Growth hormone affects gene expression and proliferation in human prostate cancer cells.

We previously showed that growth hormone (GH) receptors (GHR) are expressed in the most commonly studied human prostate cancer (PCa) cell lines and that GHR isoforms undergo differential, cell-type-specific hormonal regulation. We now report that human GH (hGH) can stimulate/modulate insulin-like growth factor (IGF) and ß-oestradiol (E(2) ) receptor (ER(ß) ) gene expressions in these cells and interact with IGF-I and E(2) to stimulate androgen-dependent LNCaP cell proliferation. We observed a cell type-dependent, differential regulation of IGF axis gene expression by GH: IGF-I was stimulated in the androgen-dependent LNCaP cells; IGF-II was stimulated in androgen-insensitive (AI) PC3 cells; the IGF-I cognate receptor, IGF-IR, was stimulated in LNCaP cells, but inhibited in PC3 cells; IGF-IIR was stimulated in both LNCaP and PC3 cells. GH also stimulated ER(ß) gene expression in LNCaP and PC3 cells, but had little or no effect on any of those genes in AI DU145 cells. The potent androgen analogue, mibolerone, also stimulated IGF-I, IGF-IR and ER(ß) , but reduced IGF-IIR mRNAs in LNCaP cells. Furthermore, triiodothyronine (T(3) ) and E(2) also stimulated the expression of those four genes in LNCaP cells, but co-administration of GH had almost no effect. Finally, we also studied the effects of GH, IGF-I and E(2) , alone or in combination, on LNCaP cell proliferation. Importantly, we demonstrated, for the first time, that although GH and IGF-I alone had no effect on LNCaP cell proliferation, concomitant administration for 96 h revealed a permissive role of GH on IGF-I-induced proliferation. GH also appeared to exert a synergistic effect on E(2) -stimulated LNCaP cell proliferation. Taken together, these findings indicate that GH via GHRs, most likely in concert with gonadal steroids, T(3) , IGF system axis and probably other hormones and growth factors, potentially plays an important role in the mechanisms underlying tumour cell growth in PCa.

Shedding of growth hormone-binding protein is inhibited by hydroxamic acid-based protease inhibitors: proposed mechanism of activation of growth hormone-binding protein secretase.

The present study describes events postulated to be involved in the regulated mechanism of proteolytic shedding of growth hormone (GH)-binding protein (GHBP). Using Chinese hamster ovary (CHO) cell lines stably transfected either with the full-length human GH receptor (hGHR) or with the cytoplasmic domain-truncated hGHR (hGHR(tr)), we show that the phorbol ester, phorbol 12-myristate 13-acetate (PMA), caused a rapid time- and dose-dependent increase in GHBP secretion, which, as expected, was matched by a corresponding decrease in cell-surface GHR. Furthermore, PMA equally enhanced GHBP release from CHO/hGHR(tr) cells, suggesting that the cytoplasmic domain of hGHR is not essential for PMA-induced shedding. PMA is known to specifically activate protein kinase C and, indeed, the stimulatory effects of PMA in both cell lines were completely inhibited by the protein kinase inhibitor, staurosporine (100 nM), suggesting that activation of protein kinase C (PKC) may mediate PMA-induced GHBP shedding. Since proteolytic cleavage of several cell-surface proteins was shown to be stimulated by modulators of PKC activity and inhibited by metalloprotease inhibitors, we studied the effects of two hydroxamic acid-based inhibitors of zinc-dependent metalloproteases, BB-3103 and Ro31-9790, on GHBP proteolysis. Pretreatment of CHO/hGHR cells with both these inhibitors reduced PMA-enhanced shedding of GHBP, in a dose-dependent manner, with IC(50) values of approximately 0.41 microM for BB-3103 and approximately 0.97 microM for Ro31-9790. In addition, these inhibitors dose-dependently reduced the shedding enhanced by the sulfhydryl alkylator, N-ethylmaleimide (NEM), with IC(50) values of approximately 0.32 microM and approximately 0.58 microM for BB-3103 and Ro31-9790 respectively. It was of interest to find out that Ro31-9790 acted not only to modulate PMA- or NEM-induced shedding processes, but also markedly reduced the spontaneous, time-dependent accumulation of GHBP released from CHO/hGHR cells growing in serum-containing medium. Taken together, these results suggest that one or more zinc-dependent metalloprotease(s), acting at the cell surface, may be involved in GHBP secretase activity. A scheme is proposed whereby at least part of the regulated maturation and/or activation of the protease activity may involve a cysteine-switch mechanism and/or PKC-dependent phosphorylation. In the long run, specific inhibitors of these processes could be applied in the regulation of GHBP levels and, thus, of GH availability and/or activity.

