Bilateral adrenal enucleation-induced changes in adenohypophyseal pro-opiomelanocortin (POMC)-related peptides synthesis and secretion: a comparative study with adrenalectomized rats.

The aim of the present study was to elucidate the modulatory effect of transient changes in endogenous glucocorticoids, occurring after bilateral adrenal enucleation (ENUC), on anterior pituitary (AP) proopiomelanocortin (POMC)-derived peptides synthesis and output in rats. For this purpose, adult female rats were either bilaterally ENUC, adrenalectomized (ADX), or sham-operated (SHAM) and killed by decapitation 2, 7, 14, and 21 days after surgery. Trunk blood was collected for measurements of ACTH, beta-endorphin (beta-END) and corticosterone (B) concentrations; APs were quickly dissected for the determination of ACTH, beta-endorphin (beta-END)-like (beta-END-LI) and gamma 3-MSH contents and adrenal glands were removed and submitted to histological study. The results indicate that ENUC and ADX increased AP POMC-related peptides synthesis and release in association with changes in the AP processing of peptides belonging to the N-terminal (gamma 3-MSH), mid (ACTH) and C-terminal (beta-LPH/ENDs) portions of POMC. While ADX abolished plasma B levels, ENUC induced a transient (day 2) decrease in plasma B concentrations which returned to SHAM levels at 7 days after surgery. These data tallied with the histological observations carried out, indicating a time-dependent regenerative process of the adrenal which was completed by three weeks after ENUC. There was a different pattern in plasma ACTH and beta-END levels between ENUC and ADX; maximal plasma peptide levels were found 7-14 days after ENUC, then falling down to SHAM values at 21 days post ENUC. Conversely, there was a constant increment in plasma peptide levels up to 21 days after ADX. At 2 days after both ENUC and ADX all peptides measured in the AP were lower than SHAM values, thus reflecting a rapid corticotrope secretion. Thereafter, 7 or more days after surgery, AP peptide content in ADX rats increased, in a time-related fashion, up to 21 days after surgery. Only beta-END-LI showed a similar AP content to that of the SHAM group, thereafter indicating a preferential cleavage of POMC to beta-END long after ADX (21 days). ENUC rats showed increased AP POMC peptides content throughout the whole time, and it was significantly different from SHAM and ADX values 14 days post-surgery. Interestingly, we found an increment in AP gamma 3-MSH, a peptide which is preferentially synthesized in the intermediate lobe of the rat pituitary, in both ENUC and ADX situations. Our results further indicate that: 1) glucocorticoids, from regenerating adrenal origin, induce a fast negative feedback mechanism on AP secretion, and 2) there might be a delayed inhibitory action of newly synthesized corticosteroids on higher levels of the central nervous system. The lack of glucocorticoids (ADX) clearly corroborates a persistent enhancement of AP POMC-related peptides synthesis and secretion. The differences in AP processing of POMC between ENUC and ADX might be due to qualitative/quantitative changes in hypothalamic ACTH secretagogues output.

Evidence for the paracrine action of islet-derived corticotropin-like peptides on the regulation of insulin release.

