Coordination-chemistry study of polypeptides. III. protonation-deprotonation equilibrium study of synthetic alphaH -corticotropin1-32. Data on the pH-dependent conformation of corticotropin.

By potentiometric equilibrium measurements and the computer evaluation of experimental data, the protonation equilibrium constants of four fragments of corticotropin (ACTH), ACTH1-32. ACTH1-28, ACTH1-14 and ACTH1-4, were determined and assigned to the corresponding functional groups. From the dependence of the protonation constants on the length of the peptide chain, it was established which functional groups participate in the formation of intramolecular hydrogen-bonds in aqueous solutions at various pH. These results indicated a pH-dependent conformation of the molecule.     

Ability of corticotropin releasing hormone to stimulate cortisol secretion independent from pituitary adrenocorticotropin

Cortisol secretion by the adrenal cortex is thought to depend upon a preceding release of pituitary ACTH. This concept ignores a large number of observations suggesting important extrapituitary influences on adrenocortical function. The present study was designed to demonstrate the contribution of these extrapituitary mechanisms in the release of cortisol induced by human corticotropin releasing hormone (hCRH) in man. In patients with proven deficiency in pituitary ACTH the functional atrophy of the adrenals had been restored by pretreatment with long-acting ACTH. Fifty-eight hours after the second and last injection of ACTH a CRH test was performed (100 micrograms hCRH intravenously). Administration of hCRH induced a small but significant increase in plasma cortisol. Surprisingly, this rise was preceded by an increase in plasma ACTH similar to the ACTH response observed in the control group. It appeared that hCRH is able to stimulate cortisol release in the absence of pituitary ACTH, presumably by stimulating extrapituitary sources of ACTH.     

Differential expression of functional adrenocorticotropic hormone receptors by subpopulations of lymphocytes

In an effort to investigate the presence of adrenocorticotropic hormone (ACTH) receptors on rat lymphocytes, cells were separated by a panning procedure into T and B cell populations. By using the radiolabeled ACTH agonist, (125I-Tyr23) phenylalanine2-norleucine4-ACTH1-24, substantial numbers of ACTH binding sites were detected on T and B lymphocytes, but not on thymocytes. Scatchard analysis revealed two types of binding sites on each cell population, one with Kd1 = 0.088 +/- 0.025 nM and one with Kd2 = 4.2 +/- 0.6 nM; however, the absolute number of binding sites per cell was different. B lymphocytes expressed approximately three times the number of Kd1 binding sites per cell when compared with T lymphocytes. However, ACTH receptor expression by these cell populations was not static as suggested by the ability to induce receptor expression via mitogens. B or T cells and thymocytes stimulated with the mitogens LPS or Con A, respectively, substantially increased their number of Kd1 binding sites per cell (approximately three-fold). Even more dramatic increases in Kd1 receptor expression (approximately 100-fold) were observed when comparing "normal" and stimulated thymocytes. To demonstrate that these ACTH binding sites were in fact functional, cAMP levels were measured in lymphocytes 10 min after exposure to varying concentrations of ACTH. Dose-dependent increases in cAMP levels were observed, with significant stimulation occurring with as little as 0.1 nM ACTH added. Taken together, these studies demonstrate the presence of functional ACTH receptors on normal, rat T and B lymphocytes.     

Effect an ACTH analog on the processes of learning and memory in rats

On the basis of the idea of the important role of neurotransmitter systems in realization of neuropeptide effects, the participation was studied of the monoaminergic systems in the mechanisms of the ACTH analogue influence on the processes of learning and memory in control animals and animals with a changed functional state of the monoaminergic systems. In parallel the influence was studied of the ACTH analogue on the content of the endogenic monoamines in various brain structures of rats. It has been shown that administration of the ACTH analogue in a dose of 10 mcg affects the elaboration and preservation of conditioned reflexes (CRs) of passive avoidance, CRs of two-side avoidance and labyrinth CRs only in conditions of changed functional state of the monoaminergic systems. Amnesia, usually elicited by 5-oxytryptophane and disulfiram is prevented by administration of the ACTH analogue. Administration of the ACTH analogue is accompanied by the intensification of serotonine metabolism in the midbrain and medulla and by an increase of noradrenaline content in the hypothlamus.     

