Direct effect of ACTH on renin release in isolated perfused guinea-pig kidneys with adrenal glands.

To investigate the direct effect of corticotropin (ACTH) on the renin-angiotensin-aldosterone system, isolated guinea-pig kidneys with adrenal glands were perfused with various doses of ACTH (0.1-1000 micrograms/l) and 0.3 mmol/l of dibutyryl cyclic AMP (cAMP) through each cannula inserted into the abdominal aorta and the inferior caval vein. Perfusate renin activity was increased in a dose-dependent manner by the addition of ACTH in a range of 0.1-1000 micrograms/l, and reached a plateau at 20 min with each dose. The perfusate cAMP level was dose-dependently increased with 10-1000 micrograms/l of ACTH. Perfusate renin activity was also markedly increased by the addition of dibutyryl cAMP. The same effects of ACTH on renin and cAMP secretions were observed in the kidney perfusion model from which the adrenal glands were excluded. Aldosterone secretion failed to respond to 0.1 micrograms/l of ACTH, and was increased by higher concentrations (1-1000 micrograms/l) in the same experiments. These results demonstrate that ACTH has a direct effect on renal renin release in a physiological concentration (0.1 micrograms/l), and that the action of ACTH is probably mediated by cAMP. The sensitivity of renin release to ACTH stimulation is no less than that of aldosterone secretion during ACTH infusion, so it is possible that ACTH is an important stimulator of the renin-angiotensin system.     

ACTH, beta-endorphin, lipotropins, and other pro-opiocortin-derived peptides: a new family of hormones (author's transl)

beta-Endorphin, a pituitary morphino-mimetic peptide, was identified in a culture medium derived from a human corticotropic adenoma. Secretion products from cultured human cells derived from a small-cell carcinoma of the lung were shown to contain a high molecular weight precursor analagous to pro-opiocortin: this molecule is a polypeptide of the order of 28,000 daltons the enzymatic processing of which leads to the coordinated and simultaneous release of different peptide fragments: ACTH, beta- and gamma-lipotropins, beta-endorphin, fragment 16 K and gamma 3-MSH. All these peptides have been identified in human plasma, and pituitary and non-pituitary tumor extracts. Their plasma concentrations vary in a parallel manner, beta-endorphin and a peptide very similar to beta-MSH have been detected in human hypothalamus.     

Effects of ACTH and ACTH fragments on DNA synthesis in guinea-pig adrenal segments kept in organ culture

Stimulation of adrenal DNA synthesis by ACTH and its fragments ACTH (Synacthen) and ACTH was investigated. Synthesis of DNA was measured as the increase in the percentage of cells in S-phase (Feulgen densitometry) in guinea-pig adrenal explants kept in organ culture and exposed to the peptides for 5 h at 37 degrees C. ACTH and its C-terminal fragment ACTH (corticotrophin-like intermediate lobe peptide) were found to be potent stimulators of in-vitro adrenal DNA synthesis. The dose-response kinetics were biphasic and optimal responsiveness was reached in both instances at 1 fmol/1-10 pmol/1 (this biological effect of ACTH has hitherto not been described). The N-terminal fragment ACTH gave only minimal responses. Thyrotrophin and LH, tested as controls, did not induce adrenal DNA synthesis. Epidermal growth factor was a potent stimulator of adrenal DNA synthesis in vitro. Our data suggest a trophic action of the C-terminal part of the corticotrophic molecule. Clear trophic effects were also found for the N-terminal part of the pro-opiomelanocortin molecule N-POC (optimum 0.1 nmol/l) and N-POC(51-62) (optimum 0.1 pmol/l). The latter observations support earlier concepts that this part of the pro-opiomelanocortin molecule has a stimulatory effect on adrenal DNA synthesis.     

Effects of etomidate on cortisol biosynthesis in isolated guinea-pig adrenal cells: comparison with metyrapone

The effects of the iv hypnotic etomidate on cortisol biosynthesis have been investigated in short term incubations of dispersed guinea-pig adrenal cells and were compared with those produced by metyrapone. Fifty percent inhibition of cortisol output was obtained at a final medium concentration of 3.5 10(-8) M (basal), 2.8 10(-8) M (ACTH-stimulated) for etomidate and of 5.10(-7) M (stimulated) for metyrapone. In the presence of etomidate, 11-deoxycortisol at 5.10(-8) M reached a peak value of 244 +/- 11% of control (mean +/- SE, n = 7). 17 alpha-Hydroxyprogesterone and progesterone were not significantly affected up to 10(-7) M, but at higher concentrations, all three precursors fell under their control values. Metyrapone induced a progressive rise of 11-deoxycortisol, from 10(-7) M upwards, to a maximum level at 10(-5) M (210 +/- 15% of control, mean +/- SE, n = 5). 17-Hydroxyprogesterone and progesterone concentrations were not significantly modified by metyrapone. The less active hypnotic L-enantiomer of etomidate had almost no inhibitory effect on cortisol production. The results obtained so far suggest that etomidate is a potent inhibitor of the mitochondrial cytochrome P-450 enzymes of the adrenal cortex, mainly the 11 beta-hydroxylase. At higher dose the cholesterol side-chain cleavage enzyme system seemed also to be affected.     

