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M G Castro

Publications and source records attributed to M G Castro.

At least 127 records · Page 7Linked to original sources
PubMed1991

Biosynthesis of corticotrophin-releasing hormone (CRH) in mouse corticotrophic tumour cells expressing the human proCRH gene: intracellular storage and regulated secretion.

The mouse corticotrophic tumour cell line AtT-20 naturally synthesizes pro-opiomelanocortin (POMC) which is proteolytically processed to N-POMC(1-76), ACTH, beta-lipotrophin and beta-endorphin. The processed products are stored in secretory vesicles and released upon stimulation with specific secretagogues. AtT-20 cells which have been stably transfected with the human corticotrophin-releasing hormone (CRH) gene store and secrete immunoreactive CRH. The present results demonstrate that the CRH precursor is proteolytically processed in the transfected cells to yield the 41 amino acid neuropeptide CRH(1-41). On stimulation with the secretagogue noradrenaline, CRH(1-41) was released into the medium, while the precursor was not. Whilst treatment of wild-type AtT-20 cells with exogenous CRH(1-41) (1 nM) caused a fourfold stimulation of ACTH release above basal levels, the peptide had no effect on ACTH release from the stably transfected cells R1 and R4. These results suggest that the endogenous CRH produced by the transfected R1 and R4 cells may cause down-regulation of their CRH receptors, and thus exogenous CRH cannot cause further stimulation of ACTH release in these cells. We propose that the CRH precursor is correctly processed in the transfected AtT-20 cells (R1 and R4) and that the foreign prohormone is sorted into the secretory pathway.

Adrenocorticotropic Hormone↗
PubMed1991

Differential effect of aging on serum levels of prolactin and alpha-melanotropin in rats.

Prolactin (PRL) and alpha-melanocyte-stimulating hormone (alpha-MSH) are the only two pituitary hormones whose basal secretion is under tonic dopaminergic inhibition exerted by the hypothalamus. In the female rat, continuous exposure to estrogens is believed to depress hypothalamic dopaminergic activity and lead to the appearance of PRL-secreting pituitary adenomas during aging. Since there is no information about the impact of aging on circulating alpha-MSH levels, it was of interest to assess and compare the serum levels of PRL and alpha-MSH in male and female rats of different ages. Young (3-4 months) and old (24-25 months) male and female Sprague-Dawley rats as well as senescent (33-35 months) females were killed by decapitation between 10 AM and 1 PM, and pituitaries were immediately removed and dissected. Hormones were measured in unextracted trunk serum by radioimmunoassay. Serum PRL levels were (mean +/- SE), 18.4 +/- 2.0, 26.8 +/- 3.8, 19.8 +/- 2.5, 43.0 +/- 7.5, and 193.5 +/- 47.6 ng/ml for young and old males, and young, old, and senescent females, respectively. Serum alpha-MSH levels were 243.2 +/- 15.2, 252.9 +/- 24.8, 320.0 +/- 31.3, 234.7 +/- 19.1, and 374.0 +/- 29.7 pg/ml for young and old males, and young, old and senescent females, respectively. Anterior pituitary and neurointermediate lobe weights increased significantly with age in both sexes, although the change was particularly conspicuous in the females. We conclude that aging does not have a major impact on circulating alpha-MSH levels in rats and that melanotrophs probably have a greater ability than prolactotrophs to withstand age-associated alterations in central regulatory mechanisms.

Aging↗
PubMed1990

Expression of biologically active human pre-procorticotropin releasing hormone in E. coli: characterization and purification.

1. Human pre-procorticotropin releasing hormone (CRH) was expressed in E. coli strain TG2 as a fusion protein with beta-galactosidase. 2. A 140 kDa band which corresponded to beta-galactosidase pre-proCRH fusion protein was identified in lysates of TG2 cells harbouring the recombinant plasmid pre-proCRH (10-196) [ph PPC (10-196)] after sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Coomassie Blue staining. The identity of the fusion protein was confirmed by Western blotting and a two-site immunoradiometric assay. 3. Purification of the fusion protein from isolated, washed and solubilized inclusion bodies was achieved by ion-exchange chromatography in the presence of 8 M urea. 4. When comparing the adrenocorticotropin-releasing activity on a molar basis, the potency of the chimeric CRH precursor was 4% of that of synthetic r/h CRH (1-41).

