Alessandro M. Capponi

Submitochondrial Distribution of Three Key Steroidogenic Proteins (Steroidogenic Acute Regulatory Protein and Cytochrome P450scc and 3β-Hydroxysteroid Dehydrogenase Isomerase Enzymes) upon Stimulation by Intracellular Calcium in Adrenal Glomerulosa Cells

In adrenal glomerulosa cells, angiotensin II (Ang II) and potassium stimulate aldosterone synthesis through activation of the calcium messenger system. The rate-limiting step in steroidogenesis is the transfer of cholesterol to the inner mitochondrial membrane. This transfer is believed to depend upon the presence of the steroidogenic acute regulatory (StAR) protein. The aim of this study was 1) to examine the effect of changes in cytosolic free calcium concentration and of Ang II on intramitochondrial cholesterol and 2) to study the distribution of StAR protein in submitochondrial fractions during activation by Ca2+ and Ang II. To this end, freshly prepared bovine zona glomerulosa cells were submitted to a high cytosolic Ca2+ clamp (600 nM) or stimulated with Ang II (10 nM) for 2 h. Mitochondria were isolated and subfractionated into outer membranes, inner membranes (IM), and contact sites (CS). Stimulation of intact cells with Ca2+ or Ang II led to a marked, cycloheximide-sensitive increase in cholesterol in CS (to 143 ± 3.2 and 151.1 ± 18.1% of controls, respectively) and in IM (to 119 ± 5.1 and 124.5 ± 6.5% of controls, respectively). Western blot analysis revealed a cycloheximide-sensitive increase in StAR protein in mitochondrial extracts of Ca2+-clamped glomerulosa cells (to 159 ± 23% of controls). In submitochondrial fractions, there was a selective accumulation of StAR protein in IM following stimulation with Ca2+ (228 ± 50%). Similarly, Ang II increased StAR protein in IM, and this effect was prevented by cycloheximide. In contrast, neither Ca2+ nor Ang II had any effect on the submitochondrial distribution of cytochrome P450scc and 3β-hydroxysteroid dehydrogenase isomerase. The intramitochondrial presence of the latter enzyme was further confirmed by immunogold staining in rat adrenal fasciculata cells and by immunoblot analysis in MA-10 mouse testicular Leydig cells. These findings demonstrate that under acute stimulation with Ca2+-mobilizing agents, newly synthesized StAR protein accumulates in IM after transiting through CS. Moreover, our results suggest that the import of StAR protein into IM may be associated with cholesterol transfer, thus promoting precursor supply to the two first enzymes of the steroidogenic cascade within the mitochondria and thereby activating mineralocorticoid synthesis.

Connexin43-dependent mechanism modulates renin secretion and hypertension

To investigate the function of Cx43 during hypertension, we studied the mouse line Cx43KI32 (KI32), in which the coding region of Cx32 replaces that of Cx43. Within the kidneys of homozygous KI32 mice, Cx32 was expressed in cortical and medullary tubules, as well as in some extra- and intraglomerular vessels, i.e., at sites where Cx32 and Cx43 are found in WT mice. Under such conditions, renin expression was much reduced compared with that observed in the kidneys of WT and heterozygous KI32 littermates. After exposure to a high-salt diet, all mice retained a normal blood pressure. However, whereas the levels of renin were significantly reduced in the kidneys of WT and heterozygous KI32 mice, reaching levels comparable to those observed in homozygous littermates, they were not further affected in the latter animals. Four weeks after the clipping of a renal artery (the 2-kidney, 1-clip [2K1C] model), 2K1C WT and heterozygous mice showed an increase in blood pressure and in the circulating levels of renin, whereas 2K1C homozygous littermates remained normotensive and showed unchanged plasma renin activity. Hypertensive, but not normotensive, mice also developed cardiac hypertrophy. The data indicate that replacement of Cx43 by Cx32 is associated with decreased expression and secretion of renin, thus preventing the renin-dependent hypertension that is normally induced in the 2K1C model.

Repression of DAX-1 and induction of SF-1 expression. Two mechanisms contributing to the activation of aldosterone biosynthesis in adrenal glomerulosa cells.

