Growth Hormone Binding Sites Research Articles

Ration Size and Protein Intake affect Circulating Growth Hormone Concentration, Hepatic Growth Hormone Binding and Plasma Insulin-Like Growth Factor-I Immunoreactivity in a Marine Teleost, the Gilthead Sea Bream (Sparus aurata)

The nutritional regulation of the growth hormone liver axis has been studied in gilthead sea bream (Sparus aurata). In a first study, fingerling fish were fed three experimental diets with varying proportions of protein (34, 45 and 55%). A 60% decrease in plasma growth hormone concentration was observed with the increase of specific growth rates and dietary protein levels. An opposite response was observed in hepatic growth hormone-binding sites and plasma insulin-like growth factor-I immunoreactivity that would reflect the insensitivity of liver to growth hormone action during relatively low protein intake. In a second study, fish were fed a commercial diet (55% protein) at different feeding levels (0, 1.2, 2.7 and 5.5 g/(100 g body wt·d). An 84% decrease in plasma growth hormone concentration was observed with the increase of specific growth rates and feeding levels from 0 to 2.7 g/(100 g body wt·d). However, significantly greater growth hormone concentration was found in fish fed 5.5 g/(100 g body wt·d) when compared with fish fed 2.7 g/(100 g body wt·d). Hepatic growth hormone-binding sites and plasma insulin-like growth factor-I immunoreactivity increased with the increase of feeding levels from 0 to 2.7 g/(100 g body wt·d), but these values were lower in fish-fed 5.5 g/(100 g body wt·d) than in those fed 2.7 g/(100 g body wt·d). The physiological importance of these results remains to be clarified, though probably it is a part of the mechanism that diminishes feed utilization for growth at high feeding levels. In any case, for fish of the same age, the increase of circulating growth hormone can be used as a reliable marker of impaired growth in marine fish such as gilthead sea bream.

Growth Hormone Can Induce Expression of Four Major Milk Protein Genes in Transfected MAC-T Cells

Growth hormone (GH) can increase milk production in cattle, and this effect was thought to be mediated by an indirect mechanism because traditional ligand binding assays failed to detect GH binding sites in the mammary gland. However, recent findings that GH receptor (GHR) mRNA and protein are expressed in the epithelial cells of the bovine mammary gland suggest that GH may directly act on these cells to affect milk production. Therefore, the objective of this study was to determine whether GH could affect milk protein gene expression, nutrient uptake, and cell proliferation in bovine mammary epithelial cells using the bovine mammary epithelial cell-derived MAC-T cells as a model. Native MAC-T cells had low expression of GHR. Thus, we transfected them with expression plasmids for GHR and signal transducer and activator of transcription 5 (STAT5), 2 key components of GHR signaling, to maximize their GH response. Growth hormone increased the expression of αS1-casein, αS2-casein, β-casein, and α-lactalbumin mRNA 16- to 117-fold in the transfected MAC-T cells, whereas it had no effect on the expression of κ-casein, β-lactoglobulin, or insulin-like growth factor I mRNA. Cotransfection analyses showed that GH also strongly induced reporter gene expression from αS1-casein, αS2-casein, β-casein, and α-lactalbumin gene promoters. Growth hormone had no effect on the uptake of 2-deoxyglucose, an unmetabolizable glucose analog, amino acids, or oleic acid; neither did it affect cell proliferation or death. These observations together with the fact that GH receptor mRNA and protein are expressed in the epithelial cells of the bovine mammary gland raise the possibility that GH might act directly on the mammary epithelial cells in cows to stimulate transcription of major milk protein genes, as part of the mechanism by which GH stimulates milk production.

Expression of growth hormone receptor and its mRNA in hepatic cirrhosis.

