The differential regulation of aldosterone output in hamster adrenal by angiotensinII and adrenocorticotropin

Aldosterone was isolated from hamster adrenal cells and was identified by high performance liquid chromatography and thermospray mass spectroscopy analysis. Basal outputs from adrenal cell suspensions were of the same order of magnitude, 8.4 ± 1.9 ng and 8.0 ± 0.7 ng/2 h/50,000 cells, for aldosterone and corticosteroid, respectively. The outputs of aldosterone and corticosteroid increased with K⁺ concentrations to reach maxima of 3.3- and 1.6-fold at 10 meq/l of K⁺. Angiotensin_II (A_II) produced dose-dependent increases in aldosterone and corticosteroid outputs with maxima of 3- and 4-fold, respectively. In contrast, ACTH induced relatively no changes in aldosterone output, whereas dose-dependent increases in corticosteroid output were found. In time study experiments, with 10⁻⁸ M A_II, aldosterone and corticosteroid outputs were maximally increased after 1 h (6-fold) and 3 h (1.8-fold), respectively. At 10⁻⁸ M, ACTH had a small stimulatory effect on aldosterone output after 6 h, whereas it provoked a gradual increase in corticosteroid output (up to 7-fold after 8 h of incubation). The effects of A_II and ACTH on adrenal cytochrome P-450_11β involved in the last steps of aldosterone formation were evaluated by c combined in vivo andin vitro experiments. The P-450_11β mRNA level was increased by a low sodium intake but not by a 24 h ACTH stimulus. These results taken together indicate that ACTH and A_II differentially regulate P-450_11β. It is postulated that these two regulatory peptides regulate the hamster adrenal steroidogenesis by different P-450 genes.     

Catecholamine secretion by hamster adrenal cells.

Abstract— Suspensions of isolated adrenal cells were prepared by digesting hamster adrenal glands with collagenase, and the secretion of catecholamine from these cells was studied. Acetylcholine (ACh) produces a dose-dependent increase in catecholamine secretion; half-maximal secretion is produced by 3 μm -ACh, and maximal secretion by 100 μm -ACh. The cholinergic receptor in these cells appears to be nicotinic, since catecholamine secretion is stimulated by the nicotinic agonists nicotine and dimeth-ylphenylpiperaziniurn, but not by the muscarinic agonists pilocarpine or oxotremorine. ACh-induced catecholamine secretion is inhibited by hexamethonium, tubocurarine, and atropine, but is not inhibited by α-bungarotoxin. ACh-induced catecholamine secretion is dependent upon the presence of extracellular Ca²⁺, and appears to occur by exocytosis, since the release of catecholamine is accompanied by the release of dopamine β-monooxygenase, but not of lactate dehydrogenase. These biochemical studies complement the morphological evidence for exocytosis in hamster adrenal glands, and indicate that catecholamine secretion from hamster chromaffin cells is similar to that from chromaffin cells of other species.     

Adrenodoxin biosynthesis by bovine adrenal cells in monolayer culture. Induction by adrenocorticotropin

The long term effect of adrenocorticotropin (ACTH) on the synthesis of adrenodoxin in bovine adrenocortical cells was investigated. Primary, confluent monolayer cultures of adult bovine adrenocortical cells were incubated in the presence or absence of ACTH (10(-6) M) for periods up to 72 h. The amount of adrenodoxin precursor synthesized in a cell-free translation system programmed with RNA isolated from ACTH-treated cells increased to approximately 3 times the control level by 36 h. Similarly, ACTH increased the rate of incorporation of [35S]methionine into mature adrenodoxin in radiolabeled adrenocortical cells, an effect that was maximal 36 h after initiation of ACTH treatment. At longer times (48-72 h), the stimulatory effect of ACTH was not maintained, and adrenodoxin synthesis in both radiolabeled cells and cell-free translation systems declined to control levels. The content of adrenodoxin in cells treated with ACTH for 36 h, as measured by electron paramagnetic resonance spectroscopy, was approximately twice that in control cells. The results indicate that ACTH induces the synthesis of adrenodoxin in bovine adrenocortical cells. Based on the present results as well as those previously reported with respect to the induction of cholesterol side chain cleavage cytochrome P-450 by ACTH (DuBois, R. N., Simpson, E. R., Kramer, R. E., and Waterman, M. R. (1981) J. Biol. Chem. 256, 7000-7005), it is proposed that the synthesis of the mitochondrial components of the adrenocortical steroid hydroxylase system is controlled by ACTH in a coordinate fashion.     

