Ovine GH Research Articles

Molecular Cloning, Expression, Sequence Characterization and Structural Insight of Bubalus bubalis Growth Hormone-Receptor.

The current study focuses on molecular cloning, expression and structural characterization of growth hormone-receptor (GHR) and its extracellular domain as growth hormone binding protein (GHBP) from the liver of Nili-Ravi buffalo (Bubalus bubalis; Bb). RNA was isolated, genes were amplified by reverse transcriptase-polymerase chain reaction and sequence was characterized. The BbGHR sequence showed three amino acid variations in the extracellular domain when compared with Indian BbGHR. For the production of full length BbGHR and BbGHBP in Escherichia coli (E. coli) BL21 (RIPL) Codon Plus, expression plasmids were constructed under the control of T7lac promoter and isopropyl β-D thiogalactopyranoside was used as an inducer. BbGHR and BbGHBP were expressed as inclusion bodies at ~ 40% and > 30% of the total E. coli proteins, respectively. The BbGHBP was solubilized and refolded by dilution method using cysteine-cystine redox potential. The recombinant BbGHBP was purified and biological activity was checked on HeLa cell lines showing increase cell proliferation in the presence of ovine GH (oGH), hence justifying the increase in the half-life of GH in the presence of BbGHBP. For the molecular interactions of oGH-BbGHBP multiple docking programs were employed to explore the subsequent interactions which showed high binding affinity and presence of large number of hydrogen bonds. Molecular Dynamics studies performed to examine the stability of proteins and exhibited stable structures along with favorable molecular interactions. This study has described the sequence characterization of BbGHR in Nili-Ravi buffaloes and hence provided the basis for the assessment of GH-GHR binding in other Bovidae species.

Nutritional status and growth hormone regulate insulin-like growth factor binding protein (igfbp) transcripts in Mozambique tilapia

Growth in teleosts is controlled in large part by the activities of the growth hormone (Gh)/insulin-like growth factor (Igf) system. In this study, we initially identified igf-binding protein (bp)1b, -2b, -4, -5a and -6b transcripts in a tilapia EST library. In Mozambique tilapia (Oreochromis mossambicus), tissue expression profiling of igfbps revealed that igfbp1b and -2b had the highest levels of expression in liver while igfbp4, -5a and -6b were expressed at comparable levels in most other tissues. We compared changes in hepatic igfbp1b, -2b and -5a expression during catabolic conditions (28days of fasting) along with key components of the Gh/Igf system, including plasma Gh and Igf1 and hepatic gh receptor (ghr2), igf1 and igf2 expression. In parallel with elevated plasma Gh and decreased Igf1 levels, we found that hepatic igfbp1b increased substantially in fasted animals. We then tested whether systemic Gh could direct the expression of igfbps in liver. A single intraperitoneal injection of ovine Gh into hypophysectomized tilapia specifically stimulated liver igfbp2b expression along with plasma Igf1 and hepatic ghr2 levels. Our collective data suggest that hepatic endocrine signaling during fasting may involve post-translational regulation of plasma Igf1 via a shift towards the expression of igfbp1b. Thus, Igfbp1b may operate as a molecular switch to restrict Igf1 signaling in tilapia; furthermore, we provide new details regarding isoform-specific regulation of igfbp expression by Gh.

Effects of early gestation GH administration on placental and fetal development in sheep

Ovine GH (oGH) is synthesized in placental tissue during maximal placental growth and development. Our objectives were to localize oGH mRNA in the placenta, and study the impact of exogenous GH on twin pregnancies during the normal window (35-55 days of gestational age; dGA) of placental expression. In situ hybridization localized oGH mRNA in uterine luminal epithelium but not in tissues of fetal origin. While maternal GH and IGF-I concentrations were increased (P<0.001) approximately tenfold, uterine, uterine fluid, placental, and fetal weights were unaffected by treatment at either 55 or 135 dGA. Fetal length, liver weight, and liver weight per kg of body weight were unaffected by maternal GH treatment. However, in the cotyledon, IGF-binding protein (BP)-1 and IGFBP-4 mRNA concentrations were increased (P<0.05), while IGFBP-2 mRNA was decreased (P<0.05). The concentration of mRNA for IGFBP-3 was unaffected by treatment. Within the caruncle, IGFBP-1 mRNA was decreased (P<0.05), while IGFBP-3 and IGFBP-4 mRNA were increased (P<0.05), and IGFBP-2 mRNA was unchanged due to GH treatment. While our data indicate that elevated maternal GH and IGF-I concentrations during early and mid-gestation do not enhance placental and fetal growth in twin pregnancies, localization of GH mRNA in uterine luminal epithelium could explain GHs transitory expression from 35 to 55 dGA, since by the end of this period the majority of the uterine luminal epithelium has fused with chorionic binucleate cells forming the placental syncytium.

