Zona Glomerulosa Mitochondria Research Articles

Sources and sites of action of calcium in the regulation of aldosterone biosynthesis.

The role of free calcium as a crucial intracellular messenger in the stimulation of aldosterone biosynthesis by various agonists is well established. Using electropermeabilized or Ca(2+)-clamped adrenal zona glomerulosa (ZG) cells, we have previously shown that Ca2+ entry into the mitochondrial matrix is required for the activation of steroidogenesis. We now describe the use of various strategies to answer the following questions: 1. Which pathway does Ca2+ follow before triggering steroidogenesis? 2. Which step of steroidogenesis is under the control of Ca2+? The first approach combined the patch-clamp method, in the perforated patch configuration, with microfluorimetry of Ca2+; in the second approach, ZG cells were transiently transfected with a chimeric cDNA encoding for the calcium-sensitive photoprotein aequorin linked to a mitochondrial targeting presequence; in a third approach, ZG mitochondria were isolated and fractionated into outer membranes, contact sites and inner membranes and the effect of prior exposure of the ZG cells to a physiologically elevated intracellular calcium concentration or to angiotensin II (Ang II) on cholesterol content was then examined in those three mitochondrial fractions. The results of these combined approaches allow us to propose the following scheme: The source of calcium which is predominantly responsible for mediating the steroidogenic effect of potassium appears to be funneled through the T-type calcium channels to close proximity of the mitochondria. This signal, as well as that triggered by Ang II, appears to be relayed within the mitochondrial matrix. This rise of mitochondrial calcium is associated with a transfer of free cholesterol from the outer to the inner mitochondrial membrane, via the contact sites. Thus the main role of the calcium messenger is to promote intramitochondrial cholesterol transfer and supply to the P450scc enzyme.

Structure and dynamics of adrenal mitochondria following stimulation with corticotropin releasing hormone.

The mitochondria of rat adrenals were investigated qualitatively and quantitatively in different functional states of the adrenal cortex. Following stimulation of the animals with corticotropin releasing hormone (CRH), the corticosterone serum levels reached a maximum 1 hour after stimulation with CRH. The amount of inner mitochondrial membrane within the zona fasciculata increased showing a biphasic time course, with a first maximum 2 hours and a second maximum 8 hours after stimulation. In contrast, a significant rise of mitochondrial volume occurred only 24 hours after CRH stimulation. Therefore, the dense vesicularization of mitochondrial cristae may constitute an early process to enhance the steroidogenic capacity of these cells. Within cells of the transition zone between zona glomerulosa and zona fasciculata, we could depict a special type of mitochondria with characteristic crescent-like cristae only seen after stimulation with CRH. This type of mitochondria may represent an intermediate form between mitochondria of zona glomerulosa and zona fasciculata underlining the impressive transformational capacity of adrenocortical mitochondria. After hypophysectomy, zona fasciculata cells contained mitochondria with tubular inner membranes, representing a hypofunctional state. In contrast, the hypofunctional state after hypophysectomy and the hyperfunctional state after stimulation of the adrenal cortex via CRH injection did not appear to correlate with the morphology of mitochondria from the zona reticularis and adrenal medulla.

Regulation of Aldosterone Synthase Cytochrome P-450 in Rat Adrenals by Angiotensin II and Potassium*

Changes in the levels of aldosterone synthase cytochrome P-450, a recently identified enzyme in rat adrenals, were studied in response to the renin-angiotensin system and K stimuli. As examined by an immunoblot technique, the zona glomerulosa mitochondria from rats fed on a low Na-normal K diet (8.6 mmol Na+ and 207 mmol K+/kg of diet) or a low Na-high K (0.2 M KCl in drinking water) diet for 4-10 days contained significantly higher amounts of aldosterone synthase cytochrome P-450 than those from rats fed on a normal diet (86 mmol Na+ and 207 mmol K+/kg of diet). Activities of the enzyme were also found to increase by about 10-fold on day 10. In concert with these changes, both plasma renin activity and plasma aldosterone concentration increased, indicating that the renin-angiotensin system was activated in these rats. Feeding with a normal Na-high K diet also induced significantly higher levels of both amount and activity of aldosterone synthase cytochrome P-450 together with an elevated serum K concentration on day 4, though they all decreased to near the control level on the following days. On the other hand, when enalapril malate, an angiotensin I-converting enzyme inhibitor, was administered to the low Na-normal K rats, the increases in the amount and activity of the enzyme as well as in plasma aldosterone concentration were suppressed altogether. However, the enalapril administration to the low Na-high K rats suppressed the increases only partially. These results indicate that the aldosterone synthase cytochrome P-450 is an ultimate target of the regulation of aldosterone biosynthesis by angiotensin II and K.