Characterization and regulation of prolactin receptors in MA-10 Leydig cells.

The aim of this study is to further characterize the prolactin receptors (PRL-R) previously reported in the murine Leydig tumor MA-10 cell line, as well as to study their homologous and heterologous regulation. Two forms of PRL-R, a high and a low molecular weight form, were revealed by studies of covalent crosslinking of 125I-human GH to cultured MA-10 cells or cell membranes and immunoprecipitation of the solubilized PRL-R complexes with polyclonal anti PRL-R antibody, followed by SDS-PAGE and autoradiography. The long form had a molecular weight of 101 kDa and was predominant when the study was performed in the presence of protease inhibitors. The short form, with a molecular weight of 39 kDa, appeared, at least in part, to be a proteolytic product of the longer form. The same size forms of PRL-R were detected by crosslinking studies in the parental C57BL/6 mouse testicular Leydig cells, indicating the physiological relevance of the MA-10 cell model to the study of Leydig cell PRL-R. Homologous down-regulation of PRL-R was demonstrated in cultured MA-10 cells exposed for 24 h to increasing concentrations of PRL. In contrast, heterologous, 3 5-fold up-regulation of PRL-R was induced by various cAMP-elevating agents, including 8-bromo-cAMP (10(-4) -10(-3) M), dibutyryl cAMP (3 x 10(-3) M) and cholera toxin (1-10 ng/ml), although not by hCG (up to 100 ng/ml). This up-regulatory effect was apparently the result of a change in affinity, since cholera toxin caused a 2.4-fold increase in PRL-R affinity, with no change in the number of binding sites. In summary, these studies provide further evidence that MA-10 Leydig cells can serve as a physiologically relevant model for the study of PRL and PRL-R interactions, both at the functional level, as shown in our previous study, and at the structural and regulatory levels as shown in the current study.

Characterization of prolactin- and growth hormone-binding proteins in milk and their diversity among species.

The present study was undertaken to identify and characterize the diversity and species distribution of soluble prolactin binding-protein (PRL-BP) and growth hormone-binding protein (PRL-BP) in mammalian milk. We previously divided mammalian serum GH-BP into four main groups and identified a GH-BP with shared lactogenic/somatogenic properties in rabbit, horse, dog, pig and cat (Type III species). Here we describe PRL-BP in milk of Type III species and show it is relatively conserved within the group, having similar characteristics in terms of binding affinity for hGH (0.74-5.5 x 10(10) M(-1)), specificity towards the lactogenic hormones and molecular weight (approximately 35 kDa), except for the more heterogeneous pig milk (approximately 43 to approximately 88 kDa) Furthermore, high affinity PRL-BP was also demonstrated in sheep milk, having pure lactogenic specificity and an Mr of approximately 35 kDa. Human milk contained a high affinity PRL-BP/GH-BP, which was recognized by both hPRL and hGH and also having an Mr of approximately 35 kDa. In rabbit milk a separate GH-BP was also detected; it was clearly distinguished from the corresponding milk PRL-BP on the basis of its Mr of approximately 44 kDa (vs. approximately 32 kDa for PRL-BP), its shared lactogenic/somatogenic hormonal specificity (vs. purely lactogenic for PRL-BP) and also on the basis of its relative resistance to heating at 56 degrees C for up to 3 h, while PRL-BP activity was completely destroyed within 30 min. This diversity of milk PRL-BP and GH-BP among mammalian species fits in with our earlier classification of serum GH-BP and also with the reported evolutionary rates of PRL and GH; this suggests these BPs may play important species-specific roles in the suckling newborn and/or maternal mammary gland, in keeping with the functions described for GH-BP.

Prolactin and MA-10 Leydig cell steroidogenesis: biphasic effects of prolactin and signal transduction.