Preparation and Characterization of Recombinant Chicken Growth Hormone (chGH) and Its Putative Antagonist chGH G119R Mutein

Synthetic cDNA of chicken GH (chGH) and its G119R mutein was synthesized after being optimized for expression in E. coli. The respective cDNAs were inserted into expression vector, expressed and found almost entirely in the insoluble inclusion bodies (IBs). The IBs were isolated, the proteins solubilized in 4.5 M urea, at pH 11.3 in presence of cysteine, refolded, and purified to homogeneity by anion-exchange chromatography on Q-Sepharose. The overall yields were 400 to 500 mg from 5 L of fermentation. Both proteins were > 98% pure, as evidenced by SDS-PAGE, and contained at least 95% monomers, as documented by gel-filtration chromatography under non-denaturing conditions. Circular dichroism analysis revealed that both proteins have identical secondary structure characteristic of cytokines, namely > 50% of alpha helix content. Chicken GH was capable of forming a 1:2 complex with recombinant oGH receptor extracellular domain, but its affinity, as determined by RRA, was 11-fold lower than that of ovine GH (oGH). Correspondingly, its bioactivity, assessed using FDC-P1 3B9 cells stably transfected with rabbit GHR, was 30-40-fold lower, whereas chGH G119R mutant did not bind to oGHR-ECD and was devoid of any biological activity in FDC-P1 3B9 cells. However, in binding experiments that were carried out using chicken liver membranes, both oGH and chGH showed similar IC(50) values in competition with (125)I-oGH, while the IC(50) of G119R mutein was 10-fold higher. These results emphasize the importance of species specificity and indicate the possibility of antagonistic activity of chGH G119R.

Multiple effects of an additional growth hormone gene in adult sheep1

Molecular genetics provides an increasing capacity to modulate the function of individual genes, but the practical implications of these technologies are still poorly understood. This study examined adult Merino or Merino-cross sheep that had an additional copy of the ovine GH gene with a modified metal-lothionine promoter, which resulted in a doubling of the plasma concentration of GH. Previous work showed that up to the age of 18 mo, GH sheep grew faster and had less s.c. fat, with only minor effects on fleece production. The present paper describes characteristics of reproduction, wool production, and animal health of these sheep during the following 2 yr of adult life. Ewes with the GH gene had a greater ovulation rate (1.78 vs. 1.35; P <0.05), but bore fewer lambs, apparently due to greater fetal loss after mating. Grease fleece weight was increased (P <0.05) due mainly to a greater content of suint (9.1 vs. 7.7 +/- 0.4%, P <0.01), which was associated with a deeper color of the raw wool. Effects on clean fleece weight and fiber diameter were not consistent between years. The GH sheep had swollen metatarsal and metacarpal joints, which was associated with a need for more frequent hoof-trimming, and more GH than control sheep died during the experiment (P <0.001). All of these changes are consistent with previously reported effects of increased plasma GH. Results of this study show that increased activity of a single gene (GH) affected several production characteristics and predisposed the animals to a number of distinct health problems, some of which developed after the normal age of genetic selection.

Ovine placental lactogen-induced heterodimerization of ovine growth hormone and prolactin receptors in living cells is demonstrated by fluorescence resonance energy transfer microscopy and leads to prolonged phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3.

