Characteristics of the response of dispersed guinea-pig adrenal cells to low doses (1-50 pg/ml) of alpha 1-24 adrenocorticotrophin

Isolated adrenal cells prepared by tryptic digestion of the guinea-pig adrenal gland are sensitive to low concentrations (less than 25 pg/ml) of adrenocorticotrophin (ACTH). Cell which have been pre-incubated for 2 h. centrifuged and resuspended in fresh culture medium prior to the introduction of 10 pg/ml ACTH for 60 min show a marked increase (328 +/- 109 nmol/l; mean +/- SD) in cortisol secretion over the control compared to freshly dispersed cells (75 +/- 45 nmol/l). Further potentiation of the ACTH effect was seen with the pre-incubated cells by suplementing the medium with calcium (8 mM) and ascorbate (2 mM) but not with theophylline (1 mM). Basal cortisol secretion was not affected by any of the additives. In the presence of 8 mM calcium and after 60 min incubation 10 pg/ml ACTH stimulated cortisol secretion from 328 nmol/l over the control to 839 +/- 382 nmol/l. The effect of ascorbate (2 mM) was to further increase the effect of ACTH at all dose levels tested (1-25 pg/ml). The concentration of ACTH required to provoke half maximal cortisol secretion decreased from 95 pg/ml with normal medium to 12 pg/ml with calcium -ascorbate supplemented medium. Using this supplemented medium the cells were sensitive to 1 pg/ml and cortisol secretion was stimulated 10-fold over the control with 50 pg/ml, a dose which saturated the system.     

ACTH-, glucose- and lactate-content of swine plasma during insulin-induced hypoglycemia]

The present investigation was undertaken to determine the content of ACTH, glucose and lactate in plasma of 4 pigs (body weight 82--118 kg) during a circadian period and during an insulin hypoglycemia test using 1 IU/kg in 3 pigs (body weight 96--118 kg) and 4 pigs (body weight 20--30 kg). The plasma ACTH level at rest was 57 +/- 27 pg/ml (Mean +/- SE) for all samples in all animals during a circadian period. Significant diurnal changes were not observed. During insulin-induced hypoglycaemia plasma ACTH rose from a mean (+/- SE) basal level of 35 +/- 15 to a maximum of 673 +/- 100 pg/ml at 60 min in heavier pigs and in lighter pigs to 395 +/- 153 at 30 min and 403 +/- 145 pg/ml at 120 min. Initial ACTH responses were evident 30 min (heavier pigs) and between 0 and 15 min (lighter pigs) after insulin administration. Plasma glucose decreased from a mean (+/- SE) basal level of 80 +/- 10 to a minimum of 6 +/- 1 mg/100 ml at 60 min (heavier pigs) and from 88 +/- 3 to 16 +/- 4 mg/100 ml at 60 min (lighter pigs). After its minimum level the glucose concentration showed a slower increment in the heavier pigs as compared to lighter animals. Plasma lactate rose from a mean (+/- SE) basal level of 19 +/- 10 to a maximum of 76 +/- 42 mg/100 ml at 120 min (heavier pigs) and from 12 +/- 3 to 37 +/- 16 mg/100 ml at 150 min (lighter group). In accordance with the changes in the blood plasma levels of ACTH, glucose and lactate, the clinical symptoms of hypoglycaemia in heavier pigs were more intensive.     

Activation of adrenal function in fetal sheep by the infusion of adrenocorticotropin to the fetus in utero

