Ultrastructural Mechanisms of Impaired Aldosterone Synthesis in Rats Exposed to DDT during Prenatal and Postnatal Development.

The study is aimed at elucidation of ultrastructural mechanisms underlying impaired aldosterone synthesis by glomerulosa cells in Wistar rats exposed to low doses of endocrine disrupter DDT during prenatal and postnatal development. Analysis of rat zona glomerulosa histology and function during the pubertal and postpubertal periods showed that exposure to endocrine disrupter DDT disturbs its development and reduced the production of aldosterone. Electron microscopy showed that changes in the aldosterone synthesis are related to impaired reorganization of the mitochondrial apparatus, one of the leading factors in the regulation of steroidogenesis, in glomerulosa cells in DDT-exposed rats during puberty.

ß-Catenin and FGFR2 regulate postnatal rosette-based adrenocortical morphogenesis.

Rosettes are widely used in epithelial morphogenesis during embryonic development and organogenesis. However, their role in postnatal development and adult tissue maintenance remains largely unknown. Here, we show zona glomerulosa cells in the adult adrenal cortex organize into rosettes through adherens junction-mediated constriction, and that rosette formation underlies the maturation of adrenal glomerular structure postnatally. Using genetic mouse models, we show loss of ß-catenin results in disrupted adherens junctions, reduced rosette number, and dysmorphic glomeruli, whereas ß-catenin stabilization leads to increased adherens junction abundance, more rosettes, and glomerular expansion. Furthermore, we uncover numerous known regulators of epithelial morphogenesis enriched in ß-catenin-stabilized adrenals. Among these genes, we show Fgfr2 is required for adrenal rosette formation by regulating adherens junction abundance and aggregation. Together, our data provide an example of rosette-mediated postnatal tissue morphogenesis and a framework for studying the role of rosettes in adult zona glomerulosa tissue maintenance and function.

Development of monoclonal antibodies against human CYP11B1 and CYP11B2.

1. The final enzymes in the biosynthesis of aldosterone and cortisol are by the cytochrome P450 CYP11B2 and CYP11B1, respectively. The enzymes are 93% homologous at the amino acid level and specific antibodies have been difficult to generate. 2. Mice and rats were immunized with multiple peptides conjugated to various immunogenic proteins and monoclonal antibodies were generated. The only peptide sequences that generated specific antibodies were amino acids 41-52 for the CYP11B2 and amino acids 80-90 for the CYP11B1 enzyme. 3. The mouse monoclonal CYP11B2-41 was specific and sensitive for use in western blots and produced specific staining of the zona glomerulosa of normal adrenal glands. The rat monoclonal CYP11B1-80 also detected a single band by western blot and detected only the zona fasciculata. Triple immunofluorescence of the adrenal demonstrated that the CYP11B1 and the CYP11B2 did not co-localize, while as expected the CYP11B1 co-localized with the 17α-hydroxylase.

Estereología renal en el cobayo (Cavia porcellus) / Renal stereology in the guinea pig (Cavia porcellus)

El cobayo (Cavia porcellus) es un roedor perteneciente al Orden Rodentia y a la Familia Caviidae, utilizado como animal de laboratorio y de consumo humano. Los parámetros cuantitativos del riñón entregan importante información de su morfofunción dada su labor en la homeostasis del organismo. El objetivo de este estudio fue describir el riñón de cobayo (Cavia porcellus), analizando las características estereológicas para futuros estudios experimentales. Se utilizaron 5 cobayos machos, obtenidos del Bioterio de la Universidad de La Frontera, Temuco, Chile. El riñón de cobayo pesó 3,2 g, aproximadamente. El riñón posee 140.298 glomérulos en total, Nv de 458 mm³, Vv de 7,89% y Sv de 3,58 mm²/ mm³. El volumen glomerular del riñón fue de 1,73 x 10(4)mm³ y el diámetro glomerular de 90 jm. Factores como especie, edad, peso corporal, peso y volumen renal, son importantes a considerar, ya que diferencian los resultados en investigaciones morfofuncionales.

