Existence of "endorphin cells" in the adenohypophysis of the cat; comparison with "corticotropin cells." Immunocytochemical study]

Indirect immunofluorescence technique with anti-17-39ACTH and anti beta-endorphin sera has allowed us to detect "corticotropic cells" in the anterior and intermediate lobes of the adenohypophysis of the male cat. The corticotropic cells of the anterior lobe are localized in the median zone; they are PAS-positive and appeared intensively coloured in dark blue with the Herlant's tetrachrome. All the cells of the intermediate lobe react with the anti-17-39ACTH serum. Using an anti-beta-endorphin serum, we have observed that all the corticotropic cells of the anterior lobe react; but in the intermediate lobe, only a part of "corticotropic cells" react with the anti-beta-endorphin serum.     

Demonstration by immunofluorescence of adenohyphysis corticotropin and melanotropin cells in Erythrocebus patas, Cercopithecus aethiops and Papio hamadryas]

Using antibodies against synthetic corticotropic hormones (1-24 ACTH and 17-39 ACTH), and melanotropic hormones (alpha-MSH and beta-MSH), it is possible to identify corticotropic and melanotropic cells in the adenohypophysis of three species of monkeys : Erythrocebus patas, Cercopithecus aethiops and Papio hamadryas. The corticotropic cells are numerous in the anterior lobe in both the adult and infant male and female monkeys of these three species. The intermediate lobe reacts with antibodies against ACTH and also with antibodies against the two MSH. In the anterior lobe, the corticotropic cells react also with anti-beta MSH antibody but not with the anti-alpha MSH antibody.     

Hypophyseal corticotropic activity during perinatal period: ontogenesis and regulation

1. In the fetal rat hypophysis, the "opio-melano-corticotropic" cells were immunocytologically revealed earlier in the pars distalis (day 16) than in the pars intermedia (day 17). At an early stage of embryonic development, some factors, possibly of nervous origin, could exert inductive influence on the differentiation and/or the increase in the number of immunoreactive cells. 2. Several forms of ACTH, characterized by their apparent molecular weight, bioactivity and immunoreactivity, can be observed when acid extracts are subjected to gel filtration on Sephadex G 50 fine columns. The ratios between these ACTH forms change during fetal development; the rise in bioactive ACTH content during the last days of gestation, is correlated with an increase of the "intermediate" and "little" molecular forms of ACTH and a parallel decrease of the "big" form. 3. The hypophyseal corticotropic activity is highest on days 18-19 of gestation. Between days 17 and 19, the cortico-stimulating activity is largely independent of the hypothalamus although the fetal hypothalamus shows light hypophyso-stimulating influence. In contrast, between days 19 and 21, the hypophyseal corticotropic activity is mostly under the hypothalamic control; the ACTH release is greatly reduced in its absence. 4. Circulating corticosteroids exert a negative feedback directly at the hypothalamic and pituitary levels. Such negative feedback could explain the decrease of the corticotropic activity of the pituitary during the last three days of gestation and the first postnatal days. 5. The fetus at the end of gestation, as the newborn, is responsive to stress; however, the extent of the pituitary response is weaker than in the adult.     

Evidence for FSH-like material in ACTH granules of certain corticotropic cells in the pituitary of the pig

Summary The ultrastructural immunocytochemical peroxidase-antiperoxidase technique used on serial sections reveals that certain corticotropic cells contain both ACTH and FSH, but not LH (ACTH/FSH cells). To determine the specificity of the anti-FSH staining in these cells, immunocytochemical absorption experiments were performed. The results indicate that (1) anti-FSH and -ACTH antisera are bound to different antigens in the corticotropic cell, and (2) anti-FSH staining is specific for a FSH-like antigen. In the ACTH/FSH cells both hormones are stored in the same secretory granules, distributed among other granules that contain only ACTH.     

Immunohistochemical study of the differentiation of the cephalic lobe of the adenohypophysis in chick embryos]

It was shown by means of indirect immunofluorescence that ACTH appeared in the cephalic lobe of the chick embryo pituitary beginning from the 8th day of the development. A new tissue-specific antigen (A3) is revealed, which is localized in the cephalic lobe of adenohypophysis and becomes manifest at the 7th day of embryogenesis.Quantitative analysis of ACTH and A3 localization in the cells of 11-day chick embryo adenohypophyses allows a conclusion that A3 is localized in corticotropic cells.     