We determined whether ACTH1-24, infused into fetal lambs at a rate that is known to cause premature labor, elicits changes in the responsiveness of the fetal adrenal glands, and alters the pattern of corticosteroid output. Plasma cortisol (F), corticosterone (B) and progesterone (P4) were measured during 72 h of infusion of saline or ACTH (10 micrograms/h) beginning on Day 127 of pregnancy. Adrenals were then dispersed into isolated cells, and the output of F, B and P4 after exogenous ACTH determined in vitro. Plasma concentrations of F and B were higher in ACTH-treated fetuses. The increment in F (5-to 7-fold) was greater than that in B (2-fold) such that the F:B ratio in plasma of ACTH-treated fetuses on Days 2 and 3 of infusion was 2.5 times higher than in controls. After 72 h of infusion, the adrenal weights in ACTH-treated fetuses (741 +/- 38 mg, +/- SEM; n = 4) were greater than in the control animals (349 +/- 11 mg). There was a significant effect of ACTH pretreatment in vivo on F output by isolated adrenal cells in vitro. Mean increments in F output after addition of ACTH1-24 (5000 pg/ml) in vitro rose from 368 +/- 235 pg/50,000 cells in controls, to 64,639 +/- 19,875 pg/50,000 cells after ACTH in vivo. There was no significant effect of ACTH in vivo on B output in vitro; the ratio of F:B output, either in the absence or presence of ACTH in vitro, was significantly higher in cells from ACTH-pretreated fetuses. There was a significant effect of in vivo ACTH on in vitro P4 output. After ACTH treatment in vivo there was an increase in the vitro output ratio of F:P4, but no change in the output ratio of B:P4. We conclude that ACTH treatment of the fetal lamb in vivo results in activation of fetal adrenal function, increased fetal adrenal responsiveness to ACTH, and directed corticosteroid biosynthesis towards cortisol. Our results are consistent with an increase in fetal adrenal 17 alpha-hydroxylase activity after ACTH treatment.     

Effects of synthetic ovine corticotropin-releasing factor (CRF) on plasma ACTH and cortisol in 31 normal human males

Responses of plasma ACTH and cortisol to corticotropin-releasing factor (CRF) were evaluated in 31 normal human males. 1.0 micrograms/ks of sterilized synthetic ovine CRF was administered to the subjects, aged 19 to 53 yr and weighing 50 to 78 kg, at between 9:30 a.m. and 10:30 a.m. as an intravenous bolus injection after an overnight fast. Blood specimens were drawn before and 15, 30, 60, 90 and 120 min after injection for later determination of plasma ACTH and cortisol concentrations by radioimmunoassays. Plasma ACTH and cortisol levels for all subjects rose significantly (p less than 0.001) from the basal level (mean +/- SEM, 26.8 +/- 4.5 pg/ml and 12.6 +/- 0.9 micrograms/dl) to peak levels (58.4 +/- 5.5 pg/ml and 22.9 +/- 1.0 micrograms/dl) at 30 min and at 60 min, respectively. Although the plasma concentrations of ACTH and cortisol thereafter declined gradually, the levels at 120 min (43.4 +/- 5.2 pg/ml and 18.9 +/- 0.9 micrograms/ml, respectively) were still significantly higher than the basal levels (p less than 0.001). Significant inverse correlations were observed between the basal levels of each hormone and the ratio of the peak level to the basal level (p less than 0.01), and the increases in plasma ACTH and cortisol concentrations were either not significant or much smaller for the individuals in whom the basal levels were higher than 65 pg/ml and 17.0 micrograms/dl, respectively. No serious subjective symptom was observed during the experimental period in any of the subjects.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of protein kinase activation in the control of steroidogenesis by adrenocorticotrophic hormone in the adrenal cortex

A method has been developed for investigation of the effect of adrenocorticotrophic hormone (ACTH) on the state of activation of a cyclic AMP-dependent protein kinase within cells of the adrenal cortex. Enzyme activity was measured in terms of the quantity of (32)P transferred from [gamma-(32)P]ATP to histone under conditions in which bound cyclic AMP did not dissociate from the regulatory subunit of the protein kinase ACTH (1x10(-2)i.u./ml) caused a rapid and complete activation of the cyclic AMP-dependent protein kinase activity within 2min of hormone addition to the isolated cells. In response to a range of ACTH concentrations a sigmoid log dose-response curve for protein kinase activation was obtained, with half-maximal stimulation attained at about 1x10(-3)i.u./ml. However, some low doses of ACTH that elicited a marked (but submaximal) steroidogenic response failed to cause a clear stimulation of protein kinase activity in isolated adrenal cells. Theophylline (2mm) potentiated the effect of ACTH on protein kinase activity. The results implicate an important role for protein kinase in ACTH action on the adrenocortical cell.     