Role of prostaglandins in aldosterone production by the human adrenal.

The subunits of purified yeast RNA polymerases I, II and III have been analyzed by two-dimensional polyacrylamide gel electrophoretic subunit mapping techniques. The results suggest that polymerases I and III have two subunits in common, the 41,000 and 20,000 dalton peptides, which are not present in polymerase III. The 14,500 dalton peptide by all criteria is identical in polymerases I, II and III. The 28,000 and 24,000 subunits appear identical in polymerases I and II but have different charge properties in polymerase III.     

A patient with a prolactinoma associated with an aldosterone producing adrenal adenoma: differences in dopaminergic regulation of PRL and aldosterone secretion.

A patient with a rare combination of prolactinoma and aldosterone producing adrenal adenoma (APA) was reported in relation to studies concerning dopaminergic regulation of PRL and aldosterone secretion. The patient is a 38-year-old female with plasma PRL and aldosterone concentrations (PAC) of 563 ng/ml and 54 ng/dl, respectively. A bolus of 10 mg of metoclopramide significantly increased plasma PRL in 6 normal subjects and in 4 patients with APA, whereas the responses were blunted in 7 patients with prolactinoma and in our patient. The response of aldosterone to metoclopramide was less than that of PRL, but similar in all studied subjects, indicating that the dopaminergic inhibition of aldosterone secretion is less than that of PRL in normal subjects and did not change in patients with APA or prolactinoma. Oral administration of 2.5 mg of bromocriptine suppressed plasma PRL significantly in all the subjects studied, but did not produce any consistent changes in PAC. Discrepancies in the response of PRL and aldosterone to metoclopramide and to bromocriptine suggest a difference in the dopaminergic regulation of PRL and aldosterone secretion in both normal subjects and patients with prolactinoma and APA. It is unlikely that reduced dopaminergic inhibition is the basis for hypersecretion of PRL and aldosterone in our patient.     

Metabolism of 11-deoxycorticosterone by hamster adrenal mitochondria.

Metabolism of 11-deoxycorticosterone (DOC) by hamster adrenal mitochondria gives 19-hydroxy-DOC and corticosterone (via 11-hydroxylation) in approximately equal yields. The ratio of 19- to 11-hydroxylation was invariant with changes in concentration of substrate or a competitive inhibitor. It is most likely, therefore, that a single 11,19-hydroxylase catalyzes both oxidations. Both primary products are further oxidized to the corresponding carbonyl analogs, 19-oxo-DOC and 11-dehydrocorticosterone, at rates that are approx. 20% of their rates of formation. The oxidation of 11-dehydrocorticosterone is catalyzed by a dehydrogenase utilizing either NAD or NADP while the oxidation of 19-hydroxy-DOC is catalyzed by an oxidase requiring NADPH. The 11-dehydrocorticosterone is stable in this enzyme preparation while 19-oxo-DOC is metabolized to two additional products, which are tentatively identified as 19-oic-DOC and 19-norcorticosterone. 19-nor-DOC was found to be hydroxylated at a rate that is 20% faster than the rate for DOC under the same conditions. It is therefore possible that 19-norcorticosterone can arise from 19-oic-DOC via decarboxylation to 19-nor-DOC and subsequent 11-hydroxylation, but the kinetics of its formation suggest that it may actually be formed directly from 19-oxo-DOC without free intermediates. 4-Androstene-3,17-dione and 17-hydroxy-DOC were also substrates for this 11,19-hydroxylase, but 18-hydroxy-DOC was not. Maintenance of hamsters on a low sodium diet had no effect on the metabolism of DOC by the isolated adrenal mitochondria.     

