ABSTRACT

The effect of somatostatin on GH-releasing factor (GRF)-induced desensitization of somatotrophs was studied in vitro. Primary cultures of rat anterior pituitary cells pretreated for 4 or 18 h with GRF(1–40) (100 nmol/l) showed a 50% or greater reduction in maximal GH release when rechallenged with 10 nmol GRF/l. Rechallenge GRF dose–response curves were either very flat, making accurate measurement of the dose giving 50% maximum stimulation (ED₅₀) impossible, or the ED₅₀ concentration was increased from 0·3 nmol/l (untreated) to 2 nmol/l (GRF pretreated). Although GRF pretreatment reduced cellular GH content by 40–50%, correction for this did not restore GRF responsiveness measured in terms of maximal GRF-stimulated/unstimulated GH release (maximal/basal ratio), or the GRF ED₅₀ concentration. Maximal/basal GH release per 4 h from GRF-pretreated cells was reduced when cells were rechallenged with forskolin (5 μmol/l) or calcium ionophore (A23187; 10 μmol/l), to the same extent as when rechallenged with 10 nmol GRF/l. Although this might be explained by a reduction in the pool of releasable GH, an alternative explanation is that pretreatment with GRF disrupts the GH release mechanism(s) at a common step(s) beyond cyclic AMP generation and Ca²⁺ influx.

Co-incubation of cells with somatostatin and GRF (100 nmol/l) partially reversed the desensitizing action of GRF during both 4- and 18-h pretreatments in a dose-dependent manner, with 1 μmol somatostatin/l being most effective. Maximal GRF (100 nmol/l)-stimulated/basal GH release was 4·4 ± 1·0 (mean ± s.e.m., n = four experiments), 1·55 ± 0·09 and 2·43 ± 0·1 for control, GRF-pretreated (4 h) and GRF plus somatostatin-pretreated cells respectively. Comparable values for cells pretreated for 18 h were 3·66 ± 0·44 (n = three experiments), 1·78 ± 0·28 and 3·04 ± 0·04 for control, GRF- and GRF plus somatostatin-pretreated cells. Somatostatin reduced the 50% depletion of cellular GH caused by GRF pretreatment to 15–20%, as well as attenuating GH released during the pretreatment period by 40 ± 5% (mean ± s.e.m., n = seven experiments). Somatostatin restored somatotroph sensitivity of GRF-desensitized cells indicating that, in addition to reversing depletion of the releasable pool of GH, the counter-regulatory hormone also prevents disruption of post-receptor cellular biochemical events which remain to be identified. These results add to the list of GRF actions inhibited by somatostatin and suggest a potentially important role for somatostatin in vivo to maintain somatotroph responsiveness to GRF.

J. Endocr. (1987) 112, 69–76

ABSTRACT

The effect of drug-induced hypo- and hyperprolactinaemia on pituitary gonadotrophin releasing hormone receptors (GnRH-R), serum and pituitary gonadotrophins (LH and FSH) and prolactin was investigated in intact adult male and female rats. Hypoprolactinaemia (serum prolactin <20% of control values) resulting from dopamine agonist (bromocriptine) infusion (4 mg/kg per day for 7 days) was accompanied by a 40–50% increase in GnRH-R in both male and female animals, though this was not accompanied by any major change in serum or pituitary LH and FSH. Hyperprolactinaemia (serum prolactin greater than ten times control values) induced by the dopamine receptor antagonist metoclopramide (65 mg/kg per day for 7 days) increased GnRH-R between 35 and 45% in both male and female rats without altering serum gonadotrophins. Domperidone (1 mg twice daily for 14 days) also increased GnRH-R by 50% but only in female rats. Both dopamine antagonists significantly increased pituitary prolactin content. Pituitary FSH increased in female rats treated with both metoclopramide and domperidone.

The stimulatory effects of bromocriptine and metoclopramide on GnRH-R in male rats were prevented by concurrent treatment with a GnRH antiserum, suggesting that the drug effects were mediated through alteration in endogenous GnRH secretion.

Induction of massive (serum prolactin > 2000 μg/l) hyperprolactinaemia in male and female rats with a transplantable prolactin-secreting pituitary tumour did not reduce GnRH-R concentration, although serum gonadotrophins were suppressed and pituitary gonadotrophin content was increased.

These results indicate a dissociation between serum prolactin concentrations and pituitary GnRH receptor content and indicate that dopamine agonist and antagonist agents can influence GnRH-R independently of prolactin, possibly by acting on central dopamine receptors responsible for catecholaminergic regulation of GnRH secretion.

