Stable expression of rat cytochrome P450 11β-Hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in MA-10 cells

Glucocorticoids and mineralocorticoids are synthesized in the adrenal cortex through the action of two different cytochrome 11β-hydroxylases, CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) which are distributed in the zona fasciculata and glomerulosa, respectively. We have created stably transfected cell lines using the Leydig tumor cell line MA-10 with CYP11B1 and CYP11B2 cDNA-containing plasmids which have a selectable gene to confer resistance to geneticin. The expression of the transfected cDNA in the cells was characterized by Northern-blot and measurement of enzymatic activity. The cell lines express the enzymes stably for many generations. CYP11B1 transfected cells converted DOC into corticosterone, 18-OH-DOC and small amounts of 18-OH-corticosterone, in a time and concentration dependent manner. Incubation of the cells with corticosterone generated 18-OH-corticosterone especially at concentrations of 30 and 100 μM. The production of 18-OH-corticosterone from corticosterone at these doses was significantly higher than incubations with similar concentrations of DOC. CYP11B2 transfected cells converted DOC into corticosterone, 18-OH-corticosterone, aldosterone and small amounts of 18-OH-DOC in a time and concentration dependent manner. They converted corticosterone into 18-OH-corticosterone and aldosterone in a time and concentration dependent manner. The absolute and relative production of aldosterone from DOC was significantly higher than when cells were incubated with corticosterone, and the ratio of aldosterone to 18-OH-corticosterone was higher at all concentrations of DOC compared to corticosterone. CYP11B2 transfected cells (but not the CYP11B1 transfected cells) transform 18-OH-DOC into 18-OH-corticosterone, but can not convert 18-OH-DOC into aldosterone. In conclusion, stably transfected MA-10 cells with the cDNAs for the CYP11B1 and CYP11B2 enzymes were prepared and their enzymatic activity studied. These cells are useful in the study of inhibitors of the specific enzymes, as well as determining the roles that each enzyme plays in zone-specific steroidogenesis in the adrenal cortex.     

Dexamethasone and corticosterone receptor sites

Summary High resolution light microscopic autoradiography was used, together with regional surveys and combined aeridine orange staining, to define in rat hippocampus cellular and subcellular sites of concentration and retention of ³H dexamethasone and to compare the topographic pattern of labeling with that of ³H corticosterone. Nuclear uptake of ³H dexamethasone in the hippocampus is demonstrated for the first time in vivo. With ³H dexamethasone, strongest nuclear radioactive labeling was observed in certain glial cells throughout the hippocampus, followed by strong nuclear labeling in most neurons in area CA1 and in the adjacent dorsolateral subiculum and weak nuclear labeling in granule cells of the dentate gyrus. Neurons in areas CA2, CA3, CA4, and in the dorsomedial subiculum and indusium griseum showed little or no nuclear labeling after ³H dexamethasone. With ³H corticosterone, strongest nuclear labeling was observed in neurons in area CA2 and in the dorsomedial subiculum and indusium griseum, followed by area CA1, then CA3 and CA4; the dentate gyrus contained scattered strongly labeled cells among cells with intermediate nuclear labeling. At the subcellular level, evidence for both nuclear and cytoplasmic accumulation of label was found. The results indicate that dexamethasone and corticosterone have both nuclear and cytoplasmic binding sites and that particular patterns of target cell distribution exist, characteristic for each agent. This suggests a differential regulation of cellular functions for the two compounds. Corticosterone nuclear binding appears to be more extensive and encompasses regions with dexamethasone binding. Whether in certain of these common regions corticosterone binds to the same receptor as dexamethasone, which seems possible, or to different receptors, remains to be clarified.     

