Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC254
The effect of inorganic phosphate on mitochondrial Ca2+ dynamics in single permeabilized ventricular myocytes of rat
J. Lee1, J. Ha1, J. Kwon1, H. Kim1, H. Baek1, C. Leem1
1. Department of Physiology, University of Ulsan College of Medicine, Seoul, Korea, Republic of.
Mitochondrial dysfunction affects cell viability because mitochondria are related to both necrosis and apoptosis following ischemia and reperfusion. Loss of ATP and increased ATP hydrolysis, impaired ionic homeostasis are recognized as key factors of mitochondrial damage in the generation of cell death. Mitochondria can absorb huge amount of Ca2+ by buffering action of inorganic phosphate (Pi). In this study, we would like to see the effect of inorganic phosphate on mitochondrial Ca2+ dynamics with newly developed quantitative method. We enzymatically isolated single ventricular myocytes of rat. All procedures were accorded with national legislation. Buthanedione monoxime was present to prevent contracture. We measured NADH, mitochondrial Ca2+ with Fura-2FF and mitochondrial membrane potential (ψm) with tetramethylrhodamine ethyl ester simultaneously. The application of 0.5 μM Ca2+ partially depolarized ψm and increased the mitochondrial matrix Ca2+. The application of 20 mM Pi only hyperpolarized ψm, and the addition of Ca2+ almost completely depolarized ψm rapidly. And also mitochondrial matrix Ca 2+ loading was substantially decreased. The K + substitution with N-methyl-D-glucamine or the application of tetraethyl ammonium partially slowed depolarization speed and these results suggests the depolarization may be caused by K+ dependent manner. The depolarization was not blocked by cyclosporine or 5-hydroxy decanoate, which implied that this was not related to mitochondrial permeability transition pore or mitochondrial ATP sensitive K+ channel activation. One of possible causes is Ca2+ -dependent K+ channel activation, however, it cannot explain all the effects. The adenylic nucleotides, ATP or ADP could protect this depolarization. In conclusions, matrix Ca2+ and Pi somehow cause the depolarization and this effect may be caused by the formation of calcium phosphate complex or cyclosporine-independent non specific pore opening. Pi plays an important role on mitochondrial Ca2+ dynamics.
Where applicable, experiments conform with Society ethical requirements