The anomalous effect of some ACTH-fragments missing the amino acid sequence 1-10 on the corticosteroidogenesis in purified isolated rat adrenal cells

The corticosteroidogenicity of ACTH-derived peptides was tested in a purified isolated rat adrenal cell system. The activity was related to the activity of standard ACTH (synthetic hACTH; potency fixed at 100). The peptides ACTH NH2 and ACTH showed steroidogenicity at pharmacological doses (potencies: 0.00067 and 0.00032, respectively). None of the peptides tested inhibited or potentiated the ACTH-induced steroidogenesis at low doses (0.5-50 000 pg). The results suggest the presence of a second center within the ACTH molecule capable of inducing steroidogenesis.     

Structurally functional organization of corticotropin: lipolytic and steroidogenic activity of some of its fragments]

The influence of ACTH fragments, possessing structural elements, common for certain groups of peptide hormones and kinins--"common" fragments and cluster of basic amino-acids--(Lys 17,18-ACTH 11-18-NH2--I; ACTH 11-13-NH2--II; NH2CO-ACTH18-20-NH2--III) on lipolytic effect of ACTH in rat isolated fat cells and on the steroidogenic effect of ACTH in isolated rat adrenal cells was studied. Fragment I exerts a steroidogenic effect (alpha=0,84) at concentrations of 1--100 microng/ml. At low concentrations (10(-8)--10(-3) microng/ml) fragment I potentiates ACTH-induced steroidogenesis. Fragment I has no effect on the lipolysis;however, it potentiates ACTH-induced lipolysis at concentrations of 10--100 microng/ml. The results obtained support our previous supposition that "common" fragments are essential secondary non-specific active sites of hormones.     

Effect of gamma 3 or gamma 2 melanocyte stimulating hormone on steroidogenesis in the fetal sheep during late gestation

We have measured circulating concentrations of gamma 3 Melanocyte Stimulating Hormone (MSH) in fetal sheep between 111 and 145 days gestation. There was no significant effect of gestational age on the fetal plasma concentrations of gamma 3 MSH throughout this period. We have examined the role of gamma-MSH related peptides in the control of fetal adrenal steroidogenesis and found no significant change in fetal plasma cortisol or pregnenolone concentrations during a 60-72 h infusion of saline, gamma 2 MSH or gamma 3 MSH in sheep between 130 and 135 days gestation. Therefore although we have demonstrated the presence of gamma MSH related peptides in fetal sheep plasma during late gestation we have failed to demonstrate a role for gamma 3 or gamma 2 MSH in the changes in fetal steroid concentrations which occur prepartum.     

Adrenocortical response to corticotropin is inhibited by gamma 3-MSH antisera in normotensive and spontaneously hypertensive rats

In order to further investigate the coordinate action of pro-corticotropin/endorphin-derived peptides on adrenal steroidogenesis, we have evaluated the effects of highly specific antisera to synthetic rat gamma 3-MSH (1-27) peptide (gamma 3-MSH Ab) on corticosterone, 18-hydroxycorticosterone and aldosterone responses to ACTH (1-24) in chronically cannulated spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). Antisera eliminated the ACTH induced rise of all three corticosteroids. It had no effect on basal corticosteroid levels. Our results offer further evidence that the potentiating action of gamma 3-MSH may play an important role in modulating ACTH induced steroidogenesis.     