Dexamethasone and corticosterone receptor sites. Differential topographic distribution in rat hippocampus revealed by high resolution autoradiography

High resolution light microscopic autoradiography was used, together with regional surveys and combined acridine orange staining, to define in rat hippocampus cellular and subcellular sites of concentration and retention of 3H dexamethasone and to compare the topographic pattern of labeling with that of 3H corticosterone. Nuclear uptake of 3H dexamethasone in the hippocampus is demonstrated for the first time in vivo. With 3H dexamethasone, strongest nuclear radioactive labeling was observed in certain glial cells throughout the hippocampus, followed by strong nuclear labeling in most neurons in area CA1 and in the adjacent dorsolateral subiculum and weak nuclear labeling in granule cells of the dentate gyrus. Neurons in areas CA2, CA3, CA4, and in the dorsomedial subiculum and indusium griseum showed little or no nuclear labeling after 3H dexamethasone. With 3H corticosterone, strongest nuclear labeling was observed in neurons in area CA2 and in the dorsomedial subiculum and indusium griseum, followed by area CA1, then CA3 and CA4; the dentate gyrus contained scattered strongly labeled cells among cells with intermediate nuclear labeling. At the subcellular level, evidence for both nuclear and cytoplasmic accumulation of label was found. The results indicate that dexamethasone and corticosterone have both nuclear and cytoplasmic binding sites and that particular patterns of target cell distribution exist, characteristic for each agent. This suggests a differential regulation of cellular functions for the two compounds. Corticosterone nuclear binding appears to be more extensive and encompasses regions with dexamethasone binding. Whether in certain of these common regions corticosterone binds to the same receptor as dexamethasone, which seems possible, or to different receptors, remains to be clarified.     

Altered secretion of corticosteroids and prolactin in adrenal regeneration hypertensive rats.

To assess the possible role of mineralocorticoids in the onset and maintenance of hypertension in adrenal regeneration hypertensive (ARH) rats, the change in plasma mineralocorticoids, with adrenal regeneration after enucleation in ARH rats was investigated and compared with those in unilaterally nephroadrenalectomized, 1% saline-fed (UNA) rats, sham-operated, 1% saline-fed (1% NaCl) rats and water-fed (water) rats. Plasma aldosterone was determined by RIA and the other mineralocorticoids were measured by HPLC. How plasma PRL, a marker of central dopaminergic activity, affected aldosterone secretion was determined by RIA. In ARH, plasma corticosterone (B), 18-OH-DOC and aldosterone levels 2 weeks after operation were as low as 20-30% of corresponding values, but the plasma DOC level was almost 100% of the corresponding value in the other groups. Four weeks after operation plasma B increased to a level comparable with that in the other groups and the plasma aldosterone level remained low. However, plasma DOC and 18-OH-DOC levels 4 weeks after operation were as high as 120-200% of corresponding values in the other groups. Six weeks after operation, the plasma aldosterone level returned to a value comparable with that in UNA and 1% NaCl and plasma DOC and 18-OH-DOC levels returned to corresponding values in the other groups. The plasma PRL level 4 weeks after operation was significantly lower in ARH than in the other groups. These results suggest that transient DOC and 18-OH-DOC increases observed in ARH may be important in the onset of hypertension, while other factors may be involved in its maintenance and that the transient central dopaminergic hyperactivity observed in ARH may be responsible for a delayed return from aldosterone deficiency.     

Influence of ACTH secreting tumor (MtTF4) on fetal rat adrenal gland steroidogenesis in vitro in prolonged pregnancy

Pregnant female rats with ACTH secreting tumor (MtTF4) have prolonged pregnancy and cannot deliver. The fetuses of tumor bearing females have in prolonged pregnancy on days 24 and 25 of pregnancy greater body weight and smaller adrenal weight as compared to intact fetuses of the 22nd day of pregnancy. The fetal adrenal glands converted to vitro 4-14C progesterone to radioactive 11-deoxycorticosterone (DOC), corticosterone (B), 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), 18-hydroxy-corticosterone (18-OH-B) and aldosterone. Fetal adrenal glands in prolonged pregnancy synthetized in vitro less amount of radioactive DOC, B and 18-OH-DOC. A negative relationship exists between the maternal corticosterone which passes the placenta to fetuses and corticosteroidogenesis of fetal adrenal glands. These results indicate the possibility that fetal rat adrenal glands with their corticosteroids participate in pregnancy and influence normal delivery.     

