Fernando E. Estivariz

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.

Coexistence of α-melanocyte-stimulating hormone and adrenocorticotrophin in all cells containing either of the two hormones in the duck pituitary

Since birds lack the pars intermedia of the pituitary gland, the presence of ACTH-, α-MSH-, and β-MSH-containing cells in the pars distalis of the duck was investigated. By using the peroxidase-anti-peroxidase unlabeled antibody enzyme technique there were found cells reacting positively with antibodies against ACTH1–24, ACTH17–39, and α-MSH. Positive reactions could not be obtained with anti-bovine β-MSH serum. The coexistence of α-MSH and ACTH in all cells containing either of the two hormones could be established.

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.

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.

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.

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)

The regulation of the cortico-melanotrophic cell activity in aves. I: Evaluation and selection of in vitro systems for testing ACTH-releasing substances in the duck pituitary

Abstract 1. 1. In order to evaluate cortico-melanotrophic cell activity in the duck pituitary. we have adapted a mammalian ACTH radioimmunoassay (RIA) for the estimation of duck ACTH released from duck pituitary tissue in vitro . 2. 2. The in vitro systems tested for the assay of corticotrophin-releasing substances in the duck were: incubation of pituitary pieces, incubation of collagenase and trypsm dispersed cells. and perfusion of collagenase dispersed cells. 3. 3. The perfusion of duck pituitary dispersed cells proved to be the most suitable for evaluating cortico-melanotropic cell activity. Dose-response curves were obtained using ACTH response to varying dilutions of duck median eminence extract with an index of precision λ of 0.04 (plus or minus 3% of the regression coefficient, within 95% confidence limits). 4. 4. Less than a hundredth dilution of a duck median eminence extract could be effectively detected in our system.

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.

Immunohistochemical demonstration of pro-γ-MSH-like substances in the pituitary gland of various vertebrate species ☆

Abstract Using the peroxidase/antiperoxidase unlabeled antibody enzyme technique pituitaries of several vertebrate species: man, rat, pig, cat, duck, snake, toad, and fish immunostained with an antibody directed against a portion of the cryptic region of the corticotropin/lipotropin common precursor. In all of them, pars distalis corticotropes and pars intermedia melanotropes were positively stained. Specificity studies and controls performed ruled out the possibility of cross-reaction with other known pituitary hormones or peptides. The wide distribution of cross-reaction with the antibody among vertebrate species suggests that a sequence of the cryptic region may be responsible for a yet undiscovered biological effect.

Chromatographic and electrophoretic characterization of melanocyte-stimulating substances in the duck pituitary

Abstract Peptides with melanotrophic activity in the duck pituitary were resolved using chromatography in Bio-Gel P-6. Four distinct peaks of activity were obtained. Three of them coincided roughly with synthetic ACTH 1–39 , pure beef β-MSH, and synthetic α-MSH. The latter accounted for most of the melanotrophic potency of the duck pituitary. α- and β-MSH-like fractions were further purified by electrophoresis in cellulose acetate strips. Only the former fraction yielded a major band which coincided again with α-MSH, whereas the other fraction was resolved into several bands with melanophoric activity, with a minor one coinciding with β-MSH. It is concluded that the bulk of melanotrophic activity of the duck pituitary corresponds to a peptide which behaves as α-MSH in the analytical systems used.