PRO-OPIOCORTIN RELATED PEPTIDES IN HUMAN PITUITARY AND ECTOPIC ACTH SECRETING TUMOURS

Basal and stimulated secretion of N-terminal pro-opiocortin (Pro-gamma-MSH), ACTH and LPH from seven pituitary and three ectopic ACTH secreting tumours have been studied in vitro using a perfused isolated cell system. The peptides were shown to be released concomitantly and in equimolar amounts. The pituitary tumours responded to stimulation with rat stalk median eminence extracts (SME) and synthetic AVP. However, peptide release from the ectopic tumours, although pulsatile, remained autonomous. Prior to surgery, gel-chromatographic profiles of plasma immunoreactive ACTH showed only one peak, which eluted in the position of 1-39 ACTH, in patients with the pituitary tumours, but there was a second peak of large molecular weight ACTH present in the plasma from those with the ectopic ACTH syndrome. This second form of ACTH could not be detected in any of the tumour cell column effluents. An eighth pituitary tumour was atypical, in its unusually large size, clinically aggressive nature and spectrum of peptide release. Although peptide release in response to stimulation with SME was similar to that observed with the other pituitary tumours, the chromatography of the plasma ACTH resembled the ectopic plasma pattern, showing two peaks of immunoreactivity.     

The effect of β-endorphin on basal and insulin-hypoglycaemia stimulated levels of hypothalamic-pituitary-adrenal axis hormones in normal human subjects

OBJECTIVE It has been demonstrated that β-endorphin reduces CRH production and hypoglycaemia-induced ACTH secretion in the rat. We aimed to determine whether supraphysiological levels of β-endorphin inhibit the ACTH and CRH response to insulin-induced hypoglycaemia in human subjects. DESIGN Plasma glucose, prolactin, cortisol, ACTH, CRH and AVP were measured at intervals over a 3-hour period. Intravenous β-endorphin 5 mg/50 ml or an equal volume of normal saline was infused between 30 and 90 minutes, with soluble insulin 0.15 units/kg administered i.v. at 60 minutes in a cross-over design. SUBJECTS Six healthy male volunteers aged 20–35 years. MEASUREMENTS Prolactin was measured by a fluoroimmunometric assay, ACTH, CRH and AVP by radioimmunoassay, and cortisol was measured by enzyme-linked immunosorbent assay. Haemodynamic measurements were recorded prior to each blood sample. Results are expressed as mean ± standard error of the mean. RESULTS β-Endorphin resulted in a significant decrease in baseline cortisol (P < 0.05) but not ACTH. Plasma glucose (P < 0.001) and CRH (P < 0.05) and PRL (P < 0.05) increased significantly during β-endorphin compared to normal saline. After insulin administration, glucose reached a similar nadir during β-endorphin and normal saline (2.1 ± 0.1 and 1.9 ± 0.15 mmol/l, respectively) but the fall in plasma glucose was delayed during β-endorphin (P < 0.01 by ANOVA). This resulted in a significantly altered time-course for the ACTH and cortisol responses (P < 0.05 for each), but no difference overall in the magnitude of the response. In contrast, neither the timing nor the magnitude of the CRH and AVP responses were affected. Prolactin also reached a similar peak value after the administration of insulin, while the haemodynamic responses to hypoglycaemia were not significantly altered during β-endorphin. CONCLUSIONS While β-endorphin has been shown to be inhibitory to basal ACTH and cortisol secretion in humans, we note a significant increase in plasma CRH in response to β-endorphin, which may be arising from a peripheral source. Intravenous β-endorphin increases plasma glucose and delays the onset of hypoglycaemia following insulin but does not result in significant inhibition of the ACTH and cortisol response. This may reflect the poor penetration of β-endorphin into the central nervous system, although a hypothalamic effect of β-endorphin is implied by the increased PRL. The significantly delayed time course in ACTH and cortisol secretion noted during β-endorphin is not explained by a later response of either CRH or AVP. Although peripheral levels of these hormones may be a relatively insensitive measure of hypothalamic function, an additional factor may influence ACTH release during hypoglycaemia.     