The effects of colchicine,vinblastine and cytochalasins on the corticotropic responsiveness and ultrastructure of inner zone adrenocortical tissue in the Pekin duck

Tissues slices superfused with medium containing no ACTH released only traces of corticosterone. Addition of ACTH to the medium caused the rate of corticosterone release to increase to a maximum about 45 min after the addition of ACTH, after which time it either remained constant or started to wane slightly. The rate of release was affected by tissue thickness; the maximum rate of corticosterone release occurred when the tissue slices were 200 microns. Stimulated adrenocortical cells had large spherical nuclei, numerous mitochondria with tubular cristae, numerous lipid droplets, and a large amount of smooth endoplasmic reticulum. Many cells had an extensive network of microfilaments adjacent to the plasma membrane and some microtubules. Prolonged superfusion caused degenerative changes in some of the cells. Both cytochalasin B and cytochalasin D, dissolved in DMSO before addition to the superfusion medium, inhibited the corticotropic responsiveness in a dose-dependent manner. Control tissue samples superfused with medium containing DMSO, but no ACTH and no cytochalasin, released significantly more corticosterone than corresponding unstimulated samples. Few or no microfilaments were observed in adrenocortical cells after treatment with cytochalasin. Neither colchicine nor vinblastine had any discernible effect on the corticotropic responsiveness. After treatment with colchicine, adrenocortical cells had an ultrastructure characteristic of inner zone stimulated cells except that they were mainly devoid of microtubules.     

Cadmium induces changes in corticotropic and prolactin cells in the Podarcis sicula lizard pituitary gland

We analyzed the effect of cadmium on corticotropic (ACTH) and prolactin (PRL) cells in the pituitary gland of the Podarcis sicula (P. sicula) lizard under chronic exposure to this metal. Adult lizards were given CdCl₂ in drinking water at the dose of 10 µg/10 g body mass for 120 days. Light microscopy was performed after histological and immunohistochemical staining, and the effects were followed at regular time intervals up to 120 days post-treatment. We detected substantial variations in the general morphology of the pituitary: unlike the control lizards in which the gland appeared compact, the treated lizards showed a glandular tissue with dilated spaces that were more extensive at 90 and 120 days. PRL and ACTH cells showed an increase in occurrence and immunostaining intensity in treated lizards in comparison with the same cells of control animals. This cellular increase peaked for PRL at 30 days in the rostral, medial and also caudal pars distalis of the gland. ACTH cells appeared to increase markedly after 60 days of treatment in both the pars distalis and the pars intermedia. Again, at 60 days small, isolated ACTH cells were also found in the caudal pars distalis in which these cells were generally absent. However, at 120 days both these cellular types showed an occurrence, distribution and morphology similar to those observed in the control lizards. In lizards, protracted oral exposure to cadmium evidently involves an alteration of the normal morphology of the gland and an inhibitory effect of ACTH and PRL cells, since they increase in occurrence and immunostaining. Yet in time the inhibitory effect of cadmium on ACTH and PRL cells falls back and their occurrence appears similar to that of the control lizard.Key words: cadmium, corticotropic and prolactin cells, lizard.     

Immunohistochemical Identification of the Adenohypophysial Cells in the Indian Paddy Field Frog,Rana limnocharis

Cells of the pituitary pars distalis (PD) and pars intermedia (PI) in the frog Rana limnocharis have been identified by an unlabelled antibody enzyme method using antisera developed in rabbit against mammalian hypophysial hormones. On the basis of their immunoreactivity, six types of cells, viz. thyrotropic (TSH), gonadotropic (GTH), prolactin (PRL), growth hormone (GH), corticotropic (ACTH) and melanotropic (MSH), cells have been recognized. GTH and PRL cells are distributed throughout the PD. GH cells usually occur in the anterodorsal and central region of the gland. Immunoreactive TSH cells are fewer in number and are localized in the ventromedian region of the PD. Cells showing immunoreactivity to ACTH 1–24 antiserum are encountered in the rostroventral part of the PD. Cells of the PI also show immunoreactivity to ACTH 1–24 antiserum. PI cells cross-react with α-MSH antiserum at all dilutions up to 1: 50 000. However, when the same antiserum was used at dilutions up to 1: 20 000, the ACTH cells of the PD also showed cross-reactivity.     

