Steroidogenesis in the human skin: 21-hydroxylation in cultured keratinocytes.

We have evaluated the metabolism of radiolabeled progesterone (P) by the microsomal fraction isolated from HaCaT keratinocytes. P was widely metabolized to different compounds that included DOC (5-7% conversion) thus demonstrated 21-hydroxylase (21-OHase) activity, a key step in adrenal synthesis of gluco- and mineralocorticoids. However, RT-PCR amplification for the CYPc21 transcript of the corresponding gene showed no evidence for gene expression in HaCaT cells suggesting that the 21-OHase enzyme present in keratinocytes is different from that described in adrenal gland. Further characterization showed that whereas estradiol stimulated markedly P metabolism by HaCaT microsomes, with generation of new unidentified compounds, Lineweaver-Burk analysis of keratinocyte 21-OHase activity showed that the K(m) and V(max) were unaffected by estrogen. The apparent K(m) was 0.6 microM without estradiol and 0.7 microM with estradiol, while the respective V(max) values were 60 and 76 nmol/l/min. To conclude, we found extensive metabolism of P in human keratinocytes, we also provide the first demonstration of 21-OHase activity in this cell system and further showed that it is coded by a gene different from the adrenal CYPc21.

Serum 18-hydroxycortisol in primary aldosteronism, hypertension, and normotensives.

This study reports the determination of plasma 18-hydroxycortisol (18-OHF) using a new and easy enzyme-linked immunosorbent assay (ELISA) method in primary aldosteronism and compares the values found in essential hypertensives and normotensive controls. In primary aldosteronism, we evaluated usefulness of plasma 18-OHF determination and the dexamethasone suppression test in the diagnosis of glucocorticoid-remediable aldosteronism using the genetic test as the gold standard. We studied 31 primary aldosteronism patients, 101 essential hypertensives, and 102 healthy normotensive controls. The plasma 18-OHF was measured using a biotin-avidin enzyme-linked assay by a new and purified polyclonal antibody. The 18-OHF value in primary aldosteronism was 6.3+/-8.05 nmol/L; this value is significantly higher than the value found in essential hypertensives and normotensive controls (2.81+/-1.42 and 2.70+/-1.41 nmol/L, respectively; P<0.0005). In primary aldosteronism, 4 of 31 patients had 18-OHF levels that were 10 times higher than the normal upper limit (2.983 nmol/L). The dexamethasone suppression test in primary aldosteronism patients was positive (serum aldosterone <4 ng/dL) in 13 of 31 cases. A chimeric CYP11B1/CYP11B2 gene was demonstrated in 4 primary aldosteronism patients, corresponding to the same cases that had higher level of 18-OHF. In conclusion, plasma 18-OHF determination by this ELISA method is reliable for detecting glucocorticoid-remediable aldosteronism, and it does so better than the dexamethasone suppression test.

Aldosterone esters and the heart.

There are clinical and experimental situations in which symptoms of mineralocorticoid excess are remediable with mineralocorticoid receptor antagonist treatment, in spite of paradoxically low levels of plasma renin and aldosterone. Several decades ago, a factor isolated from the heart was described that had mineralocorticoid properties like those of aldosterone, but much more potent. It was thought to be similar to aldosterone-18-monoacetate or -21-monoacetate, acetyl derivatives of aldosterone that are very rapidly hydrolyzed in the circulation. In our efforts to confirm and extend these observations, we extracted rat hearts and plasma harvested in a manner that would minimize hydrolysis. The product was subjected to several forms of TLC and HPLC and compared to several acetylated derivatives of aldosterone standards. We found that 68% of the aldosterone extracted from fresh myocardium corresponded to an aldosterone derivative that migrates at the same rate as aldosterone-20-monoacetate. The identity of this compound awaits definitive analysis. Tritiated aldosterone-21-monoacetate hydrolyzed to form aldosterone very rapidly; negligible monoacetate remained in blood left at 37 degrees C for 5 min or in hearts left at room temperature for 30 min. Regulation of aldosterone production serves the requirements of fluid and electrolyte homeostasis provided by transport epithelia, primarily that of the kidney. Nonepithelial actions of aldosterone would be freed of these regulatory constraints if the formation of a more potent derivative of the parent compound to which it is almost immediately hydrolyzed in the circulation were regulated within the nonepithelial target tissues.

