PACAP is transiently expressed in anterior pituitary gland of rats: in situ hybridization and cell immunoblot assay studies

In this work the expression of PACAP (pituitary adenylate cyclase activating polypeptide) in rat anterior pituitary was demonstrated for the first time using in situ hybridization. The number of cells showing PACAP signal in intact male rats was negligible similarly to that of diestrous rats. In proestrous rats sacrificed at 10h there was a moderate increase in the expression and after a decrease at 16 h and 18 h, there was a transient peak at 20 h and then the number of labeled cells was declined again (22 h). In the cell immunoblot assay study it was observed that the number of PACAP blot forming (PACAP releasing) cells in an anterior pituitary cell culture changed according to a similar pattern as the number of PACAP expressing cells. The number of blots was also the highest when the animals were sacrificed in the evening of proestrus at 20h. The results obtained by in situ hybridization and cell immunoblot assay well correlate with each other. The above-mentioned results support our hypothesis that the enhanced expression and secretion of PACAP in the pituitary gland may be involved in ceasing the LH surge.     

Time-dependent effects of starvation on serum, pituitary and hypothalamic leptin levels in rats

Leptin is produced by white adipose tissue and other cell types and is involved in both short- and long-term appetite control. Here we studied effects of starvation on serum, pituitary and hypothalamic levels of leptin during 72 h period. Each of the starved groups was sacrificed simultaneously with the group of ad libitum fed animals. The progression of the discrete starvation response phases was monitored by testing the blood glucose, free fatty acid, urea and corticosterone levels. Starvation caused biphasic increase in corticosterone and free fatty acid levels, and significant but transient decrease in urea and glucose levels. Starvation also abolished diurnal rhythm of changes in leptin concentrations in serum and hypothalamic and pituitary tissues. Only 6 h starving period was sufficient to lock serum leptin at low levels, whereas 12 h were needed to silence leptin production/secretion in hypothalamus for the whole examined period. In contrast, leptin production by pituitary tissues of starved animals required 24 h to reach minimum, followed by full recovery by the end of starvation period. These results indicate the tissue specific pattern of leptin release and suggest that the locally produced leptin could activate its receptor in pituitary cells independently of serum levels of this hormone.     

Suppression of basal plasma cortisol and adrenal androgen concentrations, but not of ACTH and cortisol responses to hCRH by low-dose dexamethasone in rhesus monkeys

Glucocorticoid treatment at replacement doses does not result in a suppression of ACTH and cortisol responses to corticotropin-releasing hormone (CRH), while basal plasma concentrations of cortisol and adrenal androgens are efficiently suppressed 34 h after starting treatment. This finding could be demonstrated in rhesus monkeys receiving a continuous infusion of dexamethasone (1 microgram/kg per h) for 48 h and confirms our observations in patients on alternate-day prednisone therapy and in patients with congenital adrenal hyperplasia on glucocorticoid replacement therapy. We conclude that the decrease of basal adrenal steroid secretion resulting from glucocorticoid replacement therapy represents an effect on hypothalamic rather than on pituitary function.     

Influence of Bcg Administration Time on the in-Vivo Migration of Leukocytes

The temporal variation in the migration of polymorphonuclear leukocytes (PMN) induced by live BCG was studied in the mouse. Ten μl of a 5 ± 10⁶ live BCG/ml suspension or sterile saline were placed on a cell trap immediately before its subcutaneous implantation at different clock times: 0100, 0500, 0900, 1300, 1700 and 2100 in animals synchronized with L(0700-1900): D(1900-0700). Eight hours later, the cell trap was removed, prepared for histological identification and counted. PMN counts in the cell trap were maximal 480 min after implantation. Nonspecific migration was thought to occur and the peak value of leukocytes of 22.8 ± 6.1 cells/10,000 μm² was obtained when the saline cell-trap was implanted at 0500. In the BCG-treated mice, a circadian rhythm was observed in the migration of leukocytes. The acrophase was at 1700. The results support the hypothesis that the circadian stage of antigen encounter influences the magnitude of the immune response.     

Influence of Bcg Administration Time on the in-Vivo Migration of Leukocytes

The temporal variation in the migration of polymorphonuclear leukocytes (PMN) induced by live BCG was studied in the mouse. Ten μl of a 5 ± 10⁶ live BCG/ml suspension or sterile saline were placed on a cell trap immediately before its subcutaneous implantation at different clock times: 0100, 0500, 0900, 1300, 1700 and 2100 in animals synchronized with L(0700-1900): D(1900-0700). Eight hours later, the cell trap was removed, prepared for histological identification and counted. PMN counts in the cell trap were maximal 480 min after implantation. Nonspecific migration was thought to occur and the peak value of leukocytes of 22.8 ± 6.1 cells/10,000 μm² was obtained when the saline cell-trap was implanted at 0500. In the BCG-treated mice, a circadian rhythm was observed in the migration of leukocytes. The acrophase was at 1700. The results support the hypothesis that the circadian stage of antigen encounter influences the magnitude of the immune response.     