Cushing's Syndrome Due to an ACTH-producing Neuroendocrine Tumour in the Nasal Roof

ABSTRACT A patient with ectopic adrenocorticotrophic hormone (ACTH) production from a neuroendocrine tumour of the nasal roof is presented. By indirect immunoperoxidase techniques the tumour cells were shown to be distinctly positive for ACTH and β-endorphin but negative for other peptides derived from pro-opiomelanocortin. Neither corticotropin releasing hormone (CRF) found in some tumours associated with ectopic Cushing's syndrome, nor gastrin immunoreactivity, which coexists with ACTH in normal rat pituitary and in rat and human gastrointestinal cells, were demonstrable in the tumour. A review of other, previously recognized locations of CRF/ACTH producing tumours is given to increase the awareness of the ectopic Cushing's syndrome, which may lack the classical features and is characterized by fulminant clinical course, extreme fatigue, weakness, pale facial swelling, oedema and hypokalaemic alkalosis.     

The evolution of vertebrate corticotrophin and melanocyte stimulating hormone.

ACTH and β-LPH appear to be synthesised in both the pars distalis and the pars intermedia. In the pars distalis they are secreted intact. In the pars intermedia ACTH acts as the precursor of α-MSH and CLIP, and β-LPH acts as the precursor for γ-LPH and β-MSH. α-MSH, CLIP and β-MSH do not exist in significant amounts in the human pituitary. α-MSH, β-MSH, ACTH, and CLIP have been characterised in the elasmobranch pituitary. Chicken ACTH has been isolated.     

Arginine vasopressin is a much more potent stimulus to ACTH release from ovine anterior pituitary cells than ovine corticotropin-releasing factor. 1. In vitro studies

Cultured rat and ovine anterior pituitary cells were treated with a range of doses (0.01-1,000 nM) of arginine vasopressin (AVP) and ovine corticotropin-releasing factor (CRF), alone or in combination, and medium and cell content of immunoreactive (ir-)ACTH determined. In rat cells, a dose-response curve to CRF was obtained, with a threshold dose of 0.1 nM; AVP was much less effective alone, but augmented CRF responses when administered with CRF. In ovine pituitary cells AVP markedly stimulated ACTH release in a dose-dependent fashion, and with a threshold of 0.1 nM; in contrast, CRF increased ACTH release over basal only at doses greater than 100 nM. In combination, subthreshold doses of AVP potentiated rat pituitary cell responses to CRF; addition of 1 nM of AVP to varying doses of CRF was more effective in terms of ACTH release than addition of 1 nM of CRF to increasing doses of AVP. In contrast, in ovine cells the addition of 1 nM CRF to increasing doses of AVP elicited a larger ACTH response than the addition of 1 nM AVP to increasing doses of CRF. Dexamethasone pretreatment (5 nM) for 48 h significantly decreased CRF potentiation of AVP-stimulated ACTH release in ovine cells. These studies confirm that CRF is a more potent stimulus of ACTH release than AVP in the rat, and establish that in contrast AVP is a much more potent stimulus of ACTH secretion than CRF in isolated ovine pituitary cells.     

Longitudinal evaluation of adrenocorticotrophin and β-lipotrophin plasma levels following bilateral adrenalectomy in patients with Cushing's disease

OBJECTIVES Bilateral adrenalectomy may be indicated in patients with Cushing's disease in whom hypercortlsolism is not resolved after pituitary microsurgery. However, Nelson's syndrome is a major long-term complication of such therapy. We have carried out a longitudinal study on patients with Cushing's disease who underwent bilateral adrenalectomy comparing plasma /Mipotrophin (β-LPH) with ACTH levels. PATIENTS AND METHODS Seven patients unsuccessfully treated by pituitary surgery for Cushing's disease underwent bilateral adrenalectomy. Blood samples were collected on days 8 and 15 and at 1, 2, 3, 6, 9,12,18 and 24 months after adrenalectomy in all patients. Five patients were followed up for the longer periods of 30,33,39,72 and 84 months respectively. Plasma ACTH and β-LPH were determined by RIA after extraction. Pituitary CT scan was done at 6-8-month intervals. RESULTS A pituitary tumour was detected in three patients at 14, 24 and 26 months after adrenalectomy respectively. The basal ACTH (range 8-21 pmol/l) began to rise between 15 and 30 days and exhibited a sharp increase with a range of 36-114 pmol/l at 3 months and a range of 53-187 pmol/l at 6-9 months after adrenalectomy. The ACTH levels in the three patients who later presented with a pituitary tumour were indistinguishable from those observed in the other patients up to 12 months after adrenalectomy. Only at 12 months or thereafter were their ACTH levels higher than in the other patients (958 ± 252 vs 205 ± 22 pmol/l). β-LPH concentrations changed in parallel with ACTH levels. The ACTH levels correlated positively with β-LPH levels (r= 0 76). CONCLUSIONS In patients with Cushing's disease undergoing bilateral adrenalectomy, plasma ACTH and β-LPH concentrations cannot predict the development of pituitary tumours until 12 months after surgery.     