To investigate the expression of growth hormone receptor (GHR) and mRNA of GHR in cirrhotic livers of rats with the intension to find the basis for application of recombinant human growth hormone (rhGH) to patients with liver cirrhosis. Hepatic cirrhosis was induced in Sprague-Dawley rats by administration of thioacetamide intraperitoneally for 9-12 weeks. Collagenase IV was perfused in situ for isolation of hepatocytes. The expression of GHR and its mRNA in cirrhotic livers was studied with radio-ligand binding assay, RT-PCR and digital image analysis. One class of specific growth hormone-binding site, GHR, was detected in hepatocytes and hepatic tissue of cirrhotic livers. The binding capacity of GHR (R(T), fmol/mg protein) in rat cirrhotic liver tissue (30.8+/-1.9) was significantly lower than that in normal control (74.9+/-3.9) at the time point of the ninth week after initiation of induction of cirrhosis (n=10, P<0.05), and it decreased gradually along with the accumulation of collagen in the process of formation and development of liver cirrhosis (P<0.05). The number of binding sites (X10(4)/cell) of GHR on rat cirrhotic hepatocytes (0.86+/-0.16) was significantly lower than that (1.28+/-0.24) in control (n=10, P<0.05). The binding affinity of GHR among liver tissue, hepatocytes of various groups had no significant difference (P>0.05). The expression of GHR mRNA (riOD, pixel) in rat cirrhotic hepatic tissues (23.3+/-3.1) was also significantly lower than that (29.3+/-3.4) in normal control (n=10, P<0.05). The growth hormone receptor was expressed in a reduced level in liver tissue of cirrhotic rats, and lesser expression of growth hormone receptors was found in a later stage of cirrhosis. The reduced expression of growth hormone receptor was partly due to its decreased expression on cirrhotic hepatocytes and the reduced expression of its mRNA in cirrhotic liver tissue.

Growth hormone (GH) action in the brain: neural expression of a GH-response gene.

The presence of growth hormone (GH) binding sites and GH-receptor (GHR)-immunoreactive proteins in the brain suggests it is a target site for GH action. This could, however, reflect the presence of GH-binding proteins (GHBP) that are not linked to intracellular signal-transduction mechanisms, rather than authentic receptors. The possibility that GH has actions in the brain therefore has been examined by determining an intracellular mediator of GH action. The mechanism of GH action involves the induction of a number of specific GH-response genes. In chickens, a novel GH-responsive gene (GHRG-1) has been identified as an intracellular marker of GH action, since this gene is not expressed in GH-resistant dwarfs with dysfunctional GHRs and in normal chickens it is upregulated by exogenous GH. In normal chickens GHRG-1 mRNA is also abundant and widespread in the brain. In the cerebellum it is specifically localized in the cerebellar folia. It is present in most cells in the granular layers of the gray matter but is present in only a small number of scattered cells in the molecular layer and white matter. Intense labeling for GHRG-1 mRNA is also present in the large Purkinje cells and their dendrites at the interface between the molecular and granular layers. Labeling is also seen in the interneuronal basket cells projecting onto the Purkinje cells. In the mid-brain, cells in the ocular nerve complex and the tractus isthmo-opticus were strongly stained for GHRG-1 mRNA, with less intense staining in the central gray. In the hypothalamus, numerous small cells in periventricular locations and ependymal cells lining the III ventricle also label for GHRG-1 mRNA. These results clearly show, for the first time, the expression of a GH-responsive gene in neural tissues. Moreover, as GH- and GHR-immunoreactivity previously has been shown to be present in the same tissues expressing GHRG-1, it is possible that GH acts as an autocrine or paracrine within the CNS.

Relative localization of the prolactin receptor binding sites for lactogenic hormones.

A monoclonal antibody termed MAb R7B4, directed to an epitope present in prolactin receptors (PRLRs), was used as a tool to map the receptor binding sites for human growth hormone (hGH), ovine prolactin (oPRL) and human placental lactogen (hPL). Although the three hormones completely inhibited the binding of each other to Nb2 cells or rat liver receptors, MAb R7B4 behaviour was different depending on the hormone tested and the receptor source. According to the MAb effects, PRLR from Nb2 cells would locate both hGH and oPRL close to R7B4 epitope, whereas hPL would bind far from the MAb binding site. On the other hand, PRLR from rat liver should bind hGH close to the R7B4 epitope but oPRL and hPL would be recognized by a separate region of the same receptor. Thus, results presented in this paper suggest that PRLR binding sites for hGH, oPRL and hPL do not exactly overlap in spite of full competition between ligands.