Responses of Y1 adrenocortical tumor cells to o-nitrophenyl sulfenyl ACTH

NPS(o-nitrophenyl sulfenyl)-ACTH stimulated steroidogenesis in Y1 cells to the same maximum level as did ACTH, but with a 60-fold lower potency than the native hormone. NPS-ACTH also stimulated the extracellular accumulation of cAMP in Y1 cells with lower potency than the unmodified hormone. The amount of cAMP accumulated in the presence of NPS-ACTH approached 75% of the maximum with ACTH. In Y1 plasma membranes, NPS-ACTH was a partial agonist of adenylate cyclase activity, witn an efficacy dependent upon the type of guanyl nucleotide present. The steroidogenic responses of two Y1(Kin) mutants and two Y1(Cyc) mutants to NPS-ACTH paralleled their responses to ACTH and reflected closely their defects in cAMP-dependent protein kinase and ACTH-sensitive adenylate cyclase activity. These data imply an obligatory role for adenylate cyclase and cAMP-dependent protein kinase activities in stimulation of steroidogenesis in Y1 cells by NPA-ACTH.     

Prostaglandins and steroidogenesis in isolated bovine adrenal cells. Effects of ACTH, prostaglandin-synthesis inhibitors, prostaglandins and prostaglandin analogs

In bovine adrenal cortex cells, dispersed without preferential loss of cells, we investigated (1) whether endogenous prostaglandins (PGs) are involved in ACTH-induced adrenal steroidogenesis, and (2) the steroidogenic effects of PGs and PG analogs. Free cells produced considerable amounts of PGE2, whereas only minute quantities of PGF2 alpha and PGA1 were synthesized. PGE2 synthesis, however, was not significantly increased when ACTH elicited a steroidogenic response in free cells. High concentrations of PG-synthesis inhibitors such as indomethacin affected both PG synthesis and steroidogenesis, whereas intermediate concentrations (10(-6) M) inhibited production of both PGE2 and aldosterone even after cAMP and cortisol response to ACTH had returned to normal values. It is concluded that endogenous PGE2 is not a link in the acute mechanism of action of trophic hormones in which cAMP is involved. Of the prostanoid structures, PGs of the E series were the most potent stimulating agents of cortisol production, although less active than ACTH. On the other hand, PGA1 induced an ACTH-like aldosterone synthesis. PGE2 was less active, and other prostanoid structures were without effect on aldosterone production. It is suggested that in pathological circumstances, PGA1 regulates aldosterone production and PGE2 increases both aldosterone and cortisol production.     

Responses of aldosterone-producing adenomas to ACTH and angiotensins.

To elucidate the control mechanism of aldosterone production in primary aldosteronism, in vivo and in vitro studies were done in 7 patients with aldosterone-producing adenomas. In the in vivo study, plasma aldosterone was stimulated more significantly by (Formula: see text), synthetic ACTH than by angiotensin II or furosemide. Diurnal variations of plasma aldosterone, which were studied in 4 patients, were similar to those seen in normal controls. In agreement with the results in the in vivo study, the in vitro study also revealed ACTH stimulated aldosterone and deoxycorticosterone (DOC) from the adenoma more markedly than angiotensin II or III. There was no adenoma which was more sensitivie to angiotenion II or III than to ACTH. From these results it is considered that changes in plasma aldosterone induced by the exogenous administration of angiotensin II or ACTH in patients with aldosterone-producing adenoma are mainly based on changes in aldosterone production in the adenoma. Furthermore, in patients with an aldosterone-producing adenoma in whom diurnal variations of plasma aldosterone similar to those in normal subjects are observed, responses of aldosterone to angiotensin II are supposed to be less than those to ACTH.     

Comparison of ACTH and ACTH precursor peptides secreted by human pituitary and lung tumour cells in vitro

The molecular forms of ACTH secreted by established human small cell lung cancer (SCLC) cells and primary cultures derived from a bronchial carcinoid tumour, a pituitary adenoma and hyperplastic pituitary tissue have been characterized by Sephadex G-75 chromatography and quantified with two novel immunoradiometric assays for ACTH and ACTH precursor peptides. Pro-opiomelanocortin (POMC; Mr 31,000) and pro-ACTH (Mr 22,000) were secreted by all cell types. No smaller peptides were identified in the culture media from SCLC and bronchial carcinoid cells, implying a deficiency in the enzymes and/or intracellular organelles required for extensive POMC processing. A more heterogeneous profile of ACTH-containing peptides was produced by cells of pituitary origin, indicating more extensive proteolytic processing of POMC. However, the major peptide secreted by cells from a large aggressive pituitary adenoma was unprocessed POMC (Mr 31,000). These results suggest that both lung and pituitary cells in vitro retain their in-vivo pattern of POMC processing and provide valuable models in which to study the regulation of ACTH synthesis and secretion.     