Immunological characterization of β-lipotropin fragments (endorphin, β-MSH,and N-fragment) from fish pituitaries

Radioimmunoassay (RIA) for each of the three segments of the β-lipotropin molecule (N-fragment, β-MSH, and β-endorphin) are described. The pituitaries of one elasmobranch and two teleost species contain the three segments in the following order of abundance: β-endorphin > β-MSH > N-fragment. By gel filtration we have shown that the majority of the β-MSH and β-endorphin immunoreactivity is accounted for by the free peptides, not by larger prohormones. Only very small amounts of immunoreactive material the size of β- or γ-LPH were found.     

OVINE CORTICOTROPHIN RELEASING FACTOR STIMULATES ACTH RELEASE FROM HUMAN CORTICOTROPHINOMA CELLS IN CULTURE; INTERACTION WITH HYDROCORTISONE AND ARGININE VASOPRESSIN

We have investigated the effects of ovine corticotrophin releasing factor (oCRF) and its interaction with hydrocortisone (HC), and arginine vasopressin (AVP) on ACTH release from human corticotrophinoma cells in culture. Tumour tissue was obtained from six patients (three with active Cushing's disease and three with Nelson's syndrome). Cultures were maintained for periods of up to six months. Ovine CRF (21 nmol) significantly (P less than 0.01) stimulated ACTH release from all tumours. Dose response (21 pmol-21 nmol) effects were observed for the three tumours investigated over 2 and 4 h. Cortisol (20 mumol) significantly (P less than 0.01) inhibited basal ACTH release from one tumour (Nelson's syndrome) by 75% over 4 h, and completely prevented the stimulatory effects of oCRF. AVP directly stimulated ACTH release from two tumours (Nelson's syndrome), and also potentiated the stimulatory action of oCRF during 30 min incubations. These data show corticotrophinoma cells from subjects with Cushing's disease and Nelson's syndrome can be directly stimulated by hypothalamic oCRF and may be potentiated by AVP. Cortisol and oCRF have been shown in one tumour to have antagonistic actions at the pituitary level.     

STUDIES ON CIRCULATING MET-ENKEPHALIN AND β-ENDORPHIN: NORMAL SUBJECTS AND PATIENTS WITH RENAL AND ADRENAL DISEASE

Studies were performed to define the responses of plasma met-enkephalin to various endocrine and pathological stimuli and to determine the relationship between plasma β-endorphin and met-enkephalin. During insulin-induced hypoglycaemia ACTH, β-LPH and β-endorphin immunoreactivity rose in parallel, but plasma met-enkephalin did not change significantly. Sephadex G75 chromatography of samples taken at the time of the peak response (45 min) confirmed the rise in both β-LPH and β-endorphin. During administration of dexamethasone, 0·5mg 6 hourly for 48 h, plasma cortisol and ACTH became undetectable at 24 h, and β-LPH and β-endorphin fell significantly by 24 h and were undetectable by 48 h; plasma met-enkephalin, however, showed no significant change. Nine adrenalectomized patients with Cushing's disease and four patients with Addison's disease had elevated plasma ACTH, β-LPH and β-endorphin but normal plasma met-enkephalin levels. Each of ten patients with renal failure had markedly elevated plasma met-enkephalin immunoreactivity which co-eluted with synthetic met-enkephalin on BioGel P4 chromatography. Trypsin and carboxypeptidase-B digestion of the P4 chromatography fractions generated met-enkephalin immunoreactivity in earlier fractions, indicating the presence of a potential high molecular weight met-enkephalin precursor. The results imply that different control mechanisms govern β-endorphin and met-enkephalin plasma levels and that the adrenal is not the only source of plasma met-enkephalin. The elevated levels of met-enkephalin in renal failure may be due to impaired clearance of met-enkephalin or of its precursor or to increased met-enkephalin production.     