The IGFBP-3 mRNA and protein levels are IGF-I-dependent and GH-independent in MG-63 human osteosarcoma cells

Growth Hormone (GH), Insulin-like Growth Factors (IGFs) and IGF-Binding Proteins which modulate the IGFs' bioavailability (e.g. IGFBP-3, -4, -5), are essential regulators of bone remodeling. In this study, MG-63 human osteosarcoma cells were used as a model system to investigate the mechanism(s) whereby IGF-I and GH control IGFBP-3 gene expression. Physiological concentrations of IGF-I (1–20 nM) induced a dose-dependent increase in the steady-state amount of IGFBP-3 mRNA (maximal stimulation: ∼9–10-fold). This increase was detectable 3 h after the onset of IGF-I treatment, was enhanced over a 24 h period, then plateaued until at least 30 h. Consistently, a dose-dependent increase in IGFBP-3 secretion (∼40–50-fold at IGF-I concentrations≥16 nM) was observed by western ligand- and immuno-blot analysis of MG-63 cells conditioned medium, and its time course was similar to that observed for IGFBP-3 transcripts. IGFBP-3 mRNA stability ( t 1/2 ∼20 h) was identical in the presence or absence of IGF-I treatment. By contrast, human (h) GH treatment (24–72 h) of MG-63 cells did not increase IGFBP-3 secretion in the conditioned medium. Ectopic expression of recombinant rat GH-R resulted in hGH-enhanced expression of GH-responsive reporter gene constructs, but did not increase endogenous IGFBP-3 gene expression, suggesting that the GH unresponsiveness was not only due to the very low level of GH binding sites at the plasma membrane level. Altogether, these results support the conclusions that in MG-63 cells (i) transcriptional rather post-transcriptional mechanisms are involved in the IGF-I-induced increase of IGFBP-3; (ii) the abundance of GH-R is very low at the plasma membrane level; (iii) the dowstream GH-signaling cascade is fully functional in this human osteosarcoma cell line; and (iv) the endogenous IGFBP-3 gene is not responsive to hGH in human MG-63 osteosarcoma cells.

Growth Hormone in the Brain: Characteristics of Specific Brain Targets for the Hormone and Their Functional Significance

During the past decade studies have shown that growth hormone (GH) may exert profound effects on the central nervous system (CNS). For instance, GH replacement therapy was found to improve the psychological capabilities in adult GH deficient (GHD) patients. Furthermore, beneficial effects of the hormone on certain functions, including memory, mental alertness, motivation, and working capacity, have been reported. Likewise, GH treatment of GHD children has been observed to produce significant improvement in many behavioral problems seen in these individuals. Studies also indicated that GH therapy affects the cerebrospinal fluid levels of various hormones and neurotransmitters. Further support that the CNS is a target for GH emerges from observations indicating that the hormone may cross the blood–brain barrier (BBB) and from studies confirming the presence of GH receptors in the brain. It was previously shown that specific binding sites for GH are present in discrete areas in the CNS of both humans and rats. Among these regions are the choroid plexus, hippocampus, hypothalmus, and spinal cord. The density of GH binding in the various brain regions was found to decline with increasing age. More recently, we were able to clone and determine the structure of several GH receptors in the rat and human brain. Although the brain receptor proteins for the hormone were shown to differ in molecular size compared to those present in peripheral tissues the corresponding transcripts did not seem to differ from their peripheral congeners. GH receptors in the hypothalamus are likely to be involved in the regulatory mechanism for hormone secretion and those located in the choroid plexus have been suggested to have a role in the receptor-mediated transport of GH across the BBB. The functions mediated by the GH receptors identified in the hippocampus are not yet known but recently it was speculated that they may be involved in the hormone's action on memory and cognitive functions.

Growth hormone, insulin-like growth factor I and cognitive function in adults.