Extraction and immunochemical characterization of delta sleep-inducing peptide-like material from the porcine pituitary and adrenal gland

The naturally occurring forms of delta sleep-inducing peptide (DSIP) are not fully identified. In the present study, porcine pituitaries and adrenal glands were extracted in water, saline or acid under various conditions and immunoreactive DSIP (IR-DSIP) quantified by radioimmunoassay. The highest concentrations were measured in anterior pituitary extracts (40.8 +/- 2.6 ng/g tissue weight) recovered using water with aprotinin. However, high performance liquid chromatography (HPLC) indicated degradation of hydrophobic forms of IR-DSIP in water extracts. Extraction in acetic acid including C18 Sep-Pak purification resulted in an elution profile of IR-DSIP in adrenal extracts with a major peak coeluting with synthetic DSIP [DSIP(1-9)], whereas anterior pituitary extract showed material of higher hydrophobicity. Approximately 30% of IR-DSIP in anterior pituitary as well as in adrenal gland extracts seemed to be glucosylated, as based on concanavalin A chromatography. One of the DSIP-immunoreactive components by immunoblotting (molecular mass 25 kDa) was identified in both pituitary and adrenal gland extracts. In conclusion, several chromatographically distinct forms of IR-DSIP are present in the porcine pituitary and adrenal gland. IR material eluting as DSIP(1-9) is present in adrenal gland extract. The procedure and solution used for tissue extraction seem to be essential in order to obtain reliable elution positions on HPLC.     

Postnatal development of TRH and TSH rhythms in the rat

We previously observed that under a 12-hour light/12-hour dark schedule (lights off at 19.00 h), adult male Sprague-Dawley rats showed a circadian rhythm for serum thyroid-stimulating hormone (TSH) with a zenith near midday. In the present work, the ontogenesis of serum TSH rhythm was determined as well as pituitary TSH variations. In addition, hypothalamic and blood TRH were measured in these rats aged 15, 25, 40 and 70 days when sacrificed. As from the first age studied (15 days), a hypothalamic thyrotropin-releasing hormone (TRH) circadian rhythm was present. The mesor and the amplitude of this hypothalamic TRH rhythm increased while the rats were growing up, in contrast with the decrease observed for these parameters as far as blood TRH circadian rhythm is concerned. The time of the acrophase moved from 17.32 h in the 15-day-old rats to 13.57 h in the 70-day-old rats, being constantly in phase opposition with the blood TRH acrophase. The low amplitude pituitary TSH circadian rhythm detected in the young rat disappeared in the adult while, in contrast, the serum TSH rhythm became consistent to reach the well-characterized circadian midday peak in the 70-day-old rats.     

Molecular pathogenesis of lipoid adrenal hyperplasia and adrenal hypoplasia congenita

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH in which the synthesis of all gonadal and adrenal cortical steroids is markedly impaired. Lipoid CAH may be caused by the defect in either the steroidogenic acute regulatory (StAR) protein or the P450scc. More than 34 different mutations in StAR gene have been identified. Clinically, most of the patients manifest adrenal insufficiency from 1 day to 2 months of age, but some patient show delayed onset of adrenal insufficiency. Affected 46, XY subjects do not show pubertal development, whereas affected 46, XX subjects undergo spontaneous feminization, breast development and cyclical vaginal bleeding at the usual age of puberty.

X-linked adrenal hypoplasia congenital (AHC) is a rare congenital adrenal disorder characterized by severe adrenal insufficiency and hypogonadotropic hypogonadism. More than 80 different several intragenic mutations of DAX-1 have been identified. The failure of pubertal development may be caused by either abnormal hypothalamic or pituitary regulation of gonadotropin secretion. In addition, although the testicular steroidogenesis is largely intact, the functional maturity of Sertoli cells and also spermatogenesis are impaired. The type of mutation does not predict clinical phenotype. Thus, unified mechanism how DAX-1 gene defect gives rise to adrenal insufficiency, hypothalamic/pituitary hypogonadism and impaired spermatogenesis remains established.     