Cloning, Molecular↗
PubMed1990

The use of inclusion bodies, isolated from Escherichia coli expressing corticotrophin-releasing hormone precursor, to raise specific antibodies against the neuropeptide moiety.

We have expressed human pre-procorticotrophin-releasing hormone (pre-proCRH) as a fusion protein to beta-galactosidase in Escherichia coli. The chimeric fusion protein was found in insoluble bacterial inclusion bodies. The inclusion bodies were isolated, purified and solubilized, and used as imunogens in rabbits to raise antibodies against the neuropeptide moiety. The antibodies generated were characterized by immunoassays and immunocytochemical techniques. The immunoassay results showed that the recombinant pre-proCRH antibodies cross-reacted with the full-length CRH precursor and several cleavage products derived from it, i.e. CRH(1-41) and CRH(36-41). They did not cross-react with the CRH antagonist CRH(9-41). Extracts of stalk median eminence from various species were also studied. The antibodies cross-reacted with extracts from ovine, bovine, human and rat tissues, exhibiting parallel displacement curves to that of synthetic rat/human CRH(1-41) used as standard. They also cross-reacted with a skin extract of the frog, a species known to contain a CRH-related peptide, i.e. sauvagine, in this tissue. The immunocytochemical studies demonstrated that the antibodies generated against recombinant human pre-proCRH labelled neurones in the rat paraventricular nucleus of the hypothalamus. They exhibited the same pattern of staining as that obtained with an antibody generated against synthetic CRH(1-41). The results indicate that these antibodies can recognize CRH(1-41) or CRH-related molecules in the hypothalamus in situ as well as in tissue extracts from several species. Hence, they will be useful tools in the study of the CRH biosynthetic pathway and its intracellular compartmentalization.

Animals↗
PubMed1989

Transmembrane signals mediating adrenocorticotropin release from mouse anterior pituitary cells.

The effect of arginine vasopressin (AVP) and corticotropin releasing factor (CRF) an adrenocorticotropin (ACTH) secretion, phosphatidylinositol breakdown and cAMP accumulation was examined in primary cultures of mouse anterior pituitary cells. AVP and CRF added alone stimulated ACTH secretion in a dose-dependent manner. At 10(-8) M concentration of peptide, AVP and CRF stimulated ACTH secretion 2.8- and 4.6-fold, respectively. AVP and CRF added in combination at equal doses gave an additive effect. CRF enhanced cAMP accumulation, but AVP had no effect on basal or CRF-induced cAMP accumulation. Both forskolin (10(-5) M) and 8-bromo-cAMP (10(-3) M) increased ACTH secretion in these cells by 2.8- and 1.7-fold, respectively. AVP induced the breakdown of phosphoinositides, and CRF alone, or in combination with AVP did not modify this effect. Phorbol 12-myristate 13-acetate (10(-7) M), dioctanoylglycerol (10(-4) M) and phospholipase C (100 mU/ml) also stimulated ACTH secretion in these cells by 4.2-, 2.4-, and 3.7-fold, respectively. Depletion of intracellular and extracellular Ca2+ decreased ACTH secretion, but had no significant effect on CRF-induced cAMP accumulation. However, AVP-induced phosphoinositide breakdown was dependent on extracellular Ca2+. These results indicate that CRF stimulates ACTH secretion via the cAMP-dependent pathway and AVP via the phosphoinositide breakdown-phospholipase C pathway. In the presence of AVP and CRF, both pathways appear to operate independently to produce an additive effect on ACTH secretion.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Regulated secretion of pro-opiomelanocortin converting enzyme and an aminopeptidase B-like enzyme from dispersed bovine intermediate lobe pituitary cells.

Coordinate secretion of two prohormone/proneuropeptide processing enzymes [pro-opiomelanocortin converting enzyme (PCE) and an aminopeptidase B-like enzyme (APBE)] and alpha-melanotropin (alpha-MSH) from bovine intermediate lobe pituitary cells was studied. Stimulation of secretion with 8-bromo-cyclic AMP produced significant increases in levels of immunoreactive alpha-MSH, PCE, and APBE. Treatment of cells with the dopaminergic agonist 2-bromo-alpha-ergocryptine resulted in significant decreases in secretion of alpha-MSH, PCE, and APBE. In neither case were there significant changes in levels of cytosolic lactic dehydrogenase or lysosomal beta-glucuronidase in the medium. The secreted PCE activity was shown to process frog and mouse pro-opiomelanocortin primarily to 23,000-Mr corticotropin (ACTH), 13,000-Mr ACTH, beta-lipotropin, a beta-endorphin-like peptide, and beta-endorphin, products comparable to those synthesized by the mouse and frog intermediate lobe in situ. The secreted enzymatic activity had a pH optimum between 4.0 and 5.0, was strongly inhibited by pepstatin A, and had an inhibitor profile similar to the purified bovine intermediate lobe PCE. The secreted APBE activity cleaved Argo-[Met]-enkephalin to [Met]-enkephalin and had a pH optimum and inhibitor profile similar to that previously reported for an activity from purified secretory vesicle fractions of bovine intermediate and neural lobes. The coordinate regulated secretion of alpha-MSH and enzyme activities (PCE and APBE) strongly indicates their colocalization in the same secretory vesicle compartment within the cell. The characteristics of the two enzymes secreted in the medium paralleled those seen in the tissue and further support their role in pro-opiomelanocortin processing in vivo.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Immunoreactive delta sleep-inducing peptide secretion from mouse dissociated, anterior pituitary cells: regulation by corticotropin-releasing factor and arginine vasopressin.

Immunoreactive delta sleep-inducing peptide (IR-DSIP) has previously been localized to the ACTH/MSH cells of the human and porcine pituitary gland. In the present report, the distribution of IR-DSIP in the mouse pituitary gland was examined by immunocytochemistry. In this species, IR-DSIP was found to be co-localized with thyroid-stimulating hormone (TSH) in anterior pituitary thyrotrophs and was also present in nerve fibers in the posterior and intermediate lobes. The effect of synthetic DSIP on IR-ACTH release from dissociated mouse anterior pituitary cells was also studied. DSIP (greater than or equal to 10(-9) M) inhibited both basal and CRF-induced IR-ACTH release from these cells. In addition, the effect of synthetic rat corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) on IR-DSIP secretion was investigated. CRF and AVP at concentrations of 10(-11)-10(-7)M inhibited release of IR-DSIP from mouse anterior pituitary cells by 63%. When CRF and AVP (10(-10)-10(-7) M) were given concomitantly, the maximal inhibition of IR-DSIP release was observed at a concentration of 10(-8)M of CRF and AVP. However, these two peptides when given together showed no additive effect on IR-DSIP secretion. These findings suggest that during CRF induction of ACTH secretion from anterior pituitary corticotrophs, CRF may also act simultaneously to inhibit DSIP secretion from the thyrotrophs.

Adrenocorticotropic Hormone↗
PubMed1989

Diminished diurnal secretion of corticosterone in aging female but not male rats.

Since very little is known about the impact of aging on adrenocortical function in female rats, it was of interest to compare the age changes in the circulating levels of adrenocorticotropin (ACTH) and corticosterone in male and female Sprague-Dawley rats, and correlate these changes with those in plasma prolactin (Prl), which is known to stimulate adrenal steroid secretion. Hormones were measured in young (3-4 months) and old (24-26 months) male rats as well as in young (3-4 months), old (25 months) and senescent (33-35 months) female rats. Sequential plasma samples were obtained from chronically cannulated animals every 30 min from 11:30 a.m. to 4:30 p.m. ACTH was measured in trunk serum--obtained between 11:30 a.m. and 1:30 p.m.--whereas corticosterone and prolactin were measured in plasma using specific RIAs for the corresponding hormones. No age changes were detected in serum ACTH in either sex. The integrated values of plasma corticosterone did not change with age in males but decreased significantly (p less than 0.05) in old and senescent as compared to young females. Plasma corticosterone was higher in young females than in young males but this sex-related difference disappeared with age. Plasma Prl increased significantly with age in both male (p less than 0.05) and female (p less than 0.001) rats but showed no significant correlation with corticosterone levels. The present results suggest that ACTH secretion does not show major age- or sex-related changes. Our corticosterone data are compatible with the idea that gonadal aging has a significant impact on plasma glucocorticoid regulation in female rats but not in males.