This review focuses on the possible contribution of the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis to cognitive function. Binding sites for GH and IGF-I are found in various areas of the brain. Their distribution suggests that GH and IGF-I contribute to the function of the hippocampus, a brain structure important for the maintenance of cognitive functions such as learning and memory. Evidence for cognitive deficits in GH-deficient individuals has been found in various studies, some of which have shown that these deficits can be reversed by GH substitution therapy. In addition to examining conditions of GH deficiency, this article reviews studies evaluating the correlation between the cognitive deficits associated with ageing and age-related decreases in GH or IGF-I secretion. Based on the available data, one might hypothesize that relative GH or IGF-I deficiency could contribute to the deterioration of cognitive functions observed in the elderly.

Correlative factors of insulin resistance in essential hypertension.

Essential Hypertension (EH) is correlated with a metabolic disturbance characterized by insulin resistance (IR). In this study, there were observed in 47 subjects with EH and 30 subjects with normal blood pressure. Serum levels of insulin-like growth factor-1 (IGF-1), serum levels of growth hormone (GH), the activity of erythrocyte insulin receptors (EIR), and ATP levels in erythrocytes, the insulin sensitivity index (ISI) was used to study the correlative factors of essential hypertension. 1. Among patients with EH, ISI, GH, and low-affinity insulin binding sites of EIRs (RT2) were found to be in significantly lower amounts, IGF-1 levels and the KD2 of the erythrocyte insulin receptors were noted to be significantly higher. Compared with the control group, there was a marked difference between EH group and the control group. However, no statistical difference was observed between the hypertensive group and the group with normal blood pressure as regards erythrocyte ATP levels, high-affinity insulin binding sites of EIRs (RT1), and the KD1 of EIRs. 2. In the hypertensive group, the ISI was negatively correlated with mean arterial blood pressure (MBP), a family history of hypertension, the body mass index (BMI), the waist-hip ratio (WHR) and IGF-1 levels (r=-0.614delta, -0.354**, -0.386**, -0.472**, -0.298*, delta p < 0.001, **p < 0.01, *p < 0.05), were positively correlated with RT2 and GH levels (r=0.301**, 0.275*, **p < 0.01, *p < 0.05). There were no statistically significant differences between ISI and age, sex, smoking history, drinking, RT1, KD1, and ATP levels in erythrocytes. 3. The ISI was used as the dependent variable in multiple linear stepwise regression analysis. MBP (X1), a family history of EH (X2), WHR (X3), GH (X4), IGF-1 (X5), RT2 (X6), and the body mass index (X7) was used as independent variables. X1, X2, X3, X5, X6, and X7 were used in the equations. The results indicate that patients with EH also tend to have IR. We suggest that MBP, a family history of hypertension, BMI, WHR, IGF-1, and RT2 might be independent factors affecting IR in cases of essential hypertension.

Generation of epitope‐specific antibodies to rat GHBP in the sheep using an interspecies switching strategy involving site‐directed mutagenesis of ovine GHBP

Site-directed antibodies to the growth hormone receptor could be potentially useful as growth hormone mimics but, in previous attempts, we found that antisera generated using peptides derived from growth hormone receptor sequences failed to recognize the intact protein. As an alternative approach to this problem, we have now adopted a strategy of epitope-switching between rat and ovine growth hormone receptors to produce rat epitopes in the correct structural context. Using site-directed mutagenesis, we altered the two dominant linear epitopes in the ovine growth hormone binding protein to the analogous sequences in rat growth hormone binding protein. Site A, between Thr28 and Leu34, is equivalent to epitope 1 in ovine growth hormone binding protein and site B, between Ser121 and Asp124, corresponds to epitope 5. The wild-type ovine growth hormone binding protein and the two mutant proteins were bacterially expressed, refolded and, following purification by metal-chelate affinity chromatography, used to raise antisera in sheep. We showed using RIA, in which wild-type ovine growth hormone binding protein acted as a competitor for the binding of rat growth hormone binding protein, that only the site A mutant protein elicited a specific anti-rat growth hormone binding protein response. This was confirmed in subsequent RIA studies using the antiserum to the site A mutant protein in which only peptides corresponding to the site A sequences in mutant ovine growth hormone binding protein and rat growth hormone binding protein, but not that in wild-type ovine growth hormone binding protein, were able to act as competitors for rat growth hormone binding protein. Antibodies specific for rat growth hormone binding protein could be separated from the antiserum to the site A mutant protein by means of affinity chromatography using immobilized wild-type ovine growth hormone binding protein to remove antibodies which cross-reacted with the ovine protein. The work lays the foundations for further studies in which the biological effects of these antibody fractions will be investigated and demonstrates an approach with general applicability in the production of antibodies directed towards specific epitopes on protein molecules.

