Glucagon of caviomorphs and other tetrapods immunohistochemically investigated with two antisera against the N- and C-terminal portions of the molecule.

Pancreatic sections from diverse tetrapods, including various species of caviomorph rodents, were immunohistochemically investigated using two antisera which reacted with the N- and C-terminal portions of the glucagon molecule. While the antiserum against the N-terminal portion stained alpha cells in all the species studied, the antiserum against the C-portion failed to stain alpha cells in two caviomorphs of the Caviidae family (guinea pig and cuis) and in one of the Octodontidae family (degu). The observations in guinea pig and degu were expected, since their glucagons differ from those of many other tetrapods in the C-terminal portion of the molecule. In this paper, the cuis was added to these two species. It is noteworthy that among the caviomorphs studied herein (nine species), immunohistochemical differences were detected only in the three above-mentioned species and did not involve higher taxa, thus suggesting that these modifications are relatively recent in the evolution of this group of rodents.

Acrolein fixation for immunostaining tyrosine-hydroxylase in paraffin sections.

Routine histological fixatives barely preserve tyrosine-hydroxylase immunoreactivity in paraffin sections. fixation in 5% acrolein in phosphate buffer, pH 7.4, resulted in good preservation of the enzyme in the tissues investigated.

Immunohistochemical investigation of tyrosine-hydroxylase in the islets of Langerhans of adult mice, rats and guinea pigs.

As an indirect way to establish whether catecholamines are actually elaborated in the islets of Langerhans of adult albino and pigmented mice and guinea pigs, or albino rats, the presence of tyrosine-hydroxylase was immunohistochemically investigated using a highly specific polyclonal serum against the enzyme. The antiserum was applied to paraffin sections of pancreases fixed either in acrolein or Bouin's fluid. Such sections were then treated with the avidin-biotin method. Tyrosine-hydroxylase was found exclusively in the beta cells of rats and in those of the two strains of mice. Results in the guinea pig varied according to whether the animals were pigmented or albino. In pigmented specimens, the enzyme was detected in beta and non-beta cells, whereas in albino animals it was undetectable in any of the islet cells. All these observations were performed in material fixed in acrolein. Bouin's fluid resulted a rather poor fixative for the detection of the enzyme in the islets. The results are compared with those of other authors who investigated the presence of monoamines in the islets of Langerhans. Since tyrosine-hydroxylase is a specific marker for catecholamine-synthesizing cells, it is concluded that insular cells have the ability to elaborate these substances.

Alpha-melanocyte-stimulating hormone stimulates sodium excretion in the salt gland of the duck.

Salt glands of ducks were induced to secrete sodium through the ingestion of salt water. In salt-adapted animals the administration of melanocyte-stimulating hormone (MSH) produced a rise in the sodium excreted by the salt gland, an effect which was not mimicked by adrenocorticotropin. Studies in vitro using incubations of gland slices and radioactive sodium ion showed that MSH increased sodium efflux, indicating that it acted directly upon the gland. We have previously observed that MSH has no effect on the pigmentary system of the duck. It is proposed that in the evolutionary process this hormone has acquired new target tissues in these birds.

Cytological differences in the localization of glucocorticoid receptor-like immunoreactivity in the normal and transplanted pituitary pars intermedia.

Glucocorticoid receptor-like immunoreactivity (GCRI) was found in the normal pituitary pars intermedia (PI) when immunohistochemistry was used. Since in previous studies we described two kinds of cells in the denervated (grafted) PI, i.e., "light cells" (overactive cells which do not contain detectable melanocyte-stimulating hormone) and "dark cells" (hypoactive cells which contain the hormone), it was decided to investigate whether different patterns of distribution of the receptors could be detected in the grafted gland when compared with the intact PI. Intact glands showed the receptors located in the nucleus. In transplanted glands, it was observed that light cells showed receptors in both the nuclei and the cytoplasm; on the other hand, dark cells displayed them in the nuclei only, as is the case in all cells of the normal PI. We had previously interpreted dark cells as dopamine-indifferent, whereas light cells were considered dopamine-sensitive. The changes in the distribution of GCR after denervation by grafting, which only affected the light cells, support the view of other authors that GCR of the pars intermedia are under the influence of dopamine and reinforce our opinion that dark cells are dopamine-indifferent.

Ammonium thiocyanate for detaching antibodies from histological specimens.

When different antigens must be demonstrated in the same structure, the permanence of former antibodies can lead to false identification of another antigen. The peroxidase-antiperoxidase method was used, followed by the oxygen acceptor ethyl-carbazole. After staining the sections, they were destained with xylene and the antibodies detached with 3 M ammonium thiocyanate; then the specimens were treated for the demonstration of the other antigen. The procedure could be repeated and thus as many as four antigens could be demonstrated without damaging the tissues. Antigens participating in the immunohistochemical staining were well-preserved after destaining and detaching the antibodies as demonstrated by their ability to react again in a second staining.

Transplantation of the pituitary pars distalis induces the corticotrophs to store melanocyte-stimulating hormone, an effect reversed by the administration of corticotropin-releasing factor.

