Angiotensin II promotes selective uptake of high density lipoprotein cholesterol esters in bovine adrenal glomerulosa and human adrenocortical carcinoma cells through induction of scavenger receptor class B type I.

Angiotensin II is one of the main physiological regulators of aldosterone biosynthesis in the zona glomerulosa of the adrenal cortex. The hormone stimulates intracellular cholesterol mobilization to the mitochondrion for steroid biosynthesis. Here we have examined whether angiotensin II also modulates exogenous lipoprotein cholesterol ester supply to the steroidogenic machinery and whether this control is exerted on the selective transport of high density lipoprotein-derived cholesterol ester to intracellular lipid droplets through the scavenger receptor class B type I. In bovine adrenal glomerulosa and human NCI H295R adrenocortical carcinoma cells, high density lipoprotein stimulated steroid production. Angiotensin II pretreatment for 24 h potentiated this response. Fluorescence microscopy of cellular uptake of reconstituted high density lipoprotein containing a fluorescent cholesterol ester revealed an initial, time-dependent narrow labeling of the cell membrane followed by an intense accumulation of the fluorescent cholesterol ester within lipid droplets. At all time points, labeling was more pronounced in cells that had been treated for 24 h with angiotensin II. Fluorescence incorporation into cells was prevented by a monoclonal antibody directed against apolipoprotein A-I. Upon quantitative fluorometric determination, cholesterol ester uptake in angiotensin II-treated bovine cells was increased to 175 +/- 15% of controls after 2 h and to 260 +/- 10% after 4 h of exposure to fluorescent high density lipoprotein. The amount of scavenger receptor class B type I protein detected in cells treated with angiotensin II for 24 h reached 203 +/- 12% of that measured in control cells (n = 3, P < 0.01). In contrast, low density lipoprotein receptors were only minimally affected by angiotensin II treatment. This increase in scavenger receptor class B type I protein was associated with a 3-fold induction of scavenger receptor class B type I mRNA, which could be prevented by actinomycin D but not by cycloheximide. Similar results were obtained in the human adenocarcinoma cell line H295R. These observations show that angiotensin II regulates the scavenger receptor class B type I-mediated selective transport of lipoprotein cholesterol ester across the cell membrane as a major source of precursor for mineralocorticoid biosynthesis in both human and bovine adrenal cells.

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 P450(scc) 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 P450(scc). In addition, we observed the anticipated stimulation of cAMP production by ACTH in the presence of leptin, suggesting that it does not interfere with intracellular ACTH signaling. In summary, our data provide evidence that the interplay existing between leptin and ACTH in vivo is mediated at least partially via a direct and opposite modulation of steroidogenic acute regulatory protein, a key factor in the adrenal steroid biosynthetic pathway. This effect of leptin could also be relevant to other steroidogenic tissues.

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.

The role of tyrosine kinases in capacitative calcium influx-mediated aldosterone production in bovine adrenal zona glomerulosa cells.

In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) involves the activation of a capacitative Ca(2+) influx through calcium release-activated calcium (CRAC) channels. In various mammalian cell systems, it has been shown that CRAC channel activation and Ca(2+) entry require tyrosine kinase activity. We have therefore examined in this work whether similar mechanisms contribute to Ang II-induced mineralocorticoid biosynthesis. In fluo-3-loaded isolated bovine glomerulosa cells, two inhibitors of tyrosine kinases, genistein and methyl-2, 5-dihydroxycinnamate (MDHC) (100 microM) prevented capacitative Ca(2+) entry elicited by Ang II (by 54 and 62% respectively), while the inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase, lavendustin A, was without effect. Similar results were observed on Ca(2+) influx triggered by thapsigargin, an inhibitor of microsomal Ca(2+) pumps. The inhibitors blocked Ang II-stimulated pregnenolone and aldosterone production in the same rank order. In addition to its specific effect on capacitative Ca(2+) influx, genistein also affected the late steps of the steroidogenic pathway, as shown by experiments in which the rate-limiting step (intramitochondrial cholesterol transfer) was bypassed with 25-OH-cholesterol (25-OH-Chol), cytosolic calcium was clamped at stimulated levels or precursors of the late enzymatic steps were supplied. In contrast, genistin, a structural analogue of genistein devoid of tyrosine kinase inhibitory activity, was almost without effect on pregnenolone or 11-deoxycorticosterone (DOC) conversion to aldosterone. These results suggest that, in bovine adrenal glomerulosa cells, Ang II promotes capacitative Ca(2+) influx and aldosterone biosynthesis through tyrosine kinase activation.

