title: Possible role for mitochondrial calcium in angiotensin II- and potassium-stimulated steroidogenesis in bovine adrenal glomerulosa cells.
abstract: In adrenal zona glomerulosa cells, the action of angiotensin II (Ang II) and of potassium (K+) on aldosterone synthesis is mediated by the Ca2+ messenger system. The major part of the steroidogenic pathway takes place inside the mitochondria, and Ca2+ must enter the mitochondrial matrix to stimulate the steroidogenic cascade. To examine how changes in the cytosolic free calcium concentration ([Ca2+]c) induced by Ang II and K+ are relayed into the mitochondrial matrix, we transfected bovine adrenal zona glomerulosa cells in primary culture with a chimeric complementary DNA encoding for the signal presequence targeting human cytochrome c oxidase subunit VIII to the matrix, linked to a complementary DNA coding for the Ca2+-sensitive photoprotein aequorin. Resting mitochondrial free calcium concentration ([Ca2+]m) amounted to 0.41 +/- 0.18 microM (n = 40). Ang II induced a concentration-dependent (EC50 = 11.3 +/- 6.0 nM), biphasic rise of [Ca2+]m. After a large transient initial peak (5.13 +/- 0.89 microM, n = 28), [Ca2+]m decreased to a plateau that remained higher than basal [Ca2+]m for several minutes in the presence of the hormone. By contrast, studies in cells transfected with cytosolic aequorin indicated that the rise of [Ca2+]c triggered by Ang II was confined to 1.34 +/- 0.26 microM (n = 17). In Ca2+-free medium, a reduced peak [Ca2+]m response to Ang II occurred without a secondary plateau. On readdition of extracellular Ca2+, in the presence of the hormone, the resulting Ca2+ influx was accompanied by small rise of [Ca2+]m. The mitochondrial uncoupler, carbonyl cyanide p-(trifluoro-methoxy)phenyl-hydrazone, prevented the Ang II-induced [Ca2+]m rise but not the [Ca2+]c response, thus demonstrating the mitochondrial location of transfected aequorin. In contrast to Ang II, K+ (13 mM) induced a sustained [Ca2+]c response, which was relayed without amplification into the mitochondrial matrix as a plateau of[Ca2+]m. This plateau of[Ca2+]m was suppressed by the addition of the dihydropyridine, nifedipine (200 nM). The inhibitor of the mitochondrial Na+/Ca2+ exchanger, CGP37157, reduced significantly the rate of decrease of [Ca2+]m following the peak induced by Ang II. In cells whose [Ca2+]c was clamped at various levels (0.05-0.860 microM) with ionomycin, a concentration-dependent stimulation of pregnenolone output was induced by Ca2+. Under these conditions, the output of pregnenolone--the early product of steroidogenesis--was markedly potentiated by CGP37157. These results suggest the existence of microdomains of high [Ca2+]c elicited by Ang II in the proximity of mitochondria. Moreover, our observations are consistent with a mitochondrial site of action for calcium in the activation of the steroidogenic cascade.
authors.affiliation: Division of Endocrinology and Diabetology, Faculty of Medicine, Geneva, Switzerland.
authors.forename: Y
authors.initials: Y
authors.lastname: Brandenburger
authors.forename: E D
authors.initials: ED
authors.lastname: Kennedy
authors.forename: C P
authors.initials: CP
authors.lastname: Python
authors.forename: M F
authors.initials: MF
authors.lastname: Rossier
authors.forename: M B
authors.initials: MB
authors.lastname: Vallotton
authors.forename: C B
authors.initials: CB
authors.lastname: Wollheim
authors.forename: A M
authors.initials: AM
authors.lastname: Capponi
mesh_terms: D000331:Aequorin / Q000235:genetics / Q000378:metabolism; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors; D002417:Cattle; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology*; D011284:Pregnenolone / Q000096:biosynthesis; D019831:Sodium-Calcium Exchanger; D013256:Steroids / Q000096:biosynthesis*; D014162:Transfection; D015384:Zona Glomerulosa / Q000166:cytology / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D013256:Steroids; D000804:Angiotensin II; D000331:Aequorin; D011284:Pregnenolone; D011188:Potassium; D002118:Calcium