The resistance of the Wistar/Furth rat strain to steroid hypertension.

We have previously reported that the Wistar/Furth (W/Fu) rat strain is resistant to mineralocorticoid hypertension. In the current study, we have examined renal mRNA levels for mineralocorticoid receptor (MR), glucocorticoid receptor (GR), renin and Na+, K(+)-ATPase in response to treatment with mineralocorticoids. Uninephrectomized male Wistar (WI) and W/Fu rats were treated with aldosterone or deoxycorticosterone acetate (DOCA) and were given 1% NaCl to drink. Rats were sacrificed after 1, 3 or 7 days of treatment. Renal MR and ATPase mRNA levels were significantly reduced in aldosterone and DOCA-treated WI rats (e.g. MR was 30% on day 3 and ATPase was 50% of control on day 7 of aldosterone treatment). Unexpectedly, GR mRNA levels paralleled the changes in MR. In W/Fu rats the level of message was either unchanged or only moderately altered by this treatment. In vivo administration of the MR antagonist RU28318 or the GR antagonist RU38486 to WI rats for 4 days reduced renal mRNA levels for both subunits of ATPase. In the W/Fu rat, this treatment resulted in no change in the alpha subunit and an increase in the beta subunit of ATPase. In preliminary studies, we have determined that the W/Fu rat is also resistant to dexamethasone-induced hypertension. These studies suggest that altered MR- and GR-mediated mechanisms may contribute to the resistance of the W/Fu rat strain to steroid-induced hypertension.

Cytochrome c oxidase in rat adrenal and liver: effects of anti-androgen treatment and studies in testicular feminized rats.

The role of androgen receptors in androgen-induced changes in rat adrenocortical and liver cytochrome c oxidase (COX) has been investigated. The anti-androgen flutamide, blunted the increase in COX activity and COX subunits II/III and IV, that is seen with androgen treatment. Testicular feminized (Tfm) rats had levels of COX activity and COX subunits II/II and IV in adrenal cortex and liver that were intermediate between the high levels found in normal male rats and the lower levels of normal female rats. These data suggest that androgen effects on adrenal and liver COX are mediated through interactions with androgen receptors known to be present in these issues. However, the observed changes in COX activity and COX subunits were not accompanied by altered levels of mRNAs encoding for COX II or COX IV.

Sex differences in cytochromes oxidase and P-45011 beta in the rat adrenal cortex.

A comparative study of cytochrome c oxidase (COX) activity and expression as well as cytochrome P-45011 beta expression has been carried out on the adrenal cortex of male and female rats. COX has also been examined in rat liver. In addition, the effect of testosterone replacement in orchiectomized male rats on adrenal COX has also been investigated. Adult male rats had higher COX activity in adrenal (255%) and liver (144%) mitochondria compared to adult female rats. Male rat adrenals and liver also had increased levels of COX II, a mitochondria-encoded COX subunit, and of COX IV, a nucleus-encoded COX subunit, as measured by Western analysis. In contrast, cytochrome P-45011 beta levels were lower (48%) in adrenal mitochondria from male rats than those of female rats. There was no significant sex difference in the level COX II and COX IV mRNAs in adrenal or liver, whereas the cytochrome P-45011 beta mRNA was 4-fold higher in female adrenals than in males. In male rats, orchiectomy caused a 23% decrease and testosterone replacement a 66% increase in adrenal COX activity. There were no corresponding changes in the levels of mRNAs encoding for COX subunits, suggesting post-transcriptional effects of testosterone on COX. These results are consistent with a regulatory role of testosterone on the expression of components of the respiratory and steroidogenic electron transport chains.

Sex differences in the steroidogenic and respiratory electron transport chains in the rat adrenal cortex.