Active steroidogenesis in the normal rat skin.

Using the radiolabeled precursors of adrenal steroids (14)C-11-deoxycorticosterone (DOC) and (14)C-progesterone ((14)C-PROG) we demonstrate that rat skin can synthesize a number of steroids. TLC separation of labeled metabolites show that among the (14)C-steroid products, two co-migrate with corticosterone (B) and 11-dehydrocorticosterone (A) standards. Thus, normal rodent skin possesses steroidogenic activity that can be shown using progesterone or DOC as primary substrates.

Metabolism of progesterone to DOC, corticosterone and 18OHDOC in cultured human melanoma cells.

We are now showing that cultured human melanoma cells can synthesize steroids such as corticosterone from progesterone or deoxycorticosterone. Corticosterone production is strongly responsive to deoxycorticosterone substrate addition (12-fold increase), but unresponsive to the adrenal stimulating factors ACTH and angiotensin II. This is the first demonstration that skin cells (malignant melanocytes) have the capability to synthesize 11-deoxycorticosterone, corticosterone, and 18-hydroxydeoxycorticosterone.

Aldosterone biosynthesis in the rat brain.

Messenger RNA (mRNA) for enzymes involved in adrenal steroid biosynthesis are expressed in the brain, and the coded enzymes have been shown to be active. The expression of mRNA for the cytochrome P-450 enzyme aldosterone synthase, crucial for the final step in the synthesis of aldosterone and the synthesis of aldosterone was studied in several anatomic areas of the rat brain. Expression of the mRNA for the aldosterone synthase was demonstrated by RT-PCR/Southern blot in adrenal, aorta, hypothalamus, hippocampus, amygdala, cerebrum, and cerebellum. Incubation of brain minces from intact and adrenalectomized rats demonstrated the synthesis of corticosterone and aldosterone from endogenous precursors. Incubations of brain minces with [1,2(3)H]-deoxycorticosterone, followed by extraction and three different successive TLCs, demonstrated the presence of labeled aldosterone, corticosterone, and 18-hydroxy-deoxycorticosterone. Incubation, in the presence of 10 microM cortisol or metyrapone, inhibited the synthesis of aldosterone or both aldosterone and corticosterone, respectively. These studies indicate that the rat brain has the enzymatic machinery for the synthesis of adrenal corticosteroids and is capable of synthesizing aldosterone. Aldosterone synthesized in the brain might play a paracrine role in the regulation of blood pressure.

Inhibition of steroidogenesis in rat adrenal cells by 18-ethynyldeoxycorticosterone: evidence for an alternative pathway of aldosterone biosynthesis.

The effect of the mechanism-based inhibitor 18-ethynyldeoxycorticosterone (18-E-DOC) on the late steps of the aldosterone biosynthetic pathway was examined in freshly isolated cells of the zona glomerulosa (ZG) and fasciculata (ZF) from rat adrenal glands. ZG synthesis of aldosterone was inhibited by 18-E-DOC in a time- and concentration-dependent manner with a Ki of approximately 0.05 microM. The maximal degree of inhibition of ZG production of aldosterone and 18-hydroxycorticosterone (18-OH-B) was approximately 80%. ZF cells, perhaps surprisingly, were found to secrete 18-OH-B at levels approximately one-third to one-fourth those of ZG cells and the Ki of 18-E-DOC inhibition of 18-OH-B secretion was approximately 10 microM for ZF cells, 200-fold higher than for ZG cells. The inhibitor had no effect on the secretion of corticosterone by either ZG or ZF, and the secretion of 18-hydroxydeoxycorticosterone (18-OH-DOC) by both the ZG and ZF was inhibited only to a minor degree. 18-E-DOC inhibited the biosynthesis of aldosterone by ZG cells incubated with 10 microM added DOC or 18-OH-DOC by approximately 75%, similar to the degree of inhibition of aldosterone biosynthesis from endogenous substrate, whereas ZF biosynthesis of 18-OH-B from either substrate was inhibited by less than 40%. ZF cells do not express aldosterone synthase, the only enzyme known to convert 18-OH-DOC into 18-OH-B. Incubation of MA-10 cells stably transfected with the cDNA of the rat aldosterone synthase with 18-E-DOC resulted in a complete inhibition of the conversion of DOC to aldosterone with a Ki of approximately 0.02 microM. In addition, transfected cells expressing 11beta-hydroxylase convert DOC to 18-OH-B in very small quantities only and cannot convert 18-OH-DOC to 18-OH-B. These data suggest that neither 11beta-hydroxylase nor aldosterone synthase are responsible for the biosynthesis of 18-OH-B by ZF cells from DOC or 18-OH-DOC, that 20% of aldosterone synthesis appears not to be attributable to the actions of aldosterone synthase and that an unknown CYP11B enzyme is also involved in the biosynthesis of 18-OH-B.