Presence of the Novel Pituitary Protein „7B2” in Bovine Chromaffin Granules: Possible Co-Release of 7B2 and Catecholamine as Induced by Nicotine

We observed the presence of the novel pituitary protein "7B2" and its release in the bovine adrenal medulla. The 7B2 concentration (mean +/- SEM) in extracts of the bovine adrenal medulla was 952 +/- 155 pg/mg tissue (n = 6). 7B2 was distributed in the chromaffin granule fraction prepared from the bovine adrenal medulla and was released by high K+ and/or nicotine from cultured cells of the bovine adrenal medulla. Co-release of 7B2 with catecholamine induced by nicotine from the cultured bovine chromaffin cells was also observed. In an analysis of the bovine adrenal medulla chromaffin granule fraction on gel permeation chromatography, there was a major peak with an apparent molecular weight of 45,000, whereas a major peak with an apparent molecular weight of 20,000 was found in that on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On reverse-phase HPLC, a major peak with a retention time of 35 min was observed in the bovine chromaffin granule fraction and in the bovine anterior pituitary extract. These findings indicate that 7B2 is a secretory protein in the bovine adrenal medulla. The possibility that 7B2 might be released with catecholamine, possibly in response to stress, warrants investigation.     

Corticotrophin-releasing factors in the hypothalamus of the developing fetal sheep.

Corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) are secreted from the hypothalamic median eminence to elicit the secretion of ACTH from the pituitary corticotrophs. During fetal development there is progressive maturation of the hypothalamic-pituitary-adrenal axis, manifest as increasing plasma ACTH and cortisol concentrations, which in species such as sheep culminates in the onset of birth. However, the precise nature of the hypothalamic signal controlling fetal pituitary ACTH secretion remains poorly understood. To investigate the ontogeny of this hypothalamic signal, the present study examined immunoreactive and bioactive ACTH-releasing factors in the developing fetal sheep hypothalamus. Immunoreactive CRH and AVP were measured by radioimmunoassay in extracts of hypothalami taken at day 70, day 100, and day 130 gestation (term = 145 days). There was a progressive and significant (P < 0.01) increase in hypothalamic CRH and AVP concentrations which was particularly marked between d100 and d130 gestation. AVP was always present in higher concentrations that CRH, although this difference was significantly reduced by day 130 gestation as the result of a large increase in the content of CRH relative to AVP. Sephadex G50 chromatography revealed that immunoreactive CRH and AVP in hypothalamic extracts existed as single molecular forms corresponding to synthetic peptides at each gestational age. In addition, these immunoreactive forms of CRH and AVP possessed significant ACTH-releasing bioactivity as measured in primary cultures of adult sheep anterior pituitary cells. Furthermore, significant bioactivity was present in high and low molecular weight fractions eluted after chromatography which did not contain any CRH or AVP immunoreactivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of placenta and maternal serum on prolactin secretion in vitro

The effects of the placenta and maternal sera on the secretion of prolactin (Prl) were examined in vitro. Placentae were obtained on each of Days 8-11 of pregnancy and extracted in 2.0% butanol-saline. To determine if these extracts could inhibit Prl secretion in vitro, dispersed anterior pituitary cells were incubated with placental extracts containing 1.0 placental equivalent obtained on each of Days 8-11 of pregnancy. Prl secretion was not affected by extracts of placentae obtained on Day 8 but was significantly inhibited by placental extracts obtained on Days 9-11 of pregnancy. In fact, progressively more mature placentae induced greater degrees of Prl inhibition. Extracts of placentae that were obtained on each of Days 8-11 of pregnancy, normalized on the basis of protein and tested for a 24-h period in the dispersed pituitary bioassay, caused the same degree of inhibition over Prl release. Additionally, placental protein from any given day (Days 8-11) of pregnancy induced a highly significant dose-dependent inhibition over Prl secretion. Equivalent amounts of a nonspecific protein, bovine serum albumin, had no effect. These findings indicate that the placenta does indeed contain a Prl inhibitory factor whose specific activity remains relatively constant between Days 8 and 11 of pregnancy. To determine if the inhibitory activity is humoral, maternal sera collected on each of Days 8-11 of pregnancy were placed in culture with dispersed pituitary cells at a concentration of 15.0%. Concomitant with gestational maturity, there was a progressively greater inhibition of Prl release. These findings indicate that the placenta may secrete a substance into the blood which suppresses Prl release directly at the level of the pituitary gland.     