The guinea pig, (Cavia porcellus) is a rodent pertaining to the Rodentia group and the Caviidae family, used as a laboratory animal and for human consumption. Quantitative parameters of the kidney provides important information of its morphofunction, given its labor in the organism's homeostasis. The aim or this study was to describe the kidney of the guinea pig (Cavia porcellus), analyzing the stereological characteristics for future experimental studies. Five male guinea pigs (Cavia porcellus) obtained from the Biotery of the Universidad de la Frontera, Temuco, Chile, were used. The kidney of the guinea pig weighed approximately 3.2g. The kidney has 140,298 total glomerulus, Nv of 458 mm³, Vv of 7.89% and Svof 3.58mm²/mm³. The glomerular volume of the kidney was of 1.73 x 10(4)mm³ and a glomerular diameter of 90 urn. Factors such as species, age, body weight and renal volume, are important to consider, as they differentiate the results in the morphofunctional investigations.

In vitro culture on Matrigel favors the long-term maintenance of rat zona glomerulosa-cell differentiated phenotype.

Zona glomerulosa (ZG) cells cultured on plastic within few days dedifferentiate losing their capacity to secrete aldosterone (ALDO) in appreciable amounts. Evidence indicates that extracellular matrix modulates the secretory behavior of adrenocortical cells cultured in vitro. Hence, we compared the morphology and function of rat ZG cells grown on plastic and Matrigel basement membrane matrix (hereinafter Matrigel) for up to 12 days. At day 3, no significant differences were observed between cells cultured on plastic and Matrigel. Starting from day 6, ZG cells cultured on plastic lost their ultrastructural differentiated features (mitochondria with tubular cristae, smooth endoplasmic reticulum cisternae and lipid droplets), exhibiting a fibroblast-like appearance. The mRNA expression of the main steroidogenic enzymes, as evaluated by real-time polymerase chain reaction, the baseline secretion of ALDO and other post-pregnenolone hormones, as evaluated by high pressure liquid chromatography, and the secretory response to ACTH, angiotensin-II and K(+), as evaluated by radioimmunoassay, displayed a time-dependent decrease. Matrigel was found to maintain unchanged both the ultrastructure and the expresion of steroidogenic enzymes of ZG cells until day 12 of culture. Baseline and agonist-stimulated steroid-hormone secretion decreased with the duration of culture on Matrigel, but was always higher than that of ZG cells grown on plastic. Hence, our study clearly indicates that the culture on Matrigel favors the maintenance of rat ZG-cell differentiated phenotype, allowing the conclusion that this technique is suitable for long-term in vitro investigations.

The effect of cytoplasmic Ca2+ signal on the redox state of mitochondrial pyridine nucleotides.

As first observed in rat adrenal glomerulosa cells, cytoplasmic Ca(2+) signal, induced by K(+), angiotensin II or vasopressin, evokes an increase in the level of reduced mitochondrial pyridine nucleotides, NADH and NADPH. Prostaglandin F(2)alpha and extracellular ATP exert similar effects in rat ovarian luteal cells. This coupling of cytoplasmic Ca(2+) concentration and mitochondrial metabolism occurs also when the stimuli are applied at physiological concentration and under conditions when no formation of high-Ca(2+) perimitochondrial microdomains may be presumed. We present evidence that low submicromolar Ca(2+) signals in the cytoplasm can increase mitochondrial Ca(2+) concentration and activate mitochondrial dehydrogenation processes. Several observations support the assumption that intramitochondrial Ca(2+) signals play a significant role in the stimulation of steroid hormone production.

Hormone-sensitive lipase deficiency in mice causes lipid storage in the adrenal cortex and impaired corticosterone response to corticotropin stimulation.

Hormone-sensitive lipase (HSL, E.C.3.1.1.3, gene designation Lipe) is reportedly the major cholesteryl esterase of adrenal cortex. Because of the potential importance of cholesteryl ester hydrolysis in steroidogenesis, gene-targeted HSL-deficient mice were assessed for adrenal cortical morphology and function. Compared with control animals, HSL deficiency results in a marked accumulation of lipid droplets both in zona glomerulosa and zona fasciculata. In the zona fasciculata, lipid accumulation was observed progressively from the outer to the inner regions, culminating near the corticomedullary junction with the formation of syncytial-lipoid structures having the appearance of degenerative cells. These morphological changes did not significantly alter the basal levels of circulating corticosterone, but following ACTH stimulation, corticosterone levels were decreased (P < 0.001). The observation of normal basal corticosterone and aldosterone levels demonstrates that some free cholesterol for steroid synthesis can be produced independently of HSL. Taken together, these results indicate that HSL-deficient mice accumulate lipid droplets in such a way as to impair acute ACTH stimulation of corticosterone secretion. Such observations are also found in some forms of congenital adrenal hyperplasia. By extension, HSL deficiency may be a cause of hereditary adrenocortical hypofunction in humans.

