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+)](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.
Poburko
Damon
D
Department of Anesthesiology, Pharmacology & Therapeutics, University of British Columbia, Vancouver, Canada V6T 1Z1; Child & Family Research Institute, Vancouver, Canada V5Z 4H4. Damon.Poburko@medecine.unige.ch
Potter
Kathryn
K
van Breemen
Edo
E
Fameli
Nicola
N
Liao
Chiu-Hsiang
CH
Basset
Olivier
O
Ruegg
Urs T
UT
van Breemen
Cornelis
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000166:cytology; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002478:Cells, Cultured; D003600:Cytosol / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006973:Hypertension / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D011687:Purines / Q000378:metabolism; D051381:Rats; D011983:Receptors, Purinergic / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*
D002021:Buffers; D015220:Calcium Channels; D011687:Purines; D011983:Receptors, Purinergic; D019831:Sodium-Calcium Exchanger; C034703:mitochondrial calcium uniporter; D049452:Green Fluorescent Proteins; D000331:Aequorin; D000255:Adenosine Triphosphate; D012964:Sodium; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium