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Corticotropin-like immunoreactivity in the brain of intact, hypophysectomized, cortisol- and metopirone-treated eels. Comparison with changes in pituitary corticotropes and brain corticotropin-releasing factor.

An ACTH-like peptidergic system was demonstrated in the brain of three teleost species by immunocytochemistry. In order to investigate the origin of brain ACTH and factors modulating its synthesis, similar techniques were applied to the brain of eels (1) submitted to hypophysectomy in order to suppress pituitary ACTH and plasma cortisol, (2) injected with cortisol to inhibit pituitary ACTH synthesis and release, and (3) injected with metopirone to block cortisol synthesis and stimulate ACTH synthesis and release. Hypophysectomized eels showed a normal distribution of immunoreactive perikarya in the ventral hypothalamus and fibers in the brain, suggesting that brain ACTH does not arise from the pituitary. In cortisol-treated eels immunostaining was markedly reduced in brain perikarya and pituitary corticotropes, suggesting a reduced synthesis. In metopirone-injected eels, one third of the animals showed an increased immunostaining in perikarya and a dense network of immunoreactive fibers, suggesting that ACTH synthesis was increased. Brain ACTH was not affected in other animals. Pituitary corticotropes were rapidly degranulated. Responses of ACTH in the brain and pituitary occur independently when cortisol synthesis is inhibited. These responses are compared to those of the corticotropin-releasing factor system in the same eels.

Adrenocorticotropic Hormone↗

Photoreactive derivatives of corticotropin. 1. Preparation and chracterization of 2,4-dinitro-5-azidophenylsulfenyl derivative of corticotropin.

A photoaffinity probe for corticotropin (ACTH) receptors was prepared by selective modification of the single tryptophan residue in ACTH. A new photoreactive agent, 2,4-dinitro-5-azidophenylsulfenyl chloride, was synthesized and used for introducing the photoreactive group into ACTH. 2,4-Dinitro-5-azidophenylsulfenyl-Trp9-ACTH (DNAPS-ACTH) was also prepared by thiolysis of 2,4-dinitrophenyl-sulfenyl-Trp9-ACTH to form 2-thiol-Trp9-ACTH and reaction of this with 2,4-dinitro-5-azidofluorobenzene. DNAPS-ACTH was characterized by ultraviolet spectra, peptide mapping, and amino acid analysis. Covalent attachment of DNAPS-ACTH to a pituitary protein fraction FI by photolysis was demonstrated by ultraviolet absorption changes as well as by the use of tritiated DNAPS-ACTH.

Adrenocorticotropic Hormone↗

Photoreactive derivatives of corticotropin. 2. Preparation and characterization of 2-nitro-4(5)-azidophenylsulfenyl derivatives of corticotropin.

Two new photoreactive arylsulfenyl chlorides, 2-nitro-4-azidophenylsulfenyl chloride (2,4-NAPS-Cl) and 2-nitro-5-azidophenylsulfenyl chloride (2,5-NAPS-Cl), have been synthesized and used for the selective modification of corticotropin (ACTH). Both reagents reacted rapidly with N-acetyltryptophanamide and ACTH under acidic conditions. The NAPS derivatives of ACTH were purified by partition chromatography and characterized by absorption spectra, amino acid analysis, and peptide mapping. The spectral changes caused by photolysis as well as the kinetics of photolysis are described. Tritiated 2,5-NAPS-ACTH was attached covalently to a pituitary protein fraction FI by photolysis. The photolabeling of FI was blocked in the presence of excess ACTH.

Adrenocorticotropic Hormone↗

Characterization of a common precursor to corticotropin and beta-lipotropin: cell-free synthesis of the precursor and identification of corticotropin peptides in the molecule.

mRNA was isolated from cultures of AtT-20/D-16v tumor cells and translated in a mRNA-dependent reticulocyte cell-free system. The corticotropin (ACTH) product was purified by a double-antibody immunoprecipitation procedure using antisera specific for the alpha(1-24) sequence of ACTH. The product is shown by sodium dodecyl sulfate/gel electrophoresis and gel filtration on guanidine-HCl columns to be homogeneous with an apparent molecular weight (Mr) of 28,500. A product with the same molecular weight is synthesized when membrane-bound polysomes from D-16v cells are allowed to complete their nascent chains in a reticulocyte cell-free system. Mr 31,000 ACTH isolated from tumor cells has been separated into three proteins of different apparent Mr:29,000, 32,000, and 34,000. The cell-free product contains the same lysine-, methionine-, and phenylalanine-labeled tryptic peptides as the Mr 29,000 ACTH synthesized in the tumor cells. Tryptic peptide analysis also reveals the presence of the alpha(1-39) sequence in the Mr 28,500 cell-free product and suggests that there is only one copy of this sequence in the Mr 28,500 molecule.

Adrenocorticotropic Hormone↗

Adrenocortical response to corticotropin is potentiated by part of the amino-terminal region of pro-corticotropin/endorphin.

Five peptides derived from pro-corticotropin/endorphin (pro-ACTH/endorphin), the pituitary corticotroph cell prohormone, were bioassayed with isolated rat adrenocortical cells: alpha- and beta-melanotropin, beta-lipotropin, beta-endorphin, and the amino-terminal region of pro-ACTH/endorphin known as "16k fragment." The effect of each on steroidogenesis was measured at potentially physiological concentrations (0.01-1 nM) in both the absence and presence of varying concentrations of ACTH-(1-24). Of the peptides tested, only 16k fragment, the amino-terminal region of pro-ACTH/endorphin, has a slight but significant potentiating effect on ACTH-(1-24) action. Prior treatment of 16k fragment with trypsin for 30 sec dramatically increases this dose-dependent synergism. Experiments performed in vivo with hypophysectomized female rats indicate that the trypsin digest of 16k fragment stimulates cholesterol ester hydrolase (cholesterol esterase; sterol-ester acylhydrolase, EC 3.1.1.13) activity in the adrenal cortex but fails to activate cholesterol side-chain cleavage. The effect of the trypsinized material can therefore be qualitatively distinguished from that of ACTH-(1-24). When both ACTH-(1-24) and the digest are administered together, a synergistic increase in serum corticosterone concentration results. We propose that a portion of 16k fragment molecule may play a hormonal role in the control of adrenocortical steroidogenesis.

Adrenal Cortex↗

Dose-response relation between plasma concentrations of corticotropin and cortisol after administration of incremental doses of cosyntropin for corticotropin stimulation testing in cats.

We compared the plasma cortisol and immunoreactive corticotropin (IR-ACTH) responses to incremental doses (1.25, 12.5 and 125 micrograms) of synthetic ACTH (cosyntropin) administered IV to 6 clinically normal cats. Mean plasma cortisol concentration increased significantly (P < 0.0001) after administration of all 3 doses of cosyntropin. After administration of the 1.25- and 12.5-microgram doses, plasma cortisol concentration peaked at 30 minutes, then decreased to values not significantly different from baseline concentration by 90 and 120 minutes, respectively. In contrast, after administration of the 125-micrograms dose, mean cortisol concentration peaked at 60 minutes and remained significantly (P < 0.05) higher than baseline values at 120 minutes. Compared with the 1.25- and 12.5-micrograms doses, administration of the 125-micrograms dose of cosyntropin induced significantly (P < 0.05) higher cortisol responses at 60, 90, and 120 minutes. Although individual cat's peak plasma cortisol concentration after administration of the 125-micrograms dose was higher than the peak value determined after administration of the 2 lower doses of cosyntropin, these differences were not statistically significant. Mean plasma IR-ACTH concentration increased significantly (P < 0.0001) and reached a maximal value at 30 minutes after administration of all 3 doses of cosyntropin. After administration of the 1.25- and 12.5-micrograms doses, plasma IR-ACTH concentration decreased to values not significantly different from baseline concentration by 60 and 120 minutes, respectively, whereas mean IR-ACTH concentration remained significantly (P < 0.05) higher than baseline values 120 minutes after administration of the 125-micrograms dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗

Effects of continuous infusion of interleukin 1 beta on corticotropin-releasing hormone (CRH), CRH receptors, proopiomelanocortin gene expression and secretion of corticotropin, beta-endorphin and corticosterone.