Chronic hypoxia induced ultrastructural changes in the rat adrenal zona glomerulosa.

The adrenal cortex plays an important role in adaptation to various forms of stress, including hypoxia. While physiological changes in the aldosterone metabolism during hypoxia have been extensively described, few studies have focused on the morphological changes in the adrenal glands under chronic hypoxia. We studied the ultrastructure of the zona glomerulosa of 6-month-old Wistar rats exposed to chronic normobaric hypoxia. Animals were divided into two groups: control (n=12) and hypoxic (n=12). In this latter group, the animals were kept at 7% O2 concentration after a gradual adaptation (21, 15, 12, 10, 8, 7 vol% O2). The duration of the study was 112 days. In comparison with normoxic rats, body weight and adrenal gland weight of hypoxic animals was significantly reduced by 18.5% (p=0.006) and 14.7% (p=0.001) respectively. The thickness of the zona glomerulosa decreased due to atrophy of cells. The main ultrastructural changes observed were: 1) a decrease in, or complete elimination of, lipid droplet content; 2) a marked increase in lysosome number; and 3) the presence of giant mitochondria. Our findings show that rats fail to adapt to severe chronic hypoxia. The ultrastructural changes in the zona glomerulosa found in the present study could reflect changes in the aldosterone pathway.

[Structure of the adrenal gland in rat strain with hereditary stress-induced arterial hypertension nursed by Wistar dams]. / Strukturnye osobennosti nadpochechnika krys linii s nasledstvennoi indutsirovannoi stressom arterial'noi gipertenziei pri vskarmlivanii samkami linii Wistar.

The comparative investigation of the adrenal structure in two groups of rats of Inherited Stress-Induced Arterial Hypertension (ISIAH) strain was conducted. The animals of the first group were nursed by normotensive Wistar rats, while those of the second (control) group were reared by their own mothers. The volume of the adrenal medulla in rats of the first group was found to exceed that in the second group. In the adrenal zona glomerulosa and zona fasciculata of 3-week-old rats of the first group parenchymal-stromal ratio and the average volume of adrenocorticocytes were lower than those in the animals of the second group. This was accompanied by a decrease in mitochondrial volume density and accumulation of lipid droplets in the cytoplasm, indicating a reduction of hormone-producing activity of endocrinocytes in the animals the first group as compared to controls. By 6 month of age, arterial pressure and quantitative parameters of adrenal medulla and cortex in the animals the first group were..... as compared to those in the second group. It is suggested that the nursing of ISIAH rat pups by normotensive Wistar dams had modulating effect on the adrenal structure and therefore on arterial hypertension development.

Effects of extracellular calcium and potassium on the sodium pump of rat adrenal glomerulosa cells.

Because the activity of the sodium pump (Na-K-ATPase) influences the secretion of aldosterone, we determined how extracellular potassium (K(o)) and calcium affect sodium pump activity in rat adrenal glomerulosa cells. Sodium pump activity was measured as ouabain-sensitive (86)Rb uptake in freshly dispersed cells containing 20 mM sodium as measured with sodium-binding benzofluran isophthalate. Increasing K(o) from 4 to 10 mM in the presence of 1.8 mM extracellular calcium (Ca(o)) stimulated sodium pump activity up to 165% and increased intracellular free calcium as measured with fura 2. Increasing K(o) from 4 to 10 mM in the absence of Ca(o) stimulated the sodium pump approximately 30% and did not increase intracellular free calcium concentration ([Ca(2+)](i)). In some experiments, addition of 1.8 mM Ca(o) in the presence of 4 mM K(o) increased [Ca(2+)](i) above the levels observed in the absence of Ca(o) and stimulated the sodium pump up to 100%. Ca-dependent stimulation of the sodium pump by K(o) and Ca(o) was inhibited by isradipine (10 microM), a blocker of L- and T-type calcium channels, by compound 48/80 (40 microg/ml) and calmidizolium (10 microM), which inhibits calmodulin (CaM), and by KN-62 (10 microM), which blocks some forms of Ca/CaM kinase II (CaMKII). Staurosporine (1 microM), which effectively blocks most forms of protein kinase C, had no effect. In the presence of A-23187, a calcium ionophore, the addition of 0.1 mM Ca(o) increased [Ca(2+)](i) to the level observed in the presence of 10 mM K(o) and 1.8 mM Ca(o) and stimulated the sodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mM Ca(o) was not reduced by isradipine but was blocked by KN-62. Thus, under the conditions that K(o) stimulates aldosterone secretion, it stimulates the sodium pump by two mechanisms: direct binding to the pump and by increasing calcium influx, which is dependent on Ca(o). The resulting increase in [Ca(2+)](i) may stimulate the sodium pump by activating CaM and/or CaMKII.