18,19-Dihydroxydeoxycorticosterone; A novel product of cytochrome P-45011β-catalyzed reaction

After incubating 18-hydroxydeoxycorticosterone (18-OH-DOC) with cytochrome P-450_11β in the reconstituted system, the products were analyzed with HPLC. There appeared two product-peaks on the chromatogram, one of which was identified as a peak of 18-hydroxycorticosterone (18-OH-B), an expected product of the 11β-hydroxylation. Another peak did not coincide with those of any known corticoids. This unidentified product was further purified, and the purified material was analyzed by gas chromatography-mass spectrometry ($\text{GC}\text{MS}$). The mass spectrum showed that the unidentified product is one of the structural isomers of 18-OH-B. A further analysis with ¹H-NMR spectrometry indicated that a proton resonance peak of 19-CH₃ in 18-OH-DOC disappeared in the product and the methyl group of the substrate seemed to be converted to -CH₂OH. These results suggested that the unidentified product generated from 18-OH-DOC by P-450_11β-linked hydroxylase system may be 18,19-dihydroxydeoxycorticosterone (18,19,21-trihydroxypregn-4-ene-3,20-dione; 18,19-diOH-DOC), a hitherto unreported corticoid.     

Gonadotropin-releasing hormone (GnRH) neurons and pathways in the rat brain

Summary Gonadotropin-releasing hormone (GnRH) neurons and their pathways in the rat brain were localized by immunocytochemistry in 6-to 18-day-old female animals, by use of thick frozen or vibratome sections, and silver-gold intensification of the diaminobenzidine reaction product. GnRH-immunoreactive perikarya were observed in the following regions: olfactory bulb and tubercle, vertical and horizontal limbs of the diagonal band of Broca, medial septum, medial preoptic and suprachiasmatic areas, anterior and lateral hypothalamus, and different regions of the hippocampus (indusium griseum, Ammon's horn). In addition to the known GnRH-pathways (preoptico-terminal, preoptico-infundibular, periventricular), we also observed GnRH-immunopositive processes in several major tracts and areas of the brain, including the medial and cortical amygdaloid complex, stria terminalis, stria medullaris thalami, fasciculus retroflexus, medial forebrain bundle, indusium griseum, stria longitudinalis medialis and lateralis, hippocampus, periaqueductal gray of the mesencephalon, and extracerebral regions, such as the lamina cribrosa, nervus terminalis and its associated ganglia. By use of the silver-gold intensification method we present Golgi-like images of GnRH perikarya and their pathways. The possible distribution of efferents from each GnRH cell group is discussed.     

Heterogeneity of steroid hormone receptor in adult rat lung cytosol

Chromatography on cellulose DEAE-52 columns revealed that the glucocorticoid receptor from rat lung cytosol consisted of a component in the 0.001 M prewash, revealed with synthetic steroids and natural mineralocorticoids, a second component eluted with 0.04 M PO₄, labelled with triamcinolone, dexamethasone, and a third moiety in the 0.06 M PO₄ region, evident with natural glucocorticoids (corticosterone, cortisol) as well as mineralocorticoids (aldosterone, deoxycorticosterone). The thrid component coelutedf with rat blood serum transcortin in double labelled experiments. Rat lung was devoid of another component in the 0.02 M PO₄ found in rat liver supernate and of the mineralocorticoid receptor evident only in rat kidney. Chromatography on Sephadex G-200 columns revealed a shift of radioactivity from a higher to a lower molecular weight region in the presence of 0.4 M KCl. Collectively, these studies indicate the subunit nature of the lung receptor as evidenced in most tissues hitherto tested. Moreover, polymorphism within a given subunit component can not be revealed by competition alone, as attempted by others, but can be revealed under selected conditions of physical separation.     

