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In the present study, the effect of GHRP-2 on GH release was evaluated in rat primary pituitary cells, and the results were compared with those elicited by GHRP-6. In the rat system, GHRP-2, like GHRP-6, acts synergistically with GRF to release GH. Co-administration of GHRP-2 and GHRP-6 at maximal concentrations had no further effect on GH release than either one alone. The GHRP's were able to desensitize cells to each other, but not to GRF. The effect of GHRP-2 was inhibited by Peptide Antagonist, but was not affected by a GRF antagonist. In conclusion, GHRP-2 was found to stimulate GH release from rat pituitary cells via the same receptor and mechanism as GHRP-6, despite the structural difference between the peptides.

Neuramide (NMD), a substance found in crude preparations of porcine stomach extract, is a viral inhibitor that also has putative immunostimulatory effects. The effects of NMD on stress-hormone (ACTH and prolactin—PRL) release were assessed in in vivo and in vitro studies. In the former, blood levels of corticosterone and PRL were measured in NMD-treated male rats. In vitro experiments were performed to evaluate the effects of NMD and three of its fractions (obtained with high performance liquid chromatography) on ACTH and PRL release from perfused rat pituitary slices. NMD increased plasma corticosterone levels in vivo and produced dose-dependent increases in in vitro pituitary release of ACTH. No effects on PRL secretion were observed in vivo or in vitro . The stimulatory effects on ACTH release were caused by the NMD fraction with a molecular weight of >5000

The influence of two buffer systems (Hepes and CO 2 HCO 3 − ) on intracellular Ca2+ ([Ca2+]i) transients evoked by TRH and by elevated K+ were studied in single, and small clusters of, clonal rat pituitary GH4C1 cells using Fura 2. The steady-state level of [Ca2+]i was virtually identical in Hepes and CO 2 HCO 3 − . In both buffers, addition of TRH induced a transient increase in [Ca2+]i which attained a significantly higher peak in Hepes (357 ± 43 nM) when compared with values measured in the presence of CO 2 HCO 3 − (184 ± 21 nM). In Hepes, the basal IP3-level was higher than in CO 2 HCO 3 − . The TRH-evoked increase in IP3 was higher in magnitude in Hepes than in CO 2 HCO 3 − , although the stimulated/basal ratio was not different between the two buffers. The buffer composition had no effect on the specific binding of 3H-TRH to the cells. Furthermore, the amplitude of the increase in [Ca2+]i evoked by 50 mM K+ was identical in both buffers. TRH and K+ had no effect on pHi in either buffer. The present results indicate that HCO3− has an influence on TRH-induced Ca2+ transient, at least in part by modifying the TRH-evoked production of IP3.

3,5-Di-iodo-l-thyronine (T2) is a naturally occurring metabolite of thyroxine (T4). Contrary to earlier findings, T2 has recently been shown to have rapid effects in rat liver and in mononuclear blood cells. In the experiments described here, T2 was tested to determine whether it has a TSH suppressive effect in rats in vivo and in rat pituitary fragments in vitro. In experiments over 2 weeks in rats in vivo, low doses of T2 (20–200 μg/100 g body weight per day) had no significant influence on body and organ weights, but significantly decreased TSH and T4 serum concentrations. At 200 μg/100 g per day, T2 suppressed TSH to 43% and T4 to 29% of control levels. At 1–15 μg/100 g per day, 3,5,3′-tri-iodo-l-thyronine (T3), used as a comparison to T2, had significant effects on TSH and T4 levels, and also on body weight. Fifteen μg T3/100 g per day decreased TSH to 44%, T4 to 25%, and body weight to 59% of control levels. In experiments over 3 months in rats in vivo, a low dose (25 μg/100 g per day) of T2 suppressed TSH to 60% and T4 to 57% of control levels and had no significant influence on other parameters. Conversely, 0·1 μg/100 g per day T3 had significant effects on body and organ weights as well as pellet intake, but a less pronounced TSH suppressive effect: TSH concentrations were unchanged and T4 concentrations were down to 80% of control values. In rat pituitary fragments in vitro, a clear suppression of TSH secretion after a TRH pulse was demonstrated. To summarise, T2 is a specific agonist in the negative feedback mechanism on TSH secretion at the pituitary level without other apparent thyromimetic effects. Journal of Endocrinology (1995) 145, 291–297