A morphometric study of subcellular responses in frog pituitary corticotropic cells to constant environmental conditions

Ultrastructural morphometry (point-counting method) was used to evaluate stereological parameters of rough endoplasmic reticulum (RER), Golgi complex, secretory granules, mitochondria and lysosomes of Rana ridibunda pituitary corticotropic (ACTH) cells in animals subjected to constant environmental conditions (CEC) in relation to temperature and photoperiod. During this experiment, corticotropic cells are particularly sensitive to the effects of changes in environmental conditions in such a way that they course through a phase of RER and Golgi complex hypertrophy after 7 days on CEC and a phase of enhancement in the amount of secretory granules (the 11th day). After 15 days on CEC, the cells seemingly recuperate almost all the control evaluate parameters. These results strongly suggest that, in frog kept in captivity, ACTH cells play an important role in the acclimation process to new constant environmental conditions.     

Functional differences between two structurally distinct types of steroidogenic cell in the avian adrenal gland

Summary There are two regions of steroidogenic cells in the duck adrenal gland. An outer, subcapsular zone (SCZ), consisting of cells with irregularly shaped nuclei, shows relatively little smooth endoplasmic reticulum and mitochondria with shelf-like cristae. This region surrounds the inner zone (IZ) of the gland which is comprised of smaller cells with rounded nuclei, a more abundant smooth endoplasmic reticulum and mitochondria with tubular cristae. When samples of tissue from these distinct regions of the gland are superfused in vitro with media containing concentrations of 1–24 ACTH ranging from 100 to 1000 ng per ml (0.034 to 0.34 M) the steroidogenic cells in both zones release corticosterone in a dose-dependent manner. The dose-responsiveness of both the SCZ and the IZ cells over this range is a complex quadratic function of the 1–24 ACTH concentration in the medium and the semilogarithmic linear portions of the dose-response curves are restricted to a narrow midrange of ACTH concentrations. Throughout the dose-response range, however, the steroidogenic cells of the IZ are more responsive to corticotropic stimulation than are the cells of the SCZ. The cells of the two zones are further distinguished by their responses to a challenge for a second time with medium containing 1–24 ACTH; the responses of the IZ cells to a second challenge were greater than those of the SCZ cells, and at a high concentration of ACTH the SCZ slices showed no significant second response.This work was supported by a grant from the National Science Foundation (PCM 79-15777) to James Cronshaw and W.N. Holmes     

Effects of bilateral olfactory bulbectomy on the anterior pituitary corticotropic cell activity in male rats.

Bilateral olfactory bulbectomy (OB) has drastic biochemical and behavioral effects and is often associated with an increase in plasma corticosterone concentrations. This experiment examined the effects of OB on adrenocorticotropin (ACTH) and corticosterone release under basal and stress conditions and on proopiomelanocortin (POMC) gene expression. Bulbectomy potentiated hypophysal ACTH and adrenal corticosterone release induced by ether stress but had no effect on ACTH release under basal conditions, despite a significant increase of circulating corticosterone. POMC gene expression was stronger (+60%) in OB rats than in sham-operated rats. These results suggest that olfactory bulbectomy substantially altered the negative feed-back exerted by glucocorticoids on anterior pituitary corticotropic cells in the male rat.     

Identification by immunofluorescence of various categories of anterior pituitary gland cells (somatotropin, prolactin, corticotropin, melanotropin, gonadotropin, and thyrotropin cells) in the spermophile (Citellus variegatus)]

Using antibodies against human STH, rat prolactin, 1-24, 17-39-synthetic ACTH, alpha- and beta-synthetic MSH, ovine LH, porcine LH beta, bovine TSH saturated with LH, it is possible to identify somatotropic, prolactin, corticotropic, melanotropic, gonadotropic and thyreotropic cells in the adenohypophysis of rodents Citellus variegatus and Graphiurus murinus. The topographical repartition and morphological characters of these cells are described.     

