Thyrotropin-releasing hormone (TRH), a hypothalamic tripeptide, is expressed in pancreatic islets at peak levels during the late gestation and early neonate period. TRH increases insulin production in cultured beta-cells, suggesting that it might play a role in regulating pancreatic beta-cell function. However, there is limited information on TRH receptor expression in the pancreas. The aim of the present study was to explore the distribution of the TRH receptor in the pancreas and its function in pancreatic beta-cells. TRH receptor type 1 (TRHR1) gene expression was detected by RT-PCR and verified by Northern blotting and immunoblotting in the beta-cell lines, INS-1 and betaTC-6, and the rat pancreatic organ. The absence of TRH receptor type 2 expression in the tissue and cells indicated the tissue specificity of TRH receptor expression in the pancreas. The TRHR1 signals (detected by in situ hybridization) were distributed not only in islets but also in the surrounding areas of the pancreatic ductal and vasal epithelia. The apparent dissociation constant value for the affinity of [(3)H]3-methyl-histidine TRH (MeTRH) is 4.19 in INS-1 and 3.09 nM in betaTC-6. In addition, TRH induced epidermal growth factor (EGF) receptor phosphorylation with a half-maximum concentration of approximately 50 nM, whereas the high affinity analogue of TRH, MeTRH, was 1 nM. This suggested that the affinity of TRH ligands for the TRH receptor influences the activation of EGF receptor phosphorylation in betaTC-6 cells. Our observations suggested that the biological role of TRH in pancreatic beta-cells is via the activation of TRHR1. Further research is required to identify the role of TRHR1 in the pancreas aside from the islets.

In rodents, the first insulin-producing cells appear in the pancreas at mid-gestation around embryonic day 11 (E11). However, on the basis of various features, such as morphology or hormonal coexpression, it is apparent that these initial insulin-expressing cells are different from those that develop after E15. In the present study, the pancreatic expression of both thyrotropin-releasing hormone (TRH) mRNA and insulin was studied during embryonic and fetal life. We report here that in the rat, while insulin mRNA is detected in the pancreas as early as E12, TRH mRNA cannot be detected before E16. At that stage and later on during fetal and early postnatal life, TRH mRNA is detected in insulin-producing cells, no signal being detected in other endocrine cell types or in exocrine tissue. It was also noted, by means of triple staining performed at E17, that the expression of TRH mRNA was restricted to insulin-expressing cells negative for glucagon, whereas the few insulin-expressing cells present at that stage, which coexpress insulin and glucagon, did not express TRH mRNA. Taken together, these data indicate that TRH is a marker of insulin-expressing cells, which develop after E15.

Thyrotropin-releasing hormone (TRH) and somatostatin (SRIH) concentrations were determined by RIA during both embryonic development and posthatch growth of the chicken. Both TRH and SRIH were already detectable in hypothalami of 14-day-old embryos (E14). Towards the end of incubation, hypothalamic TRH levels increased progressively, followed by a further increase in newly hatched fowl. SRIH concentrations remained stable from E14 to E17 and doubled between E17 and E18 to a concentration which was observed up to hatching. Plasma GH levels remained low during embryonic development, ending in a steep increase at hatching. Plasma TSH levels on the other hand decreased during the last week of the incubation. During growth, TRH concentrations further increased, whereas SRIH concentrations fell progressively towards those of adult animals. Plasma TSH levels increased threefold up to adulthood; the rise in plasma GH levels during growth was followed by a drop in adults. In conclusion, the present report shows that important changes occur in the hypothalamic TRH and SRIH concentration during both embryonic development and posthatch growth of the chicken. Since TRH and SRIH control GH and TSH release in the chicken, the hypothalamic data are compared with plasma GH and TSH fluctuations.

Thyrotropin (TSH) is secreted not as one distinct hormone, but rather as a group of isohormones which differ in their oligosaccharide composition. Although the mechanisms regulating TSH glycosylation are not fully understood, there is strong evidence that TRH plays an important role. The aim of our study was to determine the dynamic influence of TRH on TSH microheterogeneity. Sera were obtained from euthyroid volunteers (n=20) before and 30, 60, 120, 180 and 240 min after intravenous, nasal and oral administration of TRH in three independent runs (randomized order, at a time-interval of 3 weeks between each run). TSH was immuno-concentrated and analysed by isoelectric focusing (IEF) and lentil lectin affinity chromatography. TSH immunoreactivity was measured by an automated second-generation TSH immunoassay. Overall, serum TSH concentrations reached maximal values 30 min after intravenous, 60 min after nasal and 180 min after oral TRH stimulation. IEF analysis revealed 63.3+/-3.3% of pituitary standard TSH (IRP 80/558) in the neutral pH range (8>pH>6). In contrast, 30 min after TRH stimulation 80.8+/-3.7% (P<0.001) and 60 min after TRH stimulation 44.9+/-2.2% (P<0.001) of the TSH of euthyroid probands were found in this pH range, whereas 180 min after TRH stimulation 58.4+/-2.3% (P<0.001) were detected in the acidic pH range (pH<6). This shift of TSH composition in euthyroidism after TRH stimulation was confirmed by lentil lectin analysis of TSH: core-fucose content of euthyroid TSH was 73.4+/-3.8% 30 min and 22.9+/-3.2% 120 min after TRH stimulation in contrast to basal (53.3+/-1.8%; P<0.001) and pituitary standard (IRP 80/558) TSH (63.0+/-0.9%; P<0.001). In conclusion, in euthyroidism, TRH stimulation time-dependently changes the distribution pattern of the TSH isoforms from an alkaline and neutral to a more acidic one. This corresponds to the secretion of isohormones with altered bioactivity which could influence the fine-tuning of thyroid function.