The present investigation was designed to study the direct role of PRL on testicular Leydig cell steroidogenesis, using the MA-10 murine Leydig tumor cell line as a model system. We have previously reported on the presence of specific PRL binding sites in those cells, and we now demonstrate the functionality of those sites and the biological responses induced by the binding of PRL. When cultured MA-10 cells were exposed for 24 h to increasing concentrations of PRL, washed, and then subjected to a 3-h human CG (hCG) stimulation test, a clear dose-dependent biphasic effect of PRL on the steroidogenic response was observed, even though PRL had no effect on MA-10 cell proliferation: at low PRL concentrations (0.1-10 ng/ml), hCG-induced steroidogenesis was stimulated (maximal stimulation by 1 ng/ml PRL being 200-250% of control); at higher concentrations, hCG-induced steroidogenesis was inhibited (60% inhibition was achieved by 1000 ng/ml PRL). When steroidogenesis was induced with various concentrations of cholera toxin, instead of hCG, no effect of the prior exposure to increasing concentrations of PRL was observed, indicating that PRL acts either at the level of the LH/hCG receptor or at some stage proximal to adenylate cyclase. Indeed, further study revealed that 24 or 72 h exposure of MA-10 cells to PRL caused a dose-dependent reduction in hCG binding. Thus, the maximal inhibition of 62% after 72 h with 500 ng/ml PRL, may explain, at least in part, the inhibitory effects of high PRL concentrations on hCG-induced progesterone secretion. Evidence demonstrating possible involvement of a pertussis toxin-(PT-)sensitive G protein in the signal transduction mechanism of PRL receptors is also presented: 1. GTP caused a dose-dependent reduction in affinity (Ka) of PRL binding by its receptors (from Ka = 1.66 +/- 0.2 x 10(9) M(-1) for control MA-10 cell membranes to Ka 3.03 +/- 0.6 x 10(8) M(-1) for membranes incubated with 8 mM GTP). 2. Prior exposure of MA-10 cells to PRL (10 pg/ml) caused a significant reduction in the ability of a 44-kDa membrane protein to undergo PT-induced [32P]ADP-ribosylation. These results demonstrate that MA-10 Leydig cells possess highly specific and biologically functional PRL receptors mediating direct and dose-dependent biphasic effects of PRL on hCG-induced progesterone secretion. These cells thus offer a suitable model to study the mechanism(s) of PRL action and signal transduction of its receptor on a physiologically relevant differentiated function.

Ontogenesis of LH and FSH receptors in postnatal rabbit testes: age-dependent differential expression of long and short RNA transcripts.

The ontogeny of testicular LH and FSH receptors was studied in New Zealand rabbits from 20 to 180 days postpartum. The concentrations of free receptors (per mg total proteins) were very low at day 20. They increased steeply at day 30 for the LH receptor and at day 50 for the FSH receptor. Three RNA bands (1.2, 2.5 and 3 kb) were repeatedly detected on northern blots for the LH receptor and two bands (1.2 and 2.2 kb) were detected for the FSH receptor. The 1.2 kb band (which cannot give rise to full-length, membrane-anchored receptor) was present throughout the 20-180 day period for each receptor. However, the higher molecular mass bands were nearly undetectable at day 20. The 2.5 and 3 kb bands of the LH receptor increased twofold between day 20 and day 120, while the 2.2 kb band of the FSH receptor increased fivefold between day 20 and day 75. Thus the very low concentrations, or even absence, of the larger transcripts of both LH and FSH receptors were correlated with the inability to detect their cognate protein until 20 days of age. Subsequently, coordinated increases in high molecular mass transcripts and protein were observed for both receptors. Total LH receptor content increased in parallel to the previously reported increase in plasma testosterone between day 65 and day 100. FSH receptor density began to increase steeply at day 50, just at the onset of spermatogenesis. Thus, postnatal testicular development in the rabbit seems to entail the transcription of high molecular mass, translatable transcripts of the gonadotrophin receptors.

Prolactin and testicular Leydig cell function: characterization of prolactin receptors in the murine MA-10 testicular Leydig cell line.