Both steroid 11 beta-hydroxylation and cholesterol side chain cleavage occur in the mitochondria of adrenocortical cells and they require reducing power in the form of NADPH. There are direct sources of NADPH in rat adrenal mitochondria but another potential source of NADPH is the energy-linked transhydrogenase reaction. This suggests that there is a relationship between the steroidogenic and respiratory chains. We have elaborated upon this relationship by exploring the expression of cytochrome c oxidase (CO) and cytochrome P-45011 beta. We have studied the regulation of one mitochondrial-encoded (COII) and one nuclear-encoded (COIV) subunit. Normal, untreated male rats had higher basal levels of activity of CO in adrenal (255%) and liver (144%) mitochondria, compared to normal, untreated female rats. They also had increased COII (300% and 138%) and COIV (300% and 135%). Cytochrome P-45011 beta levels, however, were lower (48%) in adrenal mitochondria of male rats than those of female rats. Androgen treatment of male rats caused an increase in the activity of CO in the mitochondria of the adrenal gland with the levels being 171% of the corresponding controls. This increase in activity paralleled an increase in the levels of COII and COIV in the adrenal as measured by Western analysis. In contrast, adrenal cytochrome P-45011 beta levels were lower (68%). Androgen treatment caused no significant change in the levels of mRNA's for COII and COIV whereas cytochrome P-45011 beta mRNA was significantly lower than normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Beta-endorphin responses to metyrapone and dexamethasone in depressed patients.

Controversy continues over the characteristics of beta-endorphin secretion in depression. Beta-endorphin plasma levels were measured in 30 drug-free male patients with a DSM-III-R major depressive disorder and 21 healthy controls. Depressed patients displayed significantly lower beta-endorphin plasma levels in baseline conditions, after the single dose metyrapone test, and after the dexamethasone suppression test. The activation of hypothalamic-pituitary-adrenal (HPA) axis in depression might be due, at least in part, to low levels of beta-endorphin. These results suggest that HPA axis dysregulation in depression may involve peptides other than ACTH.

The inhibition of rat adrenal cytochrome P-45011 beta gene expression by androgens.

The synthetic androgen methylandrostenediol (MAD) and the naturally occurring one, testosterone, both bring about hypertensive cardiovascular disease when chronically administered to rats. The pathogenesis of this form of experimental hypertension is thought to result from inhibition of steroid 11 beta-hydroxylase activity. In contrast to the above androgens, 19-nortestosterone, androstenedione and dehydroepiandrosterone (DHEA) have been reported to be without effect in elevating blood pressure. To examine the mechanism(s) involved, we have in this study compared the effects of a number of androgens on adrenal cytochrome P- 45011 beta enzyme and mRNA steady state levels. These parameters were also correlated with the ability of adrenal mitochondria isolated from these groups to hydroxylate 11-deoxycorticosterone (DOC) to corticosterone and 18-hydroxy-11-deoxycorticosterone (18-hydroxy-DOC). Rats treated for seven days with 10 mg per day of dihydrotestosterone, testosterone, 19-nortestosterone or MAD showed a profound decrease in cytochrome P-45011 beta mRNA levels (to less than 20% of controls). This was accompanied by similar changes in both the level and activity of the enzyme. Androstenedione and DHEA were less potent in effecting these changes. In addition, for MAD and testosterone we tested the dose dependence of these changes and found that increasing doses (0.1 mg to 10 mg per day) of either androgen caused progressive decreases in the parameters measured. To assess selectivity we also determined the steady state level of cytochrome P-450scc mRNA in rats treated with the various androgens. In contrast to what was found with cytochrome P-45011 beta, the mRNA transcript for cytochrome P-450scc was equal to or above control levels. We conclude that, in general, the extent of inhibition of cytochrome P-45011 beta enzyme and mRNA level by a given androgen correlates with its reported facility in producing hypertension in rats. Increased secretion of DOC continues to be a likely mechanism for the development of this hypertension but with some androgens extra-adrenal effects may be involved.

Expression of adrenal cytochromes P-450 in testosterone-induced hypertension.

Chronic treatment of rats with the naturally occurring androgen, testosterone, leads to hypertension and cardiovascular disease. This effect is believed to be mediated through the adrenal gland and in particular by action on the steroid 11 beta-hydroxylase enzyme system. To study the possible mechanism of this effect, the enzyme system was examined at several time periods up to the time that hypertension develops. Rats were treated with testosterone (10 mg/day) for 3, 7, 21, and 42 days. Levels of cytochrome P-450(11) beta enzyme and messenger RNA (mRNA) were determined as well as 11 beta-hydroxylase enzyme activity. A significant decrease in enzyme activity was observed after 3 days of treatment. This correlates with a profound decrease in the level of cytochrome P-450(11) beta enzyme as determined by Western blot analysis. A large decrease in cytochrome P-450(11) beta mRNA was also observed after 3 days of treatment. All three parameters remained low throughout the treatment period. The decrease in 11 beta-hydroxylase enzyme activity appears to result from a lower enzyme level brought about by decreased concentrations of mRNA transcripts.

