GH Activity Research Articles

Estrogen enhances growth hormone receptor expression and growth hormone action in rat osteosarcoma cells and human osteoblast-like cells.

Postmenopausal bone loss is primarily due to estrogen deficiency. Recent clinical observation demonstrate that GH increases bone mass in GH deficient patients. The present study investigates whether estrogen regulates GH action and GH receptor expression in osteoblasts. 17 beta-estradiol or GH added to the culture medium as single substances did not influence rat osteosarcoma cell proliferation nor human osteoblast-like (hOB) cell proliferation. However, together they synergistically induced osteoblast proliferation (rat osteosarcoma cells 160.1 +/- 15.5% of control cells; human osteoblast-like cells 159.6 +/- 5.1% of control cells). 17 beta-estradiol stimulated 125I-GH binding and GH receptor (GHR) mRNA levels in rat osteosarcoma cells. The stimulatory effect of estradiol was time dependent, reaching a peak after 8 h of incubation with 17 beta-estradiol (binding 216.9 +/- 27.8% and mRNA 374.6 +/- 30.8% of control). The finding that estradiol stimulated 125I-GH binding was confirmed in human osteoblast-like cells. In these cells, 17 beta-estradiol (10(-12) M) increased 125I-GH binding to 203.8 +/- 3.6% of control levels. We conclude that estrogen stimulates GH activity as well as GH binding and GHR mRNA levels in osteoblasts. These findings indicate that estrogen potentiates the effect of GH at the receptor level.

Effect of Fetal Ethanol Exposure on the In Vitro Release of Growth Hormone, Somatostatin and Growth Hormone‐Releasing Factor Induced by Clonidine and Growth Hormone Feedback in Male and Female Rats

This study was designed to examine the effect of fetal ethanol (ETOH) exposure on the sensitivity of the hypothalamic-growth hormone (GH) axis to clonidine (an alpha 2-adrenoreceptor agonist) stimulation and GH feedback. During gestation, dams were fed either a liquid diet in which 36% of the calories were derived from ETOH, or pair-fed an isocaloric control liquid diet without ETOH. A second set of controls were fed lab chow ad libitum. After birth, offspring of ETOH-fed dams were cross-fostered to a separate group of ad libitum control dams. The hypothalami and pituitaries of 10-, 20-, 30-, and 50-day-old offspring were separated by age, diet, and sex; pooled 6 to 8 per chamber; and tested in a hypothalamic-pituitary coperifusion system. Chambers were perifused with either clonidine (2 x 10(-8) M) alone, which mimics the endogenous trigger for GH release, or clonidine in combination with human GH (2 x 10(-9) M) to determine sensitivity of tissue to feedback regulation. Both stimuli act at the hypothalamic level and indirectly modulate GH release via effects on hypothalamic factors. Results of this study indicate that tissue from control male rats is responsive to the clonidine-induced GH surge by 10 days of age and to feedback depression of GH release by 20 days of age. This sensitivity persists after puberty and is associated with corresponding changes in somatotropin-release inhibiting factor (SRIF) and GH-releasing factor (GRF) release (i.e., clonidine inhibits SRIF and stimulates GRF release, and human GH reverses this pattern). Fetal ETOH exposure depresses GH sensitivity to both stimuli in male pups (age x diet x drug: p < 0.002), and this depressed sensitivity is expressed by 30 days of age by reduced responses to alpha 2-adrenergic stimulation and GH feedback (drug x diet: p < 0.002 and p < 0.001 for 30 and 50 days of age, respectively). This effect of ETOH on GH release was associated with feedback insensitivity of SRIF (drug x diet: p < 0.003, at 50 days of age) and GRF [drug x diet; p < 0.044 at 30 days; clonidine vs. clonidine and GH: p > 0.05 (NS) at 50 days of age for ETOH pups]. The depressed response of GH to clonidine after puberty may be attributable to a combination of the trends toward decreased sensitivity of both SRIF and GRF at this age. The female GH axis was both less sensitive to stimuli and less effected by ETOH than corresponding tissue from male rats (sex x age x drug x diet: p < 0.011). GH release from control female pituitaries was sensitive to clonidine before, but not after, puberty and insensitive to GH feedback at both developmental stages. On the other hand, there was a specific effect of ETOH on SRIF release at 10 days of age (diet x drug: p < 0.014), and SRIF release remained sensitive to clonidine in pups from all diet groups after puberty. Because GH release was not influenced by these changes in SRIF, these findings suggest that GH release is less sensitive to SRIF in females. In conclusion, this study suggests that fetal ETOH exposure interferes with the development of the sensitivity of the GH axis to alpha 2-adrenergic stimulation and feedback in males. Thus, the male GH axis is both more sensitive to the stimuli tested in this study and more effected by ETOH than the female axis. Furthermore, the effects of ETOH on these mechanisms do not alter GH release in males until the peripubertal period. It is likely, therefore, that the GH regulatory mechanism examined in this study does not contribute to growth retardation before puberty. If the effects of ETOH on GH release contributes to growth retardation in prepubertal males and in females, it most likely involves other regulatory mechanisms. On the other hand, because the adult pattern of GH release is programmed during development, the influence of ETOH on these developmental events may influence the male pattern of GH release and GH activity in adulthood.