Immunological characterization of endorphins, adrenocorticotropin, and melanotropins in frog hypothalamus

To gain more information about the nature and regulation of opiomelanocorticotropic peptides in the frog diencephalon, radioimmunological determinations of α- and β-MSH, ACTH, β- and γ-endorphins have been performed in hypothalamic extracts. Sephadex G-50 gel filtration revealed a single peak of α-MSH-like immunoreactivity (α-MSH-LI) comigrating with synthetis α-MSH. Two peaks of ACTH-LI were observed; the major one eluting slightly before human ACTH. Using a porcine β-endorphin antiserum which exhibited 45% cross-reaction (on a molar basis) with ovine β-LPH, one major peak of β-endorphin-LI and two additional components were observed in the elution profile; none of these peaks coeluted with synthetic porcine β-endorphin. No significant β-MSH or γ-endorphin-LI was detected. To investigate a possible role of glucocorticoids on the level of opiomelanocorticotropic peptides in frog hypothalamus, 60 male frogs were treated with dexamethasone (300 μg/day) during 8 days. Dexamethasone treatment did not modify the chromatographic distribution and the total amount of α-MSH-LI and ACTH-LI in hypothalamic extracts. A slight (15%) increase in β-endorphin-LI was even observed after 8 days of dexamethasone treatment. From these results it is concluded that three classes of opiomelanocorticotropic peptides are present in the frog hypothalamus in the following order of concentration: β-endorphin-LI > α-MSH ? ACTH. In addition, circulating glucocorticoids which significantly reduce the concentration of opiomelanocorticotropic hormones in the distal lobe of the frog pituitary (S. Jégou, M. C. Tonon, F. Leboulenger, C. Delarue, J. Côté, G. Pelletier, and H. Vaudry (1981a). In “Adv. Physiol. Sci.” E. Stark, G. B. Makara, Zs. Acs, and E. Endröczi, eds., Vol. 13, pp. 129–133. Pergamon, Budapest.) do not modify the amount of these peptides in the hypothalamus.     

In-vitro effects of corticosterone, synthetic ovine corticotrophin releasing factor and arginine vasopressin on the release of adrenocorticotrophin by fetal rat pituitary glands

The in-vitro release of ACTH by fetal rat pituitary glands on days 17, 19 and 21 of pregnancy was measured using radioimmunoassay. The spontaneous release of ACTH, expressed in pg ACTH/gland per h, increased with fetal age, in correlation with the sharp rise in pituitary ACTH content. However, since pituitary ACTH content was nearly sevenfold higher at term than on day 17, while basal release of ACTH was only threefold higher, one can speculate that the spontaneous release of ACTH was proportionally greater on day 17 than on day 21 of gestation. As corticosterone, at a physiological concentration (865 nmol/l), reduced ACTH release, it was concluded that the pituitary gland was one site of the negative feedback action of the corticosteroids during fetal life. Quantities of synthetic ovine corticotrophin releasing factor (CRF) which gave concentrations of 0.3-30 nmol/l in the incubation medium induced a sharp rise in ACTH release which was log-dose dependent between 0.3 and 3 nmolCRF /1 on day 17 and between 0.3 and 30 nmolCRF /1 on days 19 and 21. The response to CRF increased with fetal age. Quantities of arginine vasopressin (AVP) which gave concentrations of 2-200 nmol/l stimulated ACTH release at all stages of gestation investigated. However, the response to AVP was much lower than that to CRF. Potentiation of CRF-induced ACTH release was not observed when whole pituitary glands from 21-day-old fetuses were incubated with AVP (20 nmol/1) + CRF ( 3nmol /1). Such results were correlated with the ontogenesis of immunoreactive vasopressin- and CRF-containing fibres in the median eminence of the rat fetus, as well as with the CRF-like immunoreactivity present in adult rat pituitary portal plasma and the AVP content of the fetal rat hypophysis.     