Adrenocorticotropic Hormone↗
PubMed1988

Pro-opiomelanocortin and pro-vasopressin converting enzyme in pituitary secretory vesicles.

Peptide hormones are synthesized from larger precursors by cleavages at paired basic residues. We have isolated a pro-hormone converting enzyme from bovine neural and intermediate lobe secretory vesicles that cleaves pro-vasopressin and pro-opiomelanocortin at Lys-Arg residues to yield vasopressin, and adrenocorticotropin/endorphin-related peptides, respectively. The enzyme from both lobes is an aspartyl protease of approximately 70,000 Da, is a glycoprotein and has an optimum pH range of 4.0-5.0. Present within the same secretory vesicles is an aminopeptidase B-like enzyme which is a metalloprotease that is inhibited by Co2+ and Zn2+. This enzyme may play a role in trimming off the N-terminal extended basic residues from peptides liberated by the pro-hormone converting enzyme.

Animals↗
PubMed1988

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.

Adrenocorticotropic Hormone↗
PubMed1988

Presence of pro-vasopressin mRNA, neurophysin and arginine vasopressin in mouse anterior pituitary cells and the AtT-20 corticotrophic tumour cell line.

Pro-vasopressin mRNA, neurophysin and arginine vasopressin (AVP) were assayed in the mouse anterior pituitary gland, in mouse anterior pituitary cells in culture and in the AtT-20 corticotrophic tumour cell line. Northern blot analysis revealed the presence of an approximately 700 base pair pro-vasopressin mRNA in anterior pituitary and AtT-20 cells. Neurophysin, identified by immunoblots, and AVP, identified by high-performance liquid chromatography and cross-reactivity with AVP antiserum, were detected in anterior pituitary cells and AtT-20 cells. Immunocytochemical staining with anti-neurophysin showed that approximately 40-45% of the dissociated anterior pituitary cells in culture and greater than 95% of the AtT-20 cells were stained. Anterior pituitary cells in culture and AtT-20 cells had a basal level of release of AVP in the 0.01-0.1 nM range. These results indicate that anterior pituitary cells and AtT-20 cells have the ability to synthesize and process pro-vasopressin to AVP and neurophysin, endogenously.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1986

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.

Adrenocorticotropic Hormone↗
PubMed1986

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.

Adrenocorticotropic Hormone↗
PubMed1986

[Effect of monoamines on ACTH secretion by dispersed perfused cells from the adenohypophysis of barcino chico duck (Anas flavirostris)].

We have pursued with the characterization of ACTH secretagogues from the avian corticomelanotrophic (CM) cell, by testing the ACTH-releasing activity of various monoamines and related drugs, using an in vitro system which uses dispersed perfused duck pituitary cells. The substances used were: noradrenaline (NA), adrenaline (A), dopamine (DA), serotonin (5-HT), phenylephrine (Phe), and isoproterenol (IP). The responses obtained with the substances assayed were compared with those obtained with dilutions of duck median eminence extracts (DME). The order of "intrinsic activities" was: NA = A greater than Phe greater than IP greater than DA = 5-HT. The substances were tested within the range 10(-9)-10(-4) M. All substances tested behaved as partial agonists with respect to DME. The "intrinsic activity" (Vmax) of the most potent agonists tested, A and NA, was 0.66 of that obtained with DME. It is concluded that in the duck, the CM cells secrete ACTH in response to A and NA (and other pharmacological substances) at doses which are compatible with a physiological role of those catecholamines acting directly upon the CM cell of the avian adenohypophysis. 5-HT and DA behaved as very weak agonists in stimulating ACTH release from duck CM cells in the system employed.

Adrenocorticotropic Hormone↗