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

Microdomains of high cytosolic free Ca(2+) 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 Ca(2+) concentration near the outer mitochondrial membrane ([Ca(2+)](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 Ca(2+)-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 II induced a transient [Ca(2+)](om) peak reaching 1.42+/-0.28 microM, which decreased immediately to the basal resting value. The peak response to Ang II was strikingly lower than the peak response of mitochondrial free Ca(2+) concentration, which increased to 5.4+/-1.2 microM. The smaller response of [Ca(2+)](om) to Ang II compared with the elevated matrix response did not result from buffering effects of the organelle, from altered mechanisms of intramitochondrial Ca(2+) transport or from differences in the affinity of the chimaeric aequorins for Ca(2+). This approach has allowed us to follow perimitochondrial Ca(2+) homeostasis in bovine glomerulosa cells under stimulation with Ca(2+)-mobilizing agonists and to reveal a strong gradient of Ca(2+) concentration between the mitochondrial matrix and the immediate environment of the organelle.

Angiotensin II negatively modulates L-type calcium channels through a pertussis toxin-sensitive G protein in adrenal glomerulosa cells.

In bovine adrenal glomerulosa cells, angiotensin II and extracellular K+ stimulate aldosterone secretion in a calcium-dependent manner. In these cells, physiological concentrations of extracellular potassium activate both T-type (low threshold) and L-type (high threshold) voltage-operated calcium channels. Paradoxically, the cytosolic calcium response to 9 mM K+ is inhibited by angiotensin II. Because K+-induced calcium changes observed in the cytosol are almost exclusively due to L-type channel activity, we therefore studied the mechanisms of L-type channel regulation by angiotensin II. Using the patch-clamp method in its perforated patch configuration, we observed a marked inhibition (by 63%) of L-type barium currents in response to angiotensin II. This effect of the hormone was completely prevented by losartan, a specific antagonist of the AT1 receptor subtype. Moreover, this inhibition was strongly reduced when the cells were previously treated for 1 night with pertussis toxin. An effect of pertussis toxin was also observed on the modulation by angiotensin II of the K+ (9 mM)-induced cytosolic calcium response in fura-2-loaded cells, as well as on the angiotensin II-induced aldosterone secretion, at both low (3 mM) and high (9 mM) K+ concentrations. Finally, the expression of both Go and Gi proteins in bovine glomerulosa cells was detected by immunoblotting. Altogether, these results strongly suggest that in bovine glomerulosa cells, a pertussis toxin-sensitive G protein is involved in the inhibition of L-type channel activity induced by angiotensin II.

Angiotensin II type 1 receptor activation modulates L- and T-type calcium channel activity through distinct mechanisms in bovine adrenal glomerulosa cells.

