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.

Serotonergic terminals in the anterior hypothalamic nucleus involved in the prolactin release during suckling.

The present studies were designed to localize within the hypothalamus and neighboring areas the serotonergic terminals which are implicated in suckling-induced PRL release. The initial experiments were performed to characterize the circulating hormone profile induced by suckling in lactating rats, previously separated from their pups. Five minutes of suckling induced an increase in serum PRL only. During these 5 min, 5-hydroxytryptamine (5-HT) and 5-hydroxyindole-3-acetic acid concentrations were determined in the pars nervosa of the pituitary gland, hypothalamic nuclei, dorsal, and median raphe nuclei. An increase by 80% (P less than 0.01) in 5-HT concentration was found only in the rostral part of the anterior hypothalamic nucleus (rNHA). In order to investigate causal effect between the altered 5-HT neuronal activity in the rNHA and the suckling-induced PRL release, serotonergic neurotoxin was bilaterally injected in the rNHA on day 1 of lactation. Litters were adjusted to eight pups each and weighed daily to determine litter growth rates. On day 8 of lactation, litters were separated from their mothers for 4 h and allowed to suckle for 5 or 15 min after which the mothers were decapitated. Litters from lesioned animals grew at a lower rate (P less than 0.0001) than control and sham-operated animals. Serum PRL increased with suckling in animals bearing the correct rNHA lesions, but the values were lower than in control and sham-operated animals after 5 (P less than 0.05) and 15 (P less than 0.01) min. Therefore we postulate that the rNHA is the site of termination of a stimulatory serotonergic pathway on PRL release induced by suckling.

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.

Chromatographic characteristics of pro-opiomelanocortin-derived peptides from the rat transplantable tumour 7315a.

Adrenocorticotrophin (ACTH) and other pro-opiomelanocortin (POMC)-derived peptides produced by the 7315a corticomammotrophic tumour have been poorly studied although they elicit profound hypertrophy and hyperplasia in the adrenal glands of recipient Buffalo rats. Tumour extracts were chromatographed on Sephadex G-75 and fractions monitored for POMC-derived peptides by four radioimmunoassay (RIA) systems: ACTH, alpha-MSH, beta-lipotrophin (beta-LPH)/endorphin and N-terminal POMC (N-POMC). Chromatograms were compared with those of pars distalis extracts from normal Buffalo rats. All four RIA systems detected immunoreactive material in tumour extracts. ACTH, beta-LPH/endorphin and N-POMC were present in approximately equimolar amounts (ACTH content 93.40 +/- 5.27 (S.E.M.) pmol/g) whereas alpha-MSH was present in smaller amounts (2.83 +/- 0.13 pmol/g). Total peptide content correlated well with tumour weight. ACTH immunoreactivity (IR) in Sephadex chromatograms was located in a large 20,000 mol. wt peak, an ACTH(1-39) peak and a smaller peak coinciding with ACTH(1-24). The latter two peaks showed biological activity consistent with ACTH(1-39) and an ACTH (1-24)-like peptide respectively. The beta-LPH/endorphin RIA revealed a peak eluting at approximately 20,000 mol. wt which could not be ascribed to any known POMC peptide containing the endorphin sequence. A beta-LPH-like peak, a beta-endorphin-like peak and a smaller-sized peak, which contained the bulk of the beta-LPH/endorphin IR, were detected; the low molecular weight peak probably representing alpha- or gamma-endorphin.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

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.

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.

An investigation on pro-opiomelanocortin and processed peptides from the teleost fish Prochilodus platensis.

Acid extracts of carefully dissected proadenohypophysis (PA) and metaadenohypophysis (MA) of the teleost Prochilodus platensis were subjected to chromatography in Sephadex G-50 after which several pro-opiomelanocortin (POMC) peptides were detected by means of three heterologous RIA systems: alpha-MSH, ACTH and beta-endorphin. Parallelism among extracts displacement curves ranged from 26% to 95% of those of the standard curves for the different systems employed. In PA chromatograms, peaks of ACTH immunoreactivity (IR) were detected at the positions of 30 kilodalton (K), 20K, 9K, a large 4.5K peak and 2K. Only one peak of beta-endorphin IR was detected at 30K. In MA chromatograms, ACTH IR detected similar peaks as in PA runs, but 4.5K peak was much smaller, whereas a large 2K peak roughly coincided with all alpha-MSH detected in the chromatograms. beta-Endorphin IR was detected mainly as a large peak coinciding with synthetic beta-endorphin in MA runs. Bioactivity was detected in both PA and MA 4.5K ACTH peaks, whereas little activity could be demonstrated associated with the 30K, 20K and 9K ACTH IR peaks. Prochilodus PAs and MAs were incubated with tritiated aminoacids and the extracts immunoprecipitated with ACTH, beta-endorphin and N-terminal POMC (N-POMC) antisera. The dissociated complexes were run in SDS polyacrylamide slab gel electrophoresis. The tritiated bands detected confirmed the results obtained with Sephadex chromatography. N-POMC immunoprecipitated peptides were located at 28K, 18K and 9K positions. The first two probably accounted for POMC and the N-POMC/ACTH intermediate respectively; the third corresponded to the mammalian 1-76N-POMC.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of amino-terminal pro-opiocortin peptides from human pituitary glands.

