Transient receptor potential channel 6-mediated, localized cytosolic [Na+] transients drive Na+/Ca2+ exchanger-mediated Ca2+ entry in purinergically stimulated aorta smooth muscle cells

Abstract

The Na/Ca exchanger (NCX) is increasingly recognized as a physiological mediator of Ca influx and significantly contributes to salt-sensitive hypertension. We recently reported that Ca influx by the NCX (1) is the primary mechanism of Ca entry in purinergically stimulated rat aorta smooth muscle cells and (2) requires functional coupling with transient receptor potential channel 6 nonselective cation channels. Using the Na indicator CoroNa Green, we now directly observed and characterized the localized cytosolic [Na] ([Na]i) elevations that have long been hypothesized to underlie physiological NCX reversal but that have never been directly shown. Stimulation of rat aorta smooth muscle cells caused both global and monotonic [Na]i elevations and localized [Na]i transients (LNats) at the cell periphery. Inhibition of nonselective cation channels with SKF-96365 (50 μmol/L) and 2-amino-4-phosphonobutyrate (75 μmol/L) reduced both global and localized [Na]i elevations in response to ATP (1 mmol/L). This effect was mimicked by expression of a dominant negative construct of transient receptor potential channel 6. Selective inhibition of NCX-mediated Ca entry with KB-R7943 (10 μmol/L) enhanced the LNats, whereas the global cytosolic [Na] signal was unaffected. Inhibition of mitochondrial Na uptake with CGP-37157 (10 μmol/L) increased both LNats and global cytosolic [Na] elevations. These findings directly demonstrate NCX regulation by LNats, which are restricted to subsarcolemmal, cytoplasmic microdomains. Analysis of the LNats, which facilitate Ca entry via NCX, suggests that mitochondria limit the cytosolic diffusion of LNats generated by agonist-mediated activation of transient receptor potential channel 6-containing channels. © 2007 American Heart Association, Inc.