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

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