A number of recent studies suggest that interleukin 1 beta (IL-1 beta) is a major mediator of hypothalamo-pituitary-adrenal (HPA) responses following infectious aggression. We investigated whether IL-1 beta mediates long-term changes in HPA activity and studied the cellular regulation of the anterior pituitary. To mimic chronically elevated IL-1 beta production thought to occur during infectious diseases, osmotic pumps (Alzet type) were implanted in the peritoneal cavity of male rats and hIL-1 beta was infused continuously at rates of 1 or 3 micrograms/day. Effects of hIL-1 beta action on plasma ACTH, beta-endorphin (beta-EP) and corticosterone (CORT) secretion and on anterior pituitary (AP), ACTH and beta-EP content were followed. In addition, hypothalamic (HT) CRH mRNA and in AP, CRH receptor (CRH-Rc) mRNA, POMC nuclear primary transcript RNA, POMC nuclear intermediate processing RNA and POMC nuclear and cytoplasmic mRNA were quantified using a highly sensitive solution hybridization nuclease protection assay. Continuous infusion of hIL-1 beta stimulated the HPA axis at varying degrees. Increased HT CRH gene expression, AP POMC gene transcription, ACTH and beta-EP release occurred only during the first 3 days of the treatment. A long-lasting enhancement of ACTH and beta-EP synthesis and of POMC gene expression resulted from activated POMC gene transcription followed by an increased POMC mRNA stability and decreased POMC mRNA turnover. In the AP, stimulation of ACTH and beta-EP secretion and POMC gene transcription disappeared after continuous IL-1 beta treatment, possibly in part due to a refractory process mediated by decreased CRH-Rc gene expression in corticotropes.

Adrenocorticotropic Hormone↗

Interaction of vasoactive intestinal peptide (VIP) and growth hormone releasing factor (GRF) with corticotropin releasing factor (CRF) on corticotropin secretion in vitro.

The possible interaction of VIP and GRF with CRF on ACTH release was examined by using superfused rat anterior pituitary fragments. ACTH was measured in effluent medium samples by RIA. Increasing concentrations of VIP and GRF (10(-8) to 10(-6)M) had no effect on spontaneous ACTH secretion but potentiated CRF (10(-10)M) induced ACTH release in a dose dependent manner. The high concentrations of VIP or GRF required to produce the effect suggest that both the peptides could interfere with a common unidentified binding site on corticotrophs or stimulate a second messenger by a paracrine action.

Adrenocorticotropic Hormone↗

Regulation of bovine adrenal cell corticotropin receptor mRNA levels by corticotropin (ACTH) and angiotensin-II (A-II).

Bovine adrenal fasciculata-reticularis cells (BAC) expressed at least four ACTH receptor (ACTH-R) mRNA transcripts, one major of 3.6 kb and three minor of 4.2, 1.8 and 1.3 kb. ACTH and A-II increased ACTH-R mRNA levels in a time- and dose-dependent manner. At maximal concentrations, ACTH caused a 2.7-fold increase in the level of the major transcript of 3.6 kb with an ED50 = 10(-11) M and A-II produced a 2.4-fold increase with an ED50 = 5 x 10(-8) M. Under our experimental conditions, the stimulatory effects of both hormones appeared to be due to post-transcriptional changes rather than to transcriptional regulation since the hormonal effects were also observed in actinomycin-treated cells. The results indicate that regulation of ACTH-R mRNA levels may be one mechanism by which ACTH and A-II regulate adrenocortical functions.