Effect of calcium on adrenocortical secretion

The authors investigated the effect of calcium on the basal cortisol secretion and on the adrenocortical secretory reserve, the indicator of which is the so-called ACTH stimulation test. They revealed that during acute hypercalcaemia there is a significant increase of the basal cortisol secretion. The adrenocortical secretory reserve is significantly reduced during hypercalcaemia, this being manifested by a smaller rise of cortisolaemia after ACTH. The Synacthen test repeated after a 48-hour interval is fully reproducible under normocalcaemia. The basal values and the response to Synacthen do not differ from the control examination. The stimulatory action of calcium can be explained indirectly via other humoral factors, the production of which is enhanced by calcium but also the function of calcium which acts as the so-called "second messenger" in steroidogenesis. Calcium has, however, probably also a direct corticotropic effect, resembling that of ACTH.     

Allometric relations of total volumes of prolactin cells and corticotropic cells to body length in the annual cyprinodont Cynolebias whitei: effects of environmental salinity,stress and ageing

Summary An analysis of the allometric relations of the total volumes occupied by prolactin (PRL) and corticotropic (ACTH) cells (PRL volume and ACTH volume, respectively) to body length and a study of the immunocytochemical staining intensity of PRL and ACTH cells were used to determine the differences in activity of PRL and ACTH cells in freshwater-reared and in saltwater-reared Cynolebias whitei during the entire lifespan of this annual cyprinodont fish. An inflection in the allometric relation of PRL volume to body length was observed in fish of one-week old. The relatively large PRL volume in younger fish may be related to PRL cell activity before hatching. No inflections were observed in the allometric relations of PRL volume and ACTH volume to body length at the onset of maturation and the onset of ageing, indicating that the increased pituitary growth in maturing and ageing C. whitei is not the result of changes in PRL or ACTH cells. The slope of the allometric relation of PRL volume to body length in freshwater-reared fish was significantly steeper than the slope in saltwater-reared fish. The PRL volume in adult freshwater-reared fish was eight times larger than that in saltwater-reared fish of the same length. The intensity of immunocytochemical staining of saltwater PRL cells was significantly reduced. These volumetric and staining differences correspond to the low functional demand put upon PRL cells in saltwater-adapted fish. In contrast, the slope of the allometric relation of ACTH volume to body length and the intensity of immunocytochemical staining of ACTH cells were similar in freshwater-reared and in saltwater-reared fish. It is concluded that the functional demand put upon ACTH cells is similar in freshwater-reared and saltwater-reared C. whitei; the involvement of ACTH cells in the osmoregulation of the fish in both environments is similar.     

Effect of neonatally injected ACTH and ACTH analogues on eye-opening of the rat.

Three-day old rats were injected subcutaneously either with natural purified pig ACTH (ACTH 1–39), ACTH 1–24, or ACTH analogues as long-acting zinc-phosphate preparations. ACTH 1–39, ACTH 1–24, ACTH 1–18, ACTH 1–16 accelerated the time of eye-opening, whereas an extract of corticosteroids produced $\text{in}$$\text{vitro}$ by excised adrenals of ACTH-treated three-day old rats was ineffective. Injections with ACTH on the twelfth day of life had no effect on eye-opening. It is concluded that a neonatal injection with ACTH or closely related analogues with markedly less corticotropic activity can accelerate the time of eye-opening. This effect is not mediated by the adrenal cortex. The sensitive period for it appears to be shortly after birth.     

Phorbol ester-induced down-regulation of protein kinase C abolishes vasopressin-mediated responses in rat anterior pituitary cells

The role of protein kinase C (PKC) in the multihormonally regulated ACTH secretory responses of rat anterior pituitary cells was examined in control cells or after pretreatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of PKC. Using affinity-purified polyclonal antiserum raised against purified rat brain PKC, immunoprecipitable PKC was demonstrated in [35S]methionine-labeled cells appearing as a doublet of 78/80 kilodaltons. Long-term treatment (24 h) of cells with 0.6 microM TPA caused the specific loss of immunologically reactive PKC. Consistently, TPA pretreatment decreased the amount of phosphatidylserine-dependent protein kinase activity measured in vitro by 90%. In control cells, vasopressin (AVP) stimulated ACTH secretion and potentiated ACTH secretion stimulated by CRF. After a 24-h treatment with 0.6 microM TPA, secretory responses to AVP and the potentiating effect of AVP on CRF action were completely abolished. In contrast, CRF action on ACTH secretion, thought to be mediated by cAMP, was unaffected. Similarly, forskolin- and 8 bromo-cAMP-induced ACTH secretion remained unchanged after TPA pretreatment. These results indicate a crucial role for PKC in mediating the effects of AVP on ACTH secretion and on the potentiating action of AVP on CRF-induced secretion from corticotropic cells of the anterior pituitary.     