Nongenomic regulation of ENaC by aldosterone

Aldosterone isinvolved in salt and water homeostasis. The main effect is thought toinvolve genomic mechanisms. However, the existence of plasma membranesteroid receptors has been postulated. We used whole cell patch clampto test the hypothesis that epithelial sodium channels (ENaC) expressedby renal collecting duct principal cells can be regulatednongenomically by aldosterone. In freshly isolated principal cells fromrabbit, aldosterone (100 nM) rapidly (<2 min) increased ENaC sodiumcurrent specifically. The aldosterone-activated current was completelyinhibited by amiloride. Aldosterone also activated ENaC in cellstreated with the mineralocorticoid receptor blocker spiranolactone.Nongenomic activation was inhibited by inclusion ofS-adenosyl-L-homocysteine in the pipettesolution, which inhibits methylation reactions. Also, the nongenomicactivation required 2 mM ATP supplementation in the pipette solution.Aldosterone did not activate any ENaC current in whole cell clamped ratcollecting duct principal cells. These functional studies areconsistent with aldosterone membrane binding studies, suggesting thepresence of a plasma membrane steroid receptor that affects cellularprocesses by mechanisms unrelated to altered gene expression.

     

The effect of estradiol on output of adrenocorticotropin and prolactin by fetal sheep anterior pituitary cells

We examined the hypothesis that estradiol (E2) would affect fetal anterior pituitary corticotroph and lactotroph function in vitro, and that any effects would be influenced by gestational age. Anterior pituitary cells from fetal sheep at day 129 (n = 4) and at day 139 (n = 5) of gestation were cultured. After 96 h in culture, cells were treated for 18 h with E2 concentrations ranging from 0 to 1000 nM, in the presence or absence of 100 nM of corticotropin-releasing hormone (CRH), cortisol, arginine vasopressin (AVP), or CRH and cortisol, to examine their effects on corticotroph function. Cells were also treated with bromocriptine or increasing concentrations of E2 to study their effects on lactotroph function. Immunoreactive (ir) adrenocorticotropin (ACTH) and prolactin in the culture medium were measured by radioimmunoassay. Levels of cellular pro-opiomelanocortin (POMC) mRNA and prolactin mRNA were determined by in situ hybridization. Immunohistochemistry was used to determine the percentage of cells that were immunopositive for ACTH (corticotrophs) or prolactin (lactotrophs). ACTH output was stimulated by CRH treatment at day 139 but not at day 129 of gestation, and cortisol attenuated this response. ACTH output by cells cultured with 10 nM E2 and 100 nM CRH, at 139 days of gestation, was greater than with CRH alone (p < 0.05). E2 did not affect basal ACTH output or ACTH output with any other treatment or levels of POMC mRNA. Prolactin output was not affected by E2 treatment. Bromocriptine significantly decreased prolactin output but not levels of prolactin mRNA. We conclude that E2 may affect CRH-stimulated fetal sheep pituitary corticotroph function late in gestation, but only within a narrow, physiological range of concentration.     

Response of adrenal tumor cells to adrenocorticotropin: site of inhibition by cytochalasin B.