Regulation of lymphokine (gamma-interferon) production by corticotropin

We have shown that corticotropin (ACTH), alpha-endorphin, and enkephalins can regulate antibody responses, which suggested a role for neuropeptides in a regulatory circuit between the immune and neuroendocrine systems. ACTH and structurally related peptides were examined here for regulation of mitogen induction of the lymphokine gamma-interferon (IFN gamma) in C57BL/6 mouse spleen cell cultures. Synthetic ACTH1-39 and a porcine pituitary extract containing ACTH activity were potent suppressors of the IFN gamma response. Synthetic ACTH1-39 suppressed the response by approximately 62% at 1 to 3 microM, whereas the porcine extract suppressed by greater than 90% at 1 to 3 microM ACTH. The greater potency of the pituitary extract was shown to be due to the presence of an additional peptide of Mr 2100 that was reactive with antibodies to the N-terminal region of ACTH (ACTH1-13), possessed potent anti-cellular activity against L cells and various transformed cells, but lacked ACTH biologic activity. The anti-cellular peptide suppressed the IFN gamma response by greater than 99% at 0.05 microM. The ACTH1-39 cleavage products, alpha-melanocyte stimulating hormone (alpha MSH; acetylated and amidated ACTH1-13), and corticotropin-like intermediate lobe peptide (CLIP; ACTH18-39) had no effect on IFN gamma production. ACTH1-24, like ACTH1-39, has full steroidogenesis activity but also had no effect on IFN gamma production, which suggests a dissociation of the immunoregulatory and steroidogenic properties of ACTH1-39. ACTH1-39, and possibly also the anti-cellular 2100 Mr peptide, is initially synthesized as the precursor polyprotein pro-opiomelanocortin (POMC). Enzymatic processing of POMC, first to the active ACTH1-39 or the anti-cellular peptide and then to the inactive smaller peptides, probably plays an important role in regulation of lymphokine and antibody production by ACTH and ACTH-related neuropeptides. This is consistent with the recent demonstration of the production of ACTH-like peptides by lymphocytes.     

Influence of E. coli endotoxin on ACTH induced adrenal cell steroidogenesis

The effect of endotoxin (lipopolysaccharide from E. coli) on isolated adrenocortical cells was examined. Lipopolysaccharide decreased the ACTH-induced steroidogenesis. This effect was shown by all corticotropin concentrations studied, and the longer the incubation time, the higher the effect produced. The rate of decrease of ACTH-induced steroidogenesis was dependent on the concentration of lipopolysaccharide in the medium. Binding of [125I]ACTH to adrenocortical cells was modified by lipopolysaccharide; this modification was related to a decrease of the ACTH-induced steroidogenesis. This effect supports the hypothesis of a direct interaction between lipopolysaccharide and the cell membrane with a concomitant distortion of the cell surface affecting the ACTH receptor sites of their environment. [14C]Lipopolysaccharide binds to isolated adrenocortical cells. Binding specificity was investigated by competitive experiments in the presence of various types of endotoxins, polypeptide hormones and proteins. Unlabelled lipopolysaccharide from the same bacterial strain and isolated under identical conditions than the labelled lipopolysaccharide exerted the strongest inhibitory activity. Unlabelled lipopolysaccharide of various strains different from that originating the labelled lipopolysaccharide exerted the less displacement. It would imply a certain kind of specificity but the decrease in the binding of lipopolysaccharide produced by ACTH and glucagon suggests the existence of non-specific interactions between lipopolysaccharide and cell membrane.     

Role of gamma-MSH peptides in the regulation of adrenal steroidogenesis

Alpha-, beta- and gamma-melanocyte stimulating hormones (MSHs) are peptides derived from the ACTH precursor, pro-opiomelanocortin. All three peptides have been highly conserved throughout evolution but their exact biological function in mammals is still largely obscure. In recent years, there has been a surge of interest in alpha-MSH and its role in the regulation of feeding. Gamma-MSH by contrast has been shown to be involved in the regulation of adrenal steroidogenesis and also has effects on the cardiovascular and renal systems. This review will provide an overview of the role that gamma-MSH peptides play in the regulation of adrenal steroidogenesis.     

Potentiation of the antagonistic effect of ACTH11-24 on steroidogenesis by synthesis of covalent dimeric conjugates

Covalent dimerization of the adrenocorticotropin fragment ACTH11-24 increases its antagonistic potency on the ACTH-induced steroidogenesis in isolated bovine fasciculata/reticularis cells by 3 orders of magnitude when the C-termini are linked via a 10 A long spacer. This strong potentiation, probably mediated by cross-linking of the receptors, was shown to be dependent on the point of attachment of the monomeric fragment to the spacer, thus providing information about the position of the binding site in the hormonal segment and about the distance of the receptors on the cell surface.     

