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B M Lewis
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I S Ismail
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B Issa
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J R Peters
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M F Scanlon
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Abstract

We have studied the effects of glucose on the release of somatostatin (SS), TRH and GHRH from incubated hypothalami of normal and genetically diabetic, Goto-Kakizaki (GK) rats. The active isomer d-glucose caused a dose-related inhibition of SS, TRH and GHRH from normal rat hypothalami over a 20-min incubation period in vitro. In contrast, in GK rats the effects of glucose on TRH and SS were significantly reduced and the effects on GHRH were abolished. These data indicate that the sensitivity of SS-, TRH- and GHRH-producing hypothalamic neurones is reduced in diabetic rats. The effect is most pronounced for GHRH release as there was no change in the release of this peptide with increasing glucose concentrations. In conclusion, it appears that the diabetic state in GK rats causes differential desensitisation (GHRH>TRH and SS) of neuronal responses to subsequent changes in glucose concentrations in vitro. This may be due to alterations in the neurotransmitter control and/or a reduction in number, affinity or function of glucose transporters on these peptidergic neurones or other intermediary neuronal pathways.

Journal of Endocrinology (1996) 151, 13–17

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N Masumoto
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Y Ikebuchi
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T Matsuoka
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K Tasaka
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A Miyake
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Y Murata
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Abstract

The synaptic membrane protein synaptosomal-associated protein (SNAP-25) has recently been implicated as one of the key proteins involved in exocytotic membrane fusion in neurons. However, the role of SNAP-25 in pituitary hormone release is not known. In this study, we determined that SNAP-25 is involved in regulated exocytosis in the clonal pituitary cell line GH4C1. SNAP-25 messenger RNA and protein were detected in GH4C1 cells by RT-PCR and immunoblot analysis, respectively. Immunofluorescence analysis indicated that SNAP-25 protein was localized in the plasma membrane. Next, to determine the function of SNAP-25 in GH4C1 cells, specific inhibitors of SNAP-25, botulinum neurotoxin (BoNT) /A or /E, and antisense SNAP-25 oligonucleotide were used. Neither BoNT/A nor BoNT/E affected thyrotropin-releasing hormone (TRH)-induced cytosolic Ca2+ increase, but both inhibited TRH-induced exocytosis. Moreover, they dose-dependently inhibited TRH-induced prolactin release. The introduction of antisense oligonucleotide into the cells also inhibited TRH-induced prolactin release. These results suggest that SNAP-25 is involved in regulated exocytosis in GH4C1 cells.

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C Hoang-Vu
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G Brabant
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H Leitolf
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A von zur Mühlen
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H Dralle
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S Schröder
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W D Sierralta
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Abstract

The aim of the present study was to evaluate in vivo the selective effects of a small increase in plasma TSH levels on thyroid function, proliferation and morphology. Chronically catheterized male Sprague–Dawley rats were stimulated i.v. over 5 days either with TRH (2 μg TRH in 100 μl 0·9% (w/v) NaCl (TRH-P) or the NaCl carrier alone (P), both given as pulses every 2 h. Control groups were cotreated i.v. with 10 μg thyroxine (T4)/100 g body weight per day (TRH-P+T4) starting 2 days before pulsatile stimulation. TSH plasma levels were approximately doubled by TRH-P (P≤0·001), T4 plasma levels significantly increased (P≤0·001) but tri-iodothyronine plasma levels did not change compared with treatment with P. No significant changes between groups were found in thyroid weight and in intrathyroidal iodine content, but the percentage of 5-bromo-2′-desoxyuridinelabelled thyrocytes as a marker of proliferation in TRH-P-treated animals was significantly increased over P or TRH-P+T4 (P≤0·001). Ultrastructural analysis of the thyroid evaluated by electron microscopy revealed a significant increase in the number of lysosomes (P≤0·001). The size of the endoplasmic reticulum (ER) in relation to the cytoplasm was significantly increased when treated with TRH-P compared with P or TRH-P+T4 (P≤0·001). Post-embedding immunogold staining revealed Tg as a major product within ER cisternae. Immunogold labelling was moderate in controls and higher densities of gold particles were obtained in TRH-P-treated animals (P≤0·001). In conclusion, short-term pulsatile TRH stimulation increasing the plasma levels of immunoreactive TSH only twofold is capable of inducing hypertrophy of the thyrocytes by gross ultrastructural changes which are paralleled by an increase in circulating T4. These data underscore the dominant role of TSH on thyroid ultrastructure within the narrow boundaries of normal physiological regulation.

