural Sciences and Engineering Research Council of Canada.
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