The gene expression of cytochrome P-450(11) beta during adrenal regeneration in the rat: studies using in situ hybridization.

Low levels of mRNA for the key adrenal steroidogenic enzyme, cytochrome P-450(11) beta, have been reported in the early stages of adrenal regeneration. To determine whether the defect in cytochrome P-450(11) beta gene expression is located in the adrenal zona fasciculata or glomerulosa, in situ hybridization techniques have been employed. Sections from 1 week regenerating adrenals were probed with sense and anti-sense cytochrome P-450(11) beta RNA. The signal was intense in the zona fasciculata cells in control adrenals but very low in those of adrenal regenerates. These data establish that the low levels of mRNA transcripts of cytochrome P-450(11) beta are localized to the zona fasciculata cells responsible for 11-deoxycorticosterone secretion in regenerating adrenals.

Effects of metyrapone and dexamethasone upon pro-gamma-MSH plasma levels in depressed patients and healthy controls.

There is current controversy over the mechanisms underlying hypothalamic-pituitary-adrenal (HPA) axis hyperactivity in depression. Pro-gamma-melanocyte-stimulating hormone (MSH), a portion of the N-terminal region of pro-opiomelanocortin, has been shown to act synergistically with adrenocorticotropic hormone (ACTH) in stimulating corticosteroid secretion both in vitro and in vivo. Pro-gamma-MSH and ACTH plasma levels were measured in 30 drug-free male patients with a DSM-IIIR major depressive disorder and 21 healthy controls. The baseline levels were similar in the two groups. After single-dose metyrapone stimulation, both hormones increased, but pro-gamma-MSH was significantly higher in control subjects than in depressives. After overnight 1-mg dexamethasone, ACTH was significantly less suppressed in depressives than controls. These results suggest that HPA axis dysregulation in depression may involve peptides other than ACTH and be more complex than previously reported.

Cytochromes P-45011 beta, P-450scc and adrenodoxin gene expression during adrenocortical regeneration in the rat.

Circulating levels of 11-deoxycorticosterone are increased during the development of adrenal-regeneration hypertension. The present studies were undertaken to examine the mechanisms involved in this increase. Plasmids containing cDNA inserts coding for cytochrome P-45011 beta, cytochrome P-450scc and adrenodoxin were used to determine, by Northern blot analysis, mRNA levels at 1, 2 and 3 weeks of adrenal regeneration. There was a striking decrease in mRNA transcripts for all three enzymes during the first week of regeneration when compared with intact adrenal tissue. Over the next 2 weeks the mRNA levels increased to 64% for P-45011 beta, to 80% for P-450scc and to 82% for adrenodoxin. Actin mRNA levels were 70% of control levels in the first week but were back to control levels by the second week. The present findings suggest that decreased expression of steroid 11 beta-hydroxylase could contribute to the increased secretion of 11-deoxycorticosterone during the early stages of adrenal regeneration.

Levels of adrenodoxin, NADPH-cytochrome P-450 reductase and cytochromes P-45011 beta, P-45021, P-450scc, in adrenal zona fasciculata-reticularis tissue from androgen-treated rats.

Treatment of rats with methylandrostenediol (MAD), an anabolic androgen, caused a profound reduction (65%) in the level of cytochrome P-450 11 beta in rat adrenocortical mitochondria as measured by immunoblots using a specific antibody. The decreases in mitochondrial cytochrome P-450scc (15%) and adrenodoxin (20%) were much less than that observed for cytochrome P-45011 beta. A 35% decrease in adrenal microsomal cytochrome P-450 21 and NADPH-cytochrome P-450 reductase levels was brought about by the treatment with MAD. The data establish that the preferential decrease in adrenal steroid 11 beta-hydroxylase activity associated with androgen treatment results from a decrease in cytochrome P-450 11 beta. This is consistent with the role of 11-deoxycorticosterone in the pathogenesis of androgen-induced hypertension in rats.

Steroidogenesis-activator polypeptide isolated from a rat Leydig cell tumor.