Time-dependent changes in adrenal cortex ultrastructure and corticosterone levels after noise exposure in male rats.

In response to a stressful stimulus, there is a marked activation of the hypothalamic-pituitary-adrenal axis leading to a release of adrenocorticotropic hormone. This, in turn, acts on the zona fasciculata of the adrenal cortex to increase corticosterone plasma levels. Given the frequency of chronic intermittent noise exposure in man, we selected loud noise to evaluate concomitant changes in the ultrastructure of the adrenal cortex and corticosterone release. Following chronic (21 days, 6 h per day) loud white noise exposure (100 dBA, 0-26 KHz), we found the zona fasciculata to be most sensitive to time-dependent ultrastructural changes. These consisted of modifications in cell compartments involved in hormone synthesis and release. On the other hand, we found a progressive increase in corticosterone plasma levels which reached a plateau 9 days after noise exposure. The significance of these changes, in relation to phenomena like sensitization to repetitive stress, are discussed. Furthermore, the present data suggest that chronic loud noise exposure might potentially lead to endocrine dysfunctions.

Association of the G protein alpha(q)/alpha11-subunit with cytoskeleton in adrenal glomerulosa cells: role in receptor-effector coupling.

In 3-day primary cultures of rat glomerulosa cells, a 30-min pre-incubation with either 10 microM colchicine (a microtubule-disrupting agent) or 10 microM cytochalasin B (a microfilament-disrupting agent) decreased angiotensin II (Ang II)-induced inositol phosphate accumulation by 50%. Moreover, both drugs decreased inositol phosphate production induced by fluoroaluminate (a nonspecific activator of all G proteins), indicating that both microtubules and microfilaments are essential for phospholipase C activation. Analysis of microfilament- and microtubule-enriched fractions and immunoprecipitation of actin and tubulin revealed that the alpha(q)/alpha11-subunit of the G(q/11) protein was associated with both structures. Ang II stimulation induced a rapid translocation of alpha(q)/alpha11, microfilaments, and microtubules to the membrane and induced a time-dependent increase in the level of alpha(q)/alpha11 associated with both microfilaments and microtubules. Moreover, double immunofluorescence staining clearly showed a colocalization of the alpha(q)/alpha11-subunit of the G(q/11) coupling protein and microfilament distribution. These associations and plasma membrane redistribution under Ang II stimulation indicate that microfilaments and microtubules are both involved in phospholipase C activation and inositol phosphate production. Moreover, our results indicate that the alpha(q)/alpha11 protein is closely associated with cytoskeletal elements and is found both at the plasma membrane level as well as on intracellular stress fibers.

Comparative stereological studies on zonation and cellular composition of adrenal glands of normal and anencephalic human fetuses. II. Cellular composition of the gland.

In our previous paper (Bocian-Sobkowska et al., 1997) we demonstrated a striking difference in development of zonation in adrenals of normal and anencephalic human fetuses. The purpose of the present study was to characterize, by means of stereology, the cellular composition of developing adrenals in the same case. Studies were performed on 11 pairs of adrenal glands from normal fetuses and 10 from anencephalic fetuses. In the studied period of development (24 to 39 weeks of intra-uterine life) the average volume of cells in normal glands increased as follows: zona glomerulosa (ZG) from 355 to 870 microns3; zona fasciculata (ZF) from 779 to 1200 microns3; fetal zone (FZ) from 2004 to 2380 microns3: and medulla (M) from 600 to 970 microns3. In anencephalic fetuses, the appropriate values were: ZG-380-680 microns3; ZF-460-680 microns3; FZ-1820-1680 microns3; and M-870-1400 microns3. At the end of the studied period the number of ZG cells in normal fetuses was two fold higher than in anencephalics, ZF cells-6-fold and in FZ-5-fold higher, while in the M the number of cells was nearly equal in both groups. During the whole investigated period of intra-uterine development the total number of adrenocortical cells in normal glands increased ca 2.5-fold, while in anencephalic glands only ca 0.5-fold, reaching at the end ca 40% of normal value. In both normal and anencephalic adrenals the number of ZG and M cells was highly correlated with ZG/M cell ratio, being slightly higher in normal glands. No such relation was demonstrated for cells of the remaining adrenocortical zones.