Effect of Fetal Ethanol Exposure on the In Vitro Release of Growth Hormone, Somatostatin and Growth Hormone-Releasing Factor Induced by Clonidine and Growth Hormone Feedback in Male and Female Rats

This study was designed to examine the effect of fetal ethanol (ETOH) exposure on the sensitivity of the hypothalamic-growth hormone (GH) axis to clonidine (an α2-adrenoreceptor agonist) stimulation and GH feedback. During gestation, dams were fed either a liquid diet in which 36% of the calories were derived from ETOH, or pair-fed an isocaloric control liquid diet without ETOH. A second set of controls were fed lab chow ad libitum. After birth, offspring of ETOH-fed dams were cross-fostered to a separate group of ad libitum control dams. The hypothalami and pituiiries of 10–, 20–, 30–, and 50-day-old offspring were separated by age, diet, and sex; pooled 6 to 8 per chamber; and tested in a hypothalamic-pituitary coperifusion system. Chambers were perifused with either clonidine (2 × 10−-8 M) alone, which mimics the endogenous trigger for GH release, or clonidine in combination with human GH (2 × 10−-9 M) to determine sensitivity of tissue to feedback regulation. Both stimuli act at the hypothalamic level and indirectly modulate GH release via effects on hypothalamic factors. Results of this study indicate that tissue from control male rats is responsive to the clonidine induced GH surge by 10 days of age and to feedback depression of GH release by 20 days of age. This sensitivity persists after puberty and is associated with corresponding changes in somatotropin-release inhibiting factor (SRIF) and OH-releasing factor (GRF) release (i.e., clonidine inhibits SRlF and stimulates GRF release, and human GH reverses this pattern). Fetal ETOH exposure depresses GH sensitivity to both stimuli in male pups (age × diet × drug: p < 0.002), and this depressed sensitivity is expressed by 30 days of age by reduced responses to α2-adrenergic stimulation and OH feedback (drug × diet: p < 0.002 and p < 0.001 for 30 and 50 days of age, respectively). This effect of ETOH on GH release was associated with feedback insensitivity of SRlF (drug × diet: p < 0.003, at 50 days of age) and GRF [drug × diet p < 0.044 at 30 days; clonidine vs. clonidine and GH: p > 0.05 (NS) at 50 days of age for ETOH pups]. The depressed response of GH to clonidine after puberty may be attributable to a combination of the trends toward decreased sensitivity of both SRlF and GRF at this age. The female GH axis was both less sensitive to stimuli and less effected by ETOH than corresponding tissue from male rats (sex × age × drug × diet: p < 0.011). OH release from control female pituitaries was sensitive to clonidine before, but not after, puberty and insensitive to GH feedback at both developmental stages. On the other hand, there was a specific effect of ETOH on SRlF release at 10 days of age (diet × drug: p < 0.014), and SRlF release remained sensitive to clonidine in pups from all diet groups after puberty. Because GH release was not influenced by these changes in SRIF, these findings suggest that GH release is less sensitive to SRlF in females. In conclusion, this study suggests that fetal ETOH exposure interferes with the development of the sensitivity of the GH axis to α2-adrenergic stimulation and feedback in males. Thus, the male GH axis is both more sensitive to the stimuli tested in this study and more effected by ETOH than the female axis. Furthermore, the effects of ETOH on these mechanisms do not alter GH release in males until the peripubertal period. It is likely, therefore, that the GH regulatory mechanism examined in this study does not contribute to growth retardation before puberty. If the effects of ETOH on GH release contributes to growth retardation in prepubertal males and in females, it most likely involves other regulatory mechanisms. On the other hand, because the adult pattern of GH release is programmed during development, the influence of ETOH on these developmental events may influence the male pattern of GH release and GH activity in adulthood.

