Measurement of perimitochondrial Ca2+ concentration in bovine adrenal glomerulosa cells with aequorin targeted to the outer mitochondrial membrane

Microdomains of high cytosolic free Ca2+ concentration in the proximity of mitochondria might have an important role in the stimulation of steroidogenesis in bovine adrenal glomerulosa cells. In the present study we have investigated local changes of free Ca2+ concentration near the outer mitochondrial membrane ([Ca2+](om)) under stimulation with angiotensin II (Ang II) and K+. Glomerulosa cells in primary culture were transfected with a recombinant cDNA encoding the N-terminal region of the human translocase protein 20 of the outer mitochondrial membrane, in frame with the Ca2+-sensitive photoprotein aequorin, This chimaeric aequorin (TomAeq) was associated with mitochondria-enriched subcellular fractions of transfected COS-7 cells and was susceptible to proteinase K, showing that it was targeted to the outer mitochondrial membrane, facing the cytosolic space. In bovine adrenal glomerulosa cells transfected with TomAeq cDNA, Ang IT induced a transient [Ca2+](om) peak reaching 1.42 +/- 0.28 mu M, which decreased immediately to the basal resting value. The peak response to Ang II was strikingly lower than the peak response of mitochondrial free Ca2+ concentration, which increased to 5.4 +/- 1.2 mu M. The smaller response of [Ca2+](om) to Ang II compared with the elevated matrix response did not result from buffering effects of the organelle, from altered mechanisms of intramitochondrial Ca2+ transport or from differences in the affinity of the chimaeric aequorins for Ca2+. This approach has allowed us to follow perimitochondrial Ca2+ homeostasis in bovine glomerulosa cells under stimulation with Ca2+-mobilizing agonists and to reveal a strong gradient of Ca2+ concentration between the mitochondrial matrix and the immediate environment of the organelle.