Journal of Endocrinology (1995) 146, 339–348

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T Pawełczyk
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M Pawlikowski
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J Kunert-Radek
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Abstract

The effect of TRH on cell proliferation in the anterior lobe of the pituitary is well known and documented. On the other hand, there are no data on the effects of TRH on the intermediate lobe of the pituitary gland. The aim of this study was to investigate the effect of TRH and its analogues (pGlu-His-Gly, pGlu-His-Gly-NH2) on cell proliferation in the intermediate pituitary lobe. The bromodeoxyuridine technique was used to detect the proliferating cells. It was found that TRH stimulated cell proliferation 24 h after a single injection at a dose of 100 μg/kg body weight. The TRH analogues did not exert any significant stimulatory effect either 12 h or 24 h after the injection.

The second experiment was carried out to distinguish the probable mechanism of the action of TRH. The effects of TSH and prolactin (PRL) on intermediate lobe cell proliferation were examined. It was found that both PRL and TSH exerted a significant stimulatory effect 24 h after a single s.c. injection of PRL at a dose of 150 IU/kg body weight or TSH at a dose 20 IU/kg body weight.

It therefore appears that the stimulatory effect of TRH on intermediate pituitary lobe cell proliferation is mediated by PRL and TSH.

Journal of Endocrinology (1996) 148, 193–196

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WJ de Greef
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WW de Herder
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CB Lambalk
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W Klootwijk
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E Sleddens-Linkels
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FH de Jong
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TJ Visser
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Recent studies have revealed that TRH-like immunoreactivity (TRH-LI) in human serum is predominantly pGlu-Glu-ProNH2 (< EEP-NH2), a peptide previously found in, among others tissues, the pituitary gland of various mammalian species. In the rat pituitary, < EEP-NH2 is present in gonadotrophs and its pituitary content is regulated by gonadal steroids and gonadotrophin-releasing hormone (GnRH). Hence, we reasoned that < EEP-NH2 in human serum may also arise, at least in part, from the pituitary, and that its secretion may correlate with that of gonadotrophins. Therefore, blood was simultaneously sampled from both inferior petrosal sinuses, which are major sites of the venous drainage of the pituitary gland, and a peripheral vein from seven patients with suspected adrenocorticotrophin-secreting pituitary tumours. In addition, in six postmenopausal and six cyclic women, peripheral vein blood was collected at 10-min intervals for 6 h, then a standard 100 micrograms GnRH test was performed. In the sera, TRH-LI was estimated by RIA with antiserum 4319, which binds most tripeptides that share the N- and C-terminal amino acids with TRH (pGlu-His-ProNH2). In addition, LH and FSH were measured in these sera by RIA. In the blood samples taken at 10-min intervals, an episodic variation in serum TRH-LI was noted and pulses of TRH-LI were detected at irregular intervals (from one to six pulses per 6 h) in five postmenopausal and six cyclic women. In general, these pulses did not coincide with those of LH and FSH, suggesting that TRH-LI is not co-secreted with gonadotrophins. Moreover, unlike LH and FSH, serum TRH-LI did not increase during the menopause or after exogenous administration of GnRH. Whereas gonadotrophin concentrations were significantly greater in the inferior petrosal sinus than in peripheral serum, there were no differences in TRH-LI concentrations between these serum samples. In conclusion, serum TRH-LI in humans seems not to be regulated by gonadal steroids or GnRH. Moreover, serum derived directly from the pituitary contained no more TRH-LI than did peripheral serum, which suggests that the human pituitary gland does not secrete significant amounts of < EEP-NH2, and therefore does not contribute significantly to serum TRH-LI concentrations. Further research is required to identify the site of origin of < EEP-NH2 in human serum.