An improved bioassay for ACTH determination.

An improved method for the bioassay of ACTH has been developed using short-term culture of adrenal cell suspensions derived from intact rats. Six separate pools of adrenals were used and cells derived from the same pool were compared after acute dispersion and overnight culture. The ED50, defined as the concentration of ACTH required to produce half maximal steroidogenesis, for cultured cells was 35.4+/-2 pg/ml compared to 240+/-60 pg/ml for cells used immediately after dispersion. The minimum concentrations of ACTH necessary to produce a response significantly above basal levels were 0.98+/-.10 pg/ml and 12.1+/-3 pg/ml respectively. Response characteristics of the cultured cells were highly reproducible. The incubation volume employed in this study was 0.25 ml, so the ED50 expressed as dose of ACTH per tube was actually 8.8 pg for the cultured cells. Such sensitivity provides requisite methodology for the measurement of ACTH under varying physiological conditions.     

Structure-function organization of ACTH: fragment ACTH 11-24--a functionally important site of the hormone molecule

The steroidogenic and lipolytic activities of ACTH fragments (ACTH11-24--I, ACTH11-19--II, ACTH11-16--III and ACTH 17-24--IV) were studied. Fragments I--IV exert a steroidogenic effect in isolated fasciculata rat adrenal cells at concentrations of 1--500 micrograms/ml. The inner activity (alpha) and concentration at which a half-maximum effect is achieved (EC50) for fragments I and IV are 0.64+/-0.09 and 0.5--2.0 micrograms/ml, for fragment III--0.49+/-0.07 and 0.7 microgram/ml, respectively. Fragments I--IV have no effect on the lipolysis in isolated rat fat cells. The results obtained are indicative of the functional importance of fragment ACTH11-24 in manifestation of steroidogenic action of ACTH and suggest that the second active site of ACTH is enclosed within this amino acid sequence.     

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.     

Basal plasma aldosterone levels and tetracosactid-induced increase of aldosterone secretion in conscious male rabbits: lack of correlation with glucocorticosteroid levels

The adrenocortical secretory activity under basal conditions and after treatment with tetracosactid (1-24ACTH) has been investigated in chronically cannulated male rabbits. Basal plasma concentrations of glucocorticosteroids (0.74 micrograms/100 ml) and aldosterone (78 pg/ml) have been determined in a greater number of animals. No significant positive correlation between basal glucocorticosteroid and aldosterone plasma levels could be found. After intravenous injection of 2.5, 5.0, 10.0 and 20.0 micrograms/kg body weight tetracosactid glucocorticosteroid concentrations were significantly elevated between 40--100 min after administration; aldosterone release, on the other hand, was significantly increased only after injection of 10.0 or 20.0 micrograms/kg body weight tetracosactid between 20--60 min after injection. After administration of high tetracosactid doses glucocorticosteroid and aldosterone plasma concentrations were significantly correlated (10.0 micrograms/kg: r = 0.62; 20.0 micrograms/kg: r = 0.26). Because of the relative insensitivity of the zona glomerulosa cells to tetracosactid administered intravenously, it is concluded that ACTH is only of minor importance in the regulation of aldosterone secretion in the rabbit.     

Stimulation of pituitary-adrenocortical system by ginseng saponin.

Effects of preparations of saponin mixture and isolated ginsenosides, extracted from the root of Panax ginseng, on plasma corticotropin (ACTH) and corticosterone concentrations in rats were determined by the radioimmunoassay and competitive protein binding method. When ginseng saponin mixture was administered to rats intraperitoneally, plasma ACTH and corticosterone increased significantly 30, 60 and 90 min after the treatment. The kinetic pattern of the increase in plasma ACTH was almost parallel to that in plasma corticosterone. Isolated ginsenoside, protopanaxadiol or protopanaxatriol glycoside, also increased plasma corticosterone. The ginseng-induced increase in plasma corticosterone was suppressed by pretreatment with dexamethasone. Thus the ginseng saponin was found to act on the hypothalamus and/or hypophysis primarily, and stimulated ACTH secretion which resulted in increased synthesis of corticosterone in the adrenal cortex.     

