Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal: aberrations in human complex I deficiency.
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.
Nijmegen Centre for Mitochondrial Disorders, Dept. of Pediatrics, Radboud Univ. Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
Henk-Jan
HJ
Visch
Werner J H
WJ
Koopman
Dimphy
D
Zeegers
Sjenet E
SE
van Emst-de Vries
Frank J M
FJ
van Kuppeveld
Lambertus W P J
LW
van den Heuvel
Jan A M
JA
Smeitink
Peter H G M
PH
Willems
D000255:Adenosine Triphosphate / Q000096:biosynthesis*; D001920:Bradykinin / Q000008:administration & dosage*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D042967:Electron Transport Complex I / Q000172:deficiency*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*
D000255:Adenosine Triphosphate; D042967:Electron Transport Complex I; D001920:Bradykinin; D002118:Calcium