Prolactin (PRL) receptor induction in cultured rat hepatocytes: dual regulation by PRL and growth hormone

Although early work implicated PRL as the pituitary factor inducing rat hepatic PRL receptors, recent studies indicated that GH, not PRL, was responsible. The roles for these two hormones were evaluated on rat hepatocytes cultured in serum-free medium supplemented with insulin (1 microgram/ml), epidermal growth factor EGF (25 ng/ml), glucagon (500 ng/ml), cholera toxin (2 ng/ml), hydrocortisone (10(-8) M), and transferrin (1 microgram/ml) and changed daily. Ovine (o) PRL, bovine (b) GH, or human (h) GH were introduced after 2-4 days of culture, and PRL receptors were measured by determining [125I]hGH binding in the presence and absence of excess oPRL in a total particulate fraction pretreated with 3 M MgCl2. The specific binding of hGH (% per 100 micrograms protein) decreased by 8- to 10-fold (female, 17.9 +/- 0.2% to 1.5%; male, 7.0 +/- 0.1% to 0.7%) after 3 days in culture. When added after 3 days, hGH induced PRL receptors in both female and male cells with the effect being more gradual in the latter. Induction occurred with 10 ng/ml hGH and was maximal [11- to 13-fold control] at 250-1000 ng/ml. bGH and oPRL also induced PRL receptors with maximal levels attained at 250-500 ng/ml oPRL (3- to 4-fold control). The combined addition of oPRL (300 ng/ml) and bGH (300 ng/ml) yielded levels of induction comparable to that seen with hGH. Although hormone treatment restored PRL receptor levels to those seen in male rats, the much higher levels of female rats were not attained. Treatment of hepatocytes with hGH, bGH, or oPRL affected neither cell number (through 10 days of culture) nor PRL receptor affinity. At supramaximal doses hGH, PRL, and bGH down-regulated PRL receptors, but this was particularly noticeable for oPRL and hGH. 17 beta-Estradiol and testosterone added to male and female hepatocytes simultaneously with hGH had little or no effect on receptor induction. We conclude that hepatic PRL receptors are induced by both PRL and GH, each acting through its own receptor. The failure to restore receptor levels to those seen in female rats attests to the importance of other modulators. This dual regulation of the PRL receptor explains the unusual potency of hGH which binds to both PRL and GH receptors.     

Reduction of lactogenic receptors in female hamster liver due to the human growth hormone analog produced by plerocercoids of the tapeworm, Spirometra mansonoides

The inductive effect of GH on hepatic lactogenic receptors is suspected of being due to a direct somatogenic action. Plerocercoid larvae of the tapeworm, Spriometra mansonoides, produce a factor that stimulates body growth, suppresses endogenous GH, and specifically displaces [125I]human (h) GH from hepatic receptors. Plerocercoid growth factor (PGF) mimics the growth-promoting actions of GH, but it has not been shown to duplicate all of the activities reported for GH. An important function of GH is its role in the maintenance of liver receptors for lactogenic hormones. This study was undertaken to determine if treatment of female hamsters with PGF would increase, decrease, or have no effect on liver receptors that bind hGH. Since hGH binds to somatogenic as well as lactogenic receptors, it was necessary to demonstrate the specificity of PGF's effects on [125I]hGH binding. PGF-treated (15 pleocercoids sc) hamsters had accelerated body growth, suppressed serum GH, and a marked reduction in [125I]hGH and [125I]ovine PRL binding to hepatic microsomes. Specific binding of [125I] bGH was unaltered by PGF treatment. The difference in [125I] hGH binding was due to a reduction in receptor number and not to receptor occupancy or reduced affinity. Serum GH was normalized after 10 days of estradiol benzoate (25 micrograms/day) injections, but the binding capacity for [125I]hGH of the PGF-treated group was less than half that of the control group. The fact that estrogen injections normalized serum GH, but not hGH binding, indicates that down-regulation of these receptors by PGF cannot be entirely explained on the basis of reduced levels of serum GH. The lack of any effect of PGF treatment on [125I]bGH binding suggests that the hepatic somatogenic receptors were not involved and that the reduction in receptors for [125I]hGH was associated with the lactogenic component of hGH.     