Comparison of structural and functional organization of adrenocorticotropic hormone and wasp kinin]

A comparative study of structural and functional organization of the polypeptides -- ACTH and wasp kinin was made. The effects of fragments Lys 17, 18-ACTH11(-18)-NH2--(I) and WK4(-12)--(II), possessing "common" fragments and a cluster of basic amino-acids, on the lipolytic and steroidogenic effects of ACTH and myotropic effects of bradykinin were studied. Both fragments I and II potentiate ACTH-induced lipolysis and steroidogenesis in isolated rat fat and adrenal cells but suppress the myotropic effect of bradykinin on guinea pig ileum. The similarity of biological effects of ACTH and WK fragments support our supposition on the similarity in structurally functional organization of these peptides.     

Effects of several pro-opiomelanocortin derived peptides on steroidogenesis in ovine and bovine adrenal cells

The effects of several peptides derived from the amino-terminal end of proopiomelanocortin (N-POMC) alone or in combination with ACTH on ovine and bovine adrenal cell steroidogenesis have been studied. Neither porcine N-POMC1-61 amide, nor gamma 3-MSH, nor been studied. Neither porcine N-POMC1-61 amide, nor gamma 3-MSH, nor Lys-gamma 3-MSH alone had any steroidogenic effect while porcine N-POMC1-80 alone had a week but significant steroidogenic effect on isolated adrenal, the effect being more pronounced on cultural adrenal cells. The potentiation by N-POMC peptides of the steroidogenic action of ACTH was investigated using both freshly isolated and cultured adrenal cells. At 10(-8) M N-POMC1-61 amide, gamma 3-MSH and Lys-gamma 3-MSH did not modify the ACTH dose-response for steroids (gluco- and mineralocorticoids) and cAMP production. Likewise, the stimulatory effect of 10(-12) M ACTH was not modified by increasing concentrations (10(-11) to 10(-8) M) of N-POMC1-61 amide or gamma 3-MSH. On the other hand, an additive effect of 10(-8) M N-POMC1-80 on the steroidogenic action of low concentration of ACTH was observed. This again was more pronounced in cultured adrenal cells. The same effects were observed when increasing concentrations of this peptide (10(-9) and 10(-8) M) were added together with 10(-12) M ACTH. This result indicates that the potentiating effects of N-POMC derived peptides on the steroidogenic effect of ACTH which has been described on rat and human adrenal, is not a general phenomenon in mammals.     

Steroid control of steroidogenesis in isolated adrenocortical cells: molecular and species specificity

The molecular and species specificity of glucocorticoid suppression of corticosteroidogenesis was investigated in isolated adrenocortical cells. Trypsin-isolated cells from male rat, domestic fowl and bovine adrenal glands were incubated with or without steroidogenic agents and with or without steroids. Glucocorticoids were measured by radioimmunoassay or fluorometric assay after 1-2 h incubation. Glucocorticoids suppressed ACTH-induced steroidogenesis of isolated rat cells with the following relative potencies: corticosterone greater than cortisol = cortisone greater than dexamethasone. The mineralocorticoid, aldosterone did not affect steroidogenesis. Suppression by glucocorticoids was acute (within 1-2 h), and varied directly with the glucocorticoid concentration. Testosterone also suppressed ACTH-induced steroidogenesis. Glucocorticoid-type steroids have equivalent suppressive potencies, thus suggesting that these steroids may induce suppression at least partly by a common mechanism. Although corticosterone caused the greatest suppression, testosterone was more potent. The steroid specificity of suppression of cyclic AMP (cAMP)-induced and ACTH-induced steroidogenesis were similar, suggesting that suppression is not solely the result of interference with ACTH receptor function or the induction of adenylate cyclase activity. Exogenous glucocorticoids also suppressed ACTH-induced steroidogenesis of cells isolated from domestic fowl and beef adrenal glands, thus suggesting that this observed suppression may be a general mechanism of adrenocortical cell autoregulation.     