J. Endocr. (1984) 102, 215–223

Measurement of pituitary gonadotrophin releasing hormone (Gn-RH) receptor content provides a qualitative index of prior exposure of the pituitary gland to endogenous Gn-RH. The effect of moderate hyperprolactinaemia (serum prolactin = 95–250 μg/l), achieved with three pituitary grafts beneath the renal capsule, on the pituitary Gn-RH receptor content and serum LH responses to gonadectomy of adult rats has been studied. In males the presence of hyperprolactinaemia for 7 days completely prevented the increase in Gn-RH receptor content 3 days after castration and inhibited the serum LH rise by 45%. By 6 days after castration, Gn-RH receptors had increased in the hyperprolactinaemic castrated animals but values were 33% lower than in sham-grafted controls, while the serum LH increase was attenuated by 30%. Pituitary LH content was also lower in grafted castrated animals 6 days after castration. Hyperprolactinaemia for 3 weeks had no effect on Gn-RH receptors or pituitary LH content of intact male rats, although basal serum LH was decreased by 50%. Hyperprolactinaemia also attenuated the increases in Gn-RH receptors, serum LH and pituitary LH which occurred 6 days after ovariectomy in female rats. In all experiments the pituitary content of prolactin was reduced by 80–90% in animals bearing pituitary grafts. These results suggest that hyperprolactinaemia restricts the Gn-RH receptor response to gonadectomy by decreasing endogenous hypothalamic Gn-RH secretion.

ABSTRACT

Collagenase-isolated mouse islets were incubated with gastrin-releasing peptide (GRP). At 5·6 mmol glucose/1, 10 nmol GRP/l increased the release of insulin (by 50%) and glucagon (by twofold), decreased the release of pancreatic polypeptide (by 35%), but did not significantly affect the release of somatostatin. At 16·7 mmol glucose/l, 10 nmol GRP/l increased glucagon release (by fivefold) and decreased pancreatic polypeptide release (by 46%), without significantly altering insulin and somatostatin release. GRP (200 nmol/l) did not affect insulin release by perifused mouse islets at 2·8 mmol glucose/l, but increased both first and second phase insulin release after a square wave increase in the glucose concentration to 11·1 mmol/l. At 5·6 mmol glucose/l, GRP (100 pmol/1–100 nmol/l) increased (by 50–70%) insulin release by the RINm5F clonal cell line. GRP did not affect glucose oxidation or the cyclic adenosine monophosphate content of RINm5F cells. However, the intracellular free Ca²⁺ concentration of RINm5F cells was rapidly and transiently increased by GRP (maximum increase of 64% about 10 s after exposure to 1 μmol GRP/l). The rise of intracellular free Ca²⁺ was approximately halved in the absence of extracellular Ca²⁺. The results suggest that GRP may contribute to the normal regulation of the endocrine pancreas. The insulin-releasing effect of GRP is mediated via increased cytosolic free Ca²⁺, derived both from an increased net influx of extracellular Ca2+ and from mobilization of intracellular Ca²⁺ stores.

Journal of Endocrinology (1990) 127, 335–340

ABSTRACT

The release of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) by isolated mouse pancreatic islets was determined during 30-min incubations at 5.6 and 16.7 mmol glucose/l in the absence and presence of gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP) and peptide histidine isoleucine (PHI) at concentrations of 1–1000 nmol/l. Insulin release was enhanced (>50%) by GIP (100–1000 nmol/l) and VIP (1 μmol/l) at 5.6 mmol glucose/l, but not at 16.7 mmol glucose/l. Glucagon release was increased by GIP (100–1000 nmol/l), and by VIP and PHI (1—1000 nmol/l) at both glucose concentrations in a dose-related manner (maximum increases > tenfold). Somatostatin release was similarly increased by GIP (10–1000 nmol/l) at both glucose concentrations. Only the highest concentration (1 μmol/l of PHI tested increased somatostatin release (twofold) at 5.6 mmol glucose/l, whereas PHI and VIP (1–1000 nmol/l reduced (>37%) somatostatin release at 16.7 mmol glucose/l. PP release was increased (49–58%) by 100–1000 nmol GIP/l, but was not significantly altered by VIP, and was reduced (39–56%) by PHI. The results indicate that GIP, VIP and PHI each stimulate glucagon release in a dose-related manner, but they exert discretely different effects on other islet hormones depending upon the dose and the prevailing glucose concentration.

Journal of Endocrinology (1990) 125, 375–379

ABSTRACT

In the long-term castrated rat the negative feedback effect of testosterone is markedly reduced and the raised levels of plasma LH seen in the castrated animals are not suppressed by physiological concentrations of plasma testosterone. In this study we have measured pituitary gonadotrophin-releasing hormone (GnRH) receptor content as well as plasma and pituitary LH on days 1, 10 and 40 after castration and noted the effect of testosterone replacement on these parameters.

We found that the negative feedback effect of physiological concentrations of testosterone on plasma and pituitary LH, pituitary GnRH receptor content and response to exogenous GnRH was attenuated 10 and 40 days after castration. It is suggested that the lack of effect of testosterone in the long-term castrated rat is due to its inability to reduce the pituitary GnRH receptor content. On increasing testosterone to supraphysiological levels, the negative feedback effect was reinstated.