Antipituitary antibodies have been reported to exist in sera of patients with autoimmune endocrine disorders. In order to investigate the pituitary antigens recognized by antipituitary antibodies, we studied the autoantigens in rat pituitary membrane and cytosolic fractions recognized by human antipituitary antibodies and anti-human pituitary hormone antibodies. Sera from 6 patients which showed positive antipituitary antibodies by immunofluorescence methods were studied. Each serum identified some proteins with 14.5, 22, 47, 49, 65, 84 and 97.5 kDa. Anti-GH antibodies and anti-PRL antibodies identified a positive band with 22 kDa, suggesting that anti-GH and anti-PRL antibodies may be present in patients' sera. These results indicate that Western blot analysis of rat pituitary antigens recognized by human antipituitary antibodies is a useful method to elucidate the pathophysiology of autoimmune endocrine disorders.

The effects of somatostatin-14 on inositol-1,4,5-trisphosphate (IP3) content were examined in rat pituitary, hippocampus and cortex homogenates. Somatostatin increased IP3 concentration in all these investigated regions in a dose- and time-dependent manner. Maximal increase of IP3 content was found in the pituitary homogenate, and in the hippocampal and cortex homogenates the IP3 argumentation was slightly smaller. Time-response studies showed that this effect declined with extended time of incubation. These results suggest that IP3 is involved in the mechanism of action of somatostatin in brain.

The growth hormone (GH) releasing effect of GH-releasing hormone (GHRH) and galanin, a 29-amino acid peptide widely distributed in mammalian CNS, was investigated in cultured rat pituitary tumor cells (GH 1 ) as compaerd to normal rat somatotrophs. GHRH stimulated dose-dependently GH secretion in normal somatotrophs but did not affect GH secretion in GH 1 cells. Galanin (1–10 μM) stimulated GH release in a concentration-dependent manner, but with lower potency as compared to GHRH, in normal rat pituitaries but was inhibitory in rat GH 1 cells. The results of this study indicate that while galanin has the ability to stimulate GH release from dispersed pituitary cells of normal rats it has potent direct inhibitory effects on GH release from tumor rat cells.

We have cloned and sequenced a novel cDNA (RPR7) encoding a receptor for pituitary adenylate cyclase activating polypeptide (PACAP). RPR7 was identified by PCR of rat pituitary cDNA, and full-length clones were isolated from a rat olfactory bulb cDNA library. When expressed in COS cells, RPR7 was functionally coupled to increases in intracellular cyclic adenosine monophosphate (cAMP) in response to stimulation by PACAP-38, PACAP-27, vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI). The order of potency of these ligands was PACAP-38 ~ PACAP-27 > VIP > PHI, suggesting that the receptor corresponds to the pharmacologically characterised PACAP Type I receptor.

Publisher Summary This chapter describes selected methods for investigating the site and mechanisms of action of cytokines on the neuroendocrine system and discusses their pitfalls, limitations, and advantages. These methods may also be employed for studying the effects of other cytokines on the secretion of various pituitary hormones. The most widely used method for studying the direct action of a bioactive substance on the anterior pituitary may be the technique of primary culture of rat pituitary cells. The primary monolayer culture of pituitary cells was first employed for examining the hormone-releasing or release-inhibiting effects of various hypophysiotropic factors. As monolayer pituitary cell cultures consistently respond to the classic hypophysiotropic hormones with the release of or the release inhibition of the respective pituitary hormone in a dose-dependent manner, the method has been considered the most reliable, specific, and convenient bioassay for the substances that affect pituitary cells by a direct action.

We first describe the presence of iodothyronine 5-deiodinase (5D) in the neural lobe of rat pituitary. 6-n-Propyl-2-thiouracil (PTU), a specific inhibitor of type-I deiodinase, had no effect, showing that 5D in neurohypophysis is of type-III isozyme, which is specific for 5-deiodination and has been found only in the brain, placenta and skin. The presence of 5D (type-III) together with our previous report of 5′-deiodinase (type-I in euthyroidism and type-II in hypothyroidism) shows that the isozymes of deiodinases in the neurohypophysis are quite similar to those in the brain. These data suggest a previously unrecognized role of thyroid hormone in posterior pituitary physiology.