Effects of synthetic ovine corticotropin-releasing factor (CRF) on ACTH release in vitro

The effect of the synthetic ovine corticotropin-releasing factor (CRF) on adrenocorticotropin (ACTH) release was examined by the perifusion method using rat anterior pituitary tissue and rat monolayer cultured pituitary cells. Quartered anterior pituitaries were placed in a chamber and perifused at a rate of 400 microliters/min with Dulbecco's modified Eagle Medium (DMEM, pH 7.4) bubbled with a mixture of 95% O2 and 5% CO2. The perifused medium was fractionated, and the ACTH concentration was measured by radioimmunoassay. In the monolayer cultured pituitary cells, the amount of ACTH released in the culture medium during three hours incubation was assayed by radioimmunoassay. ACTH was released from the perifused anterior pituitary in a dose-related manner by the pulse administration of CRF or arginine vasopressin (AVP) at the concentration of 1 ng/ml, 10 ng/ml and 100 ng/ml. A significant difference was not found between CRF- and AVP-induced ACTH release. In the monolayer cultured pituitary cells, synthetic ovine CRF induced ACTH release in a dose-related manner between 30 pg/ml and 30 ng/ml, but AVP induced a slight ACTH release. ACTH release was pulsatile during the continuous administration of 2.5 ng/ml of CRF for 150 min, although if gradually increased during the continuous administration of 10 ng/ml or 20 ng/ml of CRF. The continuous administration of AVP also caused pulsatile ACTH release at 10 ng/ml, but the ACTH release gradually decreased during the continuous administration of AVP. The interaction between CRF and AVP on ACTH release was examined by two methods. When CRF and AVP were given simultaneously, a mainly additive effect on ACTH release was observed. However, a low concentration of CRF seemed to potentiate AVP-induced ACTH release. These results show that both CRF and AVP have a significant CRF activity on the perifusion system, that AVP induced a slight ACTH release in monolayer cultured pituitary cells, and that CRF acts additively or potently with AVP to control the ACTH release from the anterior pituitary gland.     

Corticotropin and beta-endorphin-like materials are native to unicellular organisms

Multiple molecular forms of immunoreactive corticotropin (ACTH) and β-endorphin were present in extracts of a unicellular eukaryote (Tetrahymena pyriformis). One form of immunoreactive ACTH reacted similarly with two different ACTH antisera (one specific for the 11-24 sequence and the other with determinants within sequences 1-14 and 17-39) and migrated with synthetic hACTH-(1-39) in a gel filtration system. This form also exhibited ACTH bioactivity in a dispersed rat adrenal cell bioassay system, with a mean immunoassay/bioassay ratio of 1.5. Gel filtration revealed multiple size classes of immunoreactive β-endorphin; a major peak of radioreceptor activity was detected which exhibited a K_av similar to that of authentic β-endorphin. A major portion of immunoreactive β-endorphin-sized material exhibited retention times similar to those of synthetic human and camel β-endorphin upon reverse-phase high-pressure liquid chromatography. These distinctive properties and specificities would seem to exclude the presence of limited homologies with sequences present in other proteins. High molecular weight material containing both ACTH and β-endorphin antigenic determinants was also demonstrated, suggesting, but not proving, the presence of a common precursor molecule.     

Immunoreactive endorphins, lipotropins and corticotropins in a human nonpituitary tumor: evidence for a common precursor.

The immunoreactive (RIA), ACTH, LPH, alpha endorphin (alpha End) and beta End in an extract of a human pancreatic islet cell carcinoma causing the ectopic ACTH syndrome were assayed after gel exclusion chromatography. In addition to "big", "intermediate" and "little" ACTHs, beta lipotropin (beta LPH) and gamma LPH, the eluate fractions contained RIA-alpha End and -beta End. The major RIA-beta End component appeared to be h beta LPH, which has beta End as its carboxy-terminus, but significant concentrations of a component that coeluted with synthetic p beta End were also found. Small amounts of RIA-alpha End were found in two peaks, one that may have represented 1-76 h beta LPH, with alpha End as its carboxy-terminus, and one probably representing alpha End itself. RIA-ACTH, -LPH and -beta End were found in the void volume fractions. Thus, a human nonpituitary tumor that produced ACTH and LPHs also produced endorphins, all perhaps deriving from a common precursor.     