Functional compartmentalization of arginine-vasopressin-activated cyclic AMP in anterior pituitary gland: the presence of a compartment activated by prostaglandin E2

AVP(10(-8)-10(-6)M) increased ACTH as well as PGE2 release from rat anterior pituitary quarters in vitro in a concentration dependent manner. IBMX (0.1 mM), a phosphodiesterase inhibitor, increased the ACTH response to AVP. The cAMP content in pituitary tissue was increased by AVP. Cyclooxygenase inhibition by indomethacin(1.4 X 10(-5) M) or diclofenac (1.8 X 10(-5)M) led to a potentiation of AVP-evoked ACTH secretion and to a decrease in AVP-stimulated cAMP formation. PGE2(10(-6)M) significantly increased pituitary cAMP content and indomethacin did not affect cAMP levels activated by PGE2. PGE2 attenuated the AVP-induced ACTH release. These results indicate that at least two functional compartments of AVP-activated cAMP responses are involved in the AVP-induced ACTH release. One compartment is directly activated by AVP and participates in the propagation of AVP-induced ACTH release. The second compartment is activated by PGE2. The contribution of the second compartment to the regulation of ACTH secretion is not well understood since PGE2 shows an inhibitory effect on AVP-induced ACTH secretion.     

Allometric relations of total volumes of prolactin cells and corticotropic cells to body length in the annual cyprinodont Cynolebias whitei: effects of environmental salinity,stress and ageing

Summary An analysis of the allometric relations of the total volumes occupied by prolactin (PRL) and corticotropic (ACTH) cells (PRL volume and ACTH volume, respectively) to body length and a study of the immunocytochemical staining intensity of PRL and ACTH cells were used to determine the differences in activity of PRL and ACTH cells in freshwater-reared and in saltwater-reared Cynolebias whitei during the entire lifespan of this annual cyprinodont fish. An inflection in the allometric relation of PRL volume to body length was observed in fish of one-week old. The relatively large PRL volume in younger fish may be related to PRL cell activity before hatching. No inflections were observed in the allometric relations of PRL volume and ACTH volume to body length at the onset of maturation and the onset of ageing, indicating that the increased pituitary growth in maturing and ageing C. whitei is not the result of changes in PRL or ACTH cells. The slope of the allometric relation of PRL volume to body length in freshwater-reared fish was significantly steeper than the slope in saltwater-reared fish. The PRL volume in adult freshwater-reared fish was eight times larger than that in saltwater-reared fish of the same length. The intensity of immunocytochemical staining of saltwater PRL cells was significantly reduced. These volumetric and staining differences correspond to the low functional demand put upon PRL cells in saltwater-adapted fish. In contrast, the slope of the allometric relation of ACTH volume to body length and the intensity of immunocytochemical staining of ACTH cells were similar in freshwater-reared and in saltwater-reared fish. It is concluded that the functional demand put upon ACTH cells is similar in freshwater-reared and saltwater-reared C. whitei; the involvement of ACTH cells in the osmoregulation of the fish in both environments is similar.     

Some functional aspects of canine corticotrophs

To examine the regulation and functional significance of canine pituitary pars intermedia corticotrophs, ACTH and cortisol responses to CRF were studied in healthy dogs before and after treatment with dexamethasone. In addition the effects of the dopamine agonist bromocriptine and the dopamine antagonist pimozide were investigated. In the latter two instances prolactin concentrations were also measured. Finally the pituitaries were studied immunocytochemically for ACTH and alpha-MSH. No response of ACTH or cortisol to bromocriptine was observed. Pimozide caused a slight rise in ACTH levels in some dogs. However, prolactin levels significantly decreased with bromocriptine and increased with pimozide. Injection of synthetic ovine CRF to dogs was followed by sharp increases in ACTH and cortisol values. These responses were obliterated by prior treatment with dexamethasone. In 1 of 4 dogs given dexamethasone before euthanasia, there were few pars distalis cells with ACTH(1-24) immunopositivity, although persistence of ACTH(1-24) reaction was noted within cells of the pars intermedia. The results indicate that none of the CRF-induced ACTH secretion in dogs is derived from pars intermedia corticotrophs. Dosages of bromocriptine and pimozide that clearly alter prolactin secretion do not consistently affect ACTH levels.     

ACTH accelerates recovery of neuromuscular function following crushing of peripheral nerve

Adrenocorticotropin (ACTH)-treated adrenalectomized rats subjected to crush denervation recover sensation and functional movement sooner than saline-treated rats. Axonal regeneration is accelerated, the number of large endplates and the frequency of preterminal branching are increased. ACTH has no effect on either intact or denervated muscles. The ameliorative action of ACTH during regeneration is apparently neurogenic and independent of corticoids.     