In view of recent evidence for the endogenous synthesis of proopiomelanocortin (POMC) by pancreatic islets, we have assessed (1) the release of POMC-derived corticotropin (ACTH)-like peptides (ACTH-LP) from isolated perifused rat islets, and (2) the potential paracrine modulatory effect on insulin output of these putative secretagogues. Islets perifused at a glucose concentration of 3.3 mmol/L secreted ACTH-LP at 0.15 +/- 0.005 ng/islet/10 min, which was increased by 17-fold at 16.7 mmol/L glucose. Islets statically incubated with different concentrations of medium glucose plus synthetic 1-39ACTH at 55 pmol/L showed a significant increase of insulin release at 8 (by 79%) and 16 (by 119%) mmol/L glucose, but not at 4 mmol/L. To determine the possible cis-directed effects of these endogenously released islet ACTH-LP on insulin secretion, we either blocked their biological action by immunoneutralization with an ACTH-specific antiserum or prevented their receptor interaction by addition of the ACTH-inhibiting polypeptide (CIP) to the incubation medium. In the presence of 16.7 mmol/L glucose, the rate of insulin output decreased by approximately 25% upon exposure to the antiserum and by approximately 50% in the presence of CIP. The foregoing observations would therefore suggest that both (1) the elaboration of ACTH-LP by isolated perifused islets and (2) the stimulation of islet insulin release by exogenous 1-39ACTH in static incubation occur as a function of glucose concentration in the incubation medium, and that (3) the newly-secreted endogenous ACTH-LP operate in a cis mode to enhance islet insulin output in a manner analogous to that of exogenously added ACTH species. These results strongly support the view that islet-elaborated ACTH-LP are important physiological paracrine modulators of insulin secretion.

Modulation of ornithine decarboxylase activity by prolactin in seminal vesicles of the rat.

The stimulatory effect of prolactin on sexual accessory glands is well established, though the mechanism of trophic action remains poorly understood. We have therefore assessed the effect of high levels of prolactin on ornithine decarboxylase activity and polyamine content in seminal vesicles (SV) of adult rats. Hyperprolactinaemia was induced by implantation of tissue fragments of a prolactin-secreting tumour 7315a, and the rats killed at 35 days post-inoculation. Serum levels of prolactin were increased significantly (p < 0.001) in tumour-bearing rats. Levels of testosterone in serum were reduced markedly, whereas LH levels remained unchanged. SV weight in the experimental group was not significantly different from that in the corresponding control group. A clear increase in ornithine decarboxylase activity of SV was observed (p < 0.001) in the hyperprolactinaemic rats. However, there was no change in the polyamine content of SV in tumour-bearing rats, compared to the control group. These results indicate that ornithine decarboxylase activity in SV of adult rats is regulated mainly by prolactin. This experimental model may be useful for identification of some of the events involved in the trophic action of prolactin.

Glucose-induced secretion of ACTH-like products by rat pancreatic islets.

This work was performed to study the release of proopiomelanocortin (POMC)-derived peptides from isolated pancreatic islets and the effect of ACTH--a member of that peptide family--on insulin secretion. Islets were incubated with 3,3 and 16.6 mM glucose and insulin and ACTH-like products (ACTH-LP) were measured by radioimmunoassay. Glucose stimulated the simultaneous release of insulin and ACTH-LP, the ACTH-LP concentration being higher when assayed with an antibody reacting with the N-terminus of ACTH. However, the increment in this release in the presence of the higher glucose concentration was larger when measured with an antibody against the ACTH mid-portion. Thus, although the islets would release more of a smaller ACTH-LP, 16.6 mM glucose would selectively increase the release of peptides of larger molecular size. Islets incubated with different concentrations of synthetic ACTH (50-500 pg/ml) increased the release of insulin in a dose-dependent manner. These results suggest that the release of endogenous ACTH-LP could contribute to the paracrine regulation of insulin secretion.

Purification and characterization of a paired basic residue-specific yeast aspartic protease encoded by the YAP3 gene. Similarity to the mammalian pro-opiomelanocortin-converting enzyme.

Yeast cells express an alternate enzyme encoded by the YAP3 gene which can process pro-alpha-mating factor when this pheromone is overexpressed in KEX2-deficient mutants. The YAP3 gene product is an aspartic protease (YAP3) that cleaves at paired basic residues (Egel-Mittani, M., Flygenring, H.P., and Hansen, M. T. (1990) Yeast 6, 127-137). In this study, the YAP3 gene was overexpressed in the BJ 3501 strain of Saccharomyces cerevisiae. YAP3 was purified to apparent homogeneity using concanavalin A and pepstatin A affinity chromatography. The enzyme was characterized as an M(r) 68,000 glycoprotein with a pH optimum of 4.0-4.5. It was inhibited by pepstatin A and activated by 5 mM Ca2+. YAP3 cleaved at paired basic residues of mouse pro-opiomelanocortin (POMC) to yield adrenocorticotropin (ACTH) and beta-lipotropin (LPH); human beta-LPH to yield beta-endorphin-(1-31), beta-endorphin-(1-29), beta-endorphin-(1-28), gamma-LPH, and beta-melanocyte-stimulating hormone; and bovine N-POMC1-77 to yield gamma 3-melanocyte-stimulating hormone. It also cleaved the tetrabasic residues of ACTH1-39 to yield primarily ACTH1-15 and Lys-Arg-corticotropin-like intermediate lobe peptide. The physical properties, pH optimum, and specificity of YAP3 indicate that it is a homologue of the mammalian POMC-converting enzyme (EC 3.4.23.17), a paired basic residue-specific aspartic protease from bovine pituitary intermediate lobe secretory granules (Loh, Y. P., Parish, D.C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205).

Processing of adrenocorticotropin by two proteases in bovine intermediate lobe secretory vesicle membranes. A distinct acidic, tetrabasic residue-specific calcium-activated serine protease and a PC2-like enzyme.

Adrenocorticotropin (ACTH) is cleaved at the tetrabasic residue site, in pituitary intermediate lobe secretory vesicles, to yield ACTH1-17 and corticotropin-like intermediate lobe peptide (CLIP). ACTH1-17 is then converted to alpha-melanocyte-stimulating hormone (N-AcACTH1-13NH2) by first removing the Lys15-Lys16-Arg17 residues, followed by amidation of the COOH terminus and acetylation of the NH2 terminus. Bovine intermediate lobe secretory vesicle membranes were screened for proteolytic enzyme activity that will cleave the tetrabasic residues of ACTH. Two activities with pH optima of 5.0-6.0 and 7.5-8.0 were detected. The acidic, ACTH-converting enzyme cleaved ACTH1-39 at the tetrabasic residues between the Arg17-Arg18 bond to yield ACTH1-17 and CLIP, but did not cleave paired basic residues of pro-opiomelanocortin. This enzyme activity was characterized as a Ca(2+)-activated serine protease with unique specificity for the tetrabasic residues of ACTH1-39. The neutral activity preferentially generated ACTH1-17 and to a small extent ACTH1-16 from ACTH1-39 and ACTH1-24. This enzyme activity was Ca(2+)-dependent but was not inhibited by serine or aspartic protease inhibitors. The neutral activity was significantly immunodepleted by antiserum raised against bovine PC2/PC3, and together with specificity studies, suggests that the enzyme is a PC2-like serine protease. The pH optimum, distinct specificity for tetrabasic residues, and subcellular localization of the acidic ACTH-converting enzyme indicate a function of this enzyme in the in vivo conversion of ACTH1-39 to alpha-melanocyte-stimulating hormone in intermediate lobe secretory vesicles which have an acidic internal pH.

Anterograde nerve degeneration after superior cervical ganglionectomy coexists with a decrease in arginine vasopressin release in rats.

After bilateral superior cervical ganglionectomy (SCGx) in adult male rats, norepinephrine content of the pituitary neurointermediate lobe (NIL) decreased at 12-24 h after surgery to attain concentrations 40-60% of controls between 24 and 60 h after surgery. To assess arginine vasopressin (AVP) secretion during this time, plasma and NIL-AVP levels were measured by radioimmunoassay. In sham SCGx controls, plasma AVP increased about 2-fold within 6 h after surgery and decreased thereafter, to attain presurgical values by 60 h after surgery. In SCGx rats, a significant increase in plasma AVP concentration was observed at the 6th h after surgery, as compared to presurgical concentrations, with a decrease to values significantly lower than those of presurgical controls at 16-18 h after SCGx. As compared to sham-operated rats, significantly higher plasma AVP levels 6 h after surgery and significantly lower plasma AVP levels 16-24 h after surgery were found. NIL-AVP concentration in SCGx and sham-operated ras were significantly lower than presurgical levels at 6 h after surgery. SCGx rats had significantly higher amounts of AVP in NIL at 16-24 h after surgery. The changes in plasma and NIL-AVP levels found 6 or 16 h after SCGx or sham SCGx were unaffected by a prior pinealectomy. Two injections of the alpha 1-adrenoceptor blocker prazosin 45 and 90 min before sacrifice, alone or together with the beta-blocker propranolol, prevented the increase in plasma AVP found in SCGx rats 6 h after surgery, and the decrease in plasma AVP and the increase of NIL-AVP found 16 h after SCGx.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of calcium ions on the processing of pro-opiomelanocortin by bovine intermediate lobe pro-opiomelanocortin-converting enzyme.

The effect of Ca2+ on the extent and pattern of processing of pro-opiomelanocortin and an N-terminal fragment by a purified pituitary secretory vesicle, soluble aspartic endoprotease, was studied. Ca2+ stimulated the first cleavage of pro-opiomelanocortin by pro-opiomelanocortin-converting enzyme to yield 21-23 kDa adrenocorticotropin and beta-lipotropin, but its effect was minimal. The production of adrenocorticotropin from the 21-23 kDa intermediate was stimulated approximately 2.3-fold in the presence of 10 mM Ca2+, and processing of beta-lipotropin to beta-endorphin was stimulated about 1.3-1.4-fold by 5-10 mM Ca2+. The production of gamma-melanotropin-immunoreactive material from bovine N-pro-opiomelanocortin(1-77) was stimulated approximately 1.3-fold at both 100 microM and 1.5-2.0 mM Ca2+. Further characterization of the gamma-melanotropin-immunoreactive material by HPLC demonstrated that the major products were gamma 3-[Lys]melanotropin and gamma 3-melanotropin at both Ca2+ concentrations. These results indicate that pro-opiomelanocortin-converting enzyme is stimulated by Ca2+.

Differential glycosylation of N-POMC1-77 regulates the production of gamma 3-MSH by purified pro-opiomelanocortin converting enzyme. A possible mechanism for tissue-specific processing.

The amino terminus of bovine pro-opiomelanocortin (N-POMC1-77) is partially processed in the intermediate lobe of the pituitary to N-POMC1-49 and lys-gamma 3-melanotropin. Two pools of N-POMC1-77 were isolated which were differentially glycosylated at threonine45, while N-POMC1-49 isolated from bovine intermediate lobe extracts existed in a non-glycosylated form. This suggested that differential O-linked glycosylation of N-POMC1-77 may regulate cleavage at the Arg49-Lys50 processing site. We tested this hypothesis by incubating N-POMC1-77 glycoforms with purified proopiomelanocortin converting enzyme. Only non-O-glycosylated N-POMC1-77 and O-glycosylated N-POMC1-77 with truncated oligosaccharide sidechains were sensitive to cleavage and generated predominantly lys-gamma 3-melanotropin, identified by high-performance liquid chromatography. These data provide the first functional evidence to support a role for differential O-linked glycosylation in the regulation of the processing of the N-terminus of bovine POMC.

IGF1 and 2 in two models of adrenal growth.

Insulin-like growth factors (IGFs) 1 and 2 were measured in the adrenal glands of rats undergoing either compensatory growth following left unilateral adrenalectomy or adrenal regeneration following bilateral adrenal enucleation. In normal rat adrenal gland, the tissue concentration of IGF2 (7.45 +/- 0.99 pg/micrograms protein) wa higher than IGF1 (1.26 +/- 0.23 pg/micrograms protein), both peptides being more abundant in the inner zones of the adrenal gland compared to the capsule-glomerulosa. During compensatory growth of the right adrenal gland, IGF1 and 2 increased significantly compared with control right adrenal glands at 24 h following left unilateral adrenalectomy (P less than 0.001). At 68 h, the increase remained significant for IGF1 (P = 0.012). The two peptides were measured in the regenerating adrenal gland at 7, 14 and 21 days following bilateral enucleation. Whilst there was a trend towards an increase in the IGF1 and 2 content of regenerating adrenal glands, the increase was significant only for IGF2 in the left adrenal gland at 21 days following enucleation. Plasma IGF1 and 2 did not increase compared to controls during the experiments (110.97 +/- 1.95 and 46.33 ng/ml, respectively), suggesting that the changes in tissue IGF reflect increased local production during rapid growth of the adrenal gland.

Neuroendocrine alterations in nude mice with a human lung carcinoma producing pro-opiomelanocortin, corticotrophin-releasing hormone and arginine vasopressin.

A mucoepidermoid carcinoma of the lung (ICD classification 8430/3) resected from a patient with no clinical signs of pituitary-adrenal alterations was transplanted into 2-month-old athymic nu/nu nude mice, with the purpose of studying the effects exerted by the human tumour on the host hypothalamo-pituitary-adrenal axis. The tumour produces peptides derived from different regions of pro-opiomelanocortin (POMC: ACTH, 7.6 +/- 0.7; N-terminal POMC, 6.6 +/- 0.6; beta-LPH/endorphin, 7.3 +/- 0.7; and alpha-MSH;3.8 +/- 0.5 pmol/g wet tissue) and the neuropeptides corticotrophin-releasing hormone and arginine vasopressin (CRH: 3.6 +/- 0.4 and AVP: 1.1 +/- 0.2 pmol/g wet tissue). Immunohistochemical staining of consecutive sections of the tumour indicated that staining of tumour cells for the different peptides was not uniform and although some cells co-stained with CRH and AVP, POMC-positive cells appeared to be distinct from CRH and AVP cells. Tumour extracts were chromatographed on Sephadex G-75 and fractions monitored for POMC-derived peptides. A single peak with characteristics of alpha-MSH was detected. The ACTH, N-POMC and beta-LPH/endorphin radioimmunoassays (RIA) detected a peak at large molecular weight, eluting at the position expected for POMC. These RIA systems also revealed an ACTH(1-39) peak and another peak which probably correspond to 13 kDa ACTH, a peak eluting at the position of hN-POMC(1-48), a beta-LPH-like peak, and a smaller sized peak which may represent alpha- or gamma-endorphin. The ACTH, N-POMC and beta-LPH/endorphin contents of anterior lobe (AL) extracts, but not neutrointermediate lobe (NIL) extracts, showed a striking decrease in tumour-bearing (TB) nude mice. However, while no difference was seen in the alpha-MSH content of AL extract between TB and control (C) nude mice, it decreased in NIL extracts of TB animals. The contents of CRH and AVP in stalk-median eminence extracts of TB nude mice was significantly lower than that of C nude mice. Basal plasma corticosteroids were raised in TB nude mice at levels comparable to those in stressed C nude mice, and although adrenal weights did not vary between TB and C nude mice, morphological changes indicating hypertrophy were found in the adrenal glands of the host animals. It was concluded that the tumour dramatically alters the hypothalamo-pituitary-adrenal axis of the host, and that it may be a useful model for studying tumour-host interactions in ectopic hormone-producing tumours.

Increase in adrenocorticotropin release during wallerian degeneration of peripheral sympathetic neurons after superior cervical ganglionectomy of rats.

The changes in adrenocorticotropin (ACTH) release before, during and after sympathetic nerve degeneration following superior cervical ganglionectomy (SCGx) were examined in male rats. A 12-fold increase of circulating ACTH was found in both SCGx and sham-operated rats 6 h after surgery. In sham-operated rats, plasma ACTH decreased by about half 16-22 h after surgery, whereas in SCGx rats it remained at a high concentration from 16 to 54 h after surgery, attaining basal values by 120 h post-SCGx. In SCGx rats, MBH corticotropin-releasing hormone (CRH) content decreased significantly from 16 to 54 h after surgery, while in controls it remained unmodified. Significantly smaller arginine vasopressin (AVP) contents were found in MBH of SCGx rats as compared to sham-operated controls, 16-54 h after surgery. In rats exposed to ether or immobilization stress 22 h after SCGx, plasma ACTH levels were significantly higher than in controls; however, since unstressed ACTH levels were about twice as high in SCGx rats, the percent increase of ACTH was smaller in the SCGx group. A decreased response of plasma ACTH to ether or immobilization stress was found in rats 7 days after SCGx. In rats subjected to a simultaneous adrenalectomy (Adx) and SCGx or sham-SCGx, plasma ACTH levels increased to a similar extent in both groups. ACTH increase after Adx was accompanied by decreases in MBH CRH, and absence of significant changes in MBH AVP contents. Rats subjected to pinealectomy (Px) or sham-Px 1 week earlier and killed 22 h earlier exhibited similar responses in plasma ACTH and MBH CRH to SCGx regardless of pineal intactness.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of Lys-gamma 3-melanotropin from pro-opiomelanocortin 1-77 by a bovine intermediate lobe secretory vesicle membrane-associated aspartic protease and purified pro-opiomelanocortin converting enzyme.

The ability of bovine intermediate lobe secretory vesicle membrane-associated enzyme(s) and purified, soluble paired basic residue-specific, pro-opiomelanocortin converting enzyme (Loh, Y.P., Parish, D. C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205) to cleave bovine NH2-terminal pro-opiomelanocortin1-77 (N-POMC 1-77) was investigated. Purified pro-opiomelanocortin converting enzyme and an enzyme activity associated with the secretory vesicle membrane were shown to cleave bovine N-POMC1-77 to two major products: N-POMC1-49 and Lys-gamma 3-melanotropin (MSH), and one minor product, gamma 3-MSH. These products were identified by their retention times on high performance liquid chromatography, immunological characteristics, and for Lys-gamma 3-MSH, amino acid composition. The products generated indicate cleavage preferentially between Arg 49-Lys 50 of bN-POMC1-77 (where b indicates bovine), which is identical to the processing pattern found in the bovine intermediate lobe in situ. The membrane converting activity was shown to be stimulated by 5 mM Ca2+ and has a pH optimum of 4-5 and an inhibitor profile characteristic of an aspartic protease. This suggests that the membrane-associated enzyme involved is very similar or identical to the purified, soluble pro-opiomelanocortin converting enzyme, which has previously been reported to be an acidic, aspartic protease responsible for the initial steps of POMC processing. The results of this study lead to the proposal that the lack of processing of the Arg49-Lys50 site in POMC in the anterior lobe versus the intermediate lobe of the pituitary in vivo may be due to other regulatory mechanisms rather than invoking the existence in the intermediate lobe of another enzyme specific for this site, different from pro-opiomelanocortin converting enzyme.

Corticotropin-releasing factor and vasopressin production in the rat pituitary tumor 7315a: biochemical and immunohistochemical studies.

In order to investigate the production and secretion of hypothalamic factors by the prolactin and proopiomelanocortin (POMC)-derived, peptide-producing, transplantable rat pituitary tumor 7315a, we determined the concentrations of corticotropin-releasing factor (CRF)- and vasopressin (AVP)-like immunoreactivities (IR) in the tumor extracts [14.0 +/- 1.6 (SE) and 4.2 +/- 0.9 pmol/g, respectively] and incubation media (0.26 +/- 0.01 and 0.07 +/- 0.01 pmol/10(7) cells/h, respectively). Total peptide content correlated well with tumor weight. Moreover, there is a very good correlation between the CRF and AVP IR, but not as good between CRF or AVP IR and POMC peptide IR tumor contents. Tumor extracts were chromatographed on Sephadex G-75 and compared with chromatograms of stalk median eminence (SME) extracts from normal Buffalo rats. CRF IR in tumor chromatograms gave an unusual pattern of peaks. About 31% of the total CRF IR was eluted in the high molecular weight region. The major portion of CRF IR was located in a wide region of lower molecular weight. The AVP radioimmunoassay revealed a similar pattern of peaks in tumor and SME chromatograms. A propressophysin-like peak and a smaller peak coeluting with synthetic AVP were detected. Immunohistochemical staining of consecutive sections of the tumor indicated that AVP and CRF are often found in the same cell, but the CRF and AVP-producing cells are clearly distinct from the POMC peptide-producing cells.