Receptor-mediated endocytosis and degradation of bovine growth hormone in rat liver

Receptor-mediated endocytosis of radiolabelled bovine growth hormone (125I-bGH) via somatogenic receptors in the liver was studied following in vivo intraportal injection. At different times after injection, subcellular membrane fractions involved in binding (plasma membranes), endocytosis (endocytic vesicles) and degradation (lysosomes) of peptide hormones were isolated by sucrose density gradient centrifugation. These fractions were evaluated for the time-course accumulation of radiolabelled bGH and for the presence of internalized 125I-bGH-receptor complexes. These uptake studies indicate that after initial plasma membrane association of 125I-bGH, the ligand is transported in two successive endocytic compartments prior to arrival in lysosomes. The molecular weight of the somatogenic binders of male and female rat livers involved in internalization of 125I-bGH was determined to 95,000, 64,000, 55,000, 43,000 and 35,000, assuming a 1:1 binding of the hormone to the binder. These binders were seen in both endosomes and lysosomes, which suggests that growth hormone is transported to the lysosomes in a complex with its receptor. Binding and uptake of 125I-bGH was also compared in male and female rat livers, and endocytosis of 125I-bGH was compared to that of radiolabelled ovine prolactin (125I-oPrl). The specific uptake of 125I-bGH appeared not to be sexually differentiated in contrast to that of 125I-oPrl which showed a 35-fold higher uptake in female rat liver. Degradation of 125I-bGH was studied under in vitro binding assay conditions. A distinct 15,000 Da fragment was generated by plasma membrane, endosomal and lysosomal fractions. Based on protease inhibitor studies, a non-trypsin-like serine protease is suggested to be involved in the degradation of bGH. The 15,000 Da proteolytic fragment of GH can be affinity cross-linked to somatogenic binders of similar molecular weights as those involved in the binding of intact GH.     

Octreotide inhibits insulin-like growth factor-I hepatic gene expression in the hypophysectomized rat: evidence for a direct and indirect mechanism of action.

Somatostatin and somatostatin analogs are known to interact with the GH-insulin-like growth factor (IGF)-I axis by inhibiting GH secretion and consequently hepatic IGF-I production. Indirect evidence suggests that octreotide, a somatostatin analog, reduces serum IGF-I levels relatively more than expected from GH reduction, implying a GH-independent pathway of action. To study the role of octreotide in the regulation of IGF-I production, independently of endogenous GH, we used the hypophysectomized (hypox) rat to measure hepatic IGF-I expression and also employed cultured rat hepatocytes to examine whether octreotide has any direct effect on the production of IGF-I. Forty male hypox Sprague-Dawley rats were randomized into 4 groups to receive daily injections for 3 days of either saline, human GH (hGH) (100 g), octreotide (100 g twice), or both hGH (100 g) and octreotide (100 g twice). GH stimulated serum IGF-I levels to 104 +/- 10 micrograms/liter as compared to saline (26 +/- 2 micrograms/liter). Octreotide alone had no effect, but combining octreotide and hGH significantly reduced the hGH-induced rise in the IGF-I levels (52 +/- 6 micrograms/liter). The relative expression of hepatic IGF-I in the rats treated with hGH increased by 4-fold compared to that in the saline-treated rats. Octreotide administered simultaneously with hGH potently blocked the hGH-induced IGF-I expression to control levels. In cultured hepatocytes, IGF-I mRNA levels maximally stimulated by combining bGH and glucagon were significantly inhibited in the presence of octreotide at low concentrations (0.3 and 3 ng/ml) by 25% and 45%, respectively. In contrast, high concentrations of octreotide (30 and 300 ng/ml) had no significant effect on IGF-I mRNA abundance. We conclude that: 1) octreotide inhibits IGF-I serum levels and hepatic gene expression in the hypox rat; and 2) octreotide can inhibit partially the direct effects of GH and glucagon on hepatic IGF-I production.     

A single arginine residue determines species specificity of the human growth hormone receptor.

Although growth hormone (GH) receptors (GHRs) in many species bind human (h) GH as well as their own GH, the hGHR only binds primate GH. Arg43 in hGHR interacts with Asp171 of hGH. Nonprimates have a His in the position equivalent to residue 171 of primate GH and a Leu in position 43 of primate GHR. To determine whether Arg43 accounts for the species specificity of the hGHR, point mutations that changed Leu43 to Arg were introduced into the cDNAs encoding the bovine (b) GHR or the rat GH binding protein (GHBP) and these mutants or their wild-type (WT) counterparts were expressed in mouse L cells. Binding of hGH or bGH to transfected cells or to GHBP secreted into the incubation medium was assessed by displacement of 125I-labeled hGH. WT and mutant bGHR bound hGH with similar affinity, but the affinity of the mutant receptors for bGH was reduced 200-fold. Likewise, WT and mutant GHBP bound hGH with equal affinity, but only WT GHBP bound bGH. Cross-linking of 125I-labeled hGH to WT or mutant GHR produced a 141-kDa labeled complex whose appearance was blocked by unlabeled hGH, but bGH blocked cross-linking only to WT receptors. Both hGH and bGH stimulated tyrosine phosphorylation of a 95-kDa protein in cells transfected with WT GHR, but bGH was less effective in cells expressing mutant GHR. We conclude that incompatibility of Arg43 in the hGHR with His171 in nonprimate GH is the major determinant of species specificity.     