Atrial natriuretic factor inhibits the CRH-stimulated secretion of ACTH and cortisol in man.

Corticotrophic secretion of ACTH is stimulated by corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), and suppressed by glucocorticoids. In vitro and preclinical studies suggest that atrial natriuretic factor (ANF) may be a peptidergic inhibitor of pituitary-adrenocortical activity. The aim of this study was to elucidate a possible role of ANF as a modulator of ACTH release in humans. A bolus injection of 100 micrograms human CRH (hCRH) during a 30 min intravenous infusion of 5 micrograms/min human alpha atrial natriuretic factor (h alpha ANF) was administered at 19:00 to six healthy male volunteers. In comparison to saline, a blunted CRH-stimulated secretion of ACTH (mean maximum plasma level +/- SD 45 min after hCRH: saline 46.2 +/- 14.2 pg/ml, h alpha ANF 34.6 +/- 13.8 pg/ml, p-value = 0.007) and a delayed rise (10 min) in cortisol were detected. The maximum plasma cortisol levels remained nearly unchanged between saline and h alpha ANF administration (mean maximum plasma level +/- SD 60 min after hCRH: saline 182 +/- 26 ng/ml, h alpha ANF 166 +/- 54 ng/ml). No effects of h alpha ANF on basal cortisol levels were observed; in contrast, basal ACTH plasma levels were slightly reduced. Basal blood pressure and heart rate remained unaffected. In the control experiment, infusion of 3 IU AVP in the same experimental paradigm increased basal and stimulated ACTH and cortisol levels significantly in comparison to saline. These observations suggest that intravenously administered haANF inhibits the CRH-stimulated release of ACTH in man.     

Evidence for 6-keto-PGF1 alpha in adrenal cortex of the rat and effects of 6-keto-PGF1 alpha and PGI2 on adrenal cAMP levels and steroidogenesis.

Both intact cortical tissue and isolated cortical cells from the adrenal gland of the rat were analyzed for 6-keto-PGF1 alpha, the hydrolysis metabolite of PGI2, using high-performance liquid chromatography and gas chromatography-mass spectrometry. 6-Keto-PGF1 alpha was present in both incubations of intact tissue and isolated cells of the adrenal cortex, at higher concentrations than either PGF2 alpha or PGE2. Thus, the cortex does not depend upon vascular components for the synthesis of the PGI2 metabolite. Studies in vitro, using isolated cortical cells exposed to 6-keto-PGF1 alpha (10(-6)-10(-4)M), show that this PG does not alter cAMP levels or steroidogenesis. Cells exposed to PGI2 (10(-6)-10(-4)M), however, show a concentration-dependent increase of up to 4-fold in the levels of cAMP without altering cortico-sterone production, ACTH (5-200 microU/ml) increased cAMP levels up to 14-fold, and corticosterone levels up to 6-fold, in isolated cells. ACTH plus PGI2 produced an additive increase in levels of cAMP, however, the steroidogenic response was equal to that elicited by ACTH alone. Adrenal glands of the rat perfused in situ with PGI2 showed a small decrease in corticosterone production, whereas ACTH greatly stimulated steroid release. Thus, while 6-keto-PGF1 alpha is present in the rat adrenal cortex, its precursor, PGI2, is not a steroidogenic agent in this tissue although it does stimulate the accumulation of cAMP.     

Improvement and initial in vivo application of the radioimmunoassay of rat thyrocalcitonin.