Corticotropin releasing factor (CRF): effects on the release of pro-opiomelanocortin (POMC)-related peptides by human anterior pituitary cells in vitro

The effect of synthetic ovine corticotropin-releasing factor (oCRF), alpha-melanotropin (alpha-MSH) and arginine-vasopressin (AVP) on the release of pro-opiomelanocortin (POMC)-related peptides (beta-endorphin, beta-lipotropin, adrenocorticotropin, and the 12K N-terminal) was studied in primary cultures of human anterior pituitary (HAP) cells. The peptides released into the medium were measured by specific radioimmunoassays. HAP cells responded to oCRF at concentrations as low as 10 ng/ml by significantly increasing peptide secretion (p less than 0.005). AVP was also effective, but, it is only 0.1% as potent as oCRF. alpha-MSH was ineffective even at a dose of 10 micrograms/ml. Immunoreactive (IR)-beta EP, IR-beta LPH, IR-ACTH, and IR-HNT were released in approximately equimolar ratios. Partial characterization of the peptides released by HAP cells with gel chromatography on Sephadex G-50 superfine revealed two peaks of IR-beta EP (as beta EP and beta-LPH), two peaks of IR-beta LPH (as beta-LPH and gamma-LPH), one peak of IR-ACTH, and two peaks of IR-HNT. In conclusion, oCRF is a potent secretagogue for the equimolar release of ACTH, HNT and related peptides in the human pituitary gland.     

A phaeochromocytoma presenting with Cushing's syndrome associated with increased concentrations of circulating corticotrophin-releasing factor

The case is described of a 61-year-old male who presented with hypertension and Cushing's syndrome which resolved on excision of a unilateral adrenal mass. Histology of the tumour revealed a benign phaeochromocytoma which immunostained for corticotrophin-releasing factor (CRF-41) but not for ACTH. Preoperative plasma concentrations of immunoreactive CRF-41 were increased, and gradients for both CRF-41 and ACTH were demonstrated across the tumour. Post-operatively, CRF-41 was undetectable in plasma. The tumour contained high concentrations of immunoreactive CRF-41 which co-eluted with synthetic human CRF-41 on reversed-phase high-performance liquid chromatography. Tumour CRF-41 stimulated the release of ACTH in a dose-dependent manner from isolated rat anterior pituitary cells. We conclude that this tumour secreted CRF-41 and ACTH and had the capacity to produce ACTH-dependent Cushing's syndrome directly by secreting ACTH and indirectly by secreting CRF-41 to stimulate ACTH secretion from the anterior pituitary.     

Substance P stimulates arginine vasopressin and inhibits adrenocorticotropin release in vivo in the rat.

We have investigated the central effects of substance P (SP) on plasma concentrations of immunoreactive ACTH and on immunoreactive and bioactive arginine vasopressin (AVP) in the rat. The injection of SP (20 nmol) into the lateral ventricle intracerebroventricular, (i.c.v.) of ethanol-anaesthetised rats produced a prolonged antidiuresis lasting at least 30 min, associated with an increase in plasma AVP (from 7.8 +/- 0.6 to 12.5 +/- 1.9 fmol/ml, mean +/- SEM, n = 6). Concentrations of plasma ACTH were significantly decreased 30 min following SP (from 320 +/- 70 to 135 +/- 15 fmol/ml, n = 12). In rats anaesthetised with urethane, a significant decrease in plasma ACTH was observed 15 and 30 min following i.c.v. injection of SP (20 nmol); a downward trend was also observed in ACTH following a 40 nmol dose, but this was not significant. No effect of SP was observed on either basal or CRF-41-stimulated ACTH release from isolated rat anterior pituitary cells in vitro. These results demonstrate for the first time that SP exerts opposite effects upon the release of ACTH and AVP in the same animal, and suggest that these actions occur at the level of the hypothalamus.     

