Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal:: aberrations in human complex I deficiency

Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal: aberrations in human complex I deficiency. Am J Physiol Cell Physiol 291: C308-C316, 2006. First published March 22, 2006; doi:10.1152/ajpcell.00561.2005. - Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP](M)) and the rate of cytosolic Ca2+ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial Ca2+ indicator rhod-2 can be used to selectively buffer the Bk-induced increase in mitochondrial Ca2+ concentration ([Ca2+](M)) and, consequently, the Ca2+-stimulated increase in [ATP](M), thus allowing studies of how the increase in [ATP](M) and the cytosolic Ca2+ removal rate are related. Luminometry of healthy fibroblasts expressing either aequorin or luciferase in the mitochondrial matrix showed that rhod-2 dose dependently decreased the Bk-induced increase in [Ca2+](M) and [ATP](M) by maximally 80 and 90%, respectively. Digital imaging microscopy of cells coloaded with the cytosolic Ca2+ indicator fura-2 revealed that, in parallel, rhod-2 maximally decreased the cytosolic Ca2+ removal rate by 20%. These findings demonstrate that increased mitochondrial ATP production is required for accelerating cytosolic Ca2+ removal during stimulation with a Ca2+-mobilizing agonist. In contrast, complex I-deficient patient fibroblasts displayed a cytosolic Ca2+ removal rate that was already decreased by 40% compared with healthy fibroblasts. Rhod-2 did not further decrease this rate, indicating the absence of mitochondrial ATP supply to the cytosolic Ca2+ pumps. This work reveals the usefulness of rhodamine-based Ca2+ indicators in examining the role of intramitochondrial Ca2+ in mitochondrial (patho) physiology.