Previously we reported a homologous radioimmunoassy for rat thyrocalcitonin (TCT) which was sensitive enough (2--3 ng/ml serum) to measure TCT in thyroid venous blood or thyroid gland extracts but could not detect TCT in peripheral blood even after provocative challenge with iv calcium. In the present study chicken antisera to rat TCT were developed which were sufficiently sensitive (120--240 pg/ml serum) to permit initial evaluation of changes in TCT in rat peripheral blood. The following results were observed: (1) Basal serum TCT in young male Holtzman rats was undetectable, being less than 120--240 pg/ml; (2) induction of marked hypercalcemia by iv calcium increased TCT to approximately 1000--3000 pg/ml within 5 min; (3) thyroid cautery increased TCT to approximately 1000 pg/ml in 5--15 min; (4) calcium gavage (12.2 mg Ca/100 g) produced modest hypercalcemia in 30--60 min and increased serum TCT to approximately 500 pg/ml; (5) injection of isoproterenol raised serum TCT detectably; (6) injection of large doses of gastrin or pentagastrin did not produce detectable increases in TCT 5 or 30 min later. The results show that suitable antisera to rat TCT can be developed in chickens and applied to the measurement, by radioimmunoassay, of elevated circulating levels of TCT in the rat.     

Hypothalamic-pituitary disconnection in fetal sheep blocks the peripartum increases in adrenal responsiveness and adrenal ACTH receptor expression

Although it has been recognized for over a decade that hypothalamic-pituitary disconnection (HPD) in fetal sheep prevents the late gestation rise in plasma cortisol concentrations, the underlying mechanisms remain unclear. We hypothesized that reductions in adrenal responsiveness and ACTH receptor (ACTH-R) expression may be mediating factors. HPD or sham surgery was performed at 120 days of gestation, and catheters were placed for blood sampling. At approximately 138 days of gestation, fetuses were killed, and adrenals were removed for cell culture and analyses of ACTH-R mRNA and protein. After 48 h, adrenocortical cells were stimulated with ACTH for 2 h, and the medium was collected for cortisol measurement. The same cells were incubated overnight with medium or medium containing ACTH or forskolin (FSK), followed by ACTH stimulation (as above) and cortisol and cellular ACTH-R mRNA analyses. HPD prevented the late gestation increase in plasma cortisol and bioactive ACTH and reduced adrenal ACTH-R mRNA and protein levels by over 35%. HPD cells secreted significantly less cortisol than sham cells (3.2 +/- 1.2 vs. 47.3 +/- 11.1 ng.ml(-1).2 h(-1)) after the initial ACTH stimulation. Overnight incubation of HPD cells with ACTH or FSK restored cortisol responses to acute stimulation to levels seen in sham cells initially. ACTH-R mRNA levels in cells isolated from HPD fetuses were decreased by over 60%, whereas overnight incubation with ACTH or FSK increased levels by approximately twofold. Our findings indicate that the absence of the cortisol surge in HPD fetuses is a consequence, at least in part, of decreased ACTH-R expression and adrenal responsiveness.     

Effects of ovine corticotropin-releasing hormone and FK 33-824 (met-enkephalin analogue) on the secretions of proopiomelanocortin-derived N-terminal peptide, beta-lipotropin, beta-endorphin and adrenocorticotropin in patients with Addison's disease

The responses of plasma immunoreactive (IR) proopiomelanocortin (POMC)-derived N-terminal peptide (Nt), IR-beta-endorphin (Ep), IR-beta-lipotropin (LPH) and IR-ACTH levels to ovine corticotropin-releasing hormone (CRF) and FK 33-824 (Met-Enkephalin analogue) were studied in nine patients with Addison's disease. The basal plasma levels (mean +/- SE) of IR-Nt, IR-Ep, IR-LPH and IR-ACTH were significantly higher in patients with Addison's disease (4459 +/- 975 pg/ml, 132 +/- 25 pg/ml, 4425 +/- 1030 pg/ml, 553 +/- 89 pg/ml, respectively) than in the normal controls (202 +/- 38 pg/ml, 7 +/- 2 pg/ml, 101 +/- 18 pfi/ml, 53 +/- 16 pg/ml, respectively). Ovine CRF produced rapid and concomitant increases in plasma levels of IR-Nt, IR-Ep, IR-LPH and IR-ACTH. Ep and ACTH levels reached a peak at 30 min. On the other hand, Nt and LPH levels reached a peak at 60 min and these levels gradually decreased up to 120 min. The molar concentrations of these IR-peptides in plasma were changed in close parallel fashion to one another. FK 33-824 produced a pronounced and concomitant fall in IR-Nt, IR-EP, IR-LPH, and IR-ACTH levels. These results support the theory that Nt, Ep, LPH and ACTH are produced simultaneously from POMC as a common precursor in the pituitary gland and are secreted concomitantly under various conditions such as stimulation by CRF and inhibition by FK 33-824 in patients with Addison's disease.     

Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrenal cells of vitamin E-deficient rats.

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5'-triphosphate [14C]ATP to cyclic [14C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [14C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induced cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [14C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [14C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [14C]AMP continued to be formed. The in vitro addition of alpha-tocopherol reduced the inhibition of ACTH-induced cyclic [14C]AMP formation by ascorbate in Vitamin E-deficient rats. These studies suggest that alpha-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with the membrane-bound enzyme adenylate cyclase.     

Evidence that 19-hydroxyandrostenedione is secreted by the adrenal cortex and is under the control of ACTH and the renin-angiotensin system in man

Plasma 19-hydroxyandrostenedione (19-OH-A-dione) concentrations in man were evaluated using a specific and sensitive radioimmunoassay. Plasma 19-OH-A-dione concentrations (mean ± SE) in normal subjects are 151 ± 14 pg/ml (n=13) in males and 141 ± 9 pg/ml (n=14) in females. Plasma 19-OH-A-dione (mean ± SE) rises significantly during ACTH stimulation (116 ± 25 pg/ml vs 288 ± 38 pg/ml; P<0.01; n=5), declines significantly during dexamethasone suppression (180 ± 30 pg/ml vs 36 ± 14 pg/ml; P<0.01; n=4) and rises significantly during angiotensin II infusion (89 ± 10 pg/ml vs 159 ± 27 pg/ml; P<0.05; n=5). Plasma 19-OH-A-dione in the adrenal vein is much higher than that in the inferior vena cava (2076–3076 pg/ml vs 115–184 pg/ml; n=2). These results demonstrate that 19-OH-A-dione is directly secreted by the adrenal cortex and is under the control of ACTH and the renin-angiotensin system.     

Steroidogenic activity of high molecular weight forms of corticotropin.

The high molecular weight forms of adrenocorticotropic hormone (ACTH) produced by mouse pituitary tumor cells (AtT-20/D-16v) were separated from each other by gel filtration; their ability to stimulate steroidogenesis by isolated rat adrenal cortical cells was studied. Pools of pro-ACTH/endorphin. ACTH biosynthetic intermediate, and glycosylated ACTH(1--39) were obtained; on the basis of NaDodSO4-polyacrylamide gel electrophoresis, over 97% of the immunoactive ACTH was found to have the expected molecular weight. Suspension of isolated rat adrenal cortical cells were incubated overnight in tissue culture medium and used in a 2-h steroid production assay. Synthetic human ACTH(1--39) [hACTH(1--39)] was used as a bioassay and immunoassay standard; 60 pM hACTH(1--39) stimulated half-maximal production of fluoregenic steroid. The amount of pro-ACTH/endorphin, ACTH biosynthetic intermediate, or glycosylated (ACTH(1--39) added was estimated with an ACTH(17--24) immunoassay. All three high molecular weight forms of ACTH are capable of stimulating the same maximal level of steroidogenesis as hACTH(1--39). Glycosylated ACTH(1--39) is equipotent with hACTH(1--39); ACTH biosynthetic intermediate and pro-ACTH/endorphin are, respectively, 100- and 300-fold less potent than hACTH(1--39). Steroid production in response to all four forms of ACTH is linear in time. All of the different forms of ACTH stimulate the synthesis of corticosterone and related steroids; no significant production of cortisol or aldosterone was observed. beta-Lipotropin (beta LPH) and 16K fragment, which comprise the non-ACTH regions of pro-ACTH/endorphin and are secreted by the pituitary tumor cells, did not stimulate or interfere with steroidogenesis. Brief incubations of pro-ACTH/endorphin and ACTH biosynthetic intermediate with trypsin generated lower molecular weight forms of ACTH and increased biological activity 50-fold; thus, the decreased steroidogenic potency of these forms of ACTH is thought to be due to structural constraints on the ACTH(1--39)-like sequence in these larger precursor molecules     

The relationship between adrenal vascular events and steroid secretion: the role of mast cells and endothelin.