Regulation of the central nervous system-pituitary-adrenal axis in rats after neonatal treatment with monosodium glutamate.

We have studied the regulation of adrenal function in male rats treated neonatally with monosodium glutamate (MSG) and in littermate controls. When 6-7 months old, MSG-treated rats presented reduced body, adrenal and pituitary weight, obesity, atrophy of the optic nerve and damage of the arcuate nuclei (ARN) of the hypothalamus. MSG-treated rats showed increased serum corticosterone (CORT) levels under resting conditions; after ether stress the increase in serum CORT was greater in MSG animals when compared to littermate controls. Plasma ACTH followed the same trend although it reached significance after ether stress only. Both circulating CORT and ACTH were normally suppressed by dexamethasone (DEX) administration. Levels of corticosteroid binding globulin were also increased, whereas daily circadian rhythm of serum CORT was blunted. We also determined cytosolic receptors in areas suggested to participate in the negative feedback of glucocorticoids at the central level. Binding of (3H)-DEX in MSG rats was similar to controls in hippocampus, whole hypothalamus and anterior pituitary, but a significant reduction (approximately equal to 50%) was obtained after microdissection in the area normally occupied by the ARN, without changes in the ventromedial nuclei of the hypothalamus. These results suggest that the ARN may be involved in the regulation of the pituitary-adrenal axis, although the abnormalities observed in the MSG syndrome partially differ from those in rats with hippocampal damage, previously studied in our laboratory.

Further evidence that N-terminal pro-opiomelanocortin peptides are involved in adrenal mitogenesis.

In order to demonstrate the mitogenic effects of N-terminal pro-opiomelanocortin (N-POMC) peptides on the adrenal glands further, female rats with bilateral adrenal enucleation (hereafter referred to as enucleation) were hypophysectomized 11 days after enucleation and injected twice daily with 5 micrograms purified human N-POMC(1-28), ACTH(1-24) or 0.9% (w/v) NaCl. On day 14 after enucleation, rats were injected with colchicine and, after killing, their adrenal glands weighed, fixed and mitotic counts in histological sections assessed. Plasma corticosterone was measured fluorometrically. In other experiments, rats 7 days after enucleation were hypophysectomized and implanted with osmotic minipumps delivering 5 micrograms purified N-POMC(1-28) per day. On day 14 after enucleation, animals were treated as above. Collagenase-dispersed adrenal cells were incubated with purified or synthetic N-POMC(1-28) or synthetic N-POMC (1-36) (1-300 nmol/l) and [3H]thymidine incorporation into DNA was determined. Intact female rats were implanted with osmotic minipumps delivering 8 micrograms purified or synthetic N-POMC(1-28)/day, 8 micrograms synthetic N-POMC(1-36)/day or saline alone. Mitotic counts were performed on histological sections. Both s.c. injection or continuous delivery from minipumps of purified N-POMC(1-28) partially prevented the atrophy of regenerating adrenal glands after hypophysectomy (s.c. injection of N-POMC: 2.29 +/- 0.92 mitoses/section compared with 0.52 +/- 0.39 for controls; minipump delivery: 5.02 +/- 0.97 compared with 0.13 +/- 0.05; P less than 0.01 for both experiments). ACTH did not augment mitotic activity in enucleated-hypophysectomized rats but significantly increased plasma concentrations of corticosterone in s.c. injection experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal regeneration in the rat is mediated by mitogenic N-terminal pro-opiomelanocortin peptides generated by changes in precursor processing in the anterior pituitary.