The direct role of prolactin (PRL) in testicular function is still unclear, mostly because of lack of a suitable in vitro model. To establish the suitability of the MA-10 murine tumor Leydig cell line for the study of PRL receptors (PRLR) and effects on steroidogenesis, we initially characterized PRLR on cultured MA-10 cells. The specific binding (Bs) of [125I]human growth hormone (hGH) depends on time, temperature, and Mg2+ ion and protein concentrations, with absolute specificity for the lactogenic hormones hGH and ovine PRL. Bs is saturable and is to a single class of high-affinity (Ka = 3.6 x 10(9) M-1) low-capacity (Bmax = 19.5 fmol/mg protein) binding sites. The molecular weight of PRLR, determined by cross-linking to [125I]hGH, SDS-PAGE and autoradiography, is 35 kDa for the free receptor, suggesting that the short-form PRLR protein, previously described in liver and mammary glands, is that primarily found in MA-10 cells. Thus, the demonstration of specific PRL binding sites on MA-10 Leydig cells, with characteristics similar to primary Leydig cell PRLR, suggests that this cell line can serve as a good model for both the study of PRLR mechanism of action and the role of PRL in Leydig cell function.

The Hep G2 cell line in the study of growth hormone receptor/binding protein.

This study identifies specific, high affinity GH-receptors (GH-R) in human hepatoma Hep G2 cells. The binding characteristics of GH-R in the Hep G2 cells are similar to those of human liver membranes, such as the high specificity for hGH, the binding affinity (Ka = 1.7 +/- 0.5 x 10[9] M[-1]) and the molecular weight of the membrane bound GH-R (apparent 125,000 and 71,000). In addition, lower molecular weight forms (approximately 94,000 and approximately 58,000) were identified as GH-binding protein (GH-BP) in Hep G2 conditioned medium, or following incubation of Hep G2 cells, in the presence of 10 mM N-ethylmaleimide for 90 min at 30 degrees C; the latter are presumed to be shed by a proteolytic cleavage of the GH-R. Exposure of Hep G2 cells to physiologic concentrations of hGH resulted in a concentration-dependent increase in 3H-thymidine incorporation, up to 48.4 +/- 7.9% above control. In summary, the demonstration of specific, high affinity GH-R in Hep G2 cells, as well as shedding of GH-BP, suggest these cells may provide a homologous human system to study the receptor-effector interrelationship of hGH and to further our understanding of hepatocyte production of soluble GH-BP.

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.

Influence of Mg2+ on detection of somatogenic and lactogenic components of growth-hormone-binding protein in mammalian sera.

We recently classified the growth-hormone (GH)-binding protein (GH-BP) in a wide range of mammalian [including human (h)] sera and reported the existence of a major lactogenic component in GH-BP of type-III sera (rabbit, horse, dog, pig and cat), based on the capacity of bovine (b) and ovine prolactin (PRL) to displace 125I-labelled human growth hormone (hGH) binding and on direct 125I-bPRL binding studies. In this study, we demonstrate the high degree of Mg2+ dependence of the binding of the classically lactogenic hGH and bPRL, but not that of the somatogenic bGH to various mammalian sera (types I-IV). Serum GH-BP was assayed using a previously described and validated charcoal-separation assay. 125I-hGH binding to rat, ovine, bovine, rabbit, horse, dog and human sera was enhanced 1.5-2.5-fold in the presence of 70 mM Mg2+. The Mg2+ effect was concentration-dependent between 3.7 mM and 70 mM, causing a significant and proportional increase in 125I-hGH binding to serum. Like 125I-hGH, 125I-bPRL binding to type-III sera was also Mg(2+)-dependent. In contrast, 125I-bGH binding to all types of serum GH-BP was not affected by Mg2+ concentrations of up to 35 mM, while 70 mM Mg2+ slightly, but significantly, reduced (by approx. 15%) bGH binding to rabbit serum. In keeping with the Mg(2+)-dependent stimulation of lactogenic hormone binding to GH-BP, 70 mM Mg2+ caused a shift to the left in the displacement curves of hGH and bPRL competing with 125I-hGH binding to rabbit, dog, horse and human sera, while the effects of the somatogens bGH and rabbit GH were shifted to the right. Scatchard analysis of hGH displacement curves with sera from various species yielded linear plots and revealed that Mg2+ significantly increased (2.3-3.0-fold) the affinity constants, but not the binding capacities. These results demonstrate the ability of changes in Mg2+ concentration to determine the degree of differential recognition of somatogens versus lactogens by serum GH-BP. It remains to be determined whether such bivalent cation effects may account, at least in part, for the growth retardation seen in Zn2+ or Mg2+ ion deficiencies.