HEK-293T cells transiently transfected with ovine (o) GH receptor (GHR) and prolactin receptor (PRLR) constructs respectively tagged downstream with cyan or yellow fluorescent proteins were used to study ovine placental lactogen (oPL)-stimulated heterodimerization by fluorescence resonance energy transfer (FRET) microscopy. The oPL-stimulated transient heterodimerization of GHR and PRLR had a peak occurring 2.5-3 min after oPL application, whereas oGH or oPRL had no effect at all. The results indicate none or only little dimerization occurring before the hormonal stimulation. The effect of heterodimerization was studied by comparing activation of Janus kinase 2, signal transducer and activator of transcription (STAT)1, STAT3, STAT5, and MAPK in Chinese hamster ovary cells stably transfected with chimeric genes encoding receptors consisting of cytosolic and transmembrane parts of oGHR and oPRLR, extracellular domains of human granulocyte and macrophage colony-stimulating factor (hGM-CSF) receptor alpha or beta, and cells transfected with the two forms (alpha or beta) of PRLR and GHR. Functionality of those proteins was verified by hGM-CSF-induced phosphorylation of both intracellular PRLR and GHR domains and hGM-CSF-induced heterodimerization was documented by chimeric receptor coimmunoprecipitation. Homodimerization or heterodimerization of PRLRs and GHRs had no differential effect on activation of STAT5 and MAPK. However, heterodimerization resulted in a prolonged phosphorylation of STAT1 and in particular STAT3, suggesting that the heterodimerization of alpha-oGHR and beta-oPRLR is able to transduce a signal, which is distinct from that occurring on homodimeric associations.

Relationships between plasma concentrations of leptin and other metabolic hormones in GH-transgenic sheep infused with glucose

To study the regulation of leptin secretion in sheep, we infused glucose (0.32 g/h/kg for 12 h) into GH-transgenic animals ( n=8) that have chronically high plasma concentrations of ovine GH and insulin, but low body condition and low plasma leptin concentrations, and compared the responses with those in controls ( n=8). In both groups, the infusion increased plasma concentrations of glucose and insulin within 1 h and maintained high levels throughout the infusion period ( P<0.0001). Compared with controls, GH-transgenics had higher concentrations of insulin, IGF-1, GH (all P<0.0001) and cortisol ( P<0.05), but lower GH pulse frequency ( P<0.0001). Overall, leptin concentrations were lower in GH-transgenics than in controls ( P<0.01). A postprandial increase in leptin concentrations was observed in both groups, independently of glucose treatment, after which the values remained elevated in animals infused with glucose, but returned to basal levels in those infused with saline, independently of transgene status. In both GH-transgenics and controls, glucose infusion did not affect the concentrations of GH, IGF-1, or cortisol. In conclusion, GH-transgenic and control sheep show similar responses to glucose infusion for leptin and other metabolic hormones, despite differences between them in body condition and basal levels of these hormones. Glucose, insulin, GH, IGF-1 and cortisol are probably not major factors in the acute control of leptin secretion in sheep, although sustained high concentrations of GH and IGF-1 might reduce adipose tissue mass or inhibit leptin gene expression.

Sensitivity of hybrid ovine/rat GH receptors to oGH and rat GH in transfected FDC-P1 mouse myeloid cells in vitro.

We have described previously the properties of two mutant ovine growth hormone receptor extracellular domain (oGHR-ECD) proteins which were created by substituting sequences from the rat GHR at two different locations within the framework of the oGHR-ECD. The first mutation occurred at a region close to the N-terminus of oGHR-ECD between residues Thr 28 and Leu34 and created the protein T28E/N29S/N33K/L34P-oGHR-ECD, where the ovine specific residues T, N, N and L are replaced by their equivalent residues E, S, K and P from the rat protein. This site lies N-terminal to the first element of beta-strand structure in the GHR-ECD and we designated this protein as Site-A mutant. The second mutation was made between residues Serl21 andAsp 124 of oGHR-ECD to produce the protein S121T/E123D/D124E-oGHR-ECD where ovine specific residues are again replaced with the equivalent residues from the rat GHR-ECD. This region lies in a loop structure which joins the two beta-barrel domains of the GHR-ECD. This protein is designated as Site-B mutant. A subsequent analysis confirmed the N-terminal region between residues 28-34 of oGHR-ECD as an important epitope defined by antiserum raised to oGHR-ECD. Also of interest was the finding that mutation at both Sites A and B in oGHR-ECD compromised the affinity of the protein for bovine GH (50-fold for Site-A and 4-fold for Site-B). A comparison of the affinity of wild type oGHR for the highly homologous bovine GH with its affinity for rat GH indicated a 10-fold higher affinity for the ruminant GH than for rat GH. Mutation at Site-A of oGHR-ECD reduced the affinity for rat GH a further 3-fold. However, mutation at Site-B of oGHR-ECD increased the affinity for rat GH 2-3 fold. This indicated that the substitution of rat GHR residues for ovine GHR residues in this part of the protein had a beneficial effect in relation to affinity for rat GH and that this region of the GHR-ECD may contain important specificity determinants. In order to test whether these observed differences in affinity for bovine and rat GH affinities have any biological relevance, we have produced the same ovine --> rat mutations in the context of the full length ovine GHR. Transfection of the cDNAs encoding the wt or mutant GHRs into the mouse myeloid pre-B cell line FDC-P1 to create stably transfected clonal lines, has allowed an examination of the relative activities of ovine and rat GH, using a robust and high throughput bio-assay based on the reduction of a cell permeable tetrazolium salt. In the current manuscript, we report that the decrease in binding affinity previously reported for Site-A and Site-B mutant oGHR extracellular domain proteins is not reflected in compromised biological activities when the same mutations are expressed in the context of the full length oGHR protein. We discuss these findings in the context of the relationships between affinity and activity at the GHR.