Localization and quantification of binding sites for follicle-stimulating hormone, luteinizing hormone, growth hormone, and insulin-like growth factor I in sheep ovarian follicles.

In sheep, growth and development of ovarian follicles beyond 2 mm in diameter is acutely dependent on gonadotropin support. As a consequence, following hypophysectomy (HPX) or hypothalamic-pituitary stalk disconnection (HPD), growth of follicles beyond 2 mm is arrested and all follicles > 2 mm undergo atresia. Although administration of exogenous gonadotropins stimulates follicular growth and ovulation in HPD ewes, follicles in HPX ewes remain unresponsive unless growth hormone (GH) is also given. To determine whether the difference in follicular sensitivity to gonadotropins after HPD (gonadotropin sensitive) or HPX (gonadotropin insensitive) is related to the distribution and quantity of binding sites for FSH, LH, and/or insulin-like growth factor I (IGF-I), binding sites for these hormones were localized and quantified using topical autoradiography in healthy follicles from control (pituitary-intact), HPD, and HPX ewes. In addition, in situ hybridization was performed to localize mRNA for GH and FSH receptors. Irrespective of treatment, binding of FSH and mRNA for FSH receptor were greatest (p < 0.05) in the membrana granulosa; LH binding was greatest (p < 0.05) in the theca interna; and IGF-I binding was greatest (p < 0.05) in the theca externa. Although the relative number of binding sites for LH did not differ among treatments, those for FSH and IGF-I were lower (p < 0.05) in HPD and HPX ewes compared to controls. Attempts to quantify binding sites for GH were unsuccessful due to high nonspecific binding. However, mRNA for GH receptor was most abundant (p < 0.05) in the membrana granulosa and oocytes of small antral and preantral follicles. Compared to levels in controls and HPD ewes, the level of GH receptor mRNA was lower (p < 0.05) in follicles obtained from HPX ewes. On the basis of these data, failure of small antral follicles in HPX ewes to respond to exogenous gonadotropins is not due to a reduction in receptors for FSH, LH, or IGF-I. The observed reduction of mRNA for GH receptor in the membrana granulosa of follicles from HPX ewes provides evidence that GH may play an important role in early stages of folliculogenesis and that it is involved in the maintenance of sensitivity to gonadotropins.

Autoradiographic localization of growth hormone binding sites in Sparus aurata tissues using a recombinant gilthead seabream growth hormone

Growth hormone (GH) binding sites in tissues of gilthead seabream, Sparus aurata, were localized by using an in vitro autoradiographic analysis. Cryomicrotome sections of liver, spleen, kidney, heart and skeletal muscle were mounted on gelatin-coated slides and incubated with a radioiodinated recombinant gilthead seabream GH (0.54 ng/100 μl buffer) for 24 h at 4 °C. Slides were coated with autoradiographic emulsion and exposed for 1–9 weeks. The highest density of GH binding sites was found in liver. This specific binding was evident on hepatocytes. Blood cells in hepatic vascular system also showed GH binding while intrahepatic exocrine pancreas did not. In spleen, a high density of GH binding sites was found surrounding melanomacrophage centers. Splenic ellipsoid cells also showed a strong GH binding while splenic pulp exhibited a weak GH binding. Kidney GH binding sites were present in renal tubules and renal haemopoietic tissue. Cardiac muscle exhibited an intense GH binding. However, GH binding sites were not detected in body skeletal muscle. This study confirms and extends, by using autoradiographic techniques, previous results obtained by radioreceptor assays.