On the role of intra-adrenal unesterified cholesterol in the steroidogenic effect of corticotropin.

Since ACTH (corticotropin) increases intra-adrenal and intramitochondrial free cholesterol levels, the relative importance of these effects during ACTH-induced steroidogenesis was examined. Rats were treated in vivo with ACTH plus aminoglutethimide to increase free cholesterol (2--3-fold), and the latter was tested as a steroidogenic factor after removal of aminoglutethimide blockade and subsequent prolonged incubation of the cholesterol-rich adrenal sections in vitro. The increased free cholesterol served as a positive steroidogenic factor only during the early phase of the incubation when other ACTH-induced steroidogenic factors were operative. At later times of incubation, the increased free cholesterol did not, of itself, enhance steroidogenesis unless ACTH was added. These results suggest that the ACTH-induced enhancement of intraadrenal (and mitochondrial) free cholesterol may be important in determining the amount of steroids produced in response to a given ACTH stimulus, but another ACTH-induced factor is more important in initiating the steroidogenic process. This factor (? labile protein) appears to control a metabolic process which is distal to the mitochondrial uptake of free cholesterol.     

Pro-opiomelanocortin-derived peptides in the pig pituitary: alpha- and gamma 1-melanocyte-stimulating hormones and their glycine-extended forms

Pro-opiomelanocortin (POMC)-related peptides in extracts of anterior and neurointermediate pituitary lobes from pigs were characterized by gel chromatography, reversed-phase chromatography and radioimmunoassays. The peptide content was ca. 3-fold greater in the anterior lobe compared to the neurointermediate lobe (19.8 nmol POMC/anterior lobe vs 7.0 nmol/neurointermediate lobe). In the neurointermediate lobe 93% of POMC was processed to alpha-melanocyte-stimulating hormone (alpha-MSH) and analogs exclusively of low molecular weight. Most of the remaining adrenocorticotropic hormone (ACTH)-related material consisted of the glycine-extended intermediate ACTH-(1-14) and analogs. In contrast only one fourth to one third of the N-terminal part of POMC (N-POMC) was processed to amidated gamma-MSH and its C-terminal glycine-extended precursor. The relative amount of amidated gamma-MSH was the same as alpha-MSH and analogs (94%). However, more than 95% of these peptides were of high molecular weight. In the anterior lobe 2.3% of N-POMC was processed and 94% was amidated gamma-MSH of only high molecular weight. These results show that gamma-MSH and alpha-MSH are amidated to the same extent and that gamma 1-MSH and gamma 2-MSH immunoreactivity are present in both the anterior lobe and the neurointermediate lobe. The results suggest that the production of amidated peptides is not regulated by the amidation process itself but at an earlier step (e.g. at the proteolytic cleavage).     

Synergistic effects of corticotropin and insulin-like growth factor I on corticotropin receptors and corticotropin responsiveness in cultured bovine adrenocortical cells

Pretreatment of bovine adrenocortical cells with increasing concentrations of insulin-like growth factor I (IGF-I) for 3 days resulted in a dose dependent (ED50 congruent to 5 ng/ml) increment in Corticotropin (ACTH) receptors. Moreover, IGF-I pretreatment potentiated the effects of maximal active concentration of ACTH (10(-9) M) on its own receptors. Whereas ACTH (10(-9) M) or IGF-I (50 ng/ml) alone induced a 3- and 2.5-fold increase respectively in ACTH receptors, there was a 7.5 fold increase in the presence of the two peptides. This synergism between ACTH and IGF-I was also observed for the ACTH-induced cortisol response with an increase of 9-, 3- and 20-fold for cells pretreated with ACTH, IGF-I and the two peptides, respectively. However, the effects of both peptides on ACTH-induced cAMP production was only additive. The present results show that ACTH and IGF-I are potent stimulating factors on bovine adrenal cell differentiated functions and that the effects of both peptides are synergistic.     