Effects of recombinant ovine placental lactogen and recombinant ovine growth hormone on growth of lambs and milk production of ewes

The somatogenic and galactopoietic effects of recombinant ovine placental lactogen (oPL) were compared with the effects of recombinant ovine growth hormone (oGH) in post-weaned growing lambs and in lactating ewes. In two experiments that each lasted 35 days, 2-month-old lambs were given daily subcutaneous injections (0.1 mg/kg live weight) of oPL or oGH, and their daily growth rates were compared with those of non-treated control lambs. Ovine GH and oPL had similar profound ( P<0.01) growth-stimulating effects, enhancing lamb growth by 10 to 25%. In two other experiments, lactating ewes were injected with oGH or oPL (0.1 mg/kg live weight/day) for 14 days in mid-lactation. Treatment with oGH increased ( P<0.001) daily milk production by up to 55% over control ewes. Ovine PL increased ( P<0.01) milk production by up to 25%. In all experiments, treatment of lambs or lactating ewes with oGH, but not with oPL increased ( P<0.05) serum insulin-like growth factor-I concentrations. It is concluded that oPL and oGH have similar somatogenic effects in lambs. Both hormones exhibited galactopoietic effects, but oGH was considerably more potent than oPL.

Differential inhibition of recombinant bovine GH (rbGH) activity in vitro by in vivo enhancing monoclonal antibodies.

We have previously described the effects of complexing recombinant bovine growth hormone (rbGH) with the in vivo enhancing monoclonal antibodies (Mabs) OA11 and OA15 and the non-enhancing Mab OA14 on the subsequent activity of GH in different tissue culture models. We reported that all of these Mabs caused the inhibition of GH-stimulated Jak-2 tyrosine kinase phosphorylation in the GH responsive pre-adipocyte cell line 3T3-F442A. However, using the mouse myeloid cell line FDC-P1 transfected with the full length ovine GH receptor (GHR), we subsequently found that OA11 and OA14 remained inhibitory with respect to the end point measurement of GH stimulated mitogenesis but that OA15 had no inhibitory effect on GH stimulated mitogenesis in this cell line. In order to correlate longer term mitogenic effects of Mab-GH complexes with signalling events in this transfected cell line model, we now report on the effects of complexing with Mab on the subsequent GH stimulated phosphorylation of Stat5b (signal transducer and activator of transcription). In agreement with our data for the mitogenic activity of GH-Mab complexes, we found that OA11 and OA14 inhibit GH activation of Stat5b but that OA15 is not inhibitory. Further to this, the dose-response effect of both OA11 and OA14 on the GH stimulation of Stat5b in the FDC-P1-oGHR transfected cells correlates with the previously described dose-response effects for both Mabs in the context of GH stimulation of mitogenic effects. We conclude that in this oGHR transfected cell line model, Mab effects on short and long term GH signalling events are tightly correlated. The observation that neither of the in vivo enhancing Mabs--OA11 or OA15--amplifies the response to GH in our transfected cell line model, coupled with the differential nature of Mab effects on GH activity (OA11--inhibition; OA15--no effect) may argue for an in vivo mechanism for enhancement of GH activity.

Effects of complexation with in vivo enhancing monoclonal antibodies on activity of growth hormone in two responsive cell culture systems.