The objective of this research was to measure the effects of endophyte-infected tall fescue seed extract and various alkaloids associated with the endophyte on in vitro prolactin secretion by rat hemipituitaries. Rat anterior pituitaries (AP) were dissected into halves and placed in temperature-controlled culture chambers (37 degrees C). The tissue was perfused with culture media at a flow rate of 12 mL/h. After perfusion for at least 90 min with control media, AP halves were exposed to their respective treatments for 15 min before they were returned to the control media. The treatments for Exp. 1 were .01 micrograms of alpha-ergocryptine/mL of culture medium, .01 microgram of ergonovine/mL of culture medium, .01 gram-equivalents of endophyte-infected tall fescue seed/mL of culture medium, and .01 gram-equivalents of endophyte free tall fescue seed/mL of culture medium. Treatments for Exp. 2 consisted of 10(-4), 10(-6), and 10(-8) M concentrations of perloline, N-formyl loline, N-acetyl loline, N-methyl loline, and alpha-ergocryptine. alpha-Ergocryptine suppressed (P less than .10) prolactin secretion in both experiments. Ergonovine and perloline both stimulated (P less than .10) prolactin secretion. The loline alkaloids (N-formyl loline, N-acetyl loline, N-methyl loline) had no effect on prolactin secretion. The endophyte-infected seed extract treatment suppressed (P less than .10) prolactin secretion. The endophyte-free seed extract treatment had no effect on prolactin secretion. In Exp. 2, prolactin secretion from AP responded to alpha-ergocryptine treatment in a dose-dependent fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

A reverse haemolytic plaque assay (RHPA) for angiotensinogen was developed in rat hepatoma H4 cells and applied to investigate the possible secretion of angiotensinogen from rat pituitary cells in primary culture. Over a 24-hour incubation period in Cunningham chambers plaques with a mean area of 2,800 +/- 430 and 590 +/- 220 microns2/plaque (SD, n = 6) formed around all viable H4 cells and 2.8 +/- 0.59% of viable pituitary cells respectively. As a positive control PRL secretion from lactotrophs was routinely checked by the RHPA and shown to form plaques with a mean area of 4,050 +/- 1,850 microns2/plaque after a 4-hour incubation. By comparing plaque size in H4 cells with angiotensinogen release in cell culture, as quantified by radioimmunoassay, the secretion rate of angiotensinogen from pituitary cells was calculated as 22 +/- 8 ng/10(6) cells/24 h. Plaque-forming cells consisted of two morphologically distinct populations; 78% being small cells (less than 6 microns diameter) containing little cytoplasm and 22% were large (greater than 9 microns diameter) cells with an abundant cytoplasm. Immunocytochemical staining of pituitary cells after formation of plaques with anti-angiotensinogen, anti-LH and anti-PRL antiserum showed that the large plaque-forming cells were gonadotrophs and none were lactotrophs. All plaque-forming cells stained for angiotensinogen but only 44% of the viable cells which stained for angiotensinogen actually formed plaques. The possibility that cellular angiotensinogen was imported from extracellular sources was investigated by incubation of pituitary cells with pure 125I-angiotensinogen for periods up to 24 h. No uptake of the radiolabelled protein was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Previously, we demonstrated that it is feasible to use mass spectrometry for the charting of the most abundant peptides in bovine posterior/intermediate pituitary and pointed out that this opens the possibility to use mass spectrometry to study the regulation of entire peptide/protein profiles in response to drug treatment, endogenous regulators, stress, and disease [1]. Andrews et al. have shown similarly good results for charting catfish pancreatic peptides and suggested the use of mass spectrometry to investigate alterations of peptide profiles between different tissues and species [2]. The present state of the art of peptide/protein charting by mass spectrometry has recently been reviewed [3].

The separation of growth hormone-containing cytoplasmic secretory granules from the rat pituitary gland by continuous flow electrophoresis is described. The results are consistent with the hypothesis that granule subpopulations can be separated due to differences in surface charge; these, in turn, may be related to the oligomeric state of the hormone.