The three subtypes of atrial natriuretic peptide (ANP) receptors are expressed in the rat adrenal gland.

Atrial natriuretic peptide (ANP) actions are mediated by highly selective and specific receptors. Three subtypes have been characterized and cloned: ANP receptor-A (or GC-A), -B (or GC-B) and -C (the so-called clearance receptor). In rat adrenal gland, the mRNA for each subtype was detected using 35S-dUTP or digoxigenin-11-dUTP specific labeled probes, and in situ hybridization at light and electron microscopic levels respectively. The three subtypes were expressed the most abundantly in the zona glomerulosa. The amount of GC-A mRNA expression, quantified using macro-autoradiography and densitometry, was higher than the amounts of GC-B mRNA and ANPR-C mRNA both in zona glomerulosa and medullary of adrenal gland. At electron microscopic level, the three subtypes of ANPR were revealed in glomerulosa cells. A noticeable signal was also present in the medullary area, especially for GC-A mRNA, in adrenaline-containing chromaffin cells. No signal was detected in noradrenaline-containing chromaffin cells. The subcellular localization of the three mRNAs is similar: in the cytoplasmic matrix and in the euchromatin of the nucleus in each cell of glomerulosa, and in the same compartments of the adrenaline-containing chromaffin cells. These data indicate that the adrenal gland is an important target tissue for ANP action both in glomerulosa cells and adrenaline-containing chromaffin cells. The mRNA expression levels were different for each ANPR subtype.

Cytoplasmic Ca2+ signalling and reduction of mitochondrial pyridine nucleotides in adrenal glomerulosa cells in response to K+, angiotensin II and vasopressin.

We have examined the mitochondrial formation of NAD(P)H in rat adrenal glomerulosa cells. A short-term elevation of the K+ concentration from 3.6 to 8.4 mM induced a reversible increase in the formation of reduced pyridine nucleotides. Potassium applied after the addition of rotenone had no further effect, confirming that the redox signal was of mitochondrial origin. Inhibition of aldosterone synthesis by aminoglutethimide in K+-stimulated cells decreased the rate of decay of the NAD(P)H signal upon the termination of stimulation, indicating that the NADPH formed was consumed in aldosterone synthesis. When the NAD(P)H signal was measured simultaneously with the cytoplasmic free Ca2+ concentration ([Ca2+]i), elevation of the K+ concentration to 6.6 or 8.4 mM induced parallel increases in [Ca2+]i and NAD(P)H formation. The rates of increase and decrease of NAD(P)H were lower than for [Ca2+]i, confirming that the redox signal was secondary to the Ca2+ signal. Angiotensin II (100 pM-1 nM) induced an oscillatory NAD(P)H signal which usually returned to a lower baseline concentration, while a sustained signal with superimposed oscillations was observed at higher concentrations. Simultaneous measurements showed that NAD(P)H levels followed the [Ca2+]i pattern evoked by angiotensin II. Vasopressin (100 nM) also induced parallel oscillations of [Ca2+]i and NAD(P)H. A sustained rise in the extramitochondrial Ca2+ concentration to 1 microM induced a sustained elevation of the intramitochondrial Ca2+ concentration in permeabilized cells, as measured with rhod-2. A sustained rise in [Ca2+]i evoked by long-term stimulation with 8.4 mM K+ or 2.5 nM angiotensin II resulted in sustained NAD(P)H production. These Ca2+-dependent changes in the mitochondrial redox state support the biological response, i.e. aldosterone secretion by glomerulosa cells.

[Morphological studies of the adrenal zona glomerulosa cells and the thyroid and pituitary glands in streptozocin-induced experimental diabetic rats].

Changes in pathological morphology in the adrenal glomerulosa, the thyroid and pituitary glands in streptozotocin-induced experimental diabetic rats were studied by histologic, ultrastructural examination and morphometry. Experimental diabetes provoked atrophy and retrograde degeneration of organelles in the adrenal zona glomerulosa cells and the thyroid follicular epithelium. In the pituitary of diabetic rats, organelles in thyrotrophs were the same as the controls in morphometry, but TSH release was impaired. These studies indicate that pathological changes in the zone glomerulsa cells and the thyroid of diabetic rats appear to be responsible for the impaired function of these two glands. Moreover, reduced TSH release in the pituitary glands is also associated with impaired function of the thyroid gland.