11 beta-Hydroxysteroid dehydrogenase type 2 complementary deoxyribonucleic acid stably transfected into Chinese hamster ovary cells: specific inhibition by 11 alpha-hydroxyprogesterone.

The 11 beta-hydroxysteroid dehydrogenase type 2 (11 beta HSD-2) enzyme is thought to confer aldosterone specificity upon mineralocorticoid target tissues by protecting the mineralocorticoid receptor from binding by the more abundant glucocorticoids, corticosterone and cortisol. We have developed a Chinese hamster ovary cell line stably transfected with a plasmid containing the rat 11 beta HSD-2 complementary DNA. This cell line has expressed the enzyme consistently for many generations. The 11 beta HSD-2 was located primarily in the microsomes, but significant amounts also existed in the nuclei and mitochondria. The enzymatic reaction was unidirectional, oxidative, and inhibited by the product, 11-dehydrocorticosterone, with an IC50 of approximately 200 nM. The K(m) for corticosterone was 9.6 +/- 3.1 nM, and that for NAD+ was approximately 8 microM. The enzyme did not convert dexamethasone to 11-dehydrodexamethasone. Tunicamycin, an N-glycosylation inhibitor, had no effect on enzyme activity. 11 alpha-Hydroxyprogesterone (11 alpha OH-P) was an order of magnitude more potent a competitive inhibitor of the 11 beta HSD-2 than was glycyrrhetinic acid (GA) (approximate IC50 = 0.9 vs. 15 nM). 11 beta OH-P, progesterone, and GA were almost equipotent (IC50 = 10 and 6 nM, respectively), and 5 alpha-pregnandione and 5 beta-pregnandione were less potent (IC50 = 100 and 500 nM, respectively) inhibitors of the enzyme. When the inhibitory activities were examined with intact transfected cells, 11 alpha OH-P was more potent than GA (IC50 = 5 and 150 nM, respectively). 11 alpha OH-P was not metabolized by 11 beta HSD-2. We were unable to demonstrate the presence of 11 alpha OH-P in human urine. In conclusion, a cell line stably transfected with the rat 11 beta HSD-2 was created, and the enzyme kinetics, including inhibition, were characterized. 11 alpha OH-P was found to be a potent relatively specific inhibitor of the 11 beta HSD-2 enzyme. Its potential importance is that it is the most specific inhibitor of the 11 beta HSD-2 so far encountered and would aid in the study of the physiological importance of the isoenzyme.

Cloning and expression of the rat adrenal cytochrome P-450 11B3 (CYP11B3) enzyme cDNA: preferential 18-hydroxylation over 11 beta-hydroxylation of DOC.