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.     

Phorbol ester mimics ACTH action in corticoadrenal cells stimulating steroidogenesis, blocking cell cycle, changing cell shape, and inducing c-fos proto-oncogene expression

Cells of the Y-1 corticoadrenal line are: (a) functional, (b) cell cycle-arrested by adrenocorticotropic hormone (ACTH), (c) tumorigenic, and (d) c-Ki-ras overexpressing. We here report that the phorbol ester phorbol 12-myristate 13-acetate (PMA) mimics all ACTH-specific effects in Y-1 cells, namely: (a) steroid-ogenesis stimulation, (b) cell cycle block, and (c) cell shape change. In addition, both ACTH and PMA caused a rapid and transient induction of the c-fos proto-oncogene while having no effect on c-Ki-ras mRNA steady state levels. Dibutyryl cAMP, known to elicit ACTH effects in Y-1 cells, was a poor inducer of the c-fos gene. PMA pretreatment rendered Y-1 cells unresponsive to ACTH. These results suggest that protein kinase C is likely to be involved in the mechanisms of action of ACTH.     

3-D structure of mitochondrial cristae in rat adrenal cortex varies after acute stimulation with ACTH and CRH

We attempted to determine whether acute treatment with adrenocorticotropin hormone (ACTH) and corticotropin releasing hormone (CRH) affects mitochondrial morphology, as evaluated by the HRSEM and osmium maceration methods. We quantified CRH and ACTH effects on HRSEM images in rat glomerulosa and fasciculata. After ACTH or CRH treatment, mitochondrial cristae increased the number of globular expansions, whereas mitochondrial volume decreased in glomerulosa. As the morphological variations reported may be linked to increased hormonal production, further studies using parallel measurements of circulating and tissue hormones are now in progress, and may aid in clarifying their functional significance.     

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.     

Structure Based Screening for Inhibitory Therapeutics of CTLA-4 Unveiled New Insights About Biology of ACTH

Although the biology of adrenocorticotropic hormone (ACTH) protein has already been scrutinized, some functional aspects of its biology are yet to be elucidated in the context of immunological disorders. In this regard, virtual screening of a compound library was performed against the structure of Cytotoxic T-Lymphocyte Associated Protein-4 (CTLA-4) (assessed both spatially and energetically) to discover novel biological functions for ACTH. The results of virtual screening and the MD simulation demonstrated that DB01284 has high binding energy along with proper interaction orientation against CTLA-4 (FG loop) by a clamp like structure. The employed methodology was checked using confirmatory control analyses. Intriguingly, DB01284 belongs to Tetracosactide (already prescribed protein drug for clinical conditions) which is the N-terminal region of ACTH. This is the first study to reveal that ACTH protein binds to the same amino acids of CTLA-4 (FG-loop) as B7 and anti-CTLA-4 antibody binds. In light of this finding, the molecular mechanism of ACTH function in patients suffering from Cushing’s Syndrom and the immunological bases for ACTH therapy of multiple sclerosis (MS) patients could be further delineated. Moreover, this finding suggests that ACTH could also act to block CTLA-4 in the context of anticancer immune check point blockade.

     

Absence of functional and structural homology of natural and recombinant human leukocyte interferon (IFN-α) with human α-ACTH and β-endorphin

A comparison of the amino acid sequence of one human recombinant IFN-α (IFLrA) with either human β-endorphin or ACTH reveals only a minimal and insignificant degree of homology. Also, synthetic ACTH, β-endorphin and β-endorphin-(1–15) have no antiviral protective effects on human fibroblasts and cannot inhibit the neutralization of the antiviral effects of natural IFN-α by an antiserum directed against the interferon. Anti ACTH and Anti β-endorphin do not neutralize the antiviral effects of IFLrA, and radioimmunoassays of partially purified natural IFN-α and pure IFLrA do not reveal any evidence of α-MSH or β-endorphin-like material in the interferons. These results demonstrate an absence of functional and structural homology of natural and recombinant IFN-α with ACTH and β-endorphin.     

Inhibitory effect of ouabain on epinephrine-induced stimulation of adrenal corticosterone secretion in rats.