Growth Hormone Improves Immune Function and Survival in Burned Mice Infected with Herpes Simplex Virus Type 1

Recombinant human growth hormone (rhGH) is often used clinically for the treatment of burned patients to promote wound healing and to improve protein metabolism. Recently, the immunomodulatory activity of GH has been described, but it remains unclear whether immune abnormalities associated with burn are improved by rhGH administration. To determine the immunoregulatory activities of rhGH in thermal injury, the survival of burned mice infected with herpes simplex virus type 1 (HSV-1), along with interferon-γ (IFN-γ) production by splenic mononuclear cells (SMNCs), and generation of cytostatic macrophages in burned mice were examined after exogenous administration of rhGH. Data showed that the mortality after HSV-1 infection was improved in burned mice treated with a 4-mg/kg dose of rhGH every other day for five doses. Also, the decreased IFN-γ response of SMNCs from burned mice improved with rhGH therapy. Further, cytostatic macrophages were generated in burned mice treated with rhGH, whereas these macrophages were not demonstrated in burned mice treated with saline. These results indicate that rhGH can improve immune function in burned mice, specifically improving survival in HSV-1 infection, boltering IFN-γ production by SMNCs, and increasing production of cytostatic macrophages.

Long-term starvation in the European eel: general effects and responses of pituitary growth hormone-(GH) and somatolactin-(SL) secreting cells

European eels reaching the silver stage stop feeding in freshwater and during their spawning migration to the Sargasso sea (6000 km at least in the Ocean). The total duration of this exceptional fast is not well known. Few data are available on the general condition and the endocrine responses to starvation of migrating eels. In this study male (silver) and female (yellow and silver) eels were kept in freshwater without food for various lengths of time. Animals were killed after 7 months to 3 or 4 years. After a gradual decrease, the final body weight was reduced by 84% in males (52 months) and 67–69% in females (up to 4 years). The condition factor (K) followed a parallel curve. In the pituitary gland, GH cells were hypertrophied and highly stimulated. Their cross-sectional area was negatively correlated to K. Large GH cells remained well immunostained with an anti-eel GH serum after 7 to 12 months of starvation. In the leanest eels, the immunostaining was often reduced and many GH cells appeared degranulated, suggesting a low hormonal storage. In contrast, SL cells were reduced in size and number in the anterior half of the neurointermediate lobe (NIL), but showed a more heterogeneous picture in the caudal portion. GH and SL cell activities seemed to be negatively correlated in starved fish and controlled by different mechanisms. GH appears to play a major role in the long-term survival of fasted eels; SL does not seem to be involved.

A novel growth hormone response element unrelated to STAT (signal transducer and activator of transcription)-binding sites is a bifunctional enhancer.

Regulation of gene expression by GH has so far been shown to be mediated by a few cis-acting elements, most of which are signal transducer and activator of transcription (STAT)-binding sites. Here we have characterized a novel GH-response element present in the promoter of rat serine protease inhibitor (spi) genes. It consists of a 13 nucleotide-long GAGA box containing two GAGGAG repeats separated by a G, structurally unrelated to STAT-binding sites. In hepatocytes, the spi GAGA box behaves as a position-dependent bifunctional enhancer controlling basal and GH-dependent transcription. In addition, spi GAGA box oligonucleotides inhibit cell-free transcription driven by GAGA box-containing as well as GAGA box-less promoters, suggesting that the spi GAGA box interacts directly or indirectly with component(s) of the basic transcriptional machinery. Mobility shift assays showed that this GAGA box is specifically recognized by nuclear factors that are unrelated to previously characterized proteins binding to purine-rich elements or to GH-activated STATs. Finally, experiments performed with cells expressing wild type, truncated, or mutated forms of the GH receptor indicate that protein kinase Janus kinase 2 is involved in the GH-dependent activation of the spi GAGA box. These studies reveal the existence of an as yet unidentified Janus kinase-2-dependent, STAT-independent pathway in GH activation of gene expression.