We describe the properties of three monoclonal antibodies (MAbs) to ovine GH, two of which have previously been shown to enhance, in vivo, the biological activity of bovine and ovine growth hormone. We have examined the effects of these MAbs on GH activity in two appropriate GH-responsive cell culture systems, investigating both acute signalling effects (Janus-activated kinase (Jak)-2 tyrosine phosphorylation -5 min) and longer-term (MTT-formazan production -24 h) effects of hormone-antibody complexes. In the 3T3-F442A pre-adipocyte cell line (which has been demonstrated to be GH responsive), we show that complexation of recombinant bovine (rb) GH with either of the two enhancing anti-ovine GH MAbs (OA11 and OA15) and the non-enhancing MAb, OA14, attenuates the ability of GH to stimulate tyrosine phosphorylation of Jak-2 at a 5-min time point. Using the mouse myeloid cell line, FDC-P1, stably transfected with the full-length ovine GH receptor (oGHR), we demonstrate that rbGH causes a dose-dependent increase in MTT-formazan production by these cells. Further, we demonstrate that OA11 and OA14, but not OA15, cause a decrease in this stimulatory activity of rbGH over a hormone concentration range of 5-50 ng/ml at both 24 and 48 h. We conclude that the different in vitro activities of the two in vivo enhancing MAbs are most probably related to the time-courses over which these two assays are performed, and also to the relative affinities between antibody, hormone and receptor. In addition, the in vitro inhibitory activity of the enhancing MAb OA11 in both short- and long-term bioassay lends further support to an exclusively in vivo model for MAb-mediated enhancement of GH action.

Variation in Lipid Composition by Ovine Growth Hormone in Anabas testudineus (Bloch)

The hormonal control of lipid metabolism in heterotherms has received scant attention. The aim of this study was to evaluate the alterations in lipid components by ovine growth hormone (oGH) in a teleost Anabas testudineus . To study the role of oGH, three doses of oGH were given as intraperitoneal injections (0.1, 0.2, 0.5I¼g/g body wt/ day). Ovine GH at the higher doses significantly reduced serum cholesterol and LDL+VLDL, while increasing HDL. The concentrations of liver free fatty acids, triglycerides and cholesterol decreased and phospholipids increased in hormone treated specimens. Muscle triglycerides and cholesterol were increased after GH treatment. The results suggest the ability of oGH to break down neutral fat or oGH stimulates hepatic lipid mobilization in A. testudineus . Increased concentration of RNA suggests the ability of oGH to increase protein synthesis. Growth hormone appears to be antilipogenic and anabolic in A. testudineus .

Ruminant Placental Lactogens Act as Antagonists to Homologous Growth Hormone Receptors and as Agonists to Human or Rabbit Growth Hormone Receptors

Growth hormone receptor (GHR)-mediated activity of ruminant placental lactogens (PLs) and ovine (o) GH was compared, using cells transfected with full size human (h), rabbit (rb), and oGHRs. All three PLs acted as agonists in heterologous bioassays, whereas in homologous bioassays in cells transfected with oGHRs they antagonized the oGH activity. Despite these differences, oGH and PLs bound with similar affinity to the oGHR extracellular domain (oGHR-ECD), indicating that the binding occurs through hormone site I. Gel filtration of complexes between oPL and oGHR-ECD showed a 1:1 stoichiometry, confirming this conclusion. The oPL T185D and bPL T188D, which exhibited weak biological activity mediated through GHRs, behaved as site I antagonists, whereas oPL G130R and bPL G133R formed a 1:1 complex with GHR-ECDs and bound to h/rb/oGHR-ECDs with affinity similar to that of wild-type oPL. They had no agonistic activity in all models transfected with h/rb and oGHRs, but were antagonistic to all of them. In conclusion, ruminant PLs antagonize the activity of oGH in homologous systems, because they cannot homodimerize oGHRs, whereas in heterologous systems they act as agonists. The structural analysis hints that minor differences in the sequence of the GHR-ECDs may account for this difference. Since the initial step in the activity transduced through cytokine/hemapoietic receptors family is receptor homodimerization or heterodimerization, we suggest that the question of homologous versus heterologous interactions should be reexamined.

Feedback inhibition of ovine GH on GH release from the pituitary of rainbow trout.