Initial characterization and sexual dimorphism of serum growth hormone-binding protein in adult rats

The in vitro binding of 125I-bovine growth hormone (bGH) to adult rat serum was studied using Ultrogel AcA34 filtration. When analytical chromatography on a 1.6 x 100 cm column was performed, four peaks of radioactivity were revealed: the first two peaks with Mr +/- 220,000 and +/- 110,000 corresponded to bound 125I-bGH (abolished by excess of unlabeled bGH), the third corresponded to free 125I-bGH and the fourth to free Na125I (Vt). On a short (1 x 40 cm) column, bound 125I-bGH eluted as a single peak between the void volume (Vo) and the peak of free 125I-bGH. Serum 125I-bGH binding was specific, saturable, and time-dependent. Specific serum 125I-bGH binding (bound/total radioactivity x 100), calculated as the difference of binding in the absence and the presence of an excess unlabeled bGH, was higher in female than in male rats (26 +/- 2% vs. 11 +/- 1%, respectively; mean +/- SE; n = 6; P less than 0.01). Scatchard analysis revealed a binding affinity of 2 x 10(8) M-1 for both sexes, and a binding capacity of 6.4 x 10(-8) mol/liter for the female rats and 1.6 x 10(-8) mol/liter for the male rats (mean of three serum pools of three animals each). Specific binding of 125I-bGH to serum correlated significantly with 125I-bGH binding to liver homogenates (r = 0.83; n = 12; P less than 0.01). These results suggest the presence of a specific GH-binding protein in rat serum and provide further evidence for a close relationship between serum GH-binding protein and hepatic GH receptors.     

Specific binding of growth hormone to isolated chondrocytes from rabbit ear and epiphyseal plate

Chondrocytes were isolated from rabbit ear, epiphyseal and rib cartilage, and their ability to bind 125I-labeled human growth hormone (hGH) was investigated. The total binding of hormone to ear chondrocytes increased with time until 4-6 h, whereas non-specific binding did not increase. Total binding was 1-3% of total counts added, and non-specific binding was 10-20% of total binding. Unlabeled hGH, bGH and oPRL competed with the binding of labeled hGH in a dose-dependent manner with half-maximal binding at concentrations of 10-20 ng/ml (hGH) and 100 ng/ml (bGH and oPRL). The two latter hormones displaced almost all specific binding of hGH at higher concentrations. Ear chondrocytes from newborn to 4 week old rabbits all showed specific binding of hGH with no clear age dependence. Rib chondrocytes, on the other hand, showed very little specific binding. Epiphyseal chondrocytes from newborn animals yielded intermediate binding values. The finding of specific hGH binding by cultured chondrocytes supports the concept of a direct effect of GH on chondrocytes.     

Characterization and subcellular distribution of somatogenic receptor in rat liver

Binding of [125I]iodobovine GH [( 125I]iodo-bGH) to rat liver microsomes and Golgi/endosomal fractions isolated from male and female rats has been characterized. Binding of bGH to a pure somatogenic site was suggested by the finding that 50% inhibition of [125I]iodo-bGH binding required 5-130 ng bGH, rGH, or hGH/incubation, while around 500 ng rat PRL/incubation were needed to obtain the same effect. Binding of [125I]iodo-bGH to microsomes and Golgi/endosomes was time, temperature, and protein dependent. Maximal specific binding occurred at 15-16 and 15-20 h at 22 C in Golgi and microsomal membranes, respectively. Subcellular distribution studies demonstrated in the Golgi/endosomal fractions compared to the total particulate fraction, while residual microsomes devoid of Golgi/endosomal-derived components were approximately 2-fold enriched. Low levels of somatogenic receptors were detected in lysosome-enriched fractions. Removal of endogenous ligand by treating Golgi/endosomal membranes with 3 M MgCl2 increased specific binding of bGH about 2- to 3-fold. These results indicate that approximately 50% of specific somatogenic binding sites in the low density fractions represent internalized ligand-receptor complexes. The level of rat liver somatogenic receptors did not show a pronounced sex differentiation; however, an endocrine dependence of somatogenic receptor levels is suggested by the finding that livers from rats in the late stages of pregnancy had a level of somatogenic receptors exceeding that of nonpregnant rats.     