A cycloheximide-sensitive protein responsive to adenosine 3',5'-monophosphate has been postulated to participate in the regulation of cholesterol side-chain cleavage activity in steroidogenic tissues. Such a steroidogenesis activator polypeptide (SAP) had been isolated from rat adrenocortical tissue and partially characterized. Now a polypeptide with comparable chromatographic behavior and biological activity has been purified from the rat H-540 Leydig cell tumor in quantities sufficient for amino acid sequencing. The activator contains 30 amino acid residues and has a molecular weight of 3215. The synthetic construct based on this sequence is virtually equipotent with native H-540 tumor SAP in an adrenal mitochondrial cholesterol side-chain cleavage assay. Hormonal regulation of the intracellular concentration of this activator may control the rate of cholesterol metabolism in steroidogenic organs.

Gamma 3-melanotropin promotes mitochondrial cholesterol accumulation in the rat adrenal cortex.

Gamma 3-melanotropin (gamma 3-MSH) facilitates a rapid, dose-dependent, and cycloheximide-insensitive increase in the concentration of mitochondrial free cholesterol in the adrenals of hypophysectomized rats. Physiological concentrations of various synthetic and native preparations of gamma 3-MSH are potent, while gamma-MSH is not. This cholesterol accumulation coincides with the activation of cholesteryl ester hydrolysis by gamma 3-MSH, while the rates of cholesterol esterification and of mitochondrial cholesterol side-chain cleavage are unaffected. Conversely, ACTH inhibits cholesterol ester esterification. Therefore, gamma 3-MSH and ACTH together may coordinate a substantial shift in the set-point of cholesteryl ester in equilibrium cholesterol cycling toward the right. Because ACTH also activates cholesterol side-chain cleavage, this coordinate effect on the flux of cholesterol substrate is manifest as a potentiation of corticosteroidogenesis by gamma 3-MSH. These data extend our previous studies demonstrating that pro-gamma-MSH polypeptides have an endocrine influence on the rat adrenal cortex.

High density lipoprotein influences the response of rat adrenocortical cells to gamma 3-melanotropin.

Gamma 3-melanotropin (gamma 3-MSH) exhibits a marked dose-dependent synergism with ACTH1-24 on corticosterone production by cells isolated from the inner zones of the rat adrenal cortex. This phenomenon is demonstrated to best advantage when donor animals are killed after stress or pretreatment with ACTH. If adrenal cells are prepared from quiescent or hypophysectomized animals, the inclusion of high density lipoprotein (HDL) in the incubation medium is required for a significant gamma 3-MSH response. Dibutyryl cAMP can successfully substitute for ACTH1-24 in these incubations but rat low density lipoprotein does not reproduce the HDL effect. These data are consistent with our in vivo studies demonstrating that gamma 3-MSH potentiation is a product of increased cholesterol mobilization within the adrenal cortex and suggest that in the rat, a significant source of the cholesteryl ester pool which is responsive to gamma 3-MSH may derive from circulating HDL.

Control of aldosterone secretion by pituitary hormones.

Pro-gamma-melanotropins (pro-gamma-MSH) are implicated in the control of aldosterone secretion by the normal adrenal cortex and in idiopathic hyperaldosteronism. In contrast with other melanotropins (alpha- and beta-MSH), the pro-gamma-MSH increase aldosterone secretion by normal adrenals only in the presence of adrenocorticotrophic hormone (ACTH). However, in aldosteronoma cells this stimulatory effect of the pro-gamma-MSH is not ACTH-dependent. The mechanism of pro-gamma-MSH action on aldosterone biosynthesis may involve activation of cholesteryl ester hydrolase in zona glomerulosa, resulting in an increased provision of cholesterol for steroidogenesis.

Plasma immunoreactive gamma melanotropin in patients with idiopathic hyperaldosteronism, aldosterone-producing adenomas, and essential hypertension.