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A Quintanar-Stephano
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C Valverde-R
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Abstract

The question of whether thyroxine (T4) and TRH have a mitogenic effect on pituitary thyrotrophs and somatotrophs in thyroidectomized rats was investigated. Mitoses were counted in hematoxylin–eosin-stained or periodic acid–Schiff–hematoxylin-stained pituitary slides or immunostained for TSH or GH using male rats thyroidectomized for 5 months. Ten days before they were killed groups of rats were injected with different doses of T4 (0·5, 3 or 10 μg i.m. every second day for 10 days), TRH alone (100 ng s.c. three times a day for 10 days), or T4 plus TRH (same doses as above). Mitoses (stopped with colchicine) were counted in 1 mm2 areas at a magnification of × 1000. In thyroidectomized rats, mitoses were not significantly increased and treatment with TRH or 0·5 μg T4 alone in thyroidectomized rats did not affect mitotic counts. In thyroidectomized rats treated with higher doses of T4, mitoses were increased in a dose-dependent fashion. Simultaneous administration of TRH and T4 had a significant synergistic effect on pituitary mitoses in a T4 dose-dependent manner. The treatments also had differential effects on the relative percentages of cellular types in mitosis. Thus, 60% somatotrophs and 12·5% thyrotrophs were found in the euthyroid group. In thyroidectomized and thyroidectomized plus TRH groups, no somatotrophs in mitosis were seen, while thyrotrophs were 28·5% and 33·3% respectively. In thyroidectomized rats treated with low doses of T4, somatotrophs and thyrotrophs in mitosis increased to 38·4% and 80% respectively and, with simultaneous administration of a low dose of T4 plus TRH, although less effective than T4 alone, mitosis increased in somatotrophs and thyrotrophs to 11·1% and 54·5% respectively. A high dose of T4 alone did not increase the mitotic figures in somatotrophs (38·8%), while it diminished the percentage of thyrotrophs to 25%. The administration of high doses of T4 plus TRH had an opposite effect on the mitotic figures of somatotrophs and thyrotrophs and thus the percentage of somatotrophs increased to 50% while thyrotrophs decreased to 5·5%. Ten days of treatment with T4 were insufficient to reverse the histology to euthyroidism. It can be concluded that in long-standing hypothyroidism: (1) thyroid hormone replacement elicits a dose-dependent and differential proliferative response on pituitary thyrotrophs and somatotrophs, (2) TRH is devoid of mitogenic effects when administered alone and (3) the proliferative response of somatotrophs to T4 is enhanced by its co-administration with TRH, suggesting a permissive and/or synergistic effect of the thyroid hormone and TRH.

