Transient Receptor Potential Channel 6–Mediated, Localized Cytosolic [Na + ] Transients Drive Na + /Ca 2+ Exchanger–Mediated Ca 2+ Entry in Purinergically Stimulated Aorta Smooth Muscle Cells

Abstract

The Na + /Ca 2+ exchanger (NCX) is increasingly recognized as a physiological mediator of Ca 2+ influx and significantly contributes to salt-sensitive hypertension. We recently reported that Ca 2+ influx by the NCX (1) is the primary mechanism of Ca 2+ 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 2+ 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 2+ 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.