Growth hormone (GH)-binding protein regulation by estrogen, progesterone, and gonadotropins in human: the effect of ovulation induction with menopausal gonadotropins, GH, and gestation.

The recently described serum GH-binding protein (GH-BP) may reflect the GH-receptor level. To assess the serum GH-BP levels under various physiological and supraphysiological levels of sex steroids, we have evaluated its concentration in 26 patients undergoing 34 cycles of ovulation induction with either human menopausal gonadotropins (hMG)/hCG or GH/hMG/hCG. The latter ovulation induction protocol was undertaken in "Clonidine negative" patients in a prospective, randomized, crossed-over manner, between GH/hMG/hCG or hMG/hCG. The increase in GH-BP levels in patients' sera undergoing ovulation induction directly correlated with peripheral estradiol (E2) (r = 0.577; P < 0.001), and with peripheral progesterone (P4) (r = 0.542; P < 0.001; n = 174) concentrations in hMG/hCG cycles, and also in GH/hMG/hCG cycles (r = 0.669, P < 0.001 for E2, and r = 0.722, P < 0.001 for P4, n = 84). GH-BP levels did not change significantly in response to 0.15 mg Clonidine ingestion. The baseline GH-BP levels significantly correlated with the body mass index of 47 patients (r = 0.547; P < 0.001). The insulin-like growth factor-I (IGF-I) concentrations increased in correlation with increasing E2 levels up to 5500 pmol/L but decreased thereafter, at very high E2 concentrations. In gestations generated by ovulation induction with hMG/hCG or GH/hMG/hCG, longitudinal measurements of GH-BP levels showed an initial sharp increase during early gestation, followed by a gradual decrease beginning around the end of the first trimester and continuing during the mid and late trimesters. In normal spontaneous pregnancies, GH-BP levels, measured from the fifth week until term, were negatively correlated with gestational age (r = -0.581; P < 0.001; n = 84). This pattern is highly suggestive of the possibility that GH-BP is coregulated not only with E2 and P4 but also with hCG, and possibly other gonadotropins as well. Indeed, in patients with "resistant ovaries", pharmacological amounts of hMG failed to increase E2 concentrations but moderately increased GH-BP levels. These data provide good indirect evidence for coregulation of the GH-BP with both sex steroids and gonadotropins. The mid and late trimesters decrease in GH-BP levels in spite of increasing sex steroids concentrations, may be attributed to the decreasing hCG concentrations, and/or to the increasing secretion of placental lactogen (PL) and placental GH with the advancing gestation.

Growth hormone- and prolactin-binding proteins in mammalian serum.

The present study was undertaken to characterize the species specificity and diversity of lactogenic and somatogenic binding to serum among mammals and to classify GH-binding protein (GH-BP) in this group of species. Animal sera were characterized on the basis of human (h) GH and bovine (b) PRL binding levels, binding specificity toward GHs and PRLs, binding affinity constant (Ka), and the ability of a monoclonal antirabbit GH receptor antibody (MAb-7) to inhibit the binding to serum. Analyses of the results yielded a classification of the mammalian sera into five types of GH binding, which we elected to call GH-BP. The guinea pig had undetectable levels of GH-BP and was labeled type 0. Type I GH-BP was found in the mouse and the rat. hGH binding to GH-BP of type I serum was of low affinity (1.2-3.9 x 10(8) M-1) with high IC50 (approximately 100 ng/tube) and was purely somatogenic in nature. Type II GH-BP was found in the goat, sheep, and cow. Their IC50 of hGH binding was approximately 4-fold lower than that of type I GH-BP. Their binding of hGH was relatively specific and only marginally displaced by the PRLs. Type III GH-BP was found in the rabbit, horse, cat, dog, and pig. These animals' sera had high affinity binding toward hGH (4.7-9.2 x 10(9) M-1), with low IC50 (approximately 2 ng/tube) and dominant lactogenic binding. The great similarity of type III GH-BPs was further stressed by the ability of MAb-7 to inhibit [125I]hGH binding to all type III sera, but not to the other mammalian sera tested. Type IV GH-BP was found in the human and rhesus monkey sera. These were characterized by binding affinities that were intermediate between those for types II and III GH-BP and by the inability of nonprimate GHs to compete with hGH binding. To directly confirm the potent effect of the lactogenic hormones in type III GH-BP, the specific binding of bPRL to sera of type III animals was studied. [125I]bPRL-specific binding was determined under optimized assay conditions and was the highest in rabbit serum, with the following rank order being observed: rabbit >> horse = dog = pig > cat. Scatchard analysis of [125I]bPRL binding revealed linear plots with similar affinity constants (Ka) of 1.7-3.3 x 10(9) M-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of human growth hormone binding to somatogenic and lactogenic receptors by monoclonal antibodies to human growth hormone.