Journal of Endocrinology (1997) 154, 149–153

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A Lorsignol
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A Taupignon
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G Horvath
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B Dufy
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We studied the effects of TRH on the cytosolic free calcium concentration ([Ca2+]i) of female rat pituitary prolactin-secreting (lactotroph) and GH-secreting (somatotroph) cells in the early postnatal period, i.e. at postnatal days 5 and 10. [Ca2+]i of single identified lactotrophs and somatotrophs was recorded by dual-emission microspectrofluorimetry using the intracellular fluorescent calcium probe indo 1. An application of TRH (100 nM, 10 s) induced a marked [Ca2+]i increase in 65% of neonatal lactotrophs and 34% of neonatal somatotrophs while the remaining cells were unaffected. Most of the responsive cells, both lactotrophs and somatotrophs, exhibited a similar biphasic Ca2+ response, made up of an initial rapid large increase in [Ca2+]i followed by sustained [Ca2+]i fluctuations. In both cell types, removal of Ca2+ from the extracellular medium or addition of the Ca2+ channel blocker, cadmium chloride (500 microM), inhibited the second phase whereas the first phase persisted. Furthermore, in both cell types, protein kinase C (PKC) depletion by incubation in phorbol myristate acetate (1 microM) for 24 h abolished the second phase but did not inhibit the first phase. Conversely, when cells were pretreated with the Ca(2+)-ATPase inhibitor, thapsigargin (100 nM), all TRH-induced [Ca2+]i changes in both cell types disappeared. TRH therefore induces a biphasic increase in [Ca2+]i involving intra- and extracellular Ca2+ in neonatal lactotrophs and somatotrophs as it does in adult lactotrophs. The first phase is presumably due to mobilization of Ca2+ from intracellular stores whereas the second phase presumably results from a PKC-sensitive influx of Ca2+. TRH action on membrane potential was then investigated using the patch-clamp technique in the whole-cell mode. TRH-induced changes in membrane potential consisted of an initial hyperpolarization followed by depolarization and action potential firing. We also investigated TRH action on prolactin and GH secretion by neonatal pituitary cells using RIA. Surprisingly, static assays of prolactin and GH revealed only stimulation of prolactin release by TRH but no effect on GH secretion, although, as expected, GH-releasing factor was a potent agonist of GH secretion. Our results suggest that TRH regulates neonatal lactotrophs and somatotrophs differently, in that the [Ca2+]i changes do not correlate with stimulation of exocytosis in the latter cell type.

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Patricia Joseph-Bravo Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), A.P. 510-3, Cuernavaca, Morelos 62250, Mexico

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Lorraine Jaimes-Hoy Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), A.P. 510-3, Cuernavaca, Morelos 62250, Mexico

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Rosa-María Uribe Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), A.P. 510-3, Cuernavaca, Morelos 62250, Mexico

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Jean-Louis Charli Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), A.P. 510-3, Cuernavaca, Morelos 62250, Mexico

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correct Figure 2 is published in full below: Figure 2 Elements involved in HPT regulation. At the level of the paraventricular hypothalamic nucleus (PVN), Trh mRNA is transcribed, its expression is regulated by multiple effectors, processed TRH is

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G A C van Haasteren
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H van Toor
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W Klootwijk
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B Handler
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E Linkels
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P van der Schoot
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J van Ophemert
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F H de Jong
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T J Visser
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W J de Greef
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Abstract

This study describes the effects of litter size and acute suckling on the synthesis and release of hypothalamic TRH, as indirectly estimated by determination of hypothalamic prothyrotrophin-releasing hormone (proTRH) mRNA and median eminence TRH content. The effects of litter size (five or ten pups) were studied throughout lactation, while suckling-induced acute changes were analyzed on day 13 of lactation in dams with ten pups. In view of the enhanced adrenal activity during lactation and recent evidence that corticosteroids have negative effects on hypothalamic TRH, we also studied adrenalectomized (ADX) dams treated with corticosterone to maintain basal plasma corticosterone levels.

In addition to an increased plasma level of prolactin (PRL), adrenal weight and plasma corticosterone increased, while plasma TSH, tri-iodothyronine (T3), thyroxine (T4) and free T4 (FT4) levels decreased during lactation. Litter size correlated positively with plasma PRL, adrenal weight and plasma corticosterone. No effect of litter size was observed on plasma T3, but rats with ten pups had lower plasma TSH, T4 and FT4 than rats with a five-pup litter. Compared with dioestrous rats, lactating rats showed an increased hypothalamic proTRH mRNA content on day 2, but not on days 8 and 15 of lactation. Median eminence TRH in lactating rats gradually increased until day 15 and decreased thereafter. Acute suckling, after a 6-h separation of mother and pups, rapidly increased plasma PRL and corticosterone in the mothers, but had no effects on plasma TSH and thyroid hormone levels. Hypothalamic proTRH mRNA increased twofold after 0·5 h of suckling, and then gradually returned to presuckling values after 6 h. Compared with sham-operated rats, corticosterone-substituted ADX rats with ten pups had increased plasma PRL and TSH, hypothalamic proTRH mRNA and pituitary TSH β mRNA on day 15 of lactation. Moreover, while acute suckling did not enhance TSH release in sham-operated rats, it provoked not only PRL but also TSH release in corticosterone-substituted ADX dams.