The biosynthesis of glucocorticoids and mineralocorticoids in the rat adrenal cortex requires the action of two different cytochrome P450 11 beta-hydroxylases, CYP11B1 and CYP11B2, which are distributed in the zona fasciculata and glomerulosa, respectively. The existence of another cytochrome P450-11 beta gene, CYP11B3, was recently reported. Although CYP11B3 has similar gene structure and great homology to the CYP11B1 and -B2 genes, the CYP11B3 mRNA was not originally detected by reverse transcription-polymerase chain reaction (RT-PCR) and has only recently been cloned and detected from neonatal rat adrenals. Herein we demonstrate RT-PCR detection of CYP11B3 mRNA expressed in adult rat adrenal and brain tissues. The whole coding region of the CYP11B3 enzyme cDNA was cloned and sequenced. When transiently expressed in COS-7 cells the CYP11B3 converted deoxycorticosterone (DOC) to corticosterone and 18-hydroxydeoxycorticosterone, but not to 18-hydroxycorticosterone or aldosterone. It produced more 18-OH-DOC than corticosterone. A single mutation in CYP11B3 in which Gly-59 was replaced by Ser, reduced the enzymatic activity 5-6-fold. Furthermore, CYP11B3 mRNA expression is greater in neonatal, compared to adult rat adrenal glands.

Stable expression of rat cytochrome P450 11 beta-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 11 beta-hydroxylases, CYP11B1 (11 beta-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 microM. 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.

Is the circulating ouabain-like compound ouabain?

The evidence is very strong for a circulating inhibitor of the sodium, potassium ATPase in volume-expanded hypertension. Recently, this inhibitor was isolated from human plasma and identified as ouabain. We are reporting our results using a very specific and sensitive immunoassay for ouabain with which we were unable to detect or able to detect only very low levels of circulating immunoreactive ouabain. Immunoassay of 5 mL of human and rat plasma, incubation fluid from bovine and human adrenal cell cultures extracted using a C-18 solid phase column, and HPLC separation did not detect a peak corresponding to ouabain. This procedure could easily detect authentic ouabain added to these extracts at a concentration slightly below that reported to be present by others. The extract from the adrenal cultures had clearly detectable sodium, potassium ATPase using an assay based on inhibition of tritiated ouabain binding to human red cells. Extraction of bovine adrenals detected a very small amount of immunoassayable ouabain which did not elute at a time corresponding to that of ouabain. This study indicates that the postulated sodium, potassium ATPase inhibitor that circulates in plasma is not ouabain, but it is likely to be structurally similar to ouabain, as it appears to cross-react with some antibodies against ouabain.

The hybrid rat cytochrome P450 containing the first 5 exons of the CYP11B1 and last 4 exons from the CYP11B2 enzyme retains 11 beta-hydroxylase activity, but the alternative hybrid is inactive.

Human, mouse and rats have 2 different cytochrome P-450 11 beta-hydroxylases in the adrenal cortex. The classical rat 11 beta-hydroxylase or CYP11B1 enzyme hydroxylates deoxycorticosterone to corticosterone and 18-hydroxydeoxycorticosterone and is located throughout the adrenal. The second aldosterone synthase or CYP11B2 enzyme is located in the zona glomerulosa and converts deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. In rat the coding nucleotide sequence and the deduced amino acid sequences of the CYP11B1 and CYP11B2 genes are homologous by 88% and 83%, respectively. We have constructed two different hybrid cDNAs by exchanging two fragments of the rat CYP11B1 and CYP11B2 at the junction of the 5/6 exon and expressed them in COS 7 cells. The hybrid CYPH11B1 construct containing the first 5 exons of the CYP11B1 when expressed, retains 11 beta-hydroxylase activity, but cannot process corticosterone to 18-hydroxycorticosterone or aldosterone. The hybrid CYPH11B2 construct containing the first 5 exons of the CYP11B2 enzyme when expressed is inactive.

Synthesis of 18-hydroxycortisol and 18-oxocortisol in bovine adrenal zona glomerulosa mitochondria.