Adrenocorticotropic Hormone↗

Regulation of corticotropin receptor number and messenger RNA in cultured human adrenocortical cells by corticotropin and angiotensin II.

The regulation of ACTH receptor binding sites and mRNA by ACTH and angiotensin II (A-II) was studied using cultured human adrenal fasciculata reticularis cells (HAC). These cells expressed two major ACTH receptor transcripts of 1.8 and 3.4 kb and three minor ones of 4, 7, and 11 kb. ACTH increased the levels of all these transcripts in a time- and dose-dependent manner. At a maximal concentration of 10(-8) M, ACTH enhanced 21- and 4-fold the level of ACTH receptor mRNA and the number of receptors per cell, respectively. Pretreatment of HAC with A-II produced a dose-dependent enhancement of ACTH receptor mRNA that was associated with an increase of both ACTH receptor number and responsiveness to this hormone. The effects of A-II were completely blocked by an AT1 receptor subtype antagonist but not by an AT2 antagonist. The effects of ACTH together with A-II on ACTH receptor mRNA were greater than those induced by each hormone alone. These results show that ACTH receptor number and mRNA are positively regulated by the two main hormones (ACTH and A-II) which, in vivo, regulate adrenocortical functions. In addition, they also show that HAC are a target for A-II. Thus, regulation of ACTH receptors may be one mechanism by which ACTH and A-II regulate adrenocortical functions under both normal and pathological conditions.

Adrenal Cortex↗

Role of endogenous arginine vasopressin in potentiating corticotropin-releasing hormone-stimulated corticotropin secretion in man.

Exogenously administered vasopressin (VP) augments ACTH secretion stimulated by CRH. This study was performed to elucidate the role of endogenous VP in potentiating CRH-induced ACTH secretion in man. Synthetic human CRH (100 micrograms) was injected iv into seven normal men after they had been water loaded (20 mL/kg; 60 and 30 min before CRH injection; WL-CRH test) and water deprived (water restriction for 18 h before CRH injection; WD-CRH test). Blood samples were obtained before and 5, 15, 30, 60, 90, and 120 min after CRH injection at 0900 h for determination of plasma ACTH, cortisol, arginine vasopressin (AVP), CRH, and catecholamine levels and osmolality. Urine was obtained immediately before and 120 min after CRH injection for determination of osmolality. The mean plasma AVP levels were significantly higher during the WD-CRH test [1.8 +/- 0.4 (+/- SE) to 1.9 +/- 0.4 pmol/L] than during the WL-CRH test (0.6 +/- 0.1 to 0.9 +/- 0.1 pmol/L). The mean plasma ACTH and cortisol levels rose significantly from basal (4.5 +/- 0.6 pmol/L and 320 +/- 20 nmol/L, respectively) to peak values of 14.0 +/- 2.1 pmol/L at 30 min and 700 +/- 50 nmol/L at 60 min, respectively, during the WD-CRH test. During the WL-CRH test, mean basal plasma ACTH and cortisol levels were 3.5 +/- 0.7 pmol/L and 420 +/- 50 nmol/L, respectively, and reached peak values of 7.7 +/- 1.1 pmol/L at 60 min and 550 +/- 40 nmol/L at 30 min, respectively. Both the mean peak levels and integrated ACTH and cortisol responses were significantly higher during the WD-CRH than during the WL-CRH test. There was no significant difference between the plasma CRH and catecholamine concentrations in both tests. These results suggest that endogenous AVP potentiates CRH-stimulated ACTH secretion and, thus, plays a physiologically significant role in regulating CRH-stimulated ACTH and cortisol secretion in man.

Adrenocorticotropic Hormone↗

Inhibition of basal and corticotropin-releasing hormone-stimulated adenylate cyclase activity and cytosolic Ca2+ levels by somatostatin in human corticotropin-secreting pituitary adenomas.