Localized glucoprivation of hindbrain sites elicits corticosterone and glucagon secretion

Glucose is required for brain energy metabolism. Decerebration, aqueduct occlusion, and cannula mapping studies have established that glucose-sensing cells capable of eliciting feeding and adrenal medullary responses to glucoprivation are localized in the hindbrain. Glucoprivation also evokes corticosterone and glucagon secretion, but the location of receptors mediating these responses is unknown. To determine whether glucoreceptive sites controlling these responses are present in the hindbrain, we administered the antiglycolytic agent, 5-d-thioglucose (5TG, 24 microg in 200 nl) into brain stem sites through implanted cannulas and examined plasma concentrations of corticosterone and glucagon. Both hindbrain and hypothalamic sites were tested. Blood was collected remotely from intra-atrial catheters at 0, 30, 60, 90, 120, 180, and 240 min after 5TG or control injection. Caudal hindbrain 5TG injections potently increased circulating corticosterone and glucagon concentrations. For corticosterone, the mean peak response (maximum concentration minus time 0 concentration) elicited at positive sites (23 of 40 sites) was 391 ng/ml (SE = 16). For glucagon, the mean peak response at positive sites (27 of 40 sites) was 46 pg/ml (SE = 6). Glucoprivically evoked glucagon secretion was abolished by the ganglionic blocker, hexamethonium, but not by adrenal denervation. Six of twenty-five hypothalamic sites were positive for corticosterone secretion, yielding plasma levels of 279 +/- 23 ng/ml, but none of the hypothalamic injection sites elevated glucagon concentrations. Results demonstrate that receptor cells responsive to glucose deficit and capable of increasing corticosterone and glucagon concentrations exist within the hindbrain, thus further delineating central glucoregulatory neural circuitry.     

Evolution of the adaptive functions of hypophyseal hormones detected by using hypothalamic melanostatin

The content of two pituitary tropins (MSH and ACTH) has been determined in rats which were injected by the synthetic melanostatin to suppress MSH of the intermediate part of pituitary by radioimmune analysis. Besides, the function of the adrenal cortex according the content of corticosterone in various tissues was estimated. It has been revealed that the pituitary-adrenal complex activation is caused by the hypothalamic melanostatin suppression of the MSH secretion. All these testify the ACTH and MSH definite role in organisms adaptive reactions at different evolution steps.     

In vitro effect of cortisol and urotensin I on arginine vasotocin and isotocin secretion from pituitary cells of gilthead sea bream Sparus aurata

This study aimed at determining whether in vitro secretion of two neuropeptides, arginine vasotocin (AVT) and isotocin (IT), from pituitary cells of gilthead sea bream Sparus aurata was affected by cortisol and urotensin (UI). Pituitary cells were exposed to 1·4 × 10^?8, 1·4 × 10^?7 and 0·4 × 10^?6 M cortisol and 10^?12, 10^?10 and 10^?8 M UI for 6, 24 and 48 h, respectively. AVT and IT contents were determined in the culture media by high‐performance liquid chromatography (HPLC). An increase in AVT secretion and a decrease in IT secretion were observed at all cortisol doses. UI increased AVT secretion after 6 h of incubation at all doses. After 24 h, however, only the highest dose of UI still displayed an effect. IT secretion was not influenced by UI. It was thus demonstrated that cortisol does influence AVT and IT secretion from S. aurata pituitary cells, while UI regulates AVT secretion, as a component of hypothalamic–pituitary–interrenal (HPI) axis in this species.     

Effects of lipopolysaccharide on hypothalamic-pituitary-adrenal axis in vitro

The possible involvement of lipopolysaccharide (LPS) and interleukin-1β (IL-1β) and their eventual interplay in CRH and ACTH release from cultured hypothalamic and pituitary cells respectively, have been studied. IL-1β was able to activate the hypothalamo-pituitary-adrenal axis at both hypothalamic and pituitary sites; LPS showed no direct action at hypothalamic level but it was able to inhibit basal and IL-1β-induced ACTH release: this could be responsible for a blunting of the adrenal cortex response that normally occurs in septic shock syndrome.     

Circadian rhythm of corticotropin releasing factor-like immunoreactivity in rat hypothalamus

Levels of hypothalamic corticotropin releasing factor-like immunoreactivity (CRF-LI) were measured by radioimmunuoassay (RIA) over a 24 hour light-dark cycle and found to exhibit two peaks. One peak was detected at 1100 hr and a secondary smaller peak was found at 2000 hr. The trough between the two peaks was detected at 1700 hr which coincided with the peak in plasma corticosterone levels. The results are consistent with a decreased level of hypothalamic CRF-LI at 1700 hr reflecting an increased release of peptide followed successively by the release of ACTH and corticosterone.     