The ability of cytochalasin B to inhibit the steroidogenic response of mouse adrenal tumor cells (Y-1) to adrenocorticotropin (ACTH) was examined with two aims: to consider the specificity of the inhibitor and to determine at what point(s) in the steroidogenic pathway it acts. Cytochalasin B did not inhibit protein synthesis or transport of [3H]-cholesterol into the cells nor did it alter total cell concentration of ATP. Together with previous evidence, this suggests that the effects of cytochalasin observed are relatively specific in these cells. Cytochalasin inhibits the increase in conversion of [3H]cholesterol to 20alpha-[3H]dihydroprogesterone (20alpha-hydroxypregn-4-en-3-one: a major product of the steroid pathway in Y-1 cells) produced by ACTH but does not inhibit conversion of cholesterol to pregnenolone by mitochondrial and purified enzyme preparations from Y-1 cells and bovine adrenal, respectively. Cytochalasin does not inhibit the conversion of pregnenolone to 20alpha-dihydroprogesterone but was shown to inhibit increased transport of [3H]cholesterol to mitochondria resulting from the action of ACTH. These findings indicate that cytochalasin acts after cholesterol has entered the cells and before it is subjected to side-chain cleavage in mitochondria. In view of the known action of cytochalasin on microfilaments, it is proposed that these organelles are necessary for the transport of cholesterol to the mitochondrial cleavage enzyme and that at least one effect of ACTH (and cyclic AMP) is exerted upon this transport process. The specificity of the effects of cytochalasin is considered in relation to this conclusion.     

Isolated production of aldosterone by a malignant adrenal carcinoma

A 45-year-old female developed hypertension and hypokalemia. Elevated plasma aldosterone and suppressed plasma renin levels were measured with no evidence for glucocorticoid or androgen abnormalities. A left adrenal tumor was removed that showed histologic criteria for malignancy. It is commonly taught that malignant adrenal tumors are recognized by their multiple hormone production. However, isolated aldosterone production by a carcinoma can occur and requires close follow-up observation and therapy for this highly malignant tumor.     

Verapamil directly inhibits aldosterone synthesis by adrenal mitochondria in vitro

The action of verapamil, a calcium channel blocker, on the last step of aldosterone biosynthesis (transformation of 18-hydroxycorticosterone into aldosterone) was studied using duck adrenal mitochondria in the absence of regulatory factors. Results show that 10(-5) M verapamil inhibits the transformation of 18-hydroxycorticosterone into aldosterone by 52.8%. Moreover, our findings show that verapamil induces only a slight inhibition of respiratory capacity without action on respiratory control and does not displace 18-hydroxycorticosterone from cytochrome P450 11 beta which catalyses the reaction. Thus, this study does not explain the mechanism of inhibition induced by verapamil on the last step of aldosterone synthesis but it is of interest to note, for clinical use, that this inhibition is not linked to regulatory factors of aldosterone production. Since primary hyperaldosteronisms are characterized by their independence vis-á-vis regulatory factors, administration of verapamil may be particularly interesting for treatment of primary hyperaldosteronisms.     

Effects of adrenocorticotropin and cycloheximide on adrenal diglyceride kinase

We studied the effects of adrenocorticotropin (ACTH) and cycloheximide on adrenal enzymes involved in phosphatidate synthesis. Treatment of rats in vivo with ACTH induced a rapid increase in phosphatide synthesis from diglyceride and ATP in adrenal homogenates, and cycloheximide treatment prevented this increase if given before ACTH and rapidly reversed the increase if given after ACTH. The stimulatory effect of ACTH appeared to be largely due to an increase in diglyceride substrate, as kinase activity was not altered. The inhibitory effect of cycloheximide, on the other hand, appeared to be due to a decrease in diglyceride kinase activity. Neither ACTH nor cycloheximide treatment had any effect on the activity of glycerol-3'-phosphate acyltransferase or phosphatidate phosphatase. Our findings suggest that (a) ACTH increases the flow of phospholipid (and their levels) throughout the entire circular pathway, i.e., phosphatidate leads to CDP-diacylglycerol leads to inositides leads to diglycerides leads to phosphatidate, and (b) a labile protein may serve to allow entry into a recycling of diglyceride in this pathway. In addition, since cycloheximide blocked carbachol-induced increases in pancreatic and salivary glandular phosphatidate synthesis resulting from phosphatidylinositol hydrolysis and consequent diglyceride generation, the putative labile protein may have widespread importance.