Effects of dibutyryladenosine 3',5'-monophosphate on steroid biosynthesis in humans

The effects of dibutyryladenosine 3', 5'-monophosphate (DBcAMP), a nucleotide analogue, on blood pressure, serum electrolytes and plasma corticoid concentrations were investigated in 10 normotensive healthy subjects who received a constant diet containing 5-8 g sodium chloride in hospital. The systolic blood pressure did not change after infusion of 0.25 or 0.33 mg/kg/min of DBcAMP for 20 min. On the other hand, the diastolic blood pressure was significantly decreased after the infusion of DBcAMP. The levels of serum sodium and potassium were significantly decreased after the infusion of DBcAMP. After infusion of 0.25 mg/kg/min of DBcAMP for 20 min, the changes in plasma levels of 6 corticoids [progesterone, deoxycorticosterone (DOC), 18-hydroxy-deoxycorticosterone (18-OH-DOC), corticosterone, cortisol and dehydroepiandrosterone sulfate (DHEA-S] revealed no significant changes. After infusion of 0.33 mg/kg/min of DBcAMP for 20 min, the plasma levels of cortisol, corticosterone and 18-OH-DOC were significantly increased and the changes in plasma levels of aldosterone showed a tendency to increase but this was not significant. The plasma levels of DOC and DHEA-S were not appreciably changed, while the plasma levels of progesterone were significantly decreased after the infusion of 0.33 mg/kg/min of DBcAMP. It is speculated therefore that DBcAMP may act to enhance the activity of the sodium-potassium pump and to promote steroid biosynthesis dose-dependently in humans.     

Effects of prostaglandins on adrenal steroidogenesis in the rat

To elucidate the role of prostaglandins in adrenal steroidogenesis, we studied aldosterone and corticosterone responses to

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of prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α), prostacyclin (PGI₂), and arachidonic acid (AA) in collagenase dispersed rat adrenal capsular and decapsular cells. Whereas adrenocorticotrophic hormone (ACTH) and angiotensin II (AII) stimulated aldosterone production in capsular cells and ACTH stimulated corticosterone production in decapsular cells in a dose dependent fashion, aldosterone and corticosterone production were not stimulated significantly by PGE₂, PGF_2α, PGI₂, and AA. Although preincubation of dispersed adrenal cells with indomethacin ( ) markedly inhibited PGE₂ synthesis, ACTH- and AII-stimulated aldosterone production and ACTH-stimulated corticosterone production were not attenuated despite prostaglandin blockade. These results indicate that prostaglandins are unlikely to play an important role in adrenal steroidogenesis.     

Corticosteroid receptors in the avian kidney

The binding $\text{in vitro}$ of tritiated aldosterone to domestic duck ($\text{Anas platyrhynchos}$) kidney tissue has been investigated. Using tissue from animals on a normal diet, tritiated aldosterone was specifically bound to kidney cytosol with an apparent equilibrium dissociation constant of about 9 nM and number of binding sites in the 20 fmol/mg protein range. These values did not show statistically significant changes when the cytosol originated from animals with salt activated nasal glands. Kidney cytosols labeled with tritiated aldosterone sedimented with a single peak at 8S in a linear sucrose gradient (10–30%) and this peak was quenched by excess, radioinert aldosterone. Following incubation of labeled cytosols with crude nuclei, the cytosols became depleted of the label and aldosterone was translocated to the Tris-soluble and Tris-insoluble, 0,4 M KCl soluble nuclear fractions. Kidney cytosols metabolized aldosterone extensively to a compound presumed to be 3α,5β-tetrahydroaldosterone. However, only unchanged aldosterone became receptor-bound. It was concluded that the duck kidney possesses aldosterone receptors, though competition studies indicated that the specificity of these receptors might be different from those described in the mammalian kidney.     