In order to investigate the role of N-terminal proopiomelanocortin (N-POMC) in adrenal regeneration after bilateral adrenal enucleation (hereafter referred to as enucleation) rats 13 days after enucleation were injected (200 microliters s.c. plus 200 microliters i.p.) at 08.00 and 20.00 h with normal rabbit serum (NRS), an ACTH antiserum or an N-POMC antiserum. On the next day the animals were injected with colchicine, killed and mitotic figures in adrenal histological sections counted. The same treatment was given to rats 20 days after enucleation. Only the N-POMC antiserum significantly diminished adrenal mitotic activity 14 and 21 days after enucleation (P less than 0.01 and 0.05 respectively) when compared with NRS-treated enucleated rats, whereas plasma corticosterone levels in rats 14 days after enucleation were significantly (P less than 0.005) decreased only by treatment with ACTH antiserum. To determine whether the mitogenic N-POMC peptides involved in adrenal regeneration originated from the pituitary intermediate lobe, 0.9% (w/v) NaCl or ergocryptine (10 mg/kg body weight) was administered s.c. twice daily to rats between 7 and 13 days after enucleation. On day 14, adrenal mitotic activity and plasma levels of ACTH and N-POMC were not significantly different between ergocryptine and saline-treated enucleated rats, whereas alpha-MSH levels in ergocryptine-treated enucleated rats were significantly (P less than 0.02) decreased. Increases in N-POMC content of the pituitary lobe accompanied those of ACTH in animals 7, 10, 14 and 21 days after enucleation (P less than 0.01 compared with sham-treatment). Anterior lobes from rats 10 days after enucleation or from adrenalectomized rats showed raised ACTH and N-POMC levels compared with sham-treated animals, whereas alpha-MSH content in the anterior lobe of enucleated rats was significantly (P less than 0.005) decreased. Adrenalectomized animals had raised (P less than 0.005) amounts of alpha-MSH compared with sham-treated animals. Plasma levels of ACTH and N-POMC were significantly (P less than 0.01) raised in rats 10 days after enucleation or in adrenalectomized rats compared with those in sham-treated animals, whereas alpha-MSH levels were raised (P less than 0.005) only in adrenalectomized rats. Sephadex G-75 chromatography of anterior lobe extracts obtained 10 days after surgery from sham-treated, enucleated and adrenalectomized rats was performed.(ABSTRACT TRUNCATED AT 400 WORDS)

The regulation of the corticomelanotropic cell activity in aves. III--Effect of various peptides on the release of MSH from dispersed, perfused duck pituitary cells. Cosecretion of ACTH with MSH.

1. The melanotropin-releasing activity of arginine-vasopressin (AVP), arginine-vasotocin (AVT), oxitocin (OT), mesotocin (MT) and corticotropin-releasing factor (CRF) was studied in the duck using dispersed, perfused pituitary cells and a specific alpha-MSH RIA. 2. Log dose-response curves were obtained for all the peptides ranging from 5 to 100 ng/ml. All peptides behaved as partial agonists compared to duck median eminence extracts (DME). 3. AVT and MT displayed an alpha-MSH releasing capacity of 60% relative to DME whereas all other peptides behaved as weak agonists with less than 15% capacity relative to DME. 4. AVT and CRF when perfused together acted synergistically on alpha-MSH release yielding a dose response line whose slope approximated that of DME. 5. ACTH was cosecreted together with alpha-MSH in all situations studied with an ACTH to alpha-MSH molar ratio of about 10. 6. It is concluded that CRF and neurohypophyseal peptides may be physiological stimulators of both alpha-MSH and ACTH release in aves.