Comparative effects of prostaglandins and ACTH on the production of corticosterone and cyclic AMP in isolated adrenal gland cells of rats

The authors compared the effect of prostaglandin PGE2 and ACTH on corticosterone and cAMP production in isolated adrenal cells of rats. PGE2 stimulated steroidogenesis in the concentrations of 0.01-1.10 micrograms/ml and the maximum stimulation (2.5-fold) was observed in the PGE2 concentration of 0.1 micrograms/ml. ACTH stimulated steroidogenesis 12-fold as compared to PGE2. In combined addition of different PGE2 doses and the physiological ACTH dose (5 pg/ml) their steroid effects were summated. The results obtained are indicative of the fact that PGE2 is no mediator of the ACTH effect but that it is capable of potentiating this effect. Like ACTH, PGE2 raised the cAMP level in the adrenal gland. In concentrations causing a similar to ACTH increase in the cAMP level, PGE2 stimulated steroidogenesis to a lesser degree than ACTH, and in this connection a possibility of the existence of two cAMP foci in the adrenal gland is discussed. It is assumed that one of the foci is related and the other is not related with steroidogenesis, and PGE2 stimulates mainly the second focus.     

Subcellular localization of a protein produced in adrenal cortex cells in response to ACTH

We have reported previously that a protein, ib, is produced in adrenal cortex and other steroidogenic cells with the same tissue-specific peptide hormone or cAMP dose-response and the same kinetics as the increase in steroid hormone biosynthesis. In this study, we have fractionated adrenal cortex cells into subcellular components and used two-dimensional electrophoresis to characterize the proteins in these fractions. We have demonstrated previously that inhibition of cytosolic translation, e.g. by cycloheximide, prevents the production of protein ib. We also report that the production of this protein is not affected by inhibition of mitochondrial translation by chloramphenicol.     

Processing of enkephalin-containing peptides in isolated bovine adrenal chromaffin granules.

Intact chromaffin granules isolated from bovine adrenal medulla were incubated at 37 degrees C for up to 22 hr. Processing of enkephalin-containing (EC) peptides in the granules was followed by the change in their size distribution as shown by chromatography on Sephadex G-75 columns. A gradual shift toward lower molecular weight EC peptides was observed during the incubation, indicating processing of the higher molecular weight to lower molecular weight EC peptides. The total amount of [Met]-enkephalin, free and in peptide linkage, remained constant indicating that little or no nonspecific degradation occurred during the experiment. HPLC resolution of the fraction containing the low molecular weight EC peptides showed that free enkephalins as well as [Met]enkephalin-Arg6-Phe7 and [Met]enkephalin-Arg6-Gly7-Leu8 accumulated while [Met]enkephalin-Arg6 and [Met]enkephalin-Lys6 disappeared. All the above data indicate the presence of an atypical trypsin activity and the presence of a carboxypeptidase B-like activity within the granules. From the rates of accumulation of the low molecular weight EC peptides and the disappearance of the higher molecular weight EC peptides, a processing rate of 65-70 pmol/g tissue per hr was estimated, which calculates to a lifetime of 6-8 days for EC peptides in the granules. Under steady-state conditions this rate of processing appears to be too low to produce significant amounts of free enkephalins from larger EC peptides. This is well in accord with previous observations that relatively small amounts of free enkephalins are found in bovine adrenal medulla.     

Decreased adrenal androgen sensitivity to ACTH during aging

During the human aging process, basal plasma levels of cortisol and aldosterone demonstrate little change, while concentrations of adrenal androgens (AA) such as dehydroepiandrosterone (DHA), dehydroepiandrosterone sulfate (DHAS), and androstenedione (A) decrease dramatically in men and women. There is no age-related change in ACTH concentrations to explain this observation. In this study, ACTH (cosyntropin, alpha1-24 corticotropin) stimulation tests were performed on elderly subjects and controls to test the hypothesis that analogous to the aging testicular. Leydig cell, AA-producing cells of the aging adrenal gland may become less sensitive to physiological levels of ACTH, but still retain their sensitivity to elevated ACTH levels. Results showed that in the elderly subjects, basal levels and ACTH-stimulatability of cortisol and aldosterone were unchanged. In agreement with earlier studies, basal levels of AA were found to be decreased in the elderly. However, ACTH stimulation demonstrated impaired reserve in DHA and A, and a total lack of stimulatability of DHAS. These findings could be explained by an age-related loss of adrenal enzymes or cell populations which produce AA, a loss or decrease in a subpopulation of ACTH receptors specific for AA production, or loss of a pituitary factor necessary for AA secretion.     