The relationship between the structure of human growth hormone (hGH) and the hormone-receptor interaction was investigated by studying the effects of specific monoclonal antibodies (MAbs) to hGH on the binding of [125I]hGH to rabbit liver and mouse liver microsomes. Receptor binding assays were carried out using a constant dose (1 ng) of [125I]hGH and varying concentrations of MAbs. The assay was carried out in the presence of either excess ovine prolactin for the measurement of somatogenic (SOM) binding sites, or excess bovine growth hormone for the determination of lactogenic (LAC) binding sites. Anti-hGH MAbs were found to have a whole spectrum of effects on hGH binding, including inhibitory, non-effect and enhancing activities. Enhancement of the binding of [125I]hGH to both SOM and LAC receptors was observed in liver membranes of rabbit or mouse. The observed amplified signal of [125I]hGH binding to various receptors in the presence of MAb no. 8 may be due to conformational changes which occur following MAb binding to hGH. On the other hand, most of the other MAbs caused inhibition of [125I]hGH binding. A negative correlation exists between the cross-reaction of various MAbs with the N-terminus truncated forms of hGH (Met14-hGH or Met8Leu-hGH) and their respective KD/IC50 values enabled the evaluation of the crucial role of the N-terminus region in hGH binding to both LAC and SOM receptors. MAb nos 1 and 19, which are directed towards acid residues 95-134 and the C-terminus, inhibited SOM binding more potently than LAC binding. Thus, it seems that these mid-molecule and C-terminus regions are also important in hGH binding, and that they play a role in the partial overlap of SOM and LAC binding.

Effect of human growth hormone (GH)-binding protein in human serum on GH binding to rabbit liver membranes.

The sequence identity of growth hormone-binding protein (GH-BP) with the extracellular domain of GH receptors raised the possibility that circulating GH-BP might affect the binding of human GH (hGH) to its receptors, and thus, its biological effects. To test this hypothesis, we tested the effects of sera with low GH-BP levels (obtained from prepubertal children, girls with anorexia nervosa [AN], and patients with hepatic cirrhosis), normal control sera, and sera with high GH-BP levels (obtained from obese patients) on hGH binding to its receptors. GH-BP activity in patients' sera was measured by incubation with [125I]hGH and the separation of bound hGH from free hGH with dextran-coated charcoal. The effect of GH-BP was studied by preincubation of patients' sera with increasing concentrations of hGH, followed by incubation with [125I]hGH and a rabbit liver membrane preparation known to be rich in GH receptors, and finally by measuring hGH bound to the receptors. In this study, we report on the ability of GH-BP to reduce the inhibitory capacity (IC50) of hGH on [125I]hGH binding to GH receptors. The concentration of GH-BP in serum is positively correlated with the IC50 of hGH incubated with different sera on [125I]hGH binding to its receptors (n = 21; r = .886, P less than .001). In the presence of high serum GH-BP levels, such as those observed in obesity (20.13% +/- 0.71%/0.05 mL serum), the IC50 values were significantly higher than those obtained with sera containing GH-BP levels lower than those measured in human control subjects, such as from prepubertal children, AN patients, and cirrhotic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of human growth hormone therapy on GH binding protein in GH-deficient children.