ROLE OF PIT-1 IN THE GENE EXPRESSION OF GROWTH HORMONE, PROLACTIN, AND THYROTROPIN

To date, nine different mutations in the Pit-1 gene resulting in CPHD have been described in mammals. Four of these mutations alter residues important for DNA binding or alter the predicted alpha helical nature of the Pit-1 protein (A158P, R172X, E250X, and W261C). The A158P mutation, however, has minimal effects on DNA binding. Four mutations lie outside alpha helical regions (P24L, R143Q, K216E, and R271W) and do not significantly alter DNA binding either experimentally or by prediction. One mutation is a large deletion of the Pit-1 gene locus in the Jackson dwarf mouse. Mutant Pit-1 proteins that do not interfere with binding cause CPHD through interference with target gene activation and regulation. The R271W mutant acts as a dominant inhibitor of transcription of the GH and Prl genes. The A158P mutant is incapable of activating transcription from the GH-I site and has low activation of transcription of the distal enhancer and proximal promoter sites of Prl and of 320 bp of the 5' GH promoter sequence. Some mutant proteins interfere with nuclear receptors. For example, the K216E mutant has defective retinoic acid signaling on the Pit-1 gene enhancer. There is phenotypic variability in the degree of CPHD and in pituitary size in patients with Pit-1 gene mutations. Since Pit-1 has different functions in the somatotroph, lactotroph, and thyrotroph, it is not surprising that point mutations in different regions of the gene interfere in different ways with Pit-1 function. A mutant Pit-1 may be able to carry out its developmental role, but may be aberrant in GH and Prl gene activation or Pit-1 autoregulation. Study of Pit-1 mutations and their diverse pathophysiologic mechanisms should increase the understanding of anterior pituitary gland development and gene regulation in normal and disease states.

Insulin-dependent suppression in glutamyl hydrolase activity and elevated cellular methotrexate polyglutamates

Folates and antifolates are converted to polyglutamates, which are better retained in cells and may also bind more tightly to cellular proteins than the parent compounds. The regulation of the process of polyglutamate formation and breakdown is not fully clarified yet and is being studied by a number of approaches. An early observation concerning the potential regulation of polyglutamate formation was that insulin caused a marked increase in the rate and accumulation of polyglutamates of methotrexate (MTX) in rat hepatoma cells. The present study demonstrated that insulin caused a decrease in the activity of γ-glutamyl hydrolase (GH), the enzyme that degrades polyglutamates, that was inversely commensurate with the increase in the synthesis of MTX polyglutamates. The effects of insulin on GH activity with regard to concentration, time of onset, and the effect of N 6, O 2′ dibutyryl cAMP and theophylline were consistent with the reduction in GH being responsible for the increase in cellular MTX polyglutamate accumulation. Insulin addition also led to an increase in folate polyglutamates. The insulin effects were also seen with H35D cells, a subline with enhanced glutamyl hydrolase activity as a result of having been made resistant to 5, 10-dideazatetrahydrofolic acid. When H35 cells with insulin were compared with H35D cells lacking insulin, there was an 8-fold increase in GH and a 44-fold decrease in the number of γ-glutamate residues added to MTX.