Effects of adrenal medullectomy and passive immunization with an anti-CRF antiserum on the corticotropin secretion in response to stress in Long-Evans rats

Ether stress-induced ACTH and corticosterone secretion is similar in 60 days adrenal medullectomized and intact Long-Evans Rats. Passive immunisation with a 41-CRF antiserum impairs corticotropic function after stress. This decrease is more pronounced in adrenal medullectomized than in intact animals. These results suggest that epinephrine plays a minor role on the stress induced ACTH secretion. This role can be demonstrated only after immunoneutralisation of endogenous 41-CRF.     

Evidence for the involvement of nitric oxide in the control of steroid secretion by the frog adrenal gland

Nitric oxide (NO) has been found to modulate the response of rat, bovine and human adrenocortical cells to corticotropic factors. The aim of the present study was to investigate the possible involvement of NO in the control of corticosteroid secretion in the frog Rana ridibunda. Histochemical studies using the NADPH-diaphorase reaction and immunohistochemical labeling with antibodies against NO synthase (NOS) revealed that NOS is exclusively expressed in chromaffin cells. The NO donor sodium nitroprusside (SNP) and the NO synthase inhibitor Nw-nitro- -arginine ( -NO₂Arg) did not modify the spontaneous production of corticosterone and aldosterone by perifused adrenal slices. Similarly, -NO₂Arg had no effect on the secretory responses induced by ACTH, angiotensin II (AII) and endothelin-1 (ET-1). In contrast, SNP significantly inhibited the stimulatory effects of ACTH, AII and ET-1 on corticosterone and aldosterone secretion. These data provide the first evidence for a modulatory role of NO on adrenocortical cell activity in amphibians.     

Seasonal rhythm in the hypothalamo-hypophyseal-adrenal function of the chicken.

The seasonal rhythm in hypothalamo-hypophyseal-adrenal function was studied in 3-week-old, meat-hybrid chickens, bred under standard conditions, CRF content in the median eminence and ACTH content in the adenohypophysis showed the maximum in February, the minimum in August, to return practically to the February level by November. Adrenal corticosterone production was maximum in February decreased by May and stayed at the low level for the further months of the year.The increase in autumn of the CRF content in the median eminence and of the ACTH content in the adenohypophysis, with unaltered corticosterone production suggests a reduced functioning of the mechanisms regulating hypothalamic release. It is assumed that the winter maximum in the activity of the corticotropic system is due to the endogenous annual rhythm.     

Détection par immunofluorescence des cellules corticotropes et mélanotropes dans l'hypophyse de la grenouille,Rana temporaria L., au cours du développément

Summary During the embryonic and larval developmental stages of the frog, Rana temporaria L, anti- 1–24, 17–39 corticotropine, and MSH antibodies were used to define, with immunofluorescence technique, the appearence of corticotropic and melanotropic cells.A very small number of fluorescent corticotropic cells appears for the first time during the embryonic stage (10 mm), just before the differentiation of the pars intermedia. The cells are small, their large nucleus is surrounded by a fine rim of fluorescent cytoplasm.During premetamorphic stage, the anti-ACTH antibodies (anti- 1–24 and anti- 17–39 corticotropine) reveal more fluorescent cells in the whole pars distalis. The pars intermedia cells can also be visualized by both antisera.At the end of prometamorphosis and during climax the corticotropic cells show a more precise localization. As in adult frog pars distalis, they are concentrated in the rostral half of the lobe. With the same technique anti- and MSH antibodies reveal only the cells of the pars intermedia. No other cell type of the pars distalis reacts with these antibodies. This technique has the advantage to show that the ACTH and the MSH cells appear very early during the embryonic life.