Chronic administration of ouabain (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) definitely inhibited epinephrine-induced increase of adrenal corticosterone secretion. The inhibition rate increased along with frequency of ouabain administration. Increase in adrenal corticosterone synthesis and secretion by ACTH (20-50 mU/rat) administration was partially suppressed by pretreatment with chronic ouabain administration. A slight but significant increase of adrenal corticosterone secretion caused by epinephrine administration in hypophysectomized rats was also inhibited by pretreatment with ouabain administration. Chronic administration of neither phentolamine (1 mg/rat, intraperitoneally, once daily for consecutive 15 days) nor propranolol (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) caused significant changes in adrenal corticosterone secretion in response to ACTH as well as to epinephrine. Chronic administration of ouabain in rats causes not only elevated secretion of ACTH from anterior pituitary but also functional change in adrenals leading to suppression of corticosterone secretion in response to ACTH or epinephrine administration.     

The utility of antisera to different synthetic adrenocorticotrophins (ACTH) and melanotrophins (MSH) for immunocytochemical staining of the dog pituitary gland

Using the immunoperoxidase technique and specific antisera to synthetic ACTH beta (1-24), ACTH beta (17-39) and bMSHbeta1, selective immunocytochemical staining was localized in a distinctive cell type in the pars distalis and pars tuberalis of the dog pituitary gland. Except for a rare cell, the pars distalis and pars tuberalis did not stain with an anti-bMSH alpha serum. In the pars intermedia immunoreactive cells containing ACTH beta(1-24), ACTHbetap(17-39), bMSHbeta and/or bMSH alpha were observed. The specificity and validity of the antisera were demonstrated by elimination of their immunostaining capacity after prior absorption with their respective antigens, while absorption with other antigens failed to decrease staining intensity. The cytoplasm of the ACTH/MSH cells showed a positive reaction to periodic-acid-Schiff and assumed a pale aniline blue colour, whilst the granules were stained with carmoisine L and acid alizarine blue. These ACTH/MSH cells were further differentiated from other functional cell types of the pars distalis on the basis of their typical cytological features, intraglandular distribution and by immunochemical double staining. It is concluded that ACTH and MSH beta were present and most probably produced by the corticomelanotrophs of the pars distalis and pars tuberalis. In addition to corticomelanotrophs analogous to those of the pars distalis and pars tuberalis, the pars intermedia showed many cells which contain MSH alpha alone or together with MSH beta and/or ACTH.     

Pituitary-adrenocortical system in patients with Shy-Drager syndrome

We examined ACTH responses to the three main mechanisms of ACTH secretion, i.e., stress, negative feedback and circadian rhythm, in six patients with Shy-Drager syndrome and in six control subjects to determine whether or not injury to the central autonomic nervous system provokes some disturbances in ACTH secretion. The patients showed a poor cortisol response to the stress of insulin induced hypoglycemia along with a normal ACTH and urinary 17-OHCS response to metyrapone and a normal cortisol circadian rhythm. The discrepancy between the above-mentioned functional tests of ACTH secretion is rare to our knowledge. These findings suggested the existence of a glucoreceptor defect in such patients, or the possibility that the stress of insulin induced hypoglycemia stimulates ACTH secretion by way of the autonomic nervous system.     

Developmental aspects of the hypothalamic-pituitary-adrenal axis

The ovine fetal adrenal cortex and pituitary are functional secretory organs by the end of the first third of gestation (term is 142-152 days). By half-way through gestation the zona glomerulosa is mature morphologically, more than 80% of the aldosterone in fetal blood is of fetal adrenal origin, but conventional stimuli, for example, increased plasma K+ or angiotensin II, do not increase aldosterone secretion until near term. The zona fasciculata is immature histologically, relatively unresponsive to ACTH, and contributes less than 10% of the cortisol in fetal blood between 100 and 120 days of gestation. After this time the zona fasciculata cells begin to mature, to respond to ACTH and to produce an increasing proportion of the cortisol in fetal blood. A functional relationship between hypothalamus-pituitary-adrenal cortex matures over the last fifth of gestation. It is hypothesized that cortisol exerts a local effect in maturation of fetal zona fasciculata cells, such that low concentrations of ACTH have increasingly larger effects on growth and secretion of the fasciculata and that the level of negative feedback by cortisol on the hypothalamic-pituitary axis is reset. The analogy is drawn between the changes in gonadotrophin and gonadal hormones which culminates in puberty in man and the changes in ACTH and cortisol which culminate in parturition in sheep.