Annals of the New York Academy of SciencesVolume 839, Issue 1 p. 378-379 Feedback Inhibition of Ovine GH on GH Release from the Pituitary of Rainbow Trout THORLEIFUR AGUSTSSON, THORLEIFUR AGUSTSSON Fish Endocrinology Laboratory, Department of Zoophysiology, Göteborg University, Medicinaregatan 18, 413 90 Göteborg, SwedenSearch for more papers by this authorBJÖRN THRANDUR BJÖRNSSON, BJÖRN THRANDUR BJÖRNSSON Fish Endocrinology Laboratory, Department of Zoophysiology, Göteborg University, Medicinaregatan 18, 413 90 Göteborg, SwedenSearch for more papers by this author THORLEIFUR AGUSTSSON, THORLEIFUR AGUSTSSON Fish Endocrinology Laboratory, Department of Zoophysiology, Göteborg University, Medicinaregatan 18, 413 90 Göteborg, SwedenSearch for more papers by this authorBJÖRN THRANDUR BJÖRNSSON, BJÖRN THRANDUR BJÖRNSSON Fish Endocrinology Laboratory, Department of Zoophysiology, Göteborg University, Medicinaregatan 18, 413 90 Göteborg, SwedenSearch for more papers by this author First published: 07 February 2006 https://doi.org/10.1111/j.1749-6632.1998.tb10803.xRead the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume839, Issue1TRENDS IN COMPARATIVE ENDOCRINOLOGY AND NEUROBIOLOGY: FROM MOLECULAR TO INTEGRATIVE BIOLOGYMay 1998Pages 378-379 RelatedInformation

The Role of Growth Hormone in the Control of Gonadotropin Secretion in Adult Male Rats*

Although it is known that GH plays an important role in normal growth and development, its influence on the control of gonadotropin secretion is poorly understood. To address this issue, we have treated adult male rats with bovine GH via osmotic pumps (250 microg/day for 2 weeks; Exp design I) or immunized rats against ovine GH (100 microg/month for 6-7 months; Exp design II) and evaluated their neuroendocrine function. Vehicle-treated animals served as controls. Two experiments were conducted to evaluate the gonadotropin responses to: 1) GnRH (in saline) in gonad-intact rats and 2) testosterone propionate (TP; in oil) in castrated rats. Saline- or oil-injected rats served as controls. Circulating GH antibodies, LH, FSH, PRL, testosterone, and insulin-like growth factor I levels were measured by RIAs. Plasma LH levels were decreased (P < 0.025) in rats treated with GH. The plasma LH and FSH responses to GnRH treatment were similar in rats treated with either saline or GH. The suppressive effect of TP on LH secretion was attenuated (P < 0.025) in GH-treated rats on day 8 after castration. The FSH response to TP administration was similar in both subgroups of rats. Administration of GH decreased (P < 0.01) PRL secretion. Plasma testosterone levels were not altered by GH treatment. As expected, GH antibodies were detected and plasma insulin-like growth factor I levels were decreased (P < 0.001) in rats immunized against GH. The basal LH and FSH levels were higher (LH, P < 0.005; FSH, P < 0.025) in rats previously immunized against GH. The percent increase in plasma LH levels after GnRH treatment was decreased in GH-immunized animals. Furthermore, the percent increase in circulating FSH levels was higher in GH-immunized rats than in adjuvant-injected control rats. Administration of TP to adjuvant-injected castrated rats decreased plasma gonadotropin levels. However, similar treatment to rats immunized against GH failed to suppress plasma LH and FSH levels. The basal testosterone levels were not changed by immunization against GH. These results demonstrate that induction of GH excess decreases PRL and LH secretion, whereas biological neutralization of endogenous GH increased circulating gonadotropin concentrations. Thus, GH modulates the hypothalamic-pituitary function in adult male rats.

In vivo effects of the GH-releasing heptapeptide GHRP-1 in lambs.