In adrenal zona glomerulosa cells, calcium entry is crucial for aldosterone production and secretion. This influx is stimulated by increases of extracellular potassium in the physiological range of concentrations and by angiotensin II (Ang II). The high threshold voltage-activated (L-type) calcium channels have been shown to be the major mediators for the rise in cytosolic free calcium concentration, [Ca2+]c, observed in response to a depolarisation by physiological potassium concentrations. Paradoxically, both T- and L-type calcium channels have been shown to be negatively modulated by Ang II after activation by a sustained depolarisation. While the modulation of T-type channels involves protein kinase C (PKC) activation, L-type channel inhibition requires a pertussis toxin-sensitive G protein. In order to investigate the possibility of additional modulatory mechanisms elicited by Ang II on L-type channels, we have studied the effect of PKC activation or tyrosine kinase inhibition. Neither genistein or MDHC, two strong inhibitors of tyrosine kinases, nor the phorbol ester PMA, a specific activator of PKC, affected the Ang II effect on the [Ca2+]c response and on the Ba2+ currents elicited by cell depolarisation with the patch-clamp method. We propose a model describing the mechanisms of the [Ca2+]c modulation by Ang II and potassium in bovine adrenal glomerulosa cells.

Inhibitory action of mibefradil on calcium signaling and aldosterone synthesis in bovine adrenal glomerulosa cells.

Mibefradil is a new cardiovascular drug with peculiar Ca++ antagonistic properties. The most remarkable feature of mibefradil is its unique relative selectivity for T type calcium channels, a property that has been proposed to explain in part the beneficial pharmacological and clinical profiles of this drug. In adrenal glomerulosa cells, aldosterone biosynthesis and secretion in response to angiotensin II or extracellular potassium is dependent on a sustained influx of Ca++ through T type Ca++ channels. The effect of mibefradil on the steroidogenic function of glomerulosa cells was therefore investigated. Using the patch clamp technique, we found that mibefradil inhibits selectively and in a concentration-dependent manner (IC50 = 3 microM)++ T type currents in bovine glomerulosa cells. In addition to this tonic (voltage independent) inhibition, the drug also induced a shift of the steady-state inactivation curve of these channels toward hyperpolarized voltages, contributing to its efficacy to prevent Ca++ influx into the cell through T type channels. Concomitantly, mibefradil reduced the cytosolic calcium responses to potassium and angiotensin II (as assessed with fluorescent probes), without affecting the capacitative Ca++ influx, and inhibited pregnenolone and aldosterone formation. This inhibition of steroidogenesis was not exclusively due to mibefradil action on voltage-operated Ca++ channels, because this agent also partially reduced steroid synthesis induced by adrenocorticotropic hormone or forskolin, two activators of the cyclic AMP pathway. In conclusion, mibefradil is highly effective in adrenal glomerulosa cells in reducing T type channel activity and aldosterone biosynthesis, two actions that should contribute to the beneficial effect of the drug in the treatment of hypertension.

Atrial natriuretic peptide inhibits calcium-induced steroidogenic acute regulatory protein gene transcription in adrenal glomerulosa cells.