Somatostatin (SRIH) functions as an endocrine mediator in processes such as growth, immune resistance and reproduction. Five SRIH receptors (sstr1-5) have been identified in mammals, where they are expressed in both the brain and peripheral tIssues. To study the specific function of each receptor subtype, specific agonists (ag1-5) have been synthesized. The high degree of homology between mammalian and avian SRIH receptors suggests that these agonists might also be used in chickens. In this paper we describe two in vitro protocols (static incubation and perifusion system) to identify the SRIH receptors controlling the secretion of GH and TSH from the chicken pituitary. We found that basal GH or TSH secretion were never affected when SRIH or an agonist (1 microM) were added. SRIH diminished the GH as well as the TSH response to TSH-releasing hormone (TRH; 100 nM) in both systems. Our results have indicated that the SRIH actions at the level of the pituitary are regulated through specific receptor subtypes. In both the static and flow incubations, ag2 lowered the GH response to TRH, whereas stimulated TSH release was diminished by both ag2 and ag5. Ag3 and ag4 tended to increase rather than decrease the responsiveness of both pituitary cell types to TRH in perifusion studies. Our data have indicated that SRIH inhibits chicken pituitary function through sstr2 and sstr5. Only sstr2 seems to be involved in the control of chicken GH release, whereas both sstr2 and sstr5 inhibit induced GH secretion in mammals. The possible stimulatory action of ag3 and ag4 may point towards a species-specific function of sstr3 and sstr4.

Enhanced sialylation of thyrotropin (TSH) prolongs its metabolic clearance rate and thus increases the hormone's in vivo bioactivity. This has been shown for hypothyroid rats and for recombinant human TSH, but there are few data on the sialylation of human serum TSH. The aim of this work was to further study sialylated human serum TSH, its precursors bearing terminal galactose residues, and the role of pharmacological doses of thyrotropin-releasing hormone (TRH) on their secretion under different degrees of primary hypothyroidism. We analyzed serum TSH in patients with subclinical (n = 9) and overt primary hypothyroidism (n = 13) compared with euthyroid individuals (n = 12) and human standard pituitary TSH (IRP 80/558). Blood was drawn before and 30 min after intravenous administration of 200 micrograms TRH, and TSH was purified by immunoaffinity concentration. The content of sialylated (sialo-) TSH and isoforms bearing terminal galactose (Gal-TSH, asialo-Gal-TSH) was measured by Ricinus communis (RCA 120) affinity chromatography in combination with enzymatic cleavage of sialic acid residues. TSH immunoreactivity was measured by an automated second generation TSH immunoassay. Pituitary TSH contained 16.5 +/- 0.8% Gal-TSH. In euthyroid individuals the proportion of Gal-TSH was 14.6 +/- 1.9%, whereas TSH in patients with subclinical and overt primary hypothyroidism contained 23.9 +/- 3.5% (P < 0.05 vs euthyroid individuals) and 21.1 +/- 1.7% Gal-TSH respectively. The mean ratio of asialo-Gal TSH was 23.8 +/- 0.6% for pituitary TSH, 35.7 +/- 4.2% in euthyroid individuals, 48.0 +/- 3.3% in patients with subclinical, and 61.5 +/- 3.8% (P < 0.001 vs euthyroid individuals) in patients with overt primary hypothyroidism. For pituitary TSH the calculated proportion of sialo-TSH was 6.5 +/- 0.2%, for euthyroid individuals 20.3 +/- 2.8%, for patients with subclinical hypothyroidism 24.1 +/- 3.0%, and for patients with overt primary hypothyroidism 40.7 +/- 3.0% (P < 0.001 vs euthyroid individuals). The proportions of Gal-TSH, asialo-Gal-TSH, and sialo-TSH did not differ significantly before and after TRH administration in the individuals studied. Our data show that patients with subclinical and overt primary hypothyroidism have a markedly increased proportion of serum TSH isoforms bearing terminal galactose and sialic acid residues, which may represent a mechanism for the further stimulation of thyroid function. Pharmacological doses of TRH cause an increased quantity of TSH to be released, but do not significantly alter the proportion of sialylated or terminally galactosylated TSH isoforms.