The effects of CRH and somatostatin (SRIH) on adenylate cyclase (AC) activity, intracellular free calcium concentrations [( Ca2+]i) and in vitro ACTH release were investigated in six human ACTH-secreting pituitary adenomas. In all tumors, CRH induced a marked stimulation (from 69-210% at 10 nM), whereas SRIH caused a definite inhibition (from 29-50% at 100 nM) of membrane AC. When added together, CRH and SRIH caused a purely additive effect on AC. In adenomatous corticotrophs CRH (10 nM) caused [Ca2+]i to rise from 160 +/- 30 nM (mean +/- SD) to 410 +/- 95 nM. CRH-induced transients were biphasic, with an initial peak predominantly due to redistribution from intracellular Ca2+ stores and a secondary phase due to Ca2+ influx. The effects of CRH on [Ca2+]i were totally independent of the stimulation of AC. In fact, cAMP-elevating agents other than CRH did not modify [Ca2+]i. SRIH (100 nM) decreased resting [Ca2+]i (approximately 20-40%) as well as [Ca2+]i rises induced by CRH, arginine vasopressin, or high K+. The effect of SRIH on [Ca2+]i was maintained in presence of high cAMP levels, while was totally abolished after pertussis toxin pretreatment. CRH (10 nM) stimulated ACTH release (from 22.5 +/- 3.5 to 45.0 +/- 8.5 pmol/L) by an extent similar to that elicited by calcium ionophore and forskolin. By contrast, SRIH (0.1 microM) inhibited both basal and CRH-stimulated ACTH release. In conclusion, in human adenomatous corticotrophs SRIH exerts an inhibitory action by reducing both AC activity and, independently, [Ca2+]i. In this way, SRIH can efficiently counteract the stimulatory action of CRH that in these cells involves activation of both cAMP and Ca2+ pathways.

Adenoma↗

Acutely raised corticotropin levels in Addison's disease are not associated with increased plasma arginine vasopressin and corticotropin-releasing factor concentrations in peripheral plasma.

Using an intensive sampling protocol we have examined the associations of peripheral plasma arginine vasopressin (AVP) and CRF with nocturnal ACTH hypersecretion in patients with Addison's disease. Six subjects were studied during a phase of cortisol withdrawal (36 h) and after cortisol substitution, and the findings were compared to those in four normal control subjects. The pulse properties of ACTH hypersecretion at a 10-min sampling interval have also been examined in these settings, using Cluster analysis. In the period before cortisol replacement, the Addison's patients showed significantly greater ACTH peak maxima and peak increments than the control subjects [107 +/- 44 vs. 5.5 +/- 1.3 pmol/L (P = 0.009) and 57 +/- 23 pmol/L vs. 3.7 +/- 0.9 pmol/L (P < 0.05), respectively]. After cortisol replacement, a significant decrease in mean peak increment (57 +/- 24 vs. 15 +/- 5 pmol/L; P = 0.021) occurred. The mean interpulse intervals did not differ significantly between the Addison's and control subjects (59 +/- 5 vs. 59 +/- 4 min overall). Although not significant, the trend for the pulse interval to increase after oral cortisol (60 +/- 6 vs. 72 +/- 9 min) is probably a result of the extremely low levels of ACTH after oral cortisol administration, making peak detection difficult. Despite the ACTH hypersecretion in the Addison's subjects, plasma AVP levels were at no time different from those in the control subjects. Plasma CRF levels tended to be lower in the Addison's patients than in the control subjects. We conclude that in states of cortisol deficiency, such as Addison's disease, ACTH hypersecretion results from enhanced ACTH peak amplitude, without a change in the frequency of ACTH secretory pulses. The marked increase in plasma ACTH that follows acute cortisol deprivation is independent of detectable changes in peripheral plasma levels of CRF or AVP.

Addison Disease↗