Potentiating Effect of Growth Hormone on Vitellogenin Synthesis Induced by 17β-Estradiol in Primary Culture of Female Silver Eel (Anguilla anguillaL.) Hepatocytes

Previousin vivoexperiments have indicated a potentiating role of growth hormone (GH) during experimentally induced vitellogenesis by 17β-estradiol (E2) in the female silver eel (Anguilla anguillaL.). To investigate whether GH has direct hepatic actions, the effects of hypophysial-purified and recombinant GH on vitellogenin (Vg) synthesis in response to E2 were tested on primary cultures of hepatocytes. Hepatocytes were prepared from control or E2-primed eels. Addition of E2 alone into the culture medium induced both Vg synthesis and secretion in a dose- and time-related fashion. Bovine growth hormone (bGH) alone had no effect on the induction of Vg synthesis or secretion. Bovine GH enhanced thein vitroeffects of E2 on both Vg synthesis and secretion, an effect attenuated by anin vivoE2 priming which was dose-dependent with an ED50of 5 ng/ml. To investigate the specificity of GH action, purified eel and salmon GH and salmon, trout, and tilapia prolactins (PRL), as well as recombinant trout and tilapia GH, were tested, and the responses were compared to bGH. Purified salmon and homologous eel GH potentiated the vitellogenic response to E2. Recombinant GH were highly efficacious, excluding the presence of active contaminants in the potentiating effect of GH preparations. The potentiating effect of recombinant trout GH on the vitellogenic response was reduced at high doses (above 20 ng/ml), suggesting a down-regulation of GH binding sites by GH itself. Salmon PRL has minimal activity, but not trout and tilapia PRL, indicating that PRL is not an important potentiating factor on Vg synthesis in our model. It is concluded that GH acts directly on the liver to potentiate E2 induction of eel hepatic Vg synthesis. The potentiating effect of GH appears to be time- and dose-dependent and modulated as a function of hormonal status (E2 priming) of the eel.

Characterization of growth hormone binding sites in the goldfish,Carassius auratus: effects of hypophysectomy and hormone injection

A recombinant carp growth hormone (rcGH) was used to develop for a GH radioreceptor binding assay in the goldfish (Carassius auratus). Specific binding of(125)I-rcGH to goldfish liver membranes was a pH, time, temperature, and membrane protein dependent process. Scatchard and LIGAND analysis indicated a single class of high affinity and low capacity binding site, with an association constant (Ka) of 1.9×10(10) M(-1) and a maximum binding capacity (Bmax) of 9 fmol mg(-1) protein. Liver tissue displayed the highest(125)I-rcGH binding of all the tissues examined. Displacement of(125)I-rcGH with various unlabeled teleost and mammalian GHs and prolactins revealed that the goldfish hepatic binding site was highly specific for teleost GH. Intraperitoneal administration of 0.1, 1.0, and 10 μg rcGH g(-1) body weight to hypophysectomized goldfish resulted in a 27, 52, and 68% decrease in total binding sites, respectively. Injection of a high dose of rat prolactin (rPRL) (5 μg rPRL g(-1) body weight) also resulted in a 32% decrease in total binding sites. These results suggest that endogenous GH may have a role in the regulation of its own receptors in the goldfish.

Growth Hormone Receptors and Insulin-like Growth Factor I Receptors on Human Peripheral Blood Cells

Growth hormone (GH) and insulin-like growth factor I (IGF-I) may be able to modulate some functions of the immune system. In this study, we examined the expression of the receptors for GH and IGF-I on human peripheral blood cells in physiological and pathological conditions.By using two-colour analysis on flow cytometry with a biotinavidin system, there was a specific GH binding site on human peripheral lymphocytes. GH-binding was more prominent on CD20+ (=B cells) than on CD2+ (=T cells), although GH receptor messenger ribonucleic acid (mRNA) could be detected in the T cells as well as in the B cells. There was no age-dependency of GH-binding on the CD20 cells, and there was no relationship between GH-binding and serum GHbinding protein activities, which reflect the tissue levels of GH receptors.We detected αIR3, anti IGF-I receptor antibody, bindings and IGF-I receptor mRNA expression in peripheral blood cells. Based on two-colour analysis, there were relatively higher bindings of αIR3 on CD4+ and CD16+ cells, intermediate bindings on CD8+ cells and weak bindings on CD20+ cells. In cord blood cells, αIR3 bindings and IGF-I receptor mRNA were higher than in adult cells. In GH-deficient patients, αIR3 bindings were higher than in control subjects, and reverted to normal after GH therapy.These results suggest that 1) GH and IGF-I may have hormonal effects on the immune system, 2) GH receptors on lymphocytes dose not correlate with the tissue levels of GH receptors, and 3) the cellular levels of IGF-I receptors may be inversely related to the ambient IGF-I concentrations.