Atrial natriuretic peptide (ANP) is a potent inhibitor of mineralocorticoid synthesis induced in adrenal glomerulosa cells by physiological agonists activating the calcium messenger system, such as angiotensin II (Ang II) and potassium ion (K+). While the role of calcium in mediating Ang II- and K(+)-induced aldosterone production is clearly established, the mechanisms leading to blockade of this steroidogenic response by ANP remain obscure. We have used bovine adrenal zona glomerulosa cells in primary culture, in which an activation of the calcium messenger system was mimicked by a 2-h exposure to an intracellular high-calcium clamp. The effect of ANP was studied on the following parameters of the steroidogenic pathway: 1) pregnenolone and aldosterone production; 2) changes in cytosolic ([Ca2+]c) and mitochondrial ([Ca2+]m) Ca2+ concentrations, as assessed with targeted recombinant aequorin; 3) cholesterol content in outer mitochondrial membranes (OM), contact sites (CS), and inner membranes (IM); 4) steroidogenic acute regulatory (StAR) protein import into mitochondria by Western blot analysis; 5) StAR protein synthesis, as determined by [35S]methionine incorporation, immunoprecipitation, and SDS-PAGE; 6) StAR mRNA levels by Northern blot analysis with a StAR cDNA; 7) StAR gene transcription by nuclear run-on analysis. While clamping Ca2+ at 950 nM raised pregnenolone output 3.5-fold and aldosterone output 3-fold, ANP prevented these responses with an IC50 of 1 nM and a maximal effect of 90% inhibition at 10 nM. In contrast, ANP did not affect the [Ca2+]c or [Ca2+]m changes occurring under Ca2+ clamp or Ang II stimulation in glomerulosa cells. The accumulation of cholesterol content in CS (139.7 +/- 10.7% of control) observed under high-Ca2+ clamp was prevented by 10 nM ANP (92.4 +/- 4% of control). Similarly, while Ca2+ induced a marked accumulation of StAR protein in mitochondria of glomerulosa cells to 218 +/- 44% (n = 3) of controls, the presence of ANP led to a blockade of StAR protein mitochondrial import (113.3 +/- 15.0%). This effect was due to a complete suppression of the increased [35S]methionine incorporation into StAR protein that occurred under Ca2+ clamp (94.5 +/- 12.8% vs. 167.5 +/- 17.3%, n = 3). Furthermore, while the high-Ca2+ clamp significantly increased StAR mRNA levels to 188.5 +/- 8.4 of controls (n = 4), ANP completely prevented this response. Nuclear run-on analysis showed that increases in intracellular Ca2+ resulted in transcriptional induction of the StAR gene and that ANP inhibited this process. These results demonstrate that Ca2+ exerts a transcriptional control on StAR protein expression and that ANP appears to elicit its inhibitory effect on aldosterone biosynthesis by acting as a negative physiological regulator of StAR gene expression.

Angiotensin II potentiates adrenocorticotrophic hormone-induced cAMP formation in bovine adrenal glomerulosa cells through a capacitative calcium influx.

Angiotensin II (AngII) plays a crucial role in the control of aldosterone biosynthesis in adrenal glomerulosa cells through the stimulation of two distinct Ca2+ entry pathways: (1) opening of voltage-operated calcium channels, and (2) activation of a capacitative Ca2+ entry that is dependent on calcium release from intracellular pools. Adrenocorticotrophic hormone (ACTH), on the other hand, a major hormonal regulator of steroidogenesis, induces an increase in intracellular cAMP through the activation of a G-protein-coupled adenylyl cyclase. Recent studies have demonstrated that the rise in cAMP induced by ACTH can be potentiated by AngII in bovine glomerulosa cells. The aim of the present study was to investigate the mechanism of AngII action on ACTH-induced cAMP production. In primary cultures of bovine glomerulosa cells, we found that AngII (100 nM), which had no effect by itself on cAMP production, significantly potentiated maximal ACTH-induced cAMP formation in the presence of extracellular calcium (1.2 mM). In contrast, in the absence of extracellular calcium, AngII did not affect ACTH-induced cAMP production. These results suggest that calcium entry into the cell plays an important role in the activation of the cyclase by AngII. The inhibition of voltage-operated calcium channels by nicardipine, a dihydropyridine calcium antagonist blocking both low-threshold (T-type) and high-threshold (L-type) Ca2+ channels, did not significantly affect the potentiating effect of AngII. Moreover, the cAMP response to ACTH was insensitive to activation of these Ca2+ channels induced by potassium ions and, even when cytosolic free-calcium concentration ([Ca2+]c) was kept elevated with the Ca2+ ionophore, ionomycin, no stimulation of adenylyl cyclase was observed at concentrations of [Ca2+]c up to 640 nM. In contrast, thapsigargin, an activator of capacitative Ca2+ influx, mimicked the potentiating effect of AngII on ACTH-induced cAMP formation. In agreement with the characteristics of cAMP modulation by Ca2+ in these cells, the presence of type III adenylyl cyclase was observed by immunodetection in bovine glomerulosa cell membranes. In conclusion, these data suggest a tight coupling between the capacitative Ca2+ influx induced upon stimulation by either AngII or thapsigargin and a calcium-sensitive isoform of adenylyl cyclase, probably type III, in bovine glomerulosa cells.