A non-ACTH aldosterone-stimulating factor(s) has been implicated in the pathogenesis of idiopathic hyperaldosteronism (IHA). Although this factor has not been fully characterized, some evidence suggests that it may be related to a pro-gamma-melanotropin (pro-gamma-MSH), derived from the NH2-terminal region of pro-opiomelanocortin. In the present study, plasma immunoreactive (IR-) gamma-MSH levels at 0800 h in patients with IHA were evaluated (90 +/- 17 fmol/ml; range: 13-173 fmol/ml) and found to be significantly higher (P less than 0.05) than those in subjects with aldosterone-producing adenomas (33 +/- 8 fmol/ml), essential hypertension (33 +/- 6 fmol/ml), and normotensive controls (19 +/- 2 fmol/ml). Seven of nine IHA subjects had circulating IR-gamma-MSH levels above the normal range (greater than 35 fmol/ml). In plasmas sampled at 1200 h, IR-gamma-MSH was significantly higher in patients with IHA (95 +/- 26 fmol/ml) and adenomas (63 +/- 23 fmol/ml), as compared with essential hypertensives (31 +/- 6 fmol/ml) and normotensives (19 +/- 3 fmol/ml). Mean plasma IR-ACTH, plasma cortisol, and urinary cortisol levels did not differ significantly between any of these groups. In order to evaluate the effect of a pro-gamma-MSH in vitro, adrenal adenoma tissue was obtained from two patients, one with elevated IR-gamma-MSH (61 fmol/ml) and a second with low IR-gamma-MSH (12 fmol/ml). Aldosterone secretion by dispersed adenoma cells from the former, but not the latter, underwent a fourfold dose-dependent (10(-14)-10(-9) M) increase in response to human Lys-gamma 3-MSH. These data suggest that a pro-gamma-MSH may be implicated as a pathogenic factor in a subset of patients with primary aldosteronism, particularly among those differentially diagnosed as having IHA.

Immunoreactive ACTH and gamma-MSH in rat plasma during early stages of adrenal regeneration.

Recent evidence suggests that in addition to ACTH the pro-gamma-melanotropins play a role in controlling both the steroidogenic activity and growth of the adrenal cortex. By using the regenerating gland as a model for rapid adrenal growth, studies were carried out to monitor plasma ACTH and pro-gamma-melanotropins during the acute phase following adrenal enucleation. Adrenal enucleated rats (experimental group) were uninephroadrenalectomized; controls were uninephrectomized or uninephroadrenalectomized. At intervals up to 96 h after surgery, animals from each of the three groups were killed under quiescent conditions at the low and high points of their circadian rhythm. The plasma concentrations of both ACTH and 11 K gamma-melanocyte-stimulating hormone (MSH) in the adrenal enucleated rats were markedly elevated as compared with either control group at each time point monitored. However, there were no significant differences in plasma ACTH or 11 K gamma-MSH between the two control groups, and the levels of plasma 6 K gamma-MSH remained unchanged in all three groups throughout the experiment. These data are consistent with a role for pro-gamma-melanotropins in adrenal regeneration.

Cholesterol metabolism in the rat adrenal cortex: acute temporal changes following stress.

Adrenal and serum corticosterone concentrations, cholesterol association with adrenal cortical cytochrome P-450scc (the cytochrome P-450 catalyzing the conversion of cholesterol to pregnenolone), and adrenal cortical activities of cholesteryl ester hydrolase, acyl-CoA:cholesterol acyltransferase, and cholesterol side-chain cleavage have been determined at various times following stress in female rats. The paramount importance of cholesterol side-chain cleavage activation in the response to stress at the low point of the circadian rhythm is confirmed. At the high point of the circadian rhythm, there is evidence that the provision of free cholesterol for the cytochrome P-450scc enzyme system may also be controlled. The data support a coordinate action of ACTH and pro-gamma-melanotropin in controlling cholesterol metabolism in the adrenal cortex following stress.

Lys-gamma 3-melanotropin binds with high affinity to the rat adrenal cortex.

Partially purified plasma membrane preparations from the inner zones of the rat adrenal exhibit a specific and high affinity for a pro-gamma MSH peptide, synthetic rat Lys-[125I-iodo-Tyr1] gamma 3MSH, that is time and temperature dependent, reversible, and saturable. Studies with demedullated adrenals indicate that at least part of this binding is to the adrenal cortex. Scatchard analysis reveals a single class of binding sites (101 fmol/mg membrane protein) for the radioactive ligand, with an apparent Kd of 0.74 nM. However, this may understate the receptor affinity for native pro-gamma MSH(s), because the binding capacity of Lys-gamma 3MSH is impaired somewhat by iodination. Lys-[125I-iodo-Tyr1] gamma 3MSH exhibits a 100-fold higher affinity for the binding site than ACTH-(1-24), but unlike ACTH, concentrations of Lys-gamma 3MSH up to 10 microM fail to stimulate membrane-associated adenylate cyclase activity. Guanylate cyclase in this subcellular fraction also is unresponsive to Lys-gamma 3MSH. Results obtained with crude membrane fractions from other rat tissues suggest that specific pro-gamma MSH binding may not be unique to the adrenal cortex.