Expression and regulation of growth hormone (GH) and prolactin (PRL) receptors in a rat insulin producing cell line (INS-1)

We have examined the expression of growth hormone (GH) and prolactin (PRL) receptors, and the binding parameters of human GH (hGH), on the rat insulin producing cell line INS-1. We found that, like normal insulin producing β-cells, INS-1 cells express both GH and PRL receptors, and the majority of human GH (hGH) binding sites on this cell line are of lactogenic specificity. As calculated from Scatchard plots, about 6600 hGH binding sites with a K a of 6.2 × 10 8 M −1 are present per cell. Northern blot analysis showed two mRNA species of 4 and 1.6 kb for the GH receptor and, one major species of 10.5 kb for the PRL receptor. The PRL receptor mRNA was up-regulated by bovine GH (bGH), rat PRL (rPRL), in a time- and dose-dependent manner. On the contrary, bGH and rPRL, down-regulated the expression of GH receptor gene. The importance of this differential regulation in in vivo and in vitro studies are discussed.

The endocrinology of growth in carp and related species

The focus of this review is on various aspects of the endocrine control of growth by the brain neuroendocrine-growth hormone (GH)-insulin-like growth factor (IGF) axis in carp and other cyprinids, with reference to other species, including mammals, if there are particular gaps in knowledge. The neuroendocrine regulation of GH secretion in goldfish and other carp is multifactorial, with a balance of stimulatory and inhibitory inputs to somatotrophs. Somatostatin is the primary inhibitor of basal and stimulated growth hormone (GH) secretion. GH secretion is stimulated by GH-releasing factor, gonadotropin-releasing hormone, dopamine, neuropeptide Y, thyrotropin-releasing hormone and cholecystokinin. Sex steroids, in particular estradiol, influence the responsiveness of the somatotrophs to neuroendocrine factors; the responsiveness to gonadotropin-releasing hormone, neuropeptide Y, and thyrotropin-releasing hormone is increased by estradiol, whereas the responsiveness to dopamine and cholecystokinin is greatest in sexually regressed goldfish. Growth hormone, a 188-amino-acid peptide with 5 cysteine residues, stimulates growth through direct actions on some tissues, as well as by stimulation of production of insulin-like growth factor (IGF) production. The liver has a high number of GH receptors and is a primary target organ; GH binding sites have also been demonstrated in gill, intestine, kidney and gonads. GH increases the efficiency of food conversion; GH stimulates intestinal amino acid transport and intestinal mass, which may be one mechanism for the effects on food conversion. A number of factors influence GH receptor number, including GH itself, nutritional status and other hormones. IGFs have been characterized in several salmonid species using molecular biology techniques. Production of IGFs is under stimulation of GH, insulin and other hormones, and is also influenced by nutritional status and metabolic factors. The liver contains the highest concentrations of IGF, although IGFs have been found in a number of other tissues. IGFs appear to travel in the blood bound to specific binding proteins. IGF receptors have been demonstrated in only one teleost species to date. IGF-I stimulates cartilage proteoglycan synthesis; GH is dependent on IGF-I for this action. Growth rates of cultured fish may be stimulated by neuroendocrine factors added to food, administration of GH or recombinant GH preparations, or by producing GH transgenic lines of fish. Techniques for enhancing growth rates of cultured fish are in an experimental stage.