Effect of human growth hormone and insulin on [3H]thymidine incorporation, cell cycle progression, and cyclin D expression in 3T3- F442A preadipose cells

Cellular growth- and cyclin D expression-regulating activities of GH and insulin were investigated in 3T3-F442A preadipose cells under serum-free culture conditions. The present report provides evidence that the proliferative potential of 3T3-F442A cells is reduced by GH in a time- and concentration-dependent manner based on [3H]thymidine incorporation assay and cell cycle analysis. In contrast, treatment of 3T3-F442A cells with insulin resulted in cellular proliferation. The insulin-induced proliferation of 3T3-F442A cells was diminished in the presence of GH. In an effort to define biochemical events relevant to the regulatory activities of GH and insulin on the proliferation of 3T3-F442A cells, the effects of these peptides on the expression of cyclin D were studied using Western blotting. Treatment of 3T3-F442A cells with insulin led to an increase in cyclin D expression relative to that in untreated cells. The insulin-elicited expression of cyclin D was time and dose dependent. In addition, the ability of insulin to induce cyclin D expression was reduced by GH. Our experimental results indicate that proliferation of 3T3-F442A cells was regulated by GH and insulin. The regulatory effects of GH and insulin are mediated at least in part by the alternating expression of cyclin D protein.

Effect of human growth hormone and insulin on [3H]thymidine incorporation, cell cycle progression, and cyclin D expression in 3T3-F442A preadipose cells.

Cellular growth- and cyclin D expression-regulating activities of GH and insulin were investigated in 3T3-F442A preadipose cells under serum-free culture conditions. The present report provides evidence that the proliferative potential of 3T3-F442A cells is reduced by GH in a time- and concentration-dependent manner based on [3H]thymidine incorporation assay and cell cycle analysis. In contrast, treatment of 3T3-F442A cells with insulin resulted in cellular proliferation. The insulin-induced proliferation of 3T3-F442A cells was diminished in the presence of GH. In an effort to define biochemical events relevant to the regulatory activities of GH and insulin on the proliferation of 3T3-F442A cells, the effects of these peptides on the expression of cyclin D were studied using Western blotting. Treatment of 3T3-F442A cells with insulin led to an increase in cyclin D expression relative to that in untreated cells. The insulin-elicited expression of cyclin D was time and dose dependent. In addition, the ability of insulin to induce cyclin D expression was reduced by GH. Our experimental results indicate that proliferation of 3T3-F442A cells was regulated by GH and insulin. The regulatory effects of GH and insulin are mediated at least in part by the alternating expression of cyclin D protein.

Immunomodulation of the growth hormone — IGF-I axis

Abstract Immunomodulation of the growth hormone/insulin-like growth factor-I (GH/IGF-I) axis has huge potential for increasing animal production. Treatment with GH is known to increase somatic growth, muscle protein synthesis, efficiency of food utilisation and serum IGF-I levels, and to decrease carcass fat content in laboratory rodents and/or farm animals. In addition GH is also known to substantially enhance milk production in both cattle and sheep. All of these actions of GH are substantially enhanced if complexed with certain monoclonal antibodies (MAbs). The phenomenon of Mab-mediated enhancement of GH action offers three potential advantages over simple hormone treatment. Firstly, it would be difficult for GH alone to achieve the level of response promoted by GH/MAb complexes without repeated injections (three or four times per week) of massive amounts of hormone. Secondly, it is possible to produce a site-directed antiserum capable of enhancing endogenous GH activity by immunisation of livestock with an appropriate peptide, this would require only two or three injections of minute amounts of a biologically inactive peptide. Thirdly, using an active immunisation approach means that both the active constituents, namely, hormone and antibody are produced endogenously. Peptides capable of producing GH enhancing antisera have been successfully selected on the basis of computer prediction programmes and by mapping the epitope defined by a GH enhancing MAb. Current work involves investigating analogues of this epitope peptide in order to increase antigenicity without loss of specificity. Whether it will be possible to apply this approach to enhancement of IGF-I activity is as yet unknown. The mechanism(s) underlying this phenomenon is not known, although the observation that a MAb can enhance the effects of GH on chondrocyte proliferation in vitro suggests a cellular rather than a systemic mechanism. The one report of antibody-mediated enhancement of IGF-I activity utilised a polyclonal antiserum, thus it is possible that this may operate via a different mechanism. Alternative strategies such as immunoneutralisation of somatostatin and the use of antiidiotypic antibodies as GH mimics have also been proposed as a means of increasing animal production. However, to date MAb-mediated enhancement of hormone action seems to offer the best alternative to hormone treatment, whether this promise is fulfilled remains to be seen.