人参茎叶皂甙对大鼠垂体催乳素分泌的影响及其机制的探讨

本实验观察了人参茎叶皂甙(GSLS)对雄性大鼠血浆催乳素水平、垂体催乳素细胞超微结构和下丘脑中枢神经递质的影响。结果表明:5～100mg/kg的GSLS可刺激催乳素的释放,剂量加大反而无效;GSLS还可拮抗急性饥饿所致的大鼠垂体催乳素细胞超微结构的损伤;GSLS能分别使大鼠下丘脑中多巴胺和5-羟色胺含量增高和降低。结果表明,GSLS有刺激垂体催乳素分泌的作用,其机制可能与其直接作用于垂体细胞和/或经下丘脑中多巴胺和5-羟色胺含量的变化有关。     

Effects of experimental hypothyroidism on the development of the hypothalamo-pituitary-adrenal axis in the rat

Hypothyroid pups were obtained by adding methimazole in the mother's drinking water from day 15 of gestation and sacrificed at 4, 8 or 15 days. Circulating corticosterone decreased at all ages, while CBG concentrations diminished at day 4, increased at day 8 and did not change at day 15 in hypothyroid rats. As opposed to controls, plasma ACTH concentrations decreased steadily with age while there was an accumulation of ACTH in the anterior pituitary of hypothyroid 15-day-old rats. Anterior pituitary POMC contents were unaffected by the treatment. In the hypothalamic PVN, CRF mRNA levels in the total population of CRF-synthesizing cells and in the CRF+/AVP+ subpopulation were below those of controls whatever the age considered while AVP mRNA in the CRF+/AVP+ subpopulation did not change at day 4 and decreased at day 8 and 15 in hypothyroid animals. Both the number of cell bodies expressing detectable levels of CRF mRNA and the percentage of CRF and AVP colocalization decreased at day 4 and were unchanged thereafter. CRF and AVP immunoreactivity in the zona externa of the median eminence increased with age but was not affected by methimazole treatment. The concentration of AVP mRNA in the magnocellular cell bodies of the PVN and the SON as well as AVP immunoreactivity in the zona interna of the median eminence were not changed by the treatment at days 4 and 8. In hypothyroid 15-day-old rats, SON AVP mRNA increased, AVP immunoreactivity decreased while plasma osmolality was enhanced. In conclusion, our data demonstrate that experimental hypothyroidism impairs specifically the maturation of hypothalamic parvocellular CRF and AVP gene expression during the stress hyporesponsive period. These observations suggest that the physiological peak in plasma thyroxine concentrations that occur between day 8-12 may participate in the maturation of hypothalamic CRF- and AVP-synthesizing cells.     

Effects of acute ovariectomy on anterior pituitary gland follicle-stimulating hormone and luteinizing hormone secretion in the metestrous rat

Changes at the anterior pituitary gland level which result in follicle-stimulating hormone (FSH) release after ovariectomy in metestrous rats were investigated. Experimental rats were ovariectomized at 0900 h of metestrus and decapitated at 1000, 1100, 1300, 1500, 1700 or 1900 h of metestrus. Controls consisted of untreated rats killed at 0900 or 1700 h and rats sham ovariectomized at 0900 h and killed at 1700 h. Trunk blood was collected and the serum assayed for FSH and luteinizing hormone (LH) concentrations. The anterior pituitary gland was bisected. One-half was used to assay for FSH concentration. The other half was placed in culture medium for a 30-min preincubation and then placed in fresh medium for a 2-h incubation (basal FSH and LH release rates). The basal FSH release rate and the serum FSH concentration rose significantly by 4 h postovariectomy and remained high for an additional 6 h. The basal FSH release rate and the serum FSH concentration correlated positively (r=0.71 with 72 degrees of freedom) and did not change between 0900 and 1700 h in untreated or sham-ovariectomized rats. In contrast, the serum LH concentration and the basal LH release rate did not increase after ovariectomy. Ovariectomy had no significant effect on anterior pituitary gland FSH concentration. The results suggest that the postovariectomy rise in serum FSH concentration is the result, at least in part, of changes which cause an increase in the basal FSH secretion rate (secretion independent of the immediate presence of any hormones of nonanterior pituitary gland origin). The similarities between the selective rises in the basal FSH release rate and the serum FSH concentration in the ovariectomized metestrous rat and in the cyclic rat during late proestrus and estrus raise the possibility that an increase in the basal FSH release rate may be involved in many or all situations in which serum FSH concentration rises independently of LH.