Food hoarding,but not food intake,is attenuated by acute diazepam treatment in female Mongolian gerbils (Meriones unguiculatus)

This article is part of a Special Issue “Energy Balance”. Effects of γ-aminobutyric acid (GABA) on food hoarding are unknown in rodents, and the effects of energy balance and GABA have not been evaluated in females. To evaluate the role of food deprivation and GABA on food hoarding, female Mongolian gerbils were given i.p. injection of diazepam (1 mg/kg and 3 mg/kg, respectively), a GABA_A receptor agonist. Among food-deprived females, there was a bimodal pattern in the frequency of gerbils with different levels of food hoarding. High food hoarding (HFH) and low food hoarding (LFH) gerbils were analyzed. Diazepam blocked food deprivation-induced food hoarding in HFH gerbils, but not in LFH gerbils. This blockade was associated with increased cellular activation in selected brain areas, such as the nucleus accumbens (NAcc), caudate putamen (CP) and ventral tegmental area (VTA), which suggested that direct activation of GABA in the brain reward circuitry decreased food hoarding in HFH females. Moreover, diazepam increased Fos expression in field CA₂ and CA₃ of the hippocampus, but had no significant effect on Fos expression in field CA₁ and dentate gyrus (DG) of the hippocampus, indicating that the hippocampus has area-specific effects on food hoarding in HFH gerbils. Diazepam did not alter food intake in both HFH and LFH gerbils. In addition, serum corticosterone concentrations were higher in the HFH than in the LFH ones. Together, these data indicated that food deprivation increased food hoarding in female gerbils, diazepam reduced food deprivation-induced food hoarding in HFH gerbils, and that GABA might influence food hoarding via classical reward circuitry via the mesolimbic dopamine system and specific hippocampal areas.     

The measurement of 18-hydroxy-11-deoxycorticosterone in human plasma by radioimmunoassay

A radioimmunoassay method for the measurement of plasma levels of 18-hydroxy-11-deoxycorticosterone (18-OH-DOC) has been developed. The antiserum against 18-OH-DOC was produced in rabbits immunized against 18-OH-DOC-3-oxime-bovine serum albumin. Plasma (1–2 ml) was extracted with dichloromethane and chromatographed on paper. The purified extracts were incubated with antiserum at a $\text{1}\text{22,000}$ dilution for $\text{1}\text{2}$ hour at 37°C and for 2 hours at 4°C. Saturated ammonium sulfate was used to separate free from bound 18-OH-DOC. 1, 2-³H-18-OH-DOC was added to all samples to correct for losses and to determine the percent free. Pyridine (0.1%) was added to solvents to maintain the stability of 18-OH-DOC. Recovery after extraction was 58 ± 8 (S.D.)%. The accuracy and precision of the method were acceptable, and a sensitivity of 2 pg per sample enabled the measurement of very low levels of 18-OH-DOC. High specificity was demonstrated by a low blank value (0 ± 0.2 pg) and by demonstrating that alternative paper chromatography separation systems gave results not differing significantly from those obtained by the present method. The mean 8AM plasma 18-OH-DOC level was 8.5 ± 1.2 ng per 100 ml in 18 normotensive control subjects. There was a marked response of plasma 18-OH-DOC to ACTH stimulation and dexamethasone suppression and a significant increase after 3 hours upright posture.     

18-Hydroxy-11-deoxycorticosterone in hypertensive uremic patients during hemodialysis.

The present study was carried out in 25 hypertensive uremic patients on regular 4 h dialysis, 3 times a week. Plasma 18-hydroxy-11-deoxycorticosterone (18-OH-DOC), aldosterone (PA) and corticosteroids were determined by radioimmunoassay and competitive protein binding technique before and at the end of the 1st, 2nd and 3rd hour of hemodialysis. Plasma 18-HD-DOC was normal before dialysis and did not change significantly during hemodialysis, whereas body fluids and electolytes decreased progressively. No correlation was observed between blood pressure and 18-OH-DOC during dialysis. 18-OH-DOC did not correlate with PA which decreases progressively during hemodialysis and was correlated to plasma corticosteroids only at the 3rd hour of dialysis, probably on account of the enhanced influence of ACTH on the adrenal cortex.     