The bulk of Prolactin (PRL) in rat pituitary gland stored as big molecular forms in secretory granules is mostly excluded from radioimmunoassay (RIA) determinations because secretory granules remain intact after tissue homogenization. Also big PRL is little immunoreactive and must be deaggregated to monomers to allow a complete detection by RIA. Different dissociating agents, used to render PRL monomers soluble, were tested at various temperatures and pH conditions. Incubations of pituitary homogenates in buffers at neutral pH yield consistent levels of PRL, but the sole alkalinization of the media increases significantly the radioimmunoassayable PRL content. No significant increase was detected with EDTA and thiols. The 2.5 M urea was the most effective extraction agent increasing about 7-fold the quantification of PRL by RIA. Extraction of PRL with urea was enhanced at pH 9.0 and at 4 C and this combination constitutes the method of choice for a complete extraction of pituitary PRL.

Pituitary responsiveness to GHRH (1-29) NH2 (GHRH, 5 μg/kg iv) was analysed under sodium pentobarbital anesthesia (50 mg/kg ip), on days 30 and 90 in male rats orchidectomized or sham-operated 7 days earlier. Other groups of rats were orchidectomized or shamoperated on day 23 and tested on days 30, 45, 60 and 90. In the sham-operated animals, GHRH stimulated GH secretion on day 90, but not on day 30. GHRH-induced secretion was similar on days 45, 60 and 90 in orchidectomized and sham-operated animals. Orchidectomy on day 83 reduced this GHRH-induced GH secretion on day 90. In contrast, orchidectomy on day 23 enhanced the pituitary responsiveness to GHRH a week later. These results suggest that the increase in pituitary responsiveness to GHRH with age is independent of the testicular function and that the effect of orchidectomy depends on both the age of the rats at orchidectomy and the time elapsed between the orchidectomy and the administration of GHRH.

The release of pituitary gonadotrophin hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) is specifically stimulated by the decapeptide gonadotro-phin-releasing hormone (GnRH), which is secreted from the hypothalamus and is borne by blood to the pituitary gland.

Gonadotropin-releasing hormone (GnRH) is a neuropeptide which regulates hormone release from pituitary gonadotropes. The recently cloned pituitary GnRH receptor consists of a linear chain of amino acids in which seven transmembrane segments separate 4 extracellular and 3 intracellular (cytoplasmic) sequences. The rat receptor is approximately 90% homologous with that of the human.Observations made in several autoimmune diseases and experimental models suggest that anti-receptor antibodies can interfere with receptor function. The present studies were designed to test this hypothesis in female rats (n=9×3) after immunization with synthetic peptides whose overlapping sequences replicated all putative extracellular domains of the cloned human GnRH receptor (Table 1). The peptides (A-H) were conjugated to keyhole limpet hemocyanin and the conjugates were injected i.m. at monthly intervals for four months. A control group (K) received adjuvant only. Anti-peptide antibodies in the sera of immunized rats were detected by ELISA. Recognition of the “native” GnRH receptor was tested in immunocytochemistry on normal pituitary and brain tissue.

Recently a link between ethanol treatment, β-endorphin content and POMC gene expression has been reported. These studies show contrary results for the POMC gene expression in the rat pituitary due to the kind of ethanol administration. So we decided to start a more complex study to investigate the effects of streβ (single holding), chronic ethanol treatment and voluntary ethanol ingestion on the POMC mRNA synthesis in the rat pituitary. Total RNA from pituitaries of sacrified rats was used in hybridization experiments (Dot blots) with a POMC exon3-cRNA-probe (Digoxigenin-labelled). The resulting chemiluminogramms were quantified using a video-based image analysis system. The results suggest that the POMC gene expression in animals which have had the free choice between water and various alcoholic solutions was significantly more decreased than in animals chronically treated only with ethanol.

The patterns of growth hormone and proopiomelanocortin gene expression in the rat pituitary were used to detail the application of the in situ hybridization histochemistry technique in tissue sections. Parameters affecting the efficiency and resolution of hybridization have been optimized so that this technique can be used to identify sites of peptide synthesis and to study hormonal regulation of gene expression at the single cell level.