Decreased binding of growth hormone in the rat hypothalamus and choroid plexus following morphine treatment

Male Sprague-Dawley rats were continuously infused with morphine through subcutaneously implanted mini-osmotic pumps over a period of 5 days. The binding of rat growth hormone (rGH) to specific sites in choroid plexus, cortex, hypothalamus, hippocampus and striatum was determined. It was found that in the acute phase of morphine administration the density of growth hormone-binding sites was significantly decreased in choroid plexus and in hypothalamus, but not in any other of the tissues studied. When tolerance to morphine was developed, the level of growth hormone-binding was restored to control level. In the acute phase, the plasma levels of GH, as measured by radioimmunoassay, correlated negatively with the density of the binding sites in choroid plexus and hypothalamus. The decrease in growth hormone-binding in these regions of the rat brain was also confirmed by SDS-polyacrylamide gel electrophoresis of cross-linked complexes of the binding entities to 125I-labelled rGH as visualized by autoradiography. In experiments, where morphine was administrated by intermittent injections, a similar decrease in rGH-binding was observed. However, the time-course of this decrease seemed to be dependent upon the route of administration. Following intracerebroventricular (i.c.v.) injections, the binding of the hormone was already affected after 30 min, whereas the binding of rGH in brain areas after subcutaneous (s.c.) injections was affected at a later stage.

Nuclear translocation and anchorage of the growth hormone receptor.

The extracellular domain of the rabbit growth hormone (GH) receptor has previously been shown to be associated with the nucleus. However, in this species the GH binding protein (BP) is derived by proteolytic cleavage of the full-length receptor, and thus distinction between the receptor and BP is difficult. The intracellular domain of the GH receptor is required for GH-stimulated function. Thus a direct nuclear function of GH would presumably require the receptor intracellular domain in the nucleus. We have therefore characterized the rat nuclear GH receptor and BP based on their distinct antigenic identity. We show, in vivo, that the full-length receptor is associated with the nucleus, including the respective subnuclear fractions (nucleoplasm, outer nuclear membranes, inner nuclear membranes, and chromatin). In vivo, the receptor is also subject to ligand-dependent nuclear translocation. Cellular transfection of rat GH receptor cDNA resulted in the appearance of nuclear binding sites for 125I-labeled human GH not present in the untransfected parental cell line (Chinese hamster ovary (CHO), buffalo rat liver). To determine which portion of the receptor was responsible for nuclear anchorage, we examined the binding of 125I-labeled human GH to whole nuclei isolated from CHO cells expressing the full-length receptor, a receptor in which 184 amino acids had been deleted from the carboxyl-terminal intracellular domain (CHO-454) and a receptor containing only 5 of 349 amino acids in the intracellular domain (CHO-294). Nuclear binding above the level of the untransfected parental cell line was detected only in CHO-638 and CHO-454 cells, suggesting that amino acids 294-454 of the receptor are necessary for nuclear anchorage. This observation was not due to membrane contamination, as the CHO-294 cells express a membrane-bound receptor that was not anchored in the nucleus. The full-length GH receptor in receptor cDNA-transfected cells is nucleocytoplasmic in the absence of ligand but is also subject to rapid ligand-dependent nuclear translocation. The presence of the intracellular domain of the GH receptor in the nucleus allows the possibility of a direct nuclear response to GH.

Characterization of growth hormone binding sites in rat brain

The binding of 125I-labelled rat growth hormone (GH) to different areas in the brain was studied in male Sprague-Dawley rats. A high density of GH binding was found in the choroid plexus, hypothalamus, hippocampus, pituitary and spinal cord, whereas a lower binding density was observed in the cortex. Binding of the hormone to the various brain regions was age dependent. Binding was also dependent on time, pH and protein concentration. The binding affinity of the labelled hormone to choroid plexus was 4.3 per nmol/l and the binding capacity was 33.4 nmol/mg protein. The corresponding figures for binding of 125I-labelled GH to hypothalamus were 5.6 per nmol/l and 21.6 nmol/mg protein. By sodium dodecyl sulphate electrophoresis of the cross-linked hormone-receptor complexes, molecular weights of 60,000 and 61,000 were determined for the binding units in the choroid plexus and hypothalamus, respectively. It was further indicated that the binding unit for rat GH was distinct from that for prolactin.