It is concluded that suckling exerts a rapid, positive effect on hypothalamic proTRH mRNA content. However, the concurrent enhanced adrenal activity has negative effects on hypothalamic proTRH gene expression resulting in a suppressed hypophysial-thyroid axis during lactation. While TRH appears to play a role in PRL release during the first days of lactation and during acute suckling, TRH seems not important in maintaining PRL secretion during continued suckling.

Journal of Endocrinology (1996) 148, 325–336

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J M M Rondeel
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W Klootwijk
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E Linkels
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P H M Jeucken, W
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L J Hofland
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M E Everts
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P Kramer
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F H de Jong
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G A C van Haasteren
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S M Cockle
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T J Visser
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W J de Greef
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Abstract

Recent evidence shows that thyrotrophin-releasing hormone (TRH) immunoreactivity in the rat anterior pituitary gland is accounted for by the TRH-like tripeptide prolineamide-glutamyl-prolineamide (pGlu-Glu-ProNH2, <EEP-NH2). The present study was undertaken to investigate further the regulation, localization and possible intrapituitary function of <EEP-NH2. Anterior pituitary levels of <EEP-NH2 were determined between days 5 and 35 of life, during the oestrous cycle and after treatment with the luteinizing hormone-releasing hormone (LHRH) antagonist Org 30276. Treatment of adult males with the LHRH antagonist either for 1 day (500 μg/100 g body weight) or for 5 days (50 μg/100 g body weight) reduced anterior pituitary <EEP-NH2 levels by 25–30% (P<0·05 versus saline-treated controls). Anterior pituitary <EEP-NH2 increased between days 5 and 35 of life. In females, these levels were 2- to 3-fold higher (P<0·05) than in males between days 15 and 25 after birth; these changes corresponded with the higher plasma follicle-stimulating hormone (FSH) levels in the female rats. After day 25, <EEP-NH2 levels in female rats decreased in parallel with a decrease in plasma FSH. Injections with the LHRH antagonist (500 μg/100 g body weight), starting on day 22 of life, led to reduced contents of <EEP-NH2 in the anterior pituitary gland of female rats on days 26 and 30 (55 and 35% decrease respectively). Levels of <EEP-NH2 in the anterior pituitary gland did not change significantly during the oestrous cycle. Fractionation of anterior pituitary cells by unit gravity sedimentation was found to be compatible with the localization of <EEP-NH2 in gonadotrophs. In vitro, <EEP-NH2 dose-dependently inhibited TRH-stimulated growth hormone (GH) release from anterior pituitary cells obtained from neonatal rats, but no consistent effects were seen on the in vitro release of luteinizing hormone (LH), FSH, prolactin (PRL) or thyroid-stimulating hormone (TSH) under basal or TRH/LHRH-stimulated conditions. Furthermore, <EEP-NH2 did not affect the in vitro hormone release by anterior pituitary cells obtained from adult rats. In vivo, <EEP-NH2 (0·3–1·0 μg intravenously) did not affect plasma PRL, TSH, LH, FSH and GH in adult male rats. We conclude that <EEP-NH2 in the anterior pituitary gland is regulated by LHRH, is probably localized in gonadotrophs and may play a (paracrine) role in neonatal GH release.

Journal of Endocrinology (1995) 146, 293–300

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