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

Abstract

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+)](MT)) and the sub-plasmalemmal space ([Ca(2+)](subPM)) with targeted aequorins and in the bulk cytosol ([Ca(2+)](i)) with fura-2. Substitution of extracellular Na(+) by N-methyl-d-glucamine transiently increased [Ca(2+)](MT) ( approximately 2microM) and [Ca(2+)](subPM) ( approximately 1.3microM), which then decreased to sustained plateaus. In contrast, Na(+)-substitution caused a delayed and tonic increase in [Ca(2+)](i) (<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+)](subPM). These treatments also abolished the delay in the [Ca(2+)](i) response to 0Na(+) and increased its amplitude. Extracellular ATP (1mM) caused a peak and plateau in [Ca(2+)](i), 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(+)](i). 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+)](i) due to agonist-induced NCX reversal.

• Authors: Damon Poburko, Kathryn Potter, Edo van Breemen, Nicola Fameli, Chiu-Hsiang Liao, Olivier Basset, Urs T Ruegg, Cornelis van Breemen
• Journal: Cell calcium (Cell Calcium) Vol. 40 Issue 4 Pg. 359-71 (Oct 2006) ISSN: 0143-4160 [Print] Netherlands
• PMID: 16806462 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Buffers
• Calcium Channels
• Purines
• Receptors, Purinergic
• Sodium-Calcium Exchanger
• mitochondrial calcium uniporter
• Green Fluorescent Proteins
• Aequorin
• Adenosine Triphosphate
• Sodium
• Sarcoplasmic Reticulum Calcium-Transporting ATPases
• Calcium

Topics

• Adenosine Triphosphate (metabolism)
• Aequorin (genetics, metabolism)
• Animals
• Aorta (cytology)
• Buffers
• Calcium (metabolism)
• Calcium Channels (metabolism)
• Cells, Cultured
• Cytosol (chemistry)
• Green Fluorescent Proteins (genetics, metabolism)
• Hypertension (metabolism)
• Mitochondria (metabolism)
• Muscle, Smooth, Vascular (cytology)
• Myocytes, Smooth Muscle (cytology, metabolism)
• Purines (metabolism)
• Rats
• Receptors, Purinergic (metabolism)
• Sarcoplasmic Reticulum Calcium-Transporting ATPases (metabolism)
• Sodium (metabolism)
• Sodium-Calcium Exchanger (metabolism)