Angiotensin II (Ang II) and adrenocorticotropic hormone stimulate aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex through induction of the expression of the steroidogenic acute regulatory (StAR) protein, which promotes intramitochondrial cholesterol transfer. To understand the mechanism of this induction of the StAR protein, we have examined the effect of Ang II and forskolin, a mimicker of adrenocorticotropic hormone action, on two transcription factors known to modulate StARgene expression in opposite ways, DAX-1 and SF-1, in bovine adrenal glomerulosa cells in primary culture. Ang II markedly inhibited DAX-1 protein expression in a time- and concentration-dependent manner (to 38.7 ± 12.9% of controls at 3 nm after 6 h, p < 0.01), an effect that required de novo protein synthesis and ERK2/1 activation. This effect was associated with a concomitant decrease in DAX-1 mRNA and an increase in mitochondrial StAR protein levels. Similarly, forskolin dramatically repressed DAX-1 protein and mRNA expression (to 19.6 ± 1.8 and 50.3 ± 4.7% of controls, respectively,p < 0.01). Neither Ang II nor forskolin affected DAX-1 protein and mRNA stability. The aldosterone response to Ang II was markedly reduced (to 59 ± 4% of controls,p < 0.01) in transiently transfected cells overexpressing DAX-1. Whereas Ang II was without effect on SF-1 expression, forskolin significantly increased SF-1 protein and mRNA levels in a cycloheximide-sensitive manner (to 167.4 ± 16.6 and 173.1 ± 25.1% of controls after 6 h, respectively,p < 0.01). These results demonstrate that the balance between repressor and inducer function of DAX-1 and SF-1 are of critical importance in the regulation of adrenal aldosterone biosynthesis.

Decreased expression of steroidogenic acute regulatory protein: a novel mechanism participating in the leptin-induced inhibition of glucocorticoid biosynthesis.

The adipocyte-derived hormone leptin is a central modulator of food intake, metabolism and neuroendocrine functions. It is also involved in a physiological loop linking the activity of the hypothalamo-pituitary-adrenal axis and adipose tissue. At the adrenal level, leptin has been shown to antagonize the effects of ACTH on glucocorticoid biosynthesis by decreasing the expression of various enzymes of the steroid biosynthetic pathway. The steroidogenic acute regulatory protein regulates cholesterol delivery to the P450scc enzyme, a process that is rate limiting in steroid hormone biosynthesis. We have demonstrated here that leptin significantly inhibits the expression of steroidogenic acute regulatory protein in primary cultures of rat adrenocortical cells. This inhibition was observed at both the protein and mRNA levels. In contrast, leptin was not found to interfere with the expression of the cytosolic enzyme cholesterol ester hydrolase or with that of the mitochondrial enzyme P450scc. In addition, we ob...

Regulation of cholesterol supply for mineralocorticoid biosynthesis

In addition to intracellular cholesterol synthesis, plasma low- and high-density lipoproteins (LDL and HDL, respectively) are the major potential sources of a cholesterol precursor for steroid synthesis in all steroidogenic tissues. LDL- and HDL-cholesterol are taken up by cells through entirely distinct mechanisms. In the case of aldosterone production in the zona glomerulosa of the adrenal cortex, it has been assumed in the past that LDL is the major supplier of cholesterol. However, recent developments, in particular the discovery of the scavenger receptor class B type I for HDL and the characterization of its properties, have questioned this view. In fact, the nature of the challenging factor (angiotensin II or adrenocorticotropic hormone) appears to determine which pool of cholesterol is preferentially mobilized and which pathway (LDL receptor endocytosis or selective uptake through the HDL receptor) is regulated.

The control by angiotensin II of cholesterol supply for aldosterone biosynthesis

In the adrenal glomerulosa cell, aldosterone is synthesized from cholesterol, which is supplied to the cell and stored under the form of cholesterol esters, then hydrolyzed to be transferred to the mitochondrial outer membrane and finally transported to the inner membrane where the P450 side-chain cleavage enzyme will convert it to pregnenolone. Angiotensin II (AngII), one of the major physiological regulators of mineralocorticoid synthesis, appears to affect most of the steps along this cascade and thus to exert a powerful control over the use of cholesterol for aldosterone production.

Angiotensin II and calcium channels.

Sixty years after its initial discovery, the octapeptide hormone angiotensin II (AngII) has proved to play numerous physiological roles that reach far beyond its initial description as a hypertensive factor. In spite of the host of target tissues that have been identified, only two major receptor subtypes, AT1 and AT2, are currently fully identified. The specificity of the effects of AngII relies upon numerous and complex intracellular signaling pathways that often mobilize calcium ions from intracellular stores or from the extracellular medium. Various types of calcium channels (store- or voltage-operated channels) endowed with distinct functional properties play a crucial role in these processes. The activity of these channels can be modulated by AngII in a positive and/or negative fashion, depending on the cell type under observation. This chapter reviews the main characteristics of AngII receptor subtypes and of the various calcium channels as well as the involvement of the multiple signal transduction mechanisms triggered by the hormone in the cell-specific modulation of the activity of these channels.