We also found that in rats 40 days after castration, physiological and subphysiological concentrations of testosterone significantly increased pituitary GnRH receptor content and this may explain the previous findings that low concentrations of testosterone can enhance the effect of GnRH and increase plasma LH levels.

J. Endocr. (1986) 108, 441–449

ABSTRACT

The effect of synthetic human growth hormone-releasing hormone(1–40) (hGHRH-40) on the function of the endocrine pancreas and on glucose homeostasis in lean and genetically obese-diabetic (ob/ob) mice and normal rats has been examined. The addition of 1 μmol hGHRH-40/1 to incubated islets from normal lean mice increased insulin release by 90 and 37% at 5·6 and 16·7 mmol glucose/l respectively. Lower concentrations of hGHRH-40 did not affect insulin release. hGHRH-40 (1 μmol/l) increased pancreatic polypeptide release by 50% at 5·6 mmol glucose/l. A range of concentrations of hGHRH-40 (1 nmol/l–1 μmol/l) reduced glucagon release by 42–73% at 5·6 mmol glucose/l, and by 38–70% at 16·7 mmol glucose/l. Somatostatin release was increased (eightfold) by 1 μmol hGHRH-40/1 at 5·6 mmol glucose/l, but at 1 nmol hGHRH-40/l somatostatin release was reduced (by > 50%). At 16·7 mmol glucose/litre 0·01–1 μmol hGHRH-40/l increased somatostatin release (three- to fourfold), but 1 nmol hGHRH-40/l produced a reduction of 50%. In vivo, administration of hGHRH-40 (50 μg/kg body weight i.p.) to fasted lean and ob/ob mice did not alter basal plasma concentrations of glucose and insulin, or the glucose and insulin responses to a concomitant i.p. glucose challenge. Intravenous injection of hGHRH-40 (20 μg/kg body weight) to anaesthetized rats increased plasma concentrations of insulin in the hepatic portal vein. A lower dose of hGHRH-40 (0·2 μg/kg) was ineffective, and neither dose of hGHRH-40 altered plasma glucose. The results indicate that hGHRH-40 exerts dose-dependent effects on the secretion of islet hormones, but this does not appear to be sufficient to produce measurable effects on plasma glucose homeostasis.

Journal of Endocrinology (1989) 123, 19–24

ABSTRACT

The cellular mechanisms involved in GH biosynthesis have been investigated by the measurement of steady-state levels of cytosolic GH messenger RNA (mRNA) in primary cultures of rat pituitary cells using an RNA–complementary DNA (cDNA) hybridization assay. Growth hormone mRNA–cDNA hybridization increased in a linear manner with increasing cytosol concentration. Cellular GH mRNA levels rose by an average of 2·4-fold (range, 1·6–3·3; n = five experiments) after exposure to GH-ieleasing factor (GRF(1–40); 10 nmol/l) for 3 days. Treatment with GRF increased the release of GH into the culture medium, and depleted the cellular GH content by 40%. Total GH (in the medium plus cells) after GRF treatment increased by between 1·5- and 3·8-fold, a magnitude similar to the increase in GH mRNA levels. Treatment of cells with dibutyryl adenosine 3′:5′-cyclic monophosphate (1 mmol/l) or forskolin (5 μmol/l) increased the levels of cytosolic GH mRNA by between 1·6- and 4·7-fold. These agents increased GH release into the medium, depleted cellular GH content and increased total GH in the system to the same extent as GRF (10 nmol/l). These data demonstrate that cyclic adenosine nucleotides may mediate the GRF induction of GH gene transcription. In addition, we have shown that increases in the levels of cellular GH mRNA are reflected by increased GH biosynthesis, suggesting that the regulation of hormone gene transcription is one cellular site for the control of hormone biosynthesis and, ultimately, hormone available for release.

J. Endocr. (1986) 110, 51–57

Many transcription factors are expressed as multiple isoforms with distinct effects on the regulation of gene expression, and the functional consequences of structural differences between transcription factor isoforms may allow for precise control of gene expression. The pituitary transcription factor isoforms Pit-1 and Pit-1β differentially regulate anterior pituitary hormone gene expression. Pit-1 is required for the development of and appropriate hormone expression by anterior pituitary somatotrophs and lactotrophs. Pit-1β differs structurally from Pit-1 by the splice-insertion of the 26-residue β-domain in the trans-activation domain, and it differs functionally from Pit-1 in that it represses expression of the prolactin promoter in a cell-type specific manner. In order to identify signal and promoter context requirements for repression by Pit-1β, we examined its function in the presence of physiological regulatory signals as well as wild-type and mutant Pit-1-dependent target promoters. Here, we demonstrate that Pit-1β impairs recruitment of cAMP response element-binding protein (CREB)-binding protein to the promoters that it represses. In addition, we show that repression of target promoter activity, reduction in promoter histone acetylation, and decrease of CREB-binding protein recruitment all depend on promoter context. These findings provide a mechanism for promoter-specific repression by Pit-1β.