Prior research indicates that growth hormone (GH) cell differentiation can be induced prematurely by treatment with glucocorticoids in vitro and in vivo. However, the nature of these responses has not been fully characterized. In this study, the time course of corticosterone induction of GH-secreting cells in cultures of chicken embryonic pituitary cells, responsiveness of differentiated somatotrophs to GH secretagogues, localization of somatotroph precursor cells within the pituitary gland, and the effect of corticosterone on GH gene expression were determined to better define the involvement of glucocorticoids in somatotroph recruitment during development. Anterior pituitary cells from embryonic day 12 chicken embryos were cultured in 10(-9) M corticosterone for 4 to 48 h and were then subjected to reverse haemolytic plaque assays (RHPAs) for GH. Corticosterone treatment for as short as 16 h increased the percentage of GH cells compared with the control. When corticosterone was removed after 48 h and cells were cultured for an additional 3 days in medium alone, the percentage of GH secretors decreased but remained greater than the proportion of somatotrophs among cells that were never treated with corticosterone. To determine if prematurely differentiated somatotrophs were responsive to GH secretagogues, cells were exposed to corticosterone for 48 h and then subjected to GH RHPAs in the presence or absence of GH-releasing hormone (GHRH) or thyrotropin-releasing hormone (TRH). Approximately half of the somatotrophs induced to differentiate with corticosterone subsequently released more GH in response to GHRH and TRH than in their absence. The somatotroph precursor cells were localized within the anterior pituitary by culturing cells from the caudal lobe and cephalic lobe of the anterior pituitary separately. Corticosterone induction of GH cells was substantially greater in cultures derived from the caudal lobe of the anterior pituitary, where somatotroph differentiation normally occurs. GH gene expression was evaluated by ribonuclease protection assay and by in situ hybridization. Corticosterone increased GH mRNA in cultured cells by greater than fourfold. Moreover, corticosterone-induced somatotroph differentiation involved GH gene expression in cells not expressing GH mRNA previously, and the extent of somatotroph differentiation was augmented by treatment with GHRH in combination with corticosterone. We conclude that corticosterone increases the number of GH-secreting cells within 16 h, increases GH gene expression in cells formerly not expressing this gene, confers somatotroph sensitivity to GHRH and TRH, and induces GH production in a precursor population found primarily in the caudal lobe of the anterior pituitary, a site consistent with GH localization in adults. These findings support the hypothesis that glucocorticoids function to induce the final stages in the differentiation of fully functional somatotrophs from cells previously committed to this lineage.

RT-PCR followed by 5'- and 3'- rapid amplification of cDNA ends was used to clone and sequence ovine prolactin-releasing peptide (PrRP). The cDNA was characterised by short 5'- and 3'-untranslated regions and a GC-rich (71%) coding region. The nucleotide and deduced amino acid sequences for the coding region showed 95.6 and 94.9% identity with bovine PrRP but the amino acid sequence of PrRP31 was conserved between these species. Northern blot analysis and RT-PCR showed that, as in the rat, the peptide was more abundantly expressed in the brainstem than the hypothalamus. However, in the ovine hypothalamus, PrRP mRNA expression was more widespread than in the rat, with expression detected in both rostral and caudal parts of the mediobasal hypothalamus. The effects of synthetic ovine PrRP on prolactin secretion both in vitro and in vivo were also examined. In primary cultures of sheep pituitary cells, PrRP significantly (P<0.01) increased prolactin concentrations in the culture medium but the response was not observed in every experiment and was only seen when pituitary glands were dispersed with collagenase rather than trypsin. PrRP was much less potent than TRH which caused a significant (P<0.01) two- to threefold increase in prolactin concentrations in every experiment. Intravenous (10 and 50 nmol) or intracerebroventricular (10 and 50 nmol) injection of PrRP had no significant effect on either plasma prolactin concentration or pulsatile LH secretion whereas intravenous injection of TRH (10 nmol) produced a highly significant (P<0.01) and more than sevenfold stimulation of plasma prolactin concentrations. In conclusion, these results suggest that PrRP is unlikely to be an important prolactin-releasing factor in this species.

Recently, a set of proteins involved in the docking and fusion machinery of secretory organelles has been identified in anterior pituitary cells. In this study we analyzed, by Western blotting and immunocytochemistry, the expression of several proteins involved in exocytosis after long-term administration of 17beta-estradiol (E2) in Fischer 344 rats. No differences were observed in the amount of synaptosomal-associated protein of 25 kDa, synaptobrevin 2, syntaxin 1, synaptotagmin I and Rab3a in total brain homogenates from treated rats after E2 administration. In striking contrast, the levels of all of these exocytotic proteins, including cellubrevin, were notably decreased in pituitary glands of E2-treated rats. In addition, no differences were observed in the in vitro basal and 8-Br-cAMP-induced prolactin (PRL) release between pituitary cells from control and E2-treated rats, whereas TRH-induced PRL release in anterior pituitary cells from E2-treated animals was higher than in control donors. In conclusion, this study shows that protein components of the exocytotic machinery are specifically down-regulated in the pituitary gland of E2-treated Fischer 344 rats.