Differential effects of cations and guanyl nucleotides on agonist and antagonist binding to rat adrenal and uterine angiotensin II receptors.

The effects of various modulators (cations, Gpp(NH)p) of hormone-receptor interaction were tested on agonist [( 125I]angiotensin II) and antagonist (125I-[Sar1,Ala8]angiotensin II) binding to membrane particles from the rat adrenal zona glomerulosa and uterine smooth muscle. The two radioiodinated peptides labeled the same population of binding sites. Sodium ion (140 mM) induced a 2 fold increase in the affinity of adrenal angiotensin II receptors for the agonist (Ka = 2.15 nM-1, vs. 1.01 nM-1 for controls), but decreased antagonist binding by reducing the number of available receptors by up to 50% in both adrenal and uterine membrane particles. Potassium ion only inhibited antagonist binding. Calcium and magnesium ions (0-10 mM) increased agonist binding and decreased antagonist binding to adrenal and uterine angiotensin II receptors, an effect mediated by changes in both affinity and number of receptors for the two peptides. The non-hydrolyzable GTP analog, Gpp(NH)p (10(-9) - 10(-4) M) decreased the affinity of angiotensin II receptors for the agonist by up to 50%, but did not affect antagonist binding to the receptor. Thus, there were marked differences in the sensitivity of agonist and antagonist peptides of angiotensin II to the modulatory effect of cations and guanyl nucleotides on ligand-receptor interaction. It is suggested that these differences may be important in determining the activatory/inhibitory properties of angiotensin peptides.

Corticotropin-Releasing Activity of the Renin-Angiotensin System Peptides in Rat and in Man

In this study we investigated in the rat the binding and corticotropin-releasing factor (CRF) activity of various constituents of the renin-angiotensin system and the possible angiotensin II receptor changes following procedures known to alter plasma renin activity. We investigated also the CRF activity of angiotensin II in vitro and in vivo in humans. The CRF activity of peptides was studied by their ability to stimulate ACTH release from pituitary cells. Deleting amino acids from the N-terminus of angiotensin II resulted in decreased CRF activity; while the ED50 for angiotensin II was 2 nM, it increased to about 10 nM for the (2-8)-heptapeptide. Angiotensin I had a weak CRF activity, whereas the substrate angiotensinogen had no stimulatory effect even at a concentration of 100 nM. There was a strong correlation between the activation and binding properties of all peptides tested. Dietary salt load or depletion as well as dexamethasone treatment did not affect the number nor the affinity of pituitary angiotensin II receptors. Angiotensin II had a CRF activity on human pituitary cells in vitro. However, peripherally injected agiotensin II at a pressive dose of 7 ng/kg/min did not produce any ACTH release in normal male volunteers. These data suggest that angiotensin II may play a modulatory role in the physiological regulation of ACTH secretion, but this role might be attributed to the endogenous brain angiotensin II as it is not closely dependent on the angiotensin II plasma levels.

Inositol trisphosphate isomers in angiotensin II-stimulated adrenal glomerulosa cells

The stimulation of phosphoinositide metabolism by angiotensin II (Ang II) was studied in [3H]inositol-labelled bovine adrenal glomerulosa cells. After separation of the phosphoinositols by ion-exchange high-performance liquid chromatography, it was shown that the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) followed distinct kinetics. The first compound to increase upon stimulation with 10(-7) M Ang II was Ins(1,4,5)P3, which reached a maximum (250% of basal level) within 10 s. At lower concentrations of Ang II, this response was slower. The formation of Ins(1,4,5)P3 depended upon the concentration of Ang II, with an EC50 of 2.4 +/- 1.5 X 10(-9) M Ang II. The potency of Ang II in stimulating the turnover of phosphoinositides and in increasing the biosynthesis of aldosterone was very similar, whereas the peptide was ten times more potent in its ability to mobilize Ca2+. Ang II was also able to stimulate the production of Ins(1,4,5)P3 in permeabilized glomerulosa cells. This effect was mimicked by a non-hydrolysable analog of GTP (GTP gamma S), suggesting that a GTP binding protein is involved in the mechanism coupling the Ang II membrane receptor to phospholipase C. These results strengthen the view that Ins(1,4,5)P3 plays a key role as second messenger in the steroidogenic response to Ang II in adrenal glomerulosa cells.