Location of an epitope defined by an enhancing monoclonal antibody to growth hormone: some structural details and biological implications.

We have previously shown that a murine monoclonal antibody (MAb OA15) prepared against ovine GH (oGH) can enhance the somatogenic activity of bovine GH (bGH), as determined by increased incorporation of 35SO4(2-) into costal cartilage of hypopituitary Snell dwarf mice in vivo We have now established that MAb OA15 can also enhance the biological activity of oGH and porcine GH (pGH) in vivo. Using multiple pin peptide synthesis techniques a set of overlapping immobilized octamers representing the entire bGH sequence were synthesized and tested for their ability to bind MAb OA15 using an enzyme-linked immunosorbent assay. The pattern of binding showed that OA15 defined a functionally continuous epitope comprising residues 91-102. This region includes the C-terminal end of helix 2 plus a portion of the adjacent loop linking helices 2 and 3. Polyclonal antisera to a synthetic peptide representing this epitope mimicked the ability of MAb OA15 to enhance oGH, bGH, and pGH. Window size analysis showed that the heptapeptide 94-100 (SRVFTNS) represents the minimum unit to retain full recognition of MAb OA15. The fact that pGH, bGH, and oGH have identical sequences in this region also explains the ability of OA15 to both cross-react with and enhance the biological activity of each of these GHs. Replacement net analysis (where each residue in the heptamer is substituted with each of the 19 naturally occurring L-amino acids) demonstrated that residues R95 and T98 are critical for antibody binding and also indicated that the substitution of V96 with I, as found in rat GH, would permit the observed binding of OA15 to this hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro and in vivo studies of the antagonistic effects of human growth hormone analogs.

A bovine growth hormone (bGH) analog, bGH-G119R, has been shown to act as a functional antagonist of GH activity both in vitro and in vivo. In the present study, human (h) GH analogs with alterations in the third alpha-helix (G120A, G120R) and N terminus (I4A) were generated. These two regions have been reported to form binding site 2 in hGH, which is involved in in vitro dimerization of the GH receptor (GHR). The biological activities of these hGH analogs were tested in vitro by a radioreceptor assay and an assay to test the ability of the molecules to induce tyrosine phosphorylation of a 93-kDa protein in a human lymphocyte line, IM-9. The growth rate of transgenic mice that express different hGH analogs was used as an in vivo test of the activity of the molecules. The results demonstrated that G120R is a potent hGH antagonist both in vitro and in vivo. Transgenic mice expressing G120R demonstrated a growth-suppressed phenotype. However, I4A, which has been demonstrated to be a potent inhibitor of in vitro GHR dimerization, exhibited full growth promoting activity in transgenic mice. Thus, the ability of hGH analogs to induce GHR dimerization in vitro and the ability to promote growth in vivo are not directly correlated.

In vitro and in vivo studies of antagonistic effects of human growth hormone analogs.

A bovine growth hormone (bGH) analog, bGH-G119R, has been shown to act as a functional antagonist of GH activity both in vitro and in vivo. In the present study, human (h) GH analogs with alterations in the third alpha-helix (G120A, G120R) and N terminus (I4A) were generated. These two regions have been reported to form binding site 2 in hGH, which is involved in in vitro dimerization of the GH receptor (GHR). The biological activities of these hGH analogs were tested in vitro by a radioreceptor assay and an assay to test the ability of the molecules to induce tyrosine phosphorylation of a 93-kDa protein in a human lymphocyte line, IM-9. The growth rate of transgenic mice that express different hGH analogs was used as an in vivo test of the activity of the molecules. The results demonstrated that G120R is a potent hGH antagonist both in vitro and in vivo. Transgenic mice expressing G120R demonstrated a growth-suppressed phenotype. However, I4A, which has been demonstrated to be a potent inhibitor of in vitro GHR dimerization, exhibited full growth promoting activity in transgenic mice. Thus, the ability of hGH analogs to induce GHR dimerization in vitro and the ability to promote growth in vivo are not directly correlated.