Isolation of Aldosterone Synthase Cytochrome P-450 from Zona Glomerulosa Mitochondria of Rat Adrenal Cortex

A cytochrome P-450 capable of producing aldosterone from 11-deoxycorticosterone was purified from the zona glomerulosa of rat adrenal cortex. The enzyme was present in the mitochondria of the zona glomerulosa obtained from sodium-depleted and potassium-repleted rats but scarcely detected in those from untreated rats. It was undetectable in the mitochondria of other zones of the adrenal cortex from both the treated and untreated rats. The cytochrome P-450 was distinguishable from cytochrome P-45011 beta purified from the zonae fasciculata-reticularis mitochondria of the same rats. Molecular weights of the former and the latter cytochromes P-450, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 49,500 and 51,500, respectively, and their amino acid sequences up to the 20th residue from the N terminus were different from each other at least in one position. The former catalyzed the multihydroxylation reactions of 11-deoxycorticosterone giving corticosterone, 18-hydroxydeoxycorticosterone, 18-hydroxycorticosterone, and a significant amount of aldosterone as products. On the other hand, the latter catalyzed only 11 beta- and 18-hydroxylation reactions of the same substrate to yield either corticosterone or 18-hydroxydeoxycorticosterone. Thus, at least two forms of cytochrome P-450, which catalyze the 11 beta- and 18-hydroxylations of deoxycorticosterone, exist in rat adrenal cortex, but aldosterone synthesis is catalyzed only by the one present in the zona glomerulosa mitochondria.

Aldosterone biosynthesis and cytochrome P-450 11β: evidence for two different forms of the enzyme in rats

Whereas cytochrome P-450 11β has been recently shown to catalyze the two-step conversion of corticosterone to aldosterone in the bovine and porcine adrenal cortex, the identity of the enzyme involved in the two final steps of aldosterone biosynthesis in the rat adrenal cortex is as yet unknown. Mitochondria from capsular adrenals of sodium-deficient, potassium-replete rats converted corticosterone to 18-hydroxycorticosterone and aldosterone at markedly higher rates than mitochondria from capsular adrenals of sodium-replete, potassium-deficient rats. However, the same preparations exhibited no difference in the 11β-hydroxylase activity, i.e. the conversion of deoxycorticosterone to corticosterone. Only mitochondria of zona glomerulosa from rats with stimulated aldosterone biosynthesis contained a 49K. protein which showed a strong cross-reactivity with a monoclonal antibody raised against purified bovine cytochrome P-450 11β. By contrast, a crossreactive protein with a molecular weight of 51K was found in mitochondria of zona fasciculata and in mitochondria of zona glomerulosa from rats with a suppressed aldosterone biosynthesis. These findings indicate the existence of two different forms of cytochrome P-450 11β in the rat adrenal cortex, with only one of them, i.e. the 49K form, being capable of catalyzing the two final steps of aldosterone biosynthesis in situ.

Mitochondrial 11 β hydroxylation and adrenal morphology in rats bearing a mammotropic, ACTH-secreting pituitary tumor

The function and structure of adrenal mitochondria has been studied in rats bearing an ACTH, growth hormone and prolactin secreting tumor (MtT-F4). Basal levels of 11 β hydroxylation of deoxycorticosterone (DOC) in incubated mitochondria were higher in MtT-F4 than in controls ( P < 0.001). The addition of NADH and calcium to controls increased the conversion of DOC to corticosterone ( P < 0.001) but no change was observed in MtT-F4. With addition of NADPH plus calcium, the increase in controls was of a much higher magnitude than that produced in MtT-F4 ( P < 0.05). Calcium alone diminished the high levels of hydroxylation in MtT-F4. Measurement of mitochondrial swelling showed that in the presence of albumin and at 37°C swelling rates in MtT-F4 and controls were similar; however, large amplitude swelling developed in MtT-F4 when albumin was omitted and temperature was lowered to 25°C. ATPase activity was not different in mitochondria from both groups of rats either in the presence or absence of magnesium. The depressive effect of calcium, an uncoupler of oxidative phosphorylation, showed that some form of energy was needed for the high rate of basal hydroxylation in MtT-F4. The diminished response to the nucleotides indicated that probably both the P 450 hydroxylation chain and the transhydrogenase enzyme are impaired in MtT-F4. Electron microscopy of whole adrenal cortex showed a general appearance of hyperfunction. Mitochondria of zona glomerulosa contained inclusions of high electron density, and the outer membrane showed a multillaminar appearance. In zona fasciculata mitochondria showed normal structure, but different electronic density and individual variation in the number of cristae. In contrast, mitochondria of zona reticularis showed normal morphology.