Previous studies have described the close similarity of the GH binding protein to the liver membrane GH receptor. Since GH regulates its own liver receptors, we examined the effects of short- and long-term hGH therapy on GH binding protein in children with GH deficiency. Six GH-deficient children received their first hGH dose ever, and the pharmacodynamics of serum GH was followed for 12 h, along with measurements of GH binding protein activity. Over the first 6 h, serum GH and GH binding protein activity exhibited a parallel increase, followed by gradual decrease. At 8 h, some of the patients exhibited an apparent second peak in GH binding protein, despite the continuous decrease in serum hGH. During the period of hGH treatment, serum GH binding protein increased progressively over a period of 6 months. In a second uncontrolled group of 7 GH-deficient patients who had been treated with hGH for 30-36 months, GH binding protein activity was also significantly higher than pretreatment values. We suggest that the short-term pharmacodynamic changes probably represent the endogenous turnover of the GH receptor, whereas the elevated GH binding protein activity on hGH treatment may reflect up-regulation of the GH receptor.

Synergistic effect of growth hormone and gonadotropins in achieving conception in "clonidine-negative" patients with unexplained infertility.

Based on preliminary reports by others and by us of a potentiating effect of growth hormone (GH) on human menopausal gonadotropin (hMG)-induced ovulation, a study using a randomized, prospective, cross-over protocol between GH + hMG/human chorionic gonadotropin (hCG) and hMG/hCG was undertaken. The study included patients with long-standing (2-11 years) unexplained infertility with a negative or reduced GH response to clonidine (up to 150 micrograms of clonidine orally). The first cycle was randomly assigned between GH/hMG/hCG (study cycle) and hMG/hCG alone (control cycle), and after an interval cycle the patient's treatment was crossed over. All patients who completed the study had previously undergone 1-20 attempts at ovulation induction for in vitro fertilization (IVF) and 5-40 cycles of ovulation induction for in vivo fertilization at three different medical centers. Three patients conceived on the combined GH/hMG cycle, with diminution in the hMG consumption needed for ovulation induction in the study cycles. Another patient with long-standing mechanical infertility underwent 11 abortive attempts at ovulation induction with hMG for IVF but has never achieved egg retrieval. On the GH/hMG/hCG ovulation induction cycle, three mature ova were retrieved as opposed to no response and cancellation of the "hMG only" cycle. Another patient with 11 years of primary infertility who had undergone 21 previous attempts at ovulation induction and had reached follicular aspiration in only three of those cycles conceived spontaneously on the first cycle after the GH/hMG/hCG IVF/ET cycle. All four pregnancies that have been achieved by now in seven GH/hMG-treated patients ended in cesarean deliveries of four normal male neonates. No correlation was found between the follicular fluid levels of insulin-like growth factor I (IGF-I) and the fertilization rate in vitro. The peripheral IGF-I levels were significantly higher during the follicular phase of the study cycles than during the respective stage of the control cycles or the luteal phase of either cycle. A study of serum GH-binding protein (GH-BP) levels revealed gradual increases in the late follicular phase, in the luteal phase, and in early pregnancy. On the basis of this study and in keeping with earlier reports, we conclude that the addition of GH to hMG/hCG may serve as a contributory adjunct in selected patients. However, in contrast to others who could not find a correlation between the response to acute tests for GH release and the ovarian response to combined treatment, we conclude that the clonidine test can play a discriminatory role in identifying patients who may benefit from this innovative combination.

A new and convenient assay of growth hormone-binding protein activity in human serum.

The recent discovery of a GH-binding protein (GH-BP) in human serum has important implications for the distribution, metabolism, and physiological activity of human GH (hGH). In this report we describe an accurate, simple, and rapid assay for measuring GH-BP in human serum, based on the dextran-coated charcoal technique for separating bound from free hGH. The present method reduces the degree of dissociation of the GH-BP complex during the separation procedure, thus increasing accuracy, allows routine use of small working volumes of sera, and can be practically used for running large scale assays. Binding of [125I]hGH to human serum GH-BP, identified and characterized by this procedure, was time, temperature, and dose dependent; the binding was highly specific for hGH and with a high affinity (Ka, 1.08 +/- 0.19 x 10(9) L/mol). Preliminary results, expressed as percent specifically bound [125I]hGH corrected for endogenous hGH, indicate that the mean GH-BP activity in 50 microL sera from normal adult men and women was 11.32 +/- 0.45%. Significantly lower values were obtained in sera of infants, 3 days after birth (1.65 +/- 0.15%), and in short-statured children with normal GH secretion (7.38 +/- 0.22%). In patients with liver cirrhosis GH-BP levels decreased to 5.93 +/- 1.14%. The availability of a simple and convenient procedure for large scale determination of GH-BP coupled with the suggestion that this protein can reflect the GH receptor provides us with an indirect means for assessing changes in GH receptor activity in various physiological and pathological conditions.