Antagonistic effects of aldosterone on corticosterone-mediated changes in exploratory behavior of adrenalectomized rats

The effect of aldosterone administration on exploratory activity of chronic adrenalectomized (10 days) male rats was investigated. Aldosterone (30 μg/100 g body wt sc) administered 1 hr or 30 min prior to the behavioral test failed to normalize disturbed exploratory activity of adrenalectomized rats, in contrast to the restoration observed after corticosterone, the naturally occurring glucocorticoid of the rat. Administration of the mineralocorticoid 30 min prior to corticosterone prevented the normalization of the behavioral response by the latter steroid. Administration of the same dose of aldosterone 30 min prior to a tracer amount of [³H]corticosterone effectively blocked cell nuclear uptake of radioactive-labeled hormone in the hippocampus. The specific action of corticosterone on exploratory behavior corresponds with the stringent specificity of the neuronal hippocampal corticosterone receptor system. Mineralocorticoid receptors do not seem to be involved in effects on this behavior. The antagonistic action of aldosterone is probably exerted by competitive binding to the corticosterone receptor.     

Autoradiographic techniques and the localization of estrogen, androgen, and glucocorticoid in the pituitary and brain

SYNOPSIS. Autoradiographic techniques are reviewed which havebeen recommended. for the localization of diffusible substances,such as steroid hormones. Advancement in techniques, includinglow temperature tissue sectioning, section freeze-drying, anddry-mounting of sections, led to the development of the dry-mountautoradiographic technique. This progress in technique has enabledthe cellular and subcellular topotgraphic localization of steroidhormones in peripheral and central target tissues, includingthe identification of hormone target cells in the pituitaryand mapping of hormone neurons in the brain. In the pituitary,tritiated estrogen, androgen, and glucocorticoid are concentratedand retained in nuclei of certain anterior lobe cells. In thebrain, estrogens, androgens, and glucocorticoids are attractedby and concentrated in nuclei of certain neurons located mainlywithin the phylogenetically old periventricular brain. In viewof the widespread distribution of sex steroids in differentbrain areas, the generally held concept of a topographicallyconfined single or dual "sex center" is challenged. While estrogenand androgen neurons in the hypothalamus, in the preoptic-septal-parolfactoryregion, and in the amygdala overlap, or are even identical inpart, glucocorticoid neurons are more heavily concentrated inthe gyrus dentatus, hyppocampus, indusium griseum, dorsal nucleisepti lateralis and medialis, as well as in the piriform cortexand portions of the amygdala. It is conceptualized that thesteroid hormone neurons are hypophysiotropic neurons, beinginvolved in the neurosecretion of releasing factors, and thatthey represent sought for hormone "feedback" areas in the brain.This challenges the generally held view of the "hypophysiotrophicarea" in the hypothalamus as the anatomical site where releasingfactors are produced.     

Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the limbic system of the rat.

We used immunocytochemistry to obtain a complete cellular and subcellular mapping of the 1,25-dihydroxyvitamin D3 receptor protein (VDR) in the rat limbic system. We observed specific VDR immunostaining in the nucleus as well as in the perinuclear cytoplasm of neuronal cells. The limbic system consists of a variety of neuronal structures, and is known to have influence on memory, behavior, emotions and reproduction. In the hippocampal formation, we found strong nuclear staining as well as less distinguished cytoplasmic VDR staining in CA1, CA3 and CA4. The CA2 area showed a unique cytoplasmic predominance of VDR. The amygdala was found to exhibit specific patterns of VDR distribution in the various regions of the nucleus. We observed distinct differences of VDR localization within the limbic preoptic areas of the hypothalamus. Further parts of the brain we analyzed included the mammillary bodies, the indusium griseum and the cingulate cortex. The subcellular distribution of VDR in regions of the limbic system suggests a specific functional role of the receptor protein and indicates a role for calcitriol as a neuroactive steroid.     