An increase was found in the opiate activity of pituitary extracts obtained from rats injected with the neuroleptic drugs trifluoperazine and sulpiride. The increase of opiate activity, measured by bioassay, was particularly evident 2 h after the administration of sulpiride. Dexamethasone completely prevented the neuroleptic-induced effect. Trifluoperazine and sulpiride may have affected dopaminergic mechanisms regulating endorphin storage in the pituitary.

The 41-residue sequences of recently identified porcine corticotropin-releasing factors [I1e40]pCRF and [Asn40]pCRF were assembled on a benzhydrylamine resin support. Deprotection and cleavage from the resin were accomplished by HF treatment. The crude peptides were purified by HPLC. The homogeneity of the final materials, obtained in 0.2% and 0.4% overall yield for [I1e40]pCRF and [Asn40]pCRF respectively, was assessed after the isolation by HPLC and amino acid analysis. Both sequences of the synthetic 41-residue pCRF stimulated the release of corticotropin (ACTH) from superfused rat pituitary cells on a column, the responses being related to a log-dose of CRF in the range of 1–20 ng/ml. [I1e40]pCRF and [Asn40]pCRF also augmented the in vivo release of ACTH in rats pretreated with chlorpromazine, morphine and Nembutal. [I1e40]pCRF appeared to be equipotent to ovine CRF and about twice as active as [Asn40]pCRF. The data indicate that synthetic porcine [I1e40]pCRF and [Asn40]pCRF have high biological activity.

SummaryThe ACTH-releasing activity of hypothalamic extract and rat plasma was examined with the dispersed rat pituitary cell technique of Swallow and Sayers (8). Although both plasma and serum caused ACTH release which was dose-related, stress did not enhance the ACTH releasing activity. Furthermore, separation studies of plasma using ultrafiltration and gel separation suggest that the CRF activity in plasma is associated with molecules of a molecular weight greater than 15,000.The hypothalamic extract used in this study was kindly provided by the National Institute of Arthritis, Metabolism and Digestive Diseases (NIAMDD) Rat Pituitary Hormone Distribution Program.

SummaryThe effects of infusion of synthetic thytropin releasing hormone (TRH) into a hypophysial portal vessel on the release of prolactin and growth hormone (GH) were investigated in normal male rats under urethane anesthesia. Significant increases in serum prolactin and GH levels were consistently observed after the infusion of 100 ng or larger doses of synthetic TRH, but smaller doses had no effect. Pretreat-ment of rats with 50 fig estradiol in oil raised the basal levels of prolactin and GH, but the net increase after TRH remained the same for prolactin and was somewhat reduced for GH.The authors are indebted to Mrs. J. Gauthier and Mrs. M. Esslinger for their excellent technical assistance, Dr. Abba J. Kastin for his help in preparing the manuscript, and NIAMD Rat Pituitary Hormone Program for the supply of rat GH and Prolactin RIA Kits.

Camel synthetic beta-endorphin focused in three bands by isoelectrofocusing on 1 mm polyacrylamide thin gel. All the bands have opioid activity, measured on guinea pig ileum, and radioimmunologically react with beta-endorphin antiserum. Since beta-endorphin from rat pituitary gland, particularly from the neurointermediate lobe, also focused in several bands, we hypothesized that the camel peptide occurs in different conformations. A quick, simple technique based on histoelectrofocusing is proposed as a good approach to separating and measuring beta-endorphin from rat pituitary lobes. The method gives very high recovery.

Euthyroid rat pituitary cells were plated on a natural extracellular matrix (ECM) and cultured in either a medium supplemented with hormones and growth factors (SM) or in an unsupplemented medium (USM). Hormone supplementation resulted in a marked increase in the number of cells and in a less pronounced increase in the TSH content. Addition of 10% dehormonized serum (DHS) to the medium caused a further increase in the cell number as well as a significant elevation in the GH content but, surprisingly, it caused also a dramatic decrease in the TSH content. Time course experiments revealed that the serum doubled the rate of suppression of the TSH. DHS prepared from rat serum was as effective as that prepared from foetal calf serum and heat-inactivation of the serum did not change its suppressive effect on the production of TSH. This effect was confined to the serum fraction which was precipitable with 45% ammonium sulphate but did not bind to Con-A. It is suggested that a high molecular weight protein fraction of serum reduces the number of thyrotrophs in the culture and/or decreases the rate of TSH production by these cells.