Membrane-delimited G protein-mediated coupling between V1a vasopressin receptor and dihydropyridine binding sites in rat glomerulosa cells.

In rat glomerulosa cells, vasopressin stimulates intracellular calcium mobilization via at least two distinct mechanisms: the release of calcium from inositol-1,4,5-P3-sensitive stores and the activation of transmembrane calcium influx. In this study, we focused on the second mechanism through three experimental approaches. By videomicroscopically examining Fura-2-loaded cells, we demonstrate that vasopressin induces a dose-dependent and receptor-mediated calcium influx fully inhibited by either 1 microM nifedipine or a pertussis toxin pretreatment and potentiated by 1 microM BAY K 8644. Patch-clamp experiments also indicate that vasopressin stimulates L-type calcium current by 87% and only weakly inhibits T-type calcium current. To further characterize the coupling between the vasopressin receptor and the dihydropyridine calcium channel, we performed binding studies using tritiated nitrendipine. With this technique, we showed that on intact cells, vasopressin is able to increase the specific binding of tritiated nitrendipine in a dose-dependent manner (Kact = 2 nM). Pharmacological studies using a series of vasopressin analogs revealed that this effect is mediated via a V1a vasopressin receptor subtype. Furthermore, the vasopressin-stimulated nitrendipine binding was sensitive to pertussis toxin pretreatment, which affected only the maximum binding capacity of nitrendipine-binding sites. More interestingly, we demonstrate that vasopressin still increases nitrendipine binding to plasma membrane preparation and that GTP is absolutely necessary for such a hormonal effect. Altogether, these data confirm the existence of a tight and direct coupling between the V1a vasopressin receptor and a dihydropyridine calcium channel via a pertussis toxin-sensitive G protein.

Ultrastructural dynamics of mitochondrial morphology in varying functional forms of human adrenal cortical adenoma.

Adrenal cortical mitochondria display an extensive capacity to adapt morphologically to the functional state of the adrenal cortical cell. In the present study, we have used transmission electron microscopy to analyze cortical tissues from 3 normal human adrenal glands (zona fasciculata and zona glomerulosa), and from 8 steroid-secreting adrenal cortical adenomas (3 cortisol-producing, 4 aldosterone-producing, and 1 progesterone-producing tumor), correlating both clinical and biochemical features with cellular ultrastructure. The morphology of mitochondria was related to the enzyme activity and steroid-biosynthetic capacity of each tumor. Cells from aldosterone-producing adenomas demonstrated a large number of elongated tubular mitochondria with characteristic bridging of inner membranes, producing a lamellar-type pattern. Cells from cortisol-producing adenomas showed large round mitochondria with vesicular or tubulovesicular inner membranes surrounded by a characteristic dilated smooth endoplasmic reticulum. A highly unusual progesterone-producing adenoma, in which a deficiency of 21 alpha-hydroxylase activity was demonstrated, showed a peculiar type of enlarged lamellar mitochondria with bright inner matrix and a reduced number of inner membranes. Therefore, the ultrastructural characteristics of adrenal cortical mitochondria appear to be potential markers for the differentiation of steroid-producing adenomas. These studies point to the possibility of a broader use of electron microscopy in the study of adrenal tumors.

Adrenomedullin and calcitonin gene-related peptide inhibit aldosterone secretion in rats, acting via a common receptor.

Adrenomedullin (ADM) and calcitonin gene-related peptide (CGRP) did not affect either basal or ACTH-stimulated secretion of a1dosterone and corticosterone by dispersed rat capsular and inner adrenocortical cells, respectively. However, both peptides strongly depressed angiotensin-II (ANG- II)-stimulated a1dosterone production by capsular cells, the minimal effective concentration was 10(-7) M. The inhibitory effect of both ADM and CGRP was reversed by CGRP8-37, a specific CGRP1 receptor antagonist; a complete reversal was obtained with a CGRP8-37 concentration of 10(-6) M. Our findings indicate that ADM and CGRP specifically interfere with the intracellular mechanisms transducing the secretagogue signal of ANG-II, and suggest that the ADM effect is mediated by CGRP receptors