Two cystine-containing peptides isolated from human pituitaries were partially sequenced. The peptides are fragments derived from the amino-terminal of pro-opiocortin (NPOC) and have been chemically characterized as NPOC 1-28 (which lacks gamma-MSH) and NPOC 2-59. Their availability enables us to investigate new putative biological roles for the amino-terminal conserved sequence of pro-opiocortin.

Lack of glycosilation of pro-opiomelanocortin might account for the periodic acid-Schiff-negative reaction in ACTH cells of teleost fishes.

Unlike tetrapod ACTH cells, teleost ACTH cells do not react with the periodic acid-Schiff method (PAS). To find an explanation for this unique feature, chromatographic fractions obtained after filtration of pituitary extracts of Prochilodus platensis in Sephadex were immunologically analyzed. A high-molecular-weight protein which was identified as pro-opiomelanocortin (POMC) was detected. When this POMC was submitted to affinity chromatography in concanavalin binding, it was not detected. Furthermore, pituitaries incubated in media containing [3H]glucosamine or [3H]fucose did not incorporate these amino acids to the newly synthesized POMC. The results obtained strongly suggest an inability of the fish to glycosilate POMC, and this failure could account for the PAS-negative reaction in the ACTH cells.

Nature and control of peptide release from the pars intermedia.

Pro-opiocortin, the precursor of ACTH, LPH and gamma-MSH, is biosynthesized in both the cells of the pars intermedia and the corticotrophs of the pars distalis. In the pars distalis its processing does not vary significantly from species to species whereas in the pars intermedia large differences occur. The release of ACTH, beta-LPH and pro-gamma-MSH from the corticotrophs is under common positive control by hypothalamic corticotropin-releasing factor (CRF) and the nature of the peptides remains unchanged when they are secreted. The release of all five pars intermedia peptides that we have measured in vitro appears to be under tonic dopaminergic inhibition. The secreted peptides have also been identified chromatographically. The lack of unequivocal physiological function in the periphery, the diversity of the pars intermedia peptides and this common control mechanism tend to preclude a simple endocrine role for the pars intermedia. The neural effects of MSH and endorphin are well documented and specific neuronal uptake therefore cannot by dismissed. The absence of pars intermedia in the adult human pituitary suggests that such a site of synthesis of these peptides plays a minor role in learning and behaviour in a species (such as Homo sapiens) that has a highly evolved intelligence and may, instead, need to synthesize the peptides only in the brain.

The regulation of the corticomelanotropic cell activity in Aves--II. Effect of various peptides on the release of ACTH from dispersed, perfused duck pituitary cells.

We have estimated the corticotropin-releasing activity (CRA) of different neurohypophyseal peptides and synthetic corticotropin-releasing factor (CRF) in the duck, using perfused dispersed pituitary cells and an ACTH radioimmunoassay adapted to duck material. Log dose-response curves were obtained for different doses of arginine-vasopressin (AVP), arginine-vasotocin (AVT), mesotocin (MT), oxitocin (OT) and ovine CRF (oCRF) and compared to the response obtained with dilutions of duck median eminence extracts (DME). All peptides tested behaved as partial agonists compared to DME. AVT and MT were the most potent of all peptides tested, with a capacity of 60% relative to DME. CRF was a weak agonist together with AVP and OT. AVT and CRF perfused together at equal doses significantly potentiated the effect of each other, yielding a dose-response line whose slope approximated that of DME. A similar design was used to test the CRA of the same substances in the rat. The main difference in the pattern of response between the two species was the low potency displayed by all the neurohypophyseal peptides in the rat, compared with CRF which, in contrast with what occurred with the duck system, was the most potent secretagogue of all peptides tested. It is concluded that in birds, as in mammals, the control of ACTH secretion may be exerted by neurohypophyseal peptides and a CRF-like peptide acting synergistically upon the corticomelanotropic cell.

On the stimulatory nature of the control of MSH secretion in ducks.

Though birds lack the pars intermedia of the hypophysis, their pituitary glands do secrete MSH. This hormone and ACTH are elaborated in special cells of the pars distalis called corticomelanotrops. The present study was designed to ascertain whether the release of MSH in aves is under either stimulatory or inhibitory hypothalamic control. Extracts of median eminence were injected in ducks and plasma MSH was observed to rise after the injections: on the other hand, when pituitaries were ectopically grafted, significant changes in the levels of circulating MSH were not detected. Twenty days after grafting, the transplants were extirpated and incubated in media containing median eminence extracts. The extracts stimulated the release of MSH not only from grafts but also from pieces of normotopic glands. The grafts showed cells which contained ACTH but not MSH; however, they contained small amounts of MSH, detectable by RIA. The administration of ergocryptine brought about the inhibition of MSH secretion in vivo, and it is suggested that this drug acted on hypothalamic structures rather than directly on the corticomelanotrops. On the basis of the preceding results, it is concluded for the first time that in ducks the release of MSH has stimulatory control from the hypothalamus, contrarily to that occurring in almost all the animals so far investigated.

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.

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.