Regulation of mineralocorticoid biosynthesis by calcium and the StAR protein.

In adrenal zona glomerulosa cells, the calcium messenger system is the major signaling mechanism activated by physiological stimulators of aldosterone production. We present here evidence for a dual site of action of the calcium signal: 1) Calcium influx into the mitochondrion is a prerequisite to the activation of steroidogenesis. This calcium entry leads to a rise in mitochondrial calcium concentration and to an increase in intramitochondrial cholesterol transfer and Steroidogenic Acute Regulatory (StAR) protein accumulation in inner mitochondrial membranes. 2) Calcium also exerts a genomic regulatory effect by activating transcription of the StAR gene.

Duality of the voltage-dependent calcium influx in adrenal glomerulosa cells.

Both T- and L-type calcium channels are expressed in bovine adrenal glomerulosa cells and both channels are sensitive to moderate depolarizations of the cell membrane induced by angiotensin II (AngII) or physiological concentrations of extracellular K+. These channels present distinct pharmacology, L-type channels being more sensitive to dihydropyridines, whereas T channels are inhibited by lower concentrations of mibefradil, a new type of calcium antagonist currently used for treating hypertension. The activity of these channels is also differently modulated by AngII, which inhibits T channels through activation of protein kinase C and L channels through a Pertussis toxin-sensitive G protein. Finally, whereas the activity of L-type channels is directly reflected on the levels of the cytosolic calcium concentration ([Ca2+]c), T-type channels are more closely related to the control of steroidogenesis, possibly through a kind of "calcium pipeline" linking the plasma membrane to the mitochondria. In conclusion, two types of calcium channels, with distinct functions and differential modulation by AngII, are activated by agonists of aldosterone biosynthesis in adrenal glomerulosa cells. Most importantly, these channels have distinct sensitivities to currently used antihypertensive therapeutic drugs.

The acute regulation of mineralocorticoid biosynthesis: scenarios for the StAR system.

The zona glomerulosa cell of the adrenal cortex produces mineralocorticoids in response to physiological stimuli (angiotensin II and extracellular K(+)) activating the Ca(2+) messenger system. The mechanisms underlying the generation of the Ca(2+) signal have been analyzed extensively and recent developments have contributed to bridging the gap between intracellular signals and activation of the biological function. This article summarizes the current knowledge on the intracellular targets of the Ca(2+) messenger, obtained mainly in bovine glomerulosa cells. Ca(2+) appears to exert a dual effect, both at the intramitochondrial level and at the nuclear level, where it activates steroidogenic acute regulatory protein (StAR) gene transcription.

Submitochondrial distribution of three key steroidogenic proteins (steroidogenic acute regulatory protein and cytochrome p450scc and 3beta-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 3beta-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.

Possible role for mitochondrial calcium in angiotensin II- and potassium-stimulated steroidogenesis in bovine adrenal glomerulosa cells.

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.

Distinct functions of T- and L-type calcium channels during activation of bovine adrenal glomerulosa cells.

Calcium influx into adrenal glomerulosa cells is a key event during the stimulation of aldosterone secretion by physiological increases in extracellular potassium concentrations. Two types of voltage-operated calcium channels, T- and L-types, are present on bovine glomerulosa cells, but their respective functions are not yet clearly defined. Using the patch-clamp method in the perforated patch configuration combined with microfluorimetry of cytosolic calcium, we demonstrate that L-type channels are exclusively responsible for the sustained elevation of cytosolic calcium observed upon stimulation with extracellular potassium, even at low, physiological concentrations of this agonist. In contrast, aldosterone secretion appears closely related to T-type channel activity. Moreover, when the activity of each channel type is selectively modulated by pharmacological agents, such as dihydropyridines or zonisamide, the cytosolic calcium response can be clearly dissociated from the steroidogenic response. Similarly, modulation of T channel activation by protein kinase C results in a parallel inhibition of aldosterone secretion, without any effect on the levels of cytosolic free calcium. This direct functional link between T-type calcium channel activity and steroidogenesis suggests a model in which calcium entering the cell through these channels bypasses the cytosol to activate intramitochondrial steps of aldosterone biosynthesis.