Identification of growth hormone binding protein in rat serum.

The present report describes the initial characterization of a specific, high-affinity growth hormone binding protein (GH-BP) in adult male rat serum. GH-BP activity was measured by incubation of rat serum with [125I]hGH and [125I]rGH and separation of bound from free GH by dextran-coated charcoal. [125I]hGH binding to rat serum was dependent on serum concentration and incubation time, equilibrium being reached within 10 min both at 4 and 37 degrees C. Binding was rapidly and completely reversible and specific for somatogenic (but not lactogenic) hormones. Scatchard analysis yielded a linear plot with an affinity (Ka) of 1.51 +/- 0.63 x 10(8) M-1. Preliminary data obtained in various physiological conditions showed that GH-BP activity in adult male rats was 5.95 +/- 0.20%/0.1 ml serum. Significantly higher values were obtained in sera of female (21.66 + 0.79%/0.1 ml serum) and pregnant rats (23.02 +/- 1.15%/0.1 ml serum). A closer analysis of these binding values by Scatchard analysis revealed that the binding capacity in pregnant rats (50.5 +/- 5.8 pmol/0.1 ml serum) was significantly higher than in adult female estrous rats (19.2 +/- 6.5 pmol/0.1 ml serum), both being much higher than in adult male rats (2.5 +/- 0.6 pmol/0.1 ml serum). The GH-BP activity of 10-day-old rats was only approximately 63% of the adult male rat value. The presence of high-affinity GH-specific binding protein in rat serum suggests a probable action in regulation of GH activity. The detailed physiological role of rat serum GH-BP is currently being investigated.

The interrelationship of growth hormone (GH), liver membrane GH receptor, serum GH-binding protein activity, and insulin-like growth factor I in the male rat.

Indirect evidence suggests that the serum GH-binding protein (GH-BP) is related and possibly derived from the GH-receptor. GH, through its specific receptor, is the major regulator of insulin-like growth factor I (IGF-I) synthesis. The present study was undertaken to correlate serum GH-BP activity with liver plasma membrane GH receptors and their effects on serum IGF-I concentration during spontaneous pulsation of rat (r)GH in the normal male rat and after continuous delivery of human (h)GH to hypophysectomized male rats. In the first set of experiments, 45-day-old male rats were decapitated at 15 min intervals for 4 h. Serum GH-BP levels fluctuated with a 60 min lag behind the rGH levels. IGF-I pulsated over a 3-fold concentration range. IGF-I peak levels coincided with one of the rGH peaks, but its periodicity was longer than 3 h. Taken together with our previous studies on the turnover of the GH receptors, we suggest that each GH surge results in individual pulse-related turnover wave of receptor internalization and recycling. This is accompanied by a parallel increase in serum GH-BP activity. The GH and the receptor wave are responsible for an individual secretion pulse of IGF-I. In the second set of experiments male rats were hypophysectomized at 35 days of age. Four days later osmotic minipumps were implanted for continuous delivery of hGH. After 6 days of hGH treatment the rats were killed, blood was collected for hGH, GH-BP, and IGF-I determination, and the livers were removed. Plasma membranes were prepared, and lactogenic and somatogenic binding of [125I]hGH was evaluated. Removal of endogenous ligand was performed by exposing the membranes to 3 M MgCl2. Continuous administration of hGH induced a dose-dependent increase in liver membrane lactogenic and somatogenic binding. Parallel to that increase, serum GH-BP also increased in a dose-dependent manner, and the correlation between serum GH-BP and the liver membrane receptor was significant. Furthermore, hGH induced a dose-dependent increase in IGF-I concentration. There was a close correlation between IGF-I concentration and liver somatogenic receptors. It is concluded that up-regulation of the liver membrane GH receptors is accompanied by increased GH-BP and IGF-I. In both the pulsation experiment and the continuous infusion experiment, GH-BP closely correlated with the liver membrane GH receptor.