Steroidogenesis in isolated adrenal cells of rat. II. Effect of caffeine on ACTH and cyclic nucleotide-induced steroidogenesis and its relation to cyclic nucleotide phosphodiesterase (PDE)

Corticosterone production in isolated adrenal cells (IAC) of rat has been measured in response to ACTH or ribonucleoside 3′,5′-cyclic phosphate of adenosine (c-AMP), guanosine (c-GMP), inosine (c-IMP) and N⁶-2′-0 dibutyryl adenosine monophosphate (dc-AMP) in the presence and absence of caffeine. Caffeine inhibited ACTH-induced steroidogenesis in a manner independent of its effect on PDE. Study of PDE in whole adrenal homogenate showed hydrolysis of c-AMP, c-GMP and c-IMP but not of dc-AMP and other cyclic nucleotides. No PDE activity was demonstrable in IAC. High sensitivity of IAC to minute quantities of ACTH and various cyclic nucleotides may be due in part to lack of PDE activity in these preparations.     

Structure-activity relationships of monomeric and dimeric synthetic ACTH fragments in perifused frog adrenal slices

The effect of synthetic monomeric and dimeric ACTH fragments on spontaneous and ACTH(1-39)-evoked steroidogenesis in frog interrenal tissue was studied in vitro. Infusion of ACTH fragment 11-24 (10(-6) M) or its dimeric conjugates, attached either by their N-terminal, Glu(11-24)2, or their C-terminal amino acid, (11-24)2Lys, had no effect on the spontaneous release of corticosteroids. The monomer ACTH(11-24) and the dimer Glu(11-24)2 were also totally devoid of effect on the steroidogenic response to ACTH(1-39) (10(-9)M). In contrast, the (11-24)2Lys conjugate (10(-6)M) significantly decreased ACTH-induced stimulation of corticosterone and aldosterone (-63 and -62%, respectively). The dimeric conjugate of the fragment ACTH(7-24), linked through the C-terminal ends, (7-24)2Lys (10(-6)M), was also completely devoid of effect on basal steroidogenesis but caused a marked decrease of ACTH-evoked corticosterone and aldosterone release (-72 and -80%, respectively). Conversely, infusion of the dimer (1-24)2Lys gave rise to a dose-related stimulation of corticosterone and aldosterone release. The time-course of the steroidogenic response to the dimer was similar to that of ACTH(1-24). The 1-24 conjugate was 70 times less potent than the monomers ACTH(1-24) and ACTH(1-39). These results suggest that amphibian adrenocortical cells contain only one class of ACTH receptor which recognizes the 11-24 domain of ACTH with an affinity which depends on the presence of a strong potentiator segment, located at the N-terminus end of ACTH(1-39). Since the ACTH-dimers are thought to induce cross-linking of the receptors, our results suggest that aggregation of ACTH receptors causes a down-regulation of the receptors.     

Pressor and cardioaccelerator effects of gamma MSH and related peptides

We have recently demonstrated that the hypertensinogenic and natriuretic actions of ACTHI-39 can be found in a non-steroidogenic fragment of ACTH, ACTH4-10. These effects of ACTH or ACTH4-10 may be due to their ability to act as weak agonists of gamma MSH. gamma MSH is found in the 16K N-terminus of pro-opiocortin, and contains a sequence analogous to ACTH4-10, gamma MSH3-9. We investigated the cardiovascular effects of gamma 2MSH, gamma MSH3-9, and sterically restricted analogs of ACTH4-10. The results indicate that gamma MSH3-9, had essentially the same activities as ACTH4-10. The addition of five other amino acid residues to gamma MSH3-9 (gamma 2MSH) resulted in significant enhancement of pressor and cardioaccelerator activity. Steric restriction of the ACTH4-10 sequence by the substitution of a D-Phe in place of an L-Phe residue in position #7, or cyclization of the peptide by a half-Cys4, half Cys10 intramolecular disulfide-bridge derivatization, resulted in increased cardiovascular activities. Based on these data, the cardiovascular actions of ACTH4-10, gamma MSH3-9, and gamma 2MSH are predicted to be due to the assumption of a reverse-turn three-dimensional structure. The additional residues in gamma 2MSH appear to specifically enhance the cardiovascular activities of gamma MSH3-9. The results suggest the existence of a new class of hypophyseal peptides with cardiovascular activities, which require the assumption of a defined three-dimensional structure.     