Deciphering Posttranslational Processing Events in the Pituitary of a Neopterygian Fish: Cloning of a Gar Proopiomelanocortin cDNA

A cDNA that codes for the polypeptide hormone precursor proopiomelanocortin (POMC) was cloned and sequenced from a gar (Lepisosteus osseus) pituitary cDNA library. The gar POMC cDNA is 1237 bp and contains a 780-bp open reading frame. The deduced amino acid sequence for gar POMC is 259 amino acids in length. The general organization of gar POMC is very similar to that of other gnathostome POMC sequences. The β-endorphin sequence had 91% sequence identity with sockeye A β-endorphin and 71% sequence identity withXenopus laevisβ-endorphin. Three melanocyte-stimulating hormone (MSH) core sequences [HFR(W)] were detected. The gar α-MSH sequence was identical to the α-MSH sequence in rat POMC. The gar β-MSH sequence had 77% sequence identity with salmonid forms of β-MSH and 53% sequence identity with tetrapod forms of β-MSH. The γ-MSH region of gar POMC only had 26% primary sequence identity with tetrapod γ-MSH sequences. Gar γ-MSH had an incomplete MSH core sequence (HRF), an apparent internal deletion of five amino acids, and lacked flanking paired basic amino acids essential for proteolytic cleavage. The apparent degenerate nature of gar γ-MSH is discussed in light of the absence of this sequence in salmonid fish.     

Mechanism of restoration of ACTH release in rats with long-term lesions of the paraventricular nuclei

The effects of lesions in the paraventricular nucleus (PVN) on the adrenocortical response to ether stress were investigated in neurohypophysectomized and intact rats. During the first 4 days after placement of lesions in the PVN, the corticosterone response to ether stress was almost completely inhibited. It then gradually increased and, within 4-6 weeks of surgery, was restored to about 60% of that in sham-operated rats. Basal plasma concentrations of corticosterone were low in rats after placement of lesions in the PVN and/or after neurointermediate lobectomy (NILX). Corticosterone responses to ether stress were similar in groups submitted to PVN lesions and/or NILX, and lower than those in the appropriate sham-operated groups. In all lesioned groups, plasma ACTH concentrations after a combination of stressors (ether plus laparotomy) were also lower than those in the sham-operated groups. Six weeks after lesioning of the PVN, immunoreactive rat corticotrophin-releasing factor-41 (rCRF-41) concentrations in stalk-median eminence (SME) extract fell to about 5% of that in sham-operated rats, while immunoreactive arginine vasopressin (AVP) concentrations did not change. Immunohistochemistry revealed a substantial decrease in rCRF-41 immunostaining of the median eminence 6 weeks after lesioning of the PVN, though randomly located clusters of stained terminals were still seen in the whole rostro-caudal extent of the median eminence. A mixture containing synthetic rCRF-41 and AVP, in proportions similar to those in SME extracts from sham-operated rats, caused significantly less release of ACTH from anterior pituitary cell cultures than did SME extracts from sham-operated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calmodulin inhibitors decrease the CRF-and AVP-induced ACTH release in vitro: interaction of calcium-calmodulin and the cyclic AMP system

The effect of N-(6-aminohexyl)-5-chloro-naphthalene-1-sulfomide (W-7) and trifluoperazine (TFP) was examined on ACTH release from cultured rat anterior pituitary cells and pituitary halves. These drugs significantly inhibited the ACTH release induced by synthetic ovine corticotropin-releasing factor (CRF) in a dose-related manner. In pituitary halves, arginine vasopressin (AVP) at 10 and 100 ng/ml showed almost the same ACTH-releasing activity as CRF at the same concentrations. W-7 and TFP inhibited the CRF-and AVP-induced ACTH release from pituitary halves. The cyclic AMP levels in the pituitary halves were significantly increased by CRF, but not AVP. Although W-7 inhibited CRF-induced ACTH release, it did not have an effect in cyclic AMP accumulation. These results suggest that CRF exerts ACTH-releasing activity through both the calcium-calmodulin system and cyclic AMP system and that AVP stimulates ACTH release mainly through the calcium-calmodulin system.