The biosynthesis of 18-hydroxycortisol and 18-oxocortisol from cortisol was studied in calf adrenal zona glomerulosa mitochondria. Cortisol is converted to 18-hydroxycortisol and 18-oxocortisol in the same mitochondrial preparation in which corticosterone is metabolized to 18-hydroxycorticosterone and aldosterone. Cortisol and 18-hydroxycortisol interacted with mitochondria to cause a Type I differential spectrum, which was decreased by sodium dithionite. The metabolism of cortisol to 18-hydroxycortisol and 18-oxocortisol was inhibited by metyrapone in a competitive way. Cortisol was a competitive inhibitor of the transformation of corticosterone into 18-hydroxycorticosterone and aldosterone, and corticosterone was a competitive inhibitor of the transformation of cortisol into 18-hydroxycortisol and 18-oxocortisol, with a Ki very similar to the Km for the transformation of that steroid to aldosterone. These results indicate that cortisol is metabolized to 18-hydroxycortisol and 18-oxocortisol by a mitochondrial cytochrome P-450, which is the same as that which catalyzes the conversion of corticosterone into aldosterone.

Adrenal receptors for natriuretic peptides and inhibition of aldosterone secretion in calf zona glomerulosa cells in culture.

Atrial and brain natriuretic peptides specifically bind to primary cultures of calf adrenal glomerulosa cells. Binding of both natriuretic peptides to the same receptor has been proved by: a Dixon plot showing competitive effects for the binding of 125I-labeled brain natriuretic peptide in the presence of increasing concentrations of unlabeled atrial natriuretic peptide; a Scatchard plot showing a lower dissociation constant (Kd) for atrial natriuretic peptide than for brain natriuretic peptide binding, but the maximum binding (Bmax) values were the same; autoradiography of sodium dodecyl sulfate polyacrylamide gels after cross-linking of 125I-labeled atrial natriuretic peptide and 125I-labeled brain natriuretic peptide, showing the same molecular weights for both peptide receptors--a single 66-kD band in whole cells and a main band at 125 kD in membranes. C-Type atrial natriuretic peptide only slightly displaced atrial natriuretic peptide binding. Angiotensin II- and potassium-mediated stimulation of aldosterone production were inhibited strongly and to the same degree by atrial and brain natriuretic peptide but only slightly by C-type atrial natriuretic peptide. Stimulation of aldosterone production mediated by adrenocorticotropin was only partially inhibited by atrial and brain natriuretic peptide, while baseline aldosterone was not affected. These results suggest that atrial and brain natriuretic peptide bind to the same receptors and provoke the same effects on aldosterone production. The weak effects found with C-type atrial natriuretic peptide suggest that the primary culture of calf adrenal glomerulosa cells contain the guanylate cyclase A receptor.

Endothelin receptor subtypes and stimulation of aldosterone secretion.

Endothelins (ETs) are 21-amino acid peptides with two disulfide bonds that have powerful vasoactive properties. We have previously shown the presence of a specific, high-affinity, saturable receptor for porcine or human endothelin (ET-1) in cultured calf zona glomerulosa cells. ET-1 was a stimulator of aldosterone secretion although not as powerful as angiotensin II. Incubations of cultured calf zona glomerulosa cells with Sarafotoxin S6b (S6b), a snake venom that has a structure highly homologous to ET-1, stimulated aldosterone secretion with a potency similar to that of ET-1. Binding of [125I]ET-1 to the adrenal receptor gave a Kd of 0.17 +/- 0.05 nM and a Bmax of 36 +/- 8.5 fmol/well (n = 4). Displacement of [125I]ET-1 by unlabeled ETs and S6b showed that the concentrations needed to displace 50% of the tracer were 0.3 nM for ET-1, 0.3 nM for ET-2, 10 nM for S6b, and 100 nM for ET-3. Binding of [125I]S6b to cultured adrenal cells revealed a receptor with a Kd of 0.05 +/- 0.01 nM and a Bmax of 8 +/- 2 fmol/well (n = 4). Displacement of [125I]S6b by unlabeled ETs and S6b showed that the concentrations needed to displace 50% of the tracer were 0.03 nM for S6b, 0.06 nM for ET-1, 0.04 nM for ET-2, and 0.05 nM for ET-3. Unlabeled ET-1 and ET-2 preferentially down-regulated the binding of [125I]ET-1, and S6b preferentially down-regulated the binding of [125I]S6b.(ABSTRACT TRUNCATED AT 250 WORDS)

The binding of cortisol to adrenal mitochondria.