The actions of ACTH on the adrenal cortex are known to be 2-fold. In addition to increased steroidogenesis, ACTH also causes marked vasodilation, reflected by an increased rate of blood flow through the gland. Our studies, using the in situ isolated perfused rat adrenal preparation, have shown that zona fasciculata function and corticosterone secretion are closely related to vascular events, with an increase in perfusion medium flow rate causing an increase in corticosterone secretion, in the absence of any known stimulant. These observations give rise to two important questions: how does ACTH stimulate blood flow; and how does increased blood (or perfusion medium) flow stimulate steroidogenesis? Addressing the first question, we have recently identified mast cells in the adrenal capsule, and shown that Compound 48/80, a mast cell degranulator, mimics the actions of ACTH on adrenal blood flow and corticosterone secretion. We have also demonstrated an inhibition of the adrenal vascular response to ACTH in the presence of disodium cromoglycate, which prevents mast cell degranulation. We conclude, therefore, that ACTH stimulates adrenal blood flow by its actions on mast cells in the adrenal capsule. Addressing the second question, we looked at the role of endothelin in the rat adrenal cortex. Endothelin 1, 2 and 3 caused significant stimulation of steroid secretion by collagenase dispersed cells from both the zona glomerulosa and the zona fasciculata. A sensitive response was seen, with significant stimulation at an endothelin concentration of 10(-13) mol/l or lower. Endothelin secretion by the in situ isolated perfused rat adrenal gland was measured using the Amersham assay kit. Administration of ACTH (300 fmol) caused an increase in the rate of immunoreactive endothelin secretion, from an average of 28.7 +/- 2.6 to 52.6 +/- 6 fmol/10 min (P less than 0.01, n = 5). An increase in immunoreactive endothelin secretion was also seen in response to histamine, an adrenal vasodilator, which stimulates corticosterone secretion in the intact gland, but has no effect on collagenase-dispersed cells. From these data we conclude that endothelin may mediate the effects of vasodilation on corticosterone secretion, and this mechanism may explain some of the differences in response characteristics between the intact gland and dispersed cells.     

Inhibitory effects on steroid production from isolated adrenal cells of rhesus monkey (Macaca mulatta) of pro-opiomelanocorticotrophic peptides

Adrenal glands from Rhesus monkeys (Macaca mulatta) of 160 days gestation, newborn, 2 months-old infants or 6 months-old infants were excised and prepared, by a collagenase digestion, as a cell suspension. The cells were incubated with 10 pg/ml, 100 pg/ml or 1 ng/ml of a peptide of the ACTH/pro-opiomelanocortin 'family', 57K, 31K, 20K, alpha MSH, ovine-CLIP or gamma LPH either in the presence or absence of 166 pg/ml ACTH1-39. The production by cortisol and androstenedione was measured by radioimmunoassay. Using the steroid production by aliquots of the cell suspension with either no stimulating agent or ACTH1-39 alone as controls, the net influence of these different peptides on basal or ACTH1-39-stimulated production was observed. alpha MSH, ovine-CLIP and gamma LPH had no influence on either basal or stimulated cortisol or androstenedione production. Corticotrophic peptides of 57K, and 20K and pro-opiomelanocortin each had a steroidogenic activity alone, in all age groups. In the fetal and newborn monkeys' adrenal cells, peptides of 57K and 20K at 1 ng/ml had an inhibitory influence on ACTH1-39 stimulated cortisol and androstenedione production. The influence of the 20K peptide is partially inhibitory as the steroidogenic potential of this peptide is not additive with that of ACTH1-39. These results show that, as observed in other species, that the ACTH/pro-opiomelanocortin range of peptides are inhibitory to the action of ACTH1-39 in the developing adrenal.