Electrophysiological responses to angiotensin II of isolated rat adrenal glomerulosa cells

The membrane response of isolated rat glomerulosa cells to the application of angiotensin II (A II) has been studied using intracellular voltage measurements. The membrane response is biphasic. The first, brief phase involves an increase in membrane conductance and a hyperpolarization from the resting membrane potential. The second, long-lasting phase is characterized by a large decrease in membrane conductance and a depolarization from the resting membrane potential. The reversal potential for the second phase is -94 +/- 1.2 mV, and a linear relationship between reversal potential and external K+ indicates that the A II-mediated response is predominantly inhibition of K+ permeability. The A II response can be elicited when external Ca2+ is replaced by Sr2+ or Ba2+, but the response is inhibited when Mn2+ is added to the bath or when stimulated in a Ca2+-free solution. A II appears to inhibit at least two conductances, when the cell is stimulated by long current steps. External application of A II inhibited the Ca2+ regenerative response found in glomerulosa cells in a dose-dependent manner. The rate of rise of the regenerative response was greatly attenuated by A II; half-maximal inhibition was produced by about 10(-9) M A II. In addition, rectification, evident at voltages more positive than -60 mV during current stimulation, was also inhibited. In conclusion, A II causes rat glomerulosa cells to depolarize due to the inhibition of resting K+ permeability. Action potential activity is not observed during A II-mediated membrane depolarization; rather, both Ca2+ and K+ conductances appear to be inhibited during A II application.     

Cyproteroneacetate and ACTH adrenal function.

Cyproteroneacetate, an antiandrogenic and gonadotropin-inhibiting steroid, has a marked ACTH suppressive effect. In rats, adrenal atrophy and severe impairment of ACTH and corticosterone responses to stress are induced by a 10-day treatment with 3-0.75 mg/100 g BW cyproteroneacetate/day. Two weeks after cessation of treatment, the ACTH adrenal system has not yet recovered. The ACTH suppression is evident 6 h after a single dose. In 25 human volunteers, a single dose of 200 mg cyproteroneacetate impaired their ACTH and 11-deoxycorticosteroid response to 1 g metyrapone. A similar impairment was seen in 12 women on sequential treatment with cyproteroneacetate and ethinyl estradiol. In 4 out of 11 children treated for precocious puberty, random plasma ACTH and cortisol measurements, cortisol responses to ACTH, and ACTH and cortisol responses to insulin-induced hypoglycemia revealed severely impaired ACTH adrenal function. Questionable impairment was found in 2 out of 11 and normal function in 5 out of 11 children. In 10 patients with endogenous elevated plasma ACTH, 10 days of treatment with cyproteroneacetate, in addition to the steroid substitution, diminished the morning plasma ACTH levels. It is concluded that cyproteroneactate has a pronounced ACTH-suppressive effect. The individual susceptibility of treated patients varies and the effect is dose dependent. A cortisol-like effect must be assumed, because cyproteroneacetate-treated animals and patients under therapy can withstand stress situations without signs of adrenal insufficiency. ACTH adrenal function must, however, be closely watched in treated patients and steroid cover must be considered in conditions of stress. Great care has to be taken when the drug, with its own "stress-protective" effect, is withdrawn. The recovery of ACTH adrenal function may take several months.     

ACTH induces TIMP-1 expression and inhibits collagenase in adrenal cortex cells

To identify genes that are induced by corticotropin (ACTH) in adrenal cortex cells, we carried out a differential hybridization screening of adrenal cortex cDNA libraries. Some of the clones we identified represented tissue inhibitor of metalloproteinase 1 (TIMP-1) mRNA. We examined ACTH dependence of the expression of TIMP-1 in vitro in cultured bovine adrenocortical cells, and in ACTH-treated rats. Northern blot analysis of total RNA from cells showed that the level of TIMP-1 mRNA increases sharply within 3h after ACTH stimulation. Since TIMP-1 inhibits some cell matrix metalloproteinases (MMPs) of the collagenase type, we examined the effect of ACTH on collagenase activity in bovine adrenocortical cells. Exposure of confluent cultures to ACTH for 24h showed dose-dependent inhibition of collagenase activity. Northern blot analysis of total RNA from rat adrenal zona fasciculata-reticularis and zona glomerulosa showed that in both of these zones TIMP-1 expression was induced within 12h after ACTH injection. Long-term (9 days) treatment with ACTH increased TIMP-1 mRNA levels nearly sixfold in zona fasciculata-reticularis. Overall, our results show that ACTH causes induction of TIMP-1 and suppression of collagenase activity, and suggest that ACTH may modulate the activities of MMPs and hence cell matrix remodeling.