Development of homologous radioimmunoassays for equine growth hormone and equine prolactin and their application to the detection of circulating levels of hormone in horse plasma

Highly purified and well-characterised preparations of equine prolactin and growth hormone from equine pituitary glands were employed to set up highly sensitive and specific homologous radioimmunoassays (RIA) for the measurement of hormone in horse plasma. The limit of sensitivity of the GH RIA was 1.2 ng/ml with mean intra- and inter-assay coefficients of variation (CV) of 6.6 and 10%, respectively. The sensitivity of the equine prolactin (ePRL) RIA was 0.5 ng/ml with mean intra and inter-assay CV of 9.1 and 15.6%, respectively. Dose-response curves of a crude pituitary gland extract and plasma samples collected from a mare and foal were parallel to the standards and the PRL RIA was clinically validated by administration of thyrotropin-releasing hormone (TRH). Plasma samples taken at 15 min intervals over 24 h from lactating mares gave 24 h mean GH values in the range 5.5 to 7.95 ng/ml. Large intermittent elevations of GH activity were detected. The mean 24 h PRL concentrations were between 3.2-10.4 ng/ml in the lactating animals, with higher concentrations earlier in lactation. Long episodic bursts of PRL were detected.

Growth hormone-binding protein (GH-BP) levels in follicular fluid from human preovulatory follicles: correlation with serum GH-BP levels.

Serum GH-binding protein (GH-BP), which is identical with the extracellular domain of the GH-receptor, has important implications for the distribution and physiological activity of GH and may enable evaluation of GH-receptor function. Recent studies suggest that GH plays an important role in the modulation of ovarian function and GH-receptors/BPs are found in the female reproductive system. The purpose of the present study was to investigate the presence of GH-BP in human follicular fluid (FF) and compare the levels of FF GH-BP with those detectable in serum, in 46 women undergoing in vitro fertilization. Levels of GH-BP were determined by incubation of [125I]hGH with 50 microL FF or serum, in the absence or presence of excess hGH and specific binding was expressed as a percentage of the total counts per min incubated. The mean GH-BP level in patient FF was 12.39 +/- 0.63% (mean +/- SE) and this correlated significantly with serum GH-BP levels (r = 0.82; P < 0.001). The binding of hGH to FF was dose dependent, highly specific, and of high affinity and low capacity. The affinity constants (Ka) obtained by Scatchard analysis for hGH binding to patient's FFs and sera were not significantly different. Furthermore, we have analyzed the sodium dodecyl sulfate gel electrophoretic pattern of GH-BP in FF and serum by both the ligand-blot technique and after cross-linking with [125I]hGH. Our results show that there is close similarity between serum and FF GH-BPs. Significantly lower FF GH-BP levels were measured in patients older than 36 yr compared to younger women (P < 0.05), whereas increased values were obtained both in patients with elevated E2 concentrations in serum (> 7000 pmol/L) and in FF (> 2200 nmol/L), (P < 0.02 and P < 0.05, respectively). This first demonstration of GH-BP in FF is expected to increase our understanding of the possible direct effect of GH on ovarian steroidogenesis, and suggests a possible regulatory role for GH-BP in folliculogenesis.

BIOLOGICAL ACTIVITY Of GROWTH HORMONE (GH) IN THE FIRST MONTHS OF LIFE

The aim of our study was to evaluate GH levels using RIA and bioassay in the 1 months of life. Serum samples from 22 normal full-term newborns were collected at the 5th day and then at the 1st and 4th titis B immunization). In the same serum sample we evaluated both radioimmunological activity of GH and biological activity of GH using Nb2 rat lymphoma cell bioassay. High GH concentratins measured by RIA (32.72±3.95 ng/ml) and Nb2 bioassay (3.29±3.95 U/ml) were observed in neonates at the 5th day of life. At the 1st and 4th month we found a significant decrease (p<0.001) of radioimmunological (of 75, 27% and 92.29%, respectively). A significant correlation (p<0.01) between GH values measured by RIA and Nb2 bioassay was observed at the 5th day, but not at the 1st and 4th month of life, since the biological activity of GH decreased less than the radioimmunological 1 during the 1 months of life. Our results indicate a good agreement between immunoactivity and bioactivity of GH in the first days of life, but not later.