Binding of tritiated cortisol to adrenal zona glomerulosa mitochondria was studied and compared with that of corticosterone. Cortisol was shown to bind specifically to the inner membrane of zona glomerulosa mitochondria. Corticosterone and cortisol had similar apparent association constants (Ka) and concentrations of binding sites. The methodology was validated by obtaining similar Ka from both binding plots and kinetic data. Cortisol binding was inhibited by pretreatment with sodium dithionite, and displaced by deoxycorticosterone, corticosterone, 18-hydroxy-corticosterone, 11 beta-hydroxy-18-ethynyl-progesterone and metyrapone, but not by cholesterol. These results suggest that cortisol and corticosterone bind to the same cytochrome P-450.

Rat mesenteric artery endothelial cells in culture secrete ET-1.

Endothelial cells were harvested by the collagenase perfusion of isolated mesenteric arteries of rats and cultured. An endothelin peptide was detected in the supernatant of these cells by an antibody which recognizes ET-1 but not "rat" endothelin (ET-3). Culture media was extracted using a C-8 solid phase column and subjected to reverse phase HPLC using a system that separates all known endothelins and immunoreactive endothelins measured using another antibody which recognizes all endothelins. The main immunoreactive peak co-eluted with ET-1. We could not detect any ET-2, ET-3 or Vasoactive Intestinal Contractor. A smaller immunoreactive peak of unknown structure that eluted earlier than ET-1 was also detected. In conclusion, rat endothelial cells secrete a peptide of similar chromatographic and immunoreactive properties as ET-1.

Synthesis of 18-hydroxycortisol and 18-oxocortisol in bovine adrenal slices.

The adrenal cortex shows a histological and functional zonation which allows it to function as two distinct glands. Aldosterone is produced only in the outer area, or zona glomerulosa and cortisol is produced in the zona fasciculata. Adrenal slices (200 mu) were prepared from cores of beef adrenals using a McIlwain Tissue Slicer and incubated. Cortisol, corticosterone, aldosterone, 18-hydroxycorticosterone, 18-hydroxycortisol and 18-oxocortisol were measured by radioimmunoassay. Aldosterone and 18-hydroxycorticosterone were produced primarily in the outer slices of the adrenal. Cortisol and corticosterone were produced throughout the adrenal with the production of cortisol in the first layer probably due to contamination with fasciculata. 18-Hydroxycortisol and 18-oxocortisol were produced primarily in the outer slices. The coexistence of cytochrome P-450 corticosterone methyl oxidase I and II and the 17 alpha-hydroxylase in the outer slices leads to the synthesis of these two hybrid steroids.

Endothelin binding to cultured calf adrenal zona glomerulosa cells and stimulation of aldosterone secretion.

Endothelins are a group of potent vasoconstrictors whose structure was deduced from genomic DNA. ET-1 was first isolated from culture supernatants from porcine endothelial cells and ET-3 was identified from a rat DNA library. We report on the binding of 125I-ET-1 to zona glomerulosa cells in culture and on its ability to stimulate aldosterone secretion. Cultured calf adrenal zona glomerulosa cells have saturable, high affinity [Kd = 1.00 +/- 0.17 X 10(-10) M (SEM)] receptors which bind ET-1 in a temperature and time dependent manner. Binding was specific and angiotensin II, vasopressin, ANP, BNP, apamin, calcium channel agonists or antagonists did not interact with the receptor. ET-3 displaced 125I-ET-1 from the receptor with a relative potency of 0.39 +/- 0.1% (SEM) that of ET-1. ET-1 incubated with cultured glomerulosa cells stimulated aldosterone secretion in a dose dependent manner but it was less potent than angiotensin II. ET-3 had less than 1% the relative potency of ET-1 stimulating aldosterone secretion. This data suggest that ET-1 is an independent stimulator of aldosterone secretion and we are speculating that it might be important in those situations, like in malignant hypertension, where endothelial damage might result in increased ET-1 production.