The novel synthetic growth hormone-releasing heptapeptide GHRP-1 is reported to be more potent than growth hormone-releasing hormone (GHRH) in eliciting GH release in vivo in rats and man. However, in ovine pituitary cells in primary culture in a perifusion system, GHRP-1 was 10-fold less active than GHRH. The purpose of this work was to study the effect of GHRP-1 in sheep in vivo. Ovine GH release stimulated by either GHRP-1 or GHRH(1-29)NH2, in eight pre-ruminant lambs, was determined. GHRP-1 was administered at doses of 1.2, 2.4 and 6 nmole/kg by i.v. bolus, and GHRH(1-29)NH2 at 0.3 nmole/kg. Mean+/-s.e.m. peak GH levels in the plasma after injection of saline, 1.2, 2.4, 6 nmole/kg GHRP-1 and 0.3 nmole/kg GHRH were 2.2+/-0.9, 9.3+/-2.5, 8.8+/-2.4, 35.1+/-5.8 and 51.6+/-10.5 ng/mL, respectively. As spontaneous 20 ng/mL peaks were observed, only peaks above this level can be considered as significant. The highest dose of GHRP-1 (6 nmole/kg) elicited oGH release, but its action was surpassed by GHRH 0.3 nmole/kg. Furthermore GHRP-1 and GHRH appear to behave inversely when response amplitudes are considered. Animals exhibiting a strong reaction to GHRH-1, show a correspondingly weak reaction to GHRH and vice-versa. This may reflect differences in intracellular mechanisms at the pituitary level. Our data support the results in vitro that in sheep GHRP-1 is a weaker stimulant of GH secretion than GHRH.

Guinea pig serum contains a specific high affinity growth hormone-binding protein with novel ligand specificity.

Previous workers have suggested that guinea pig serum does not contain a GH-binding protein (GHBP) or that it is defective. The current studies, however, have identified and characterized the presence of GH-binding activity in guinea pig serum using gel chromatography to separate bound and free hormone. The detection of GH-binding activity is critically reliant on the type of radioligand used to measure binding. Clear identification of GH-binding activity was demonstrated with [125I]ovine GH (oGH), but specific binding could not be measured with [125I]human GH. The novel specificity was also shared by guinea pig liver membrane GH receptor (GHR) and cytosol GHBP, suggesting structural similarity in the GH-binding domain between the GHR and soluble GHBPs. The binding of oGH was dependent on serum concentration (5 microl serum produced 16.03 +/- 0.5% specific binding; mean +/- SEM; n = 11) and incubation time (equilibrium was reached by approximately 6 h at 21 C) and was completely reversible (t(1/2), approximately 2 h). Scatchard analysis revealed linear plots with an affinity constant (Ka) of 0.59 +/- 0.09 x 10(9) M(-1) and a capacity of 23,181 +/- 4,474 fmol/ml serum. Similar association constants were obtained for liver membrane GHR (0.79 +/- 0.22 x 10(9) M(-1)) and cytosol GHBPs (0.99 +/- 0.15 x 10(9) M(-1)), but the capacity, when expressed as femtomoles per g tissue, was significantly increased (4-fold) in cytosol (4,303 +/- 505) over that in membranes (1,071 +/- 257). There was no sex difference in Ka or level of GHBP in guinea pig serum. Surprisingly, the level of GH-binding activity was very low to undetectable in pregnant guinea pig serum. Characterization of the native structure of guinea pig GHBPs has indicated the presence of several proteins that are structurally distinct. Although the distribution of GH-binding activity covered a large Mr range (approximately 70-350 kDa) the major form of the circulating GHBP identified by gel chromatography had an apparent native Mr of 150-170 kDa. Partially purified GHBP (approximate Mr, 170 kDa) was covalently cross-linked to [125I]oGH and subjected to nonreducing SDS-PAGE. Specific GHBP complexes of 158 and 85 kDa were detected, suggesting that the partially purified GHBP complex may be composed of a smaller GHBP associated noncovalently with a non-GH-binding protein. "Pore limit" native PAGE (cathodic and anodic) revealed the presence of specific GHBPs of 363, 158, 74, and 55 kDa, which cross-hybridized with the rat liver membrane GHR monoclonal antibody mAb 263 but not with the rat serum GHBP-specific mAb 4.3. Interestingly, although GH binding was undetectable in pregnant guinea pig serum, Western immunoblot analysis with mAb 263 demonstrated the presence of a major immunoreactive GHBP band of 105 kDa in addition to 158- and 55-kDa GHBPs. The data indicate that the GHBPs are immunologically related to the rat membrane GHR, but provide no evidence to support the presence of a hydrophilic tail sequence homologous to that in the rat GHBP. These studies have identified in guinea pig serum GHBPs that exhibit novel ligand specificity, structural heterogeneity, and an immunological relationship to the rat liver membrane GHR. The identification of serum GHBP and the novel ligand specificity, which is also expressed by the liver membrane GHR, argue against the view that the guinea pig has a defective GHBP.

