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 steroidogen esis in bovine adrenal glomerulosa cells. In the present study we have inve stigated local changes of free Ca2+ concentration near the outer mitochondr ial membrane ([Ca2+](om)) under stimulation with angiotensin II (Ang II) an d K+. Glomerulosa cells in primary culture were transfected with a recombin ant cDNA encoding the N-terminal region of the human translocase protein 20 of the outer mitochondrial membrane, in frame with the Ca2+-sensitive phot oprotein aequorin, This chimaeric aequorin (TomAeq) was associated with mit ochondria-enriched subcellular fractions of transfected COS-7 cells and was susceptible to proteinase K, showing that it was targeted to the outer mit ochondrial membrane, facing the cytosolic space. In bovine adrenal glomerul osa cells transfected with TomAeq cDNA, Ang IT induced a transient [Ca2+](o m) peak reaching 1.42 +/- 0.28 mu M, which decreased immediately to the bas al 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 wi th the elevated matrix response did not result from buffering effects of th e organelle, from altered mechanisms of intramitochondrial Ca2+ transport o r from differences in the affinity of the chimaeric aequorins for Ca2+. Thi s approach has allowed us to follow perimitochondrial Ca2+ homeostasis in b ovine glomerulosa cells under stimulation with Ca2+-mobilizing agonists and to reveal a strong gradient of Ca2+ concentration between the mitochondria l matrix and the immediate environment of the organelle.