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.

Calcium stimulates intramitochondrial cholesterol transfer in bovine adrenal glomerulosa cells.

In adrenal glomerulosa cells, angiotensin II (Ang II) stimulates aldosterone synthesis through rises of cytosolic calcium ([Ca2+]c). The rate-limiting step in this process is the transfer of cholesterol to the inner mitochondrial membrane, where it is converted to pregnenolone by the P450 side chain cleavage enzyme. The aim of the present study was to examine the effect of changes in [Ca2+]c and of Ang II on intramitochondrial cholesterol distribution. Freshly prepared bovine zona glomerulosa cells were submitted to a cytosolic Ca2+ clamp (600 nM) or stimulated with Ang II (10 nM). Mitochondria were isolated and subfractionated into outer membranes (OM), inner membranes (IM), and contact sites (CS). Cholesterol content was determined by the cholesterol oxidase assay. Stimulation of intact cells with Ca2+ led to a marked decrease in cholesterol content of OM (to 54 +/- 24% of controls, n = 5) and to a concomitant increase of cholesterol in CS and IM (to 145 +/- 14%, n = 5). When glomerulosa cells were exposed to Ang II, a marked increase of cholesterol in CS occurred (to 172 +/- 16% of controls, n = 5). No significant changes were detected in OM cholesterol, suggesting a stimulation of cholesterol supply to the mitochondria in response to Ang II. Cycloheximide specifically and significantly reduced Ca2+-activated cholesterol transfer to CS and IM. In conclusion, our data indicate that one of the main functions of the Ca2+ messenger is to increase cholesterol supply to the P450 side chain cleavage enzyme by enhancing endogenous intermembrane cholesterol transfer to a mitochondrial site containing the enzymes responsible for the initial steps of the steroidogenic cascade.

Demonstration of an angiotensin II-induced negative feedback effect on aldosterone synthesis in isolated rat adrenal zona glomerulosa cells.

Although both angiotensin II (Ang II) and potassium ion (K+) induce marked elevations of cytosolic free calcium concentration, [Ca2+]c, in adrenal zona glomerulosa cells-an effect which is thought to trigger aldosterone synthesis-Ang II is also known to reduce the sustained [Ca2+]c rise induced by K+. We have examined whether this effect of Ang II on the calcium messenger system is reflected at the level of the final biological response, aldosterone synthesis. In superfused isolated rat glomerulosa cells, K+ (8 mM) induced a sustained, 60-fold increase in aldosterone production. In contrast, the maximal response to Ang II (10 nM) amounted to only 10 times the basal production. When added subsequent to K+ stimulation, Ang II provoked an immediate and dramatic drop in aldosterone synthesis, to levels obtained with Ang II alone. Under conditions of maximal K+ stimulation, this effect depended upon Ang II concentration, while the well-known synergistic effect was observed with submaximal concentrations of both agonists. The inhibitory effect of Ang II could be reproduced with dioctanoylglycerol, a selective activator of protein kinase C. By contrast, the aldosterone response to adrenocorticotropic hormone (ACTH) was not affected by Ang II. At submaximal concentrations of ACTH, the steroidogenic effect of Ang II was even additive to that of ACTH. Thus, we have shown that, under conditions of maximal stimulation, Ang II exerts a profound inhibition of steroidogenesis in K(+)-stimulated rat adrenal glomerulosa cells. This counter-regulatory mechanism may ensure adequate levels of aldosterone production in vivo.