Basal Secretion of Growth Hormone in Sheep is not Influenced by GH Feedback

Autoregulation of GH secretion in sheep was examined by intravenous and intracerebroventricular (ICV) administration of human and bovine GH. Intravenous administration of hGH at either 2 mg or 10 mg per sheep failed to alter radioimmunoassayable plasma ovine GH concentrations or plasma somatostatin. Human GH also failed to alter plasma oGH when given at a dose of 50 μg icv. Bovine GH (at 50 μg or 500 μg) ICV did not affect basal plasma oGH or somatostatin, nor did pretreatment with 50 μg bGH significantly alter GH response to clonidine stimulation, although the change in plasma GH with stimulation was only half that of controls. These data do not support a major autoregulatory effect on GH secretion in the sheep.

The role of protein kinase C in GH secretion induced by GH-releasing factor and GH-releasing peptides in cultured ovine somatotrophs.

The involvement of protein kinase C (PKC) in the action of GH-releasing factor (GRF) and synthetic GH-releasing peptides (GHRP-2 and GHRP-6) was investigated in ovine somatotrophs in primary culture. In partially purified sheep somatotrophs, GRF and GHRP-2 caused translocation of PKC activity from the cytosol to the cell membranes and caused GH release in a dose- and time-dependent manner. GHRP-6 did not cause PKC translocation. The PKC inhibitors, calphostin C, staurosporine and chelerythrine, partially reduced GH release in response to GRF and GHRP-2 at doses which selectively inhibit PKC activity. These inhibitors totally abolished GH release caused by phorbol 12-myristate 13-acetate (PMA). Down-regulation of PKC by the treatment of cells with phorbol 12,13-dibutyrate for 16 h caused a significant (P < 0.001) reduction in total PKC activity and totally abolished PKC translocation in response to a challenge with GRF, GHRP-2 or PMA. In addition, down-regulation abolished GH release in response to GRF, GHRP-2 or GHRP-6. Treatment of cells with H89, a selective PKA inhibitor, totally blocked GH release caused by either GRF or GHRP-2 and partially reduced PMA-induced GH release. H89 had no effect on PKC translocation caused by GRF, GHRP-2 or PMA and did not affect GH release caused by GHRP-6. These data suggest that GHRP-2 and GRF activate PKC in addition to stimulating adenylyl cyclase activity. Although the cAMP-protein kinase A (PKA) pathway is the major signalling pathway employed by GRF and GHRP-2, the activation of PKC may potentiate signalling via the cAMP-PKA pathway in ovine GH secretion. Importantly, the effect of PMA in increasing the secretion of GH from ovine somatotrophs is effected, in part, by up-regulation of the cAMP-PKA pathway. We conclude that there is cross-talk between the PKC pathway and the cAMP-PKA pathway in ovine somatotrophs during the action of GRF or GHRP.

Drinking in Atlantic Salmon Presmolts and Smolts in Response to Growth Hormone and Salinity

Drinking rate in freshwater Atlantic salmon presmolts (about 0.1 ml/kg/h) was unaffected by daily injections of ovine GH (50 μg/fish) for a week but upon transfer to sea water an immediate and full drinking response was developed compared to saline treated fish (3.34 ± 0.16 vs. 2.23 ± 0.27 ml/kg/h). Smolting did not affect drinking rates in freshwater but after 7 days in sea water, salmon smolts imbibed 3.88 ± 0.25 ml/kg/h, significantly higher than the rate for saline injected presmolts ( p < 0.05, one-way ANOVA), but not significantly different from oGH treated presmolts. Smolting and oGH treatment were without effect on plasma Na + levels in freshwater fish and 7 days after transfer to sea water both groups showed a better regulation of plasma Na + levels compared to saline treated presmolts. Atlantic salmon smolts showed higher levels of plasma Cl − than presmolts in freshwater, and after 7 days in sea water, both oGH presmolts and smolts showed significantly lower levels of plasma Cl − than saline injected presmolts. GH treatment in freshwater presmolts improved hypoosmoregulatory capacity following transfer to seawater and these results are discussed in relation to the physiology of smolting, and control of drinking.