Rapid report: a novel technique for quantitative measurement of free Ca2+ concentration in rat heart mitochondria.
1. The free mitochondrial Ca2+ concentration ([Ca2+]m) in rat heart mitochondria was measured quantitatively by loading the mitochondria with fura-2 and then injecting them into Xenopus laevis oocytes. 2. When oocytes were incubated with a physiological solution, the free cytosolic Ca2+ concentration ([Ca2+]c) in the oocytes was 82 +/- 11 nM (n = 20, mean +/- s.e.m.) and the [Ca2+]m of the injected rat heart mitochondria was 116 +/- 10 nM (n = 18, mean +/- s.e.m.). 3. Inhibition of the oocyte endoplasmic reticular Ca2+-ATPase with thapsigargin produced a transient increase in averaged [Ca2+]c at sub-micromolar concentrations. 4. Injection of cardiac mitochondria blunted the peak and prolonged the duration of thapsigargin-induced [Ca2+]c transients as a result of Ca2+ sequestration by the cardiac mitochondria. 5. These results demonstrate that the present technique provides a new approach for studying [Ca2+]m regulation quantitatively under physiological environments. Furthermore, it clearly shows that cardiac mitochondria can modify the shape of thapsigargin-induced cytosolic Ca2+ pulses in Xenopus oocytes.
Sheu
S S
SS
Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester, Rochester, NY 14642-8711, USA. sheus@pharmacol.rochester.edu
Sharma
V K
VK
D000818:Animals; D002118:Calcium / Q000032:analysis*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes; D016257:Fura-2; D006575:Heterocyclic Compounds, 3-Ring; D066298:In Vitro Techniques; D018613:Microscopy, Confocal; D008929:Mitochondria, Heart / Q000737:chemistry*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D012430:Ruthenium Red / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014982:Xenopus laevis
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; C068483:rhod-2; D012430:Ruthenium Red; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium; D016257:Fura-2