Corticotrophs of the pituitary pars distalis do not contain immunohistochemically detectable alpha-melanocyte-stimulating hormone (MSH). After grafting the glands beneath the renal capsule for 25 days, this hormone could be demonstrated in corticotrophs, coexisting with corticotropin. The administration of corticotropin-releasing factor deprived these cells of the content of MSH but did not apparently affect the presence of adrenocorticotropin (ACTH). It is suggested that the histological appearance of MSH in the corticotrophs may be due to a diminished rate of corticotropin release, which may provide time for splitting the former hormone in amounts larger than the negligible ones normally present in the pars distalis, or to the hypothetical fact that some hypothalamic factor may inhibit the cleavage of ACTH into MSH.

Secretion of melanocyte-stimulating hormone and adrenocorticotropin from transplanted pituitary pars intermedia in stressed and nonstressed rats.

Rats bearing kidney grafts of the pituitary pars intermedia were divided into three groups: unstressed, acutely stressed, and chronically stressed. Corresponding sham-operated rats were used for comparisons. Twenty days after grafting, the rats were sacrificed and alpha-melanocyte-stimulating hormone (alpha-MSH), adrenocorticotropin (ACTH), and corticosterone were estimated in plasma. The adrenal/body weight ratio and DNA content of the glands were also investigated. The following results were obtained: MSH was found not to be increased in unstressed rats, but it was in grafted animals subjected to acute and chronic swimming stress. ACTH and corticosterone rose in all three groups. Adrenal/body weight ratio and DNA content increased only in grafted chronically stressed rats. Moreover, plasma corticosterone was found higher in grafted hypophysectomized rats than in non-grafted hypophysectomized animals. Administration of ergocryptine to nonstressed grafted rats induced a decrease in the blood content of ACTH and MSH, indicating that the grafts were the source of a part of the circulating ACTH. On the other hand, the fall in MSH levels could show the effect of the drug upon the pars intermedia. Comparison of the ratios of both hormones released in incubations showed that grafts secreted more ACTH than MSH; on the other hand, when intact neurointermediate lobes were incubated, MSH predominated over ACTH. For the first time it is demonstrated that the pars intermedia can secrete ACTH in vivo. Nevertheless, the ability to secrete this hormone is not a property of normal intact pars intermedia, but it manifests in the transplantations probably due to the overactivity of light cells induced by chronic stoppage of dopaminergic inhibition.

Two kinds of cells in grafts of pituitary pars intermedia and their probable dependence on dopamine.

When histological sections of noninnervated kidney grafts of pituitary pars intermedia were immunohistochemically processed for the demonstration of either alpha melanocyte-stimulating hormone or adrenocorticotropic hormone, chromophilic (dark) and chromophobic (light) cells could be detected. The latter, which were not detected in the intact intermedia, were interpreted as hyperactive cells, since they lacked secretory granules and displayed an increased nuclear area. The administration of ergocryptine prevented the development of light cells. Moreover, after chronic stress (swimming), some cells appeared less intensely granulated than dark cells. The results suggest that the pars intermedia is composed of parenchymal cells with different sensitivities to dopamine. Light cells would be subject to a greater dopamine-induced inhibitory influence than dark cells. The appearance of partially degranulated cells after stress points to a participation of the grafts in this event and indicates that innervation of the intermedia is not an indispensable condition for its contribution to stress.

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.

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.

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.

[Effect of monoamines on ACTH secretion by dispersed perfused cells from the adenohypophysis of barcino chico duck (Anas flavirostris)]. / Effecto de monoaminas sobre la secreción de ACTH en células dispersas, perfundidas, de la adenohipofisis del pato barcino chico (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.

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.

Effecto de monoaminas sobre la secreción de ACTH en células dispersas, perfundidas, de la adenohipofisis del pato barcino chico (Anas flavirostris). / [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 [quot ]intrinsic activities[quot ] 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 [quot ]intrinsic activity[quot ] (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.

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)

Catecholamine metabolizing enzymes and synthesis of dopamine in normal and grafted pituitary partes distales.

The presence of enzymatic activity (tyrosine hydroxylase, dopa-decarboxylase, dopamine-beta-hydroxylase, monoamine oxydase and catechol-O-methyl transferase), as well as dopamine (DA) content and DA synthesis from tyrosine and dopa, were investigated in intact rats partes distales and in grafts (both estrogenized and nonestrogenized). Counts of prolactin cells showed the following regression in the number of these cells: estrogenized grafts greater than nonestrogenized grafts greater than intrasellar intact glands. Tyrosine hydroxylase activity was not found in intact glands, but this enzyme was detected in the two types of grafts. An approximate correlation could be established between the number of prolactin cells and the diverse enzyme activities. Dopamine was not synthesized from tyrosine in intact glands, but it occurred in the transplants. However, when dopa was used, both intact and grafted glands produced dopamine. Estrogen administration decreased dopamine content in all the glands investigated. The significance of these results in relation to the physiology of the pars distalis is discussed.