Mitochondria buffer NCX-mediated Ca2+-entry and limit its diffusion into vascular smooth muscle cells

The reverse-mode of the Na+/Ca2+-exchanger (NCX) mediates Ca2+-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca2+-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca2+] was measured in mitochondria ([Ca2+]MT) and the sub-plasmalemmal space ([Ca2+]subPM) with targeted aequorins and in the bulk cytosol ([Ca2+]i) with fura-2. Substitution of extracellular Na+ by N-methyl-d-glucamine transiently increased [Ca2+]MT (∼2 μM) and [Ca2+]subPM (∼1.3 μM), which then decreased to sustained plateaus. In contrast, Na+-substitution caused a delayed and tonic increase in [Ca2+]i (<100 nM). Inhibition of Ca2+-uptake by the sarcoplasmic reticulum (SR) (30 μM cyclopiazonic acid) or mitochondria (2 μM FCCP or 2 μM ruthenium red) enhanced the elevation of [Ca2+]subPM. These treatments also abolished the delay in the [Ca2+]i response to 0Na+ and increased its amplitude. Extracellular ATP (1 mM) caused a peak and plateau in [Ca2+]i, and only the plateau was inhibited by KB-R7943 (10 μM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na+]i. Mitochondria normally exhibited a transient elevation of [Ca2+] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10 μM) unmasked an agonist-induced increase in mitochondrial Ca2+-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca2+]i due to agonist-induced NCX reversal.