The adrenal and vascular effects of angiotensin II and III in sodium depleted rats

The effects of angiotensin II and angiotensin III on mean arterial pressure, serum aldosterone, and serum corticosterone were studied in normal and sodium depleted, conscious rats. In normal rats, angiotensin III was 76% (p > 0.10) as potent as angiotensin II on aldosterone release but only 31% (p < 0.001) as potent on blood pressure. Following sodium depletion, the pressor responses to both angiotensin II and III were reduced (p < 0.001) (65% and 86% respectively). In addition, the release of aldosterone by both peptides was potentiated by sodium depletion as indicated by an increase in the slope of the dose-response curves. However, in the sodium depleted rats, angiotensin III was only 20% (p < 0.001) as potent as angiotensin II in stimulating aldosterone release. Small changes in serum corticosterone were noted following infusions of both peptides, but unlike the case with aldosterone, sodium depletion did not alter the serum corticosterone responses to the peptides. These $\text{in}$$\text{vivo}$ experiments taken with $\text{in}$$\text{vitro}$ studies support the interpretation that angiotensin III could function to control aldosterone release in altered sodium states either as a circulating hormone if present in concentrations far in excess of those of angiotensin II or as a local hormone formed in the adrenal from angiotensin II.     

Dopaminergic control of aldosterone: modulation of the response of rat adrenal zona glomerulosa cells to alpha-Msh by pretreatment with bromocriptine or metoclopramide

Bromocriptine treatment in rats (3 mg/kg per day, 7 days) significantly reduced alpha-msh and aldosterone plasma levels 2 hrs after the final treatment in animals on low, normal and high sodium diets. Alpha-MSH dose response curves for corticosterone and 18-hydroxydeoxycorticosterone (18-OH-DOC) in subsequently incubated glomerulosa cells gave stimulation at lower concentrations of alpha-MSH (10(-10) moles per litre) than in cells from untreated animals (10(-9) moles per 1). Curves for aldosterone (ald) and 18-hydroxycorticosterone (18-OH-B) were also affected in cells from animals on a low sodium diet. Fasciculata-reticularis cell responses to ACTH were unaffected. Metoclopramide (4 mg/kg per day, 7 days) elevated plasma alpha-MSH, although ald was unaffected, but inhibited the glomerulosa cell response to alpha-MSH in vitro. Acute dopaminergic responses in plasma ald may be mediated through alpha-MSH in rats, but chronically alpha-MSH may down- regulate glomerulosa cell alpha-MSH receptors. It is unlikely that alpha-MSH mediates the adrenocortical response to sodium depletion.     

Mechanisms of enhanced angiotensin II–stimulated signal transduction in vascular smooth muscle by aldosterone

We tested the hypothesis that mineralocorticoids potentiate angiotensin II–stimulated phospholipase C activation through an increased number of angiotensin II receptors in cultured rat aortic vascular smooth muscle cells. Exposure of cells to aldosterone for 24 h resulted in concentration-dependent increases in angiotensin II receptor binding. Via studies of angiotensin II displacement by non-peptide receptor antagonists, both basal and upregulated angiotensin II receptors were found to be of the AT₁, subtype. Incubation with 1 μM aldosterone resulted in 50%–100% enhancement of angiotensin II (100 nM)–stimulated diacylglycerol formation and intracellular calcium mobilization. Exposure to 100 nM 1,25-(OH)₂ VitD₃, which did not upregulate angiotensin II receptors, did not potentiate stimulated inositol phosphate formation. Incubation with aldosterone resulted in potentiation of inositol phosphate formation upon receptor occupation (100 nM angiotensin II) but not upon post-receptor stimulation (25 mM NaF/10 μM AlCl₃). Aldosterone did not increase basal phospholipase C activity or content of the inositol trisphosphate precursor phosphatidylinositol-4,5-bisphosphate. These data are consistent with the hypothesis that aldosterone potentiates angiotensin II–stimulated, phospholipase C-dependent intracellular signals solely by coupling to an increased number of angiotensin II receptors. This mechanism may contribute to the sensitized vascular responses to angiotensin II observed in states of mineralocorticoid excess. © 1994 Wiley-Liss, Inc.