The Clinical Significance of Growth Hormone Binding Protein

The clinical significance of Growth Hormone Binding Protein (GHBP) is seen in at least three distinct, yet interrelated ways. First, GHBP serves as a serum marker for the GH-receptor level: it is absent in most patients with GH-receptor deficient Laron type dwarfism; it is low in liver cirrhosis, hypothyroidism and malnutrition, where GH-receptor insufficiency has been shown; it is high in hyperthyroidism and obesity, where GH-receptor is increased: and it increases with GH therapy, as reported for rat GH-receptor. Secondly, GHBP slows serum GH clearance and, hence, sustains higher GH levels; serum GHBP correlates negatively with GH disappearance rate, and was suggested as a pharmacological enhancer of GH activity. Finally, GHBP competes with the GH-R on GH binding, and interferes with GH biological activity: in vitro GHBP decreases GH activity, in the rat increasing levels of GHBP result in GH resistance, and in human serum, increasing levels of GHBP diminish GH bioactivity, as measured in vitro by the potency to displace hGH from GH-receptor. We suggest an algorithm to correct RIA-GH levels for the interference effect of GHPB on its biological activity. Our study of normal short children and GHD disclosed negative correlation with serum GH and higher levels in girls then in boys. GHBP did not predict growth response to hGH therapy. It is concluded that the significance of GHBP and its impact on GH bioavailibility and activity is complex, and involves consideration of the GH-receptors, GH volume distribution, GH clearance and competition with the GH-receptors.

Pituitary adenylate cyclase-activating polypeptide stimulates calcium mobilization in amphibian pituitary cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily. Although PACAP is a potent stimulator of adenylate cyclase activity in the adenohypophysis, the precise target cells for PACAP in the anterior pituitary remain unknown. The aim of the present study was to investigate whether PACAP could stimulate calcium mobilization in individual cells of the pituitary and to determine the type of cells that responded to PACAP. Enzymatically dispersed frog distal pituitary cells were plated on photoetched coverslips and cultured for 3-7 days. The cells were loaded with the fluorescent calcium indicator indo-1, and changes in intracellular calcium concentrations ([Ca2+]i) were monitored using dual wavelength microfluorimetry. The individual cells were localized with the aid of the alpha/numeric grid of the coverslips and identified retrospectively by immunofluorescence. Approximately 45% of GH and PRL cells and 25% of ACTH and TSH cells responded to PACAP (10(-5) M) ejection by an elevation of [Ca2+]i. Only 16% of gonadotropes were stimulated by PACAP. The time course of [Ca2+]i variations showed three different patterns: transient spikes, sustained stimulations, and oscillatory responses. In addition, heterogenous responses were observed within each cell type. These data provide evidence for the involvement of calcium mobilization in the mechanism of action of PACAP on pituitary cells. The results also indicate that in frogs, PACAP may stimulate the secretory activity of GH and PRL cells and, to a lesser extent, ACTH, TSH, and gonadotrope cells.

Somatotropic and lactotropic receptors in transgenic mice expressing human or bovine growth hormone genes.

The somatotropic and lactotropic receptors were studied in liver microsomal preparations from transgenic mice carrying the human growth hormone (hGH) or bovine growth hormone (bGH) gene fused to mouse metallothionein-I (MT) or phosphoenolpyruvate carboxykinase promoter/regulator (PEPCK). Specificity studies indicated that, similarly to normal mice, liver microsomes from the transgenic animals possess a mixed population of somatotropic and lactotropic binding sites. In transgenic animals of both sexes, the binding capacity of somatotropic receptors was significantly increased without corresponding changes in affinity. Expression of the MT-hGH hybrid gene was associated with the induction of somatotropic receptors which was approximately twice as great as that measured in animals expressing the MT-bGH hybrid gene. The binding capacity of lactotropic receptors in liver microsomes (quantitated by the use of labelled ovine prolactin) was increased 2-3 fold in transgenic females and approximately 10-fold in transgenic males as compared to the respective normal controls. We conclude that lifelong excess of GH up-regulates hepatic GH and prolactin receptors, and that lactogenic activity of GH is not essential for induction of prolactin receptors in the liver of transgenic mice.