ACTH-induced prostaglandin biosynthesis from 3H-arachidonic acid by adrenocortical cells.

Prostaglandins biosynthesized from 3H-arachidonic acid by trypsin-dispersed cat adrenocortical cells were isolated by silicic acid and thin layer chromatography. PGE, PGF, and a third component with mobility properties indistinguishable from either PGA or PGB were identified both in cortical cell homogenates and incubation medium. Concentrations of ACTH (125-250 muU) which stimulate steroidogenesis enhanced the conversion of labeled precursor to all three of these prostaglandins. These findings provide further evidence for the proposal that prostaglandins function as a critical link in ACTH-induced steroidogenesis.     

Presence of adrenocorticotropin-potentiating factors in porcine thyroid glands

An extract of porcine thyroid gland in 0.1 N acetic acid exerted dose-dependent potentiation of ACTH-induced corticosterone production in isolated rat adrenal cells. The extract by itself manifested no steroidogenic activity. Upon gel-filtration of the extract, potentiating activities were demonstrated in three main peaks with molecular weights of about 10,000, 5,000 and 2,000. These findings indicate the presence of heterogeneous forms of ACTH-potentiating factors in the thyroid. Significant enhancement of ACTH-induced steroidogenesis was readily apparent with three gel-filtration fractions at a lower concentration of ACTH (4.75 pM). At this concentration, dose-dependent potentiation was observed with these three fractions. Enhanced corticosterone production responses by cells preincubated with the thyroid extract were observed and the results indicated the existence of potentiating mechanisms other than inhibition of ACTH proteolysis. The lack of T4, T3 and thyroglobulin in this activity suggests that the activity resides in other constituents of the thyroid.     

Ovine fetal adrenal maturation at term and during fetal ACTH administration: evidence that the modulating effect of cortisol may involve cAMP

Exogenous ACTH1-24 promotes adrenal maturation in fetal sheep, and this effect appears to be modulated in part by cortisol (Challis et al. 1985). We have examined whether similar changes in adrenal metabolism of progesterone occur with ACTH-induced labour as at spontaneous term and whether the site of cortisol modulation is on adrenal steroidogenesis or at the level of cAMP generation. Chronically catheterized fetal sheep were infused in utero for 100 h between days 127 and 131 of pregnancy with P-ACTH, P-ACTH + metopirone, P-ACTH + metopirone + cortisol, or saline. After 100 h the metabolism of [3H]progesterone was measured in adrenal homogenates. Similar incubations were performed with adrenal tissue from fetal sheep at day 130 of pregnancy and at spontaneous labour. In the treatment groups of sheep, cAMP output by dispersed adrenal cells in response to ACTH added in vitro was also determined. Similar qualitative patterns of [3H]progesterone metabolism were found in adrenal homogenates after in vivo ACTH or at term. At both times there was an increase in cortisol and in total 17 alpha-hydroxycorticosteroid accumulation and also evidence for increased activity of 11 beta-hydroxylase enzyme. The formation of total 17 alpha-hydroxycorticosteroids was not affected significantly by concurrent treatment in vivo with metopirone +/- cortisol. The accumulation of cAMP in vitro was increased after in vivo ACTH, attenuated after ACTH + metopirone, but statistical significance over controls was restored after ACTH + metopirone + cortisol treatment. We conclude that ACTH-induced labour and spontaneous parturition in sheep is associated with qualitatively similar changes in progesterone metabolism by the fetal adrenal gland.(ABSTRACT TRUNCATED AT 250 WORDS)