Growth hormone binding to specific receptors stimulates growth and function of cloned insulin-producing rat insulinoma RIN-5AH cells

Binding of 125I-labeled human GH (hGH) to a cloned rat insulin-producing cell line RIN-5AH in monolayer culture was studied along with some physiological effects of the hormone on these cells. Binding was time and temperature dependent, and steady state binding was observed in 60 min at 37 C with [125I]hGH at 4.2 pM, whereas at 24 C, binding had not reached a steady state after 120 min. The binding was largely reversible, since 80% of initially bound [125I]hGH dissociated from the cells upon incubation in hGH-free buffer for 120 min. Half-maximal binding was obtained when cells were incubated in the presence of 3.0 X 10(-10) M unlabeled hGH. Rat GH as well as human placental lactogen were able to compete for binding sites, but with less affinity. Other non-GH peptides at 6.7 micrograms/ml did not affect [125I]hGH binding. Scatchard analysis revealed curvilinear plots, and approximately 2700 high affinity binding sites were calculated. Culture of RIN-5AH in the presence of 1 microgram/ml hGH for 4 days resulted in an 80% increase in insulin content as well as an 18% increase in cell number and DNA and protein content compared to those in cells cultured in the absence of hGH. The dose dependence of the insulinotropic effect showed that half-maximal and maximal stimulation were observed in cells cultured in the presence of 10 and 100 ng/ml, respectively. Insulin release to the medium during the 4-day culture period was not affected by hGH. These data suggest that GH, through binding to specific receptors in the cell membrane, directly stimulates proliferation and function of pancreatic beta-cells.     

Characterization and modulation of growth hormone and prolactin binding in mouse liver.

The specific binding of 125I-labeled insulin, human hormone ([125I]hGH), bovine growth hormone ([125I]bGH), and ovine prolactin ([125I]oPRL) was studied in mouse liver membranes. [125I]hGH and [125I]oPRL bound to adult liver membranes. Pregnancy increased the specific binding of [125I]hGH but not that of [125I]oPRL. [125I]hGH was displaced from membranes of pregnant mice by hGH, oPRL, and bGH, but only by hGH and oPRL from liver membranes of nonpregnant mice. Significant specific binding of [125I]bGH was seen only in pregnancy. The binding of [125I]bGH to pregnant mouse liver membranes increased with increasing concentration of either membrane protein or [125I]bGH. Both the specific binding and dissociation of [125I]bGH were greatly influenced by the time and temperature of incubation. Binding of [125I]bGH was inhibited by growth hormones, including hGH and rat GH, and not by lactogenic hormones (various prolactins and human placental lactogen), ACTH, glucagon, or insulin. The inhibition of [125I]hGH binding by hGH and bGH, in the presence of excess (2 mug/ml) of PRL, was very similar to that seen with [125I]bGH. Scatchard plots of displacement dose-response curves obtained under steady state conditions of 4C were nonlinear and very similar with either [125I]bGH or [125I]hGH. This contrasted with the linear Scatchard plots obtained from displacement dose-response curves of either [125I]oPRL or [125I]hGH in the presence of excess (2 mug/ml) bGH. Termination of pregnancy, either naturally or by hysterectomy, reduced [125I]bGH specific binding to nonpregnant levels by 24 to 36 h. Estrogen administration did not increase [125I]bGH binding in hepatic membranes. Nonpregnant mice possess hepatic lactogen binding sites which are uninfluenced by pregnancy. GH specific binding sites are markedly augmented during pregnancy. The close correlation between the level of these sites and pregnancy suggests that they are regulated by a product of the fetoplacental unit.     

Relationship between binding and biological effects of human growth hormone in rat adipocytes

Insulin-like responses to human GH (hGH) were produced in adipocytes isolated from the epididymal fat of normal rats 3 h after excision of the tissues. Insulin-like responses consisted of increased oxidation of glucose and incorporation of its carbons into total lipid, increased oxidation of L-[1-14C]leucine, and antagonism of the lipolytic actions of epinephrine. Refractoriness to these effects of hGH in the fourth hour of incubation was produced by the addition of as little as 3 ng/ml hGH as soon as possible after excision of the tissues. These cells also responded to the delayed lipolytic effect of hGH in the presence of theophylline. The cells were found to have high affinity, low capacity, specific binding sites for 125I-labeled hGH. Monoiodination of hGH did not interfere with its capacity to produce biological responses. Specific binding equilibrated rapidly and appeared to saturate at about 100 ng/ml. In cells that were capable of exhibiting an insulin-like response to hGH, rat and ovine GH successfully competed with [125I]hGH for binding sites, but porcine insulin, at a concentration of 100 mU/ml, failed to reduce the binding of [125I]hGH, indicating that GH does not produce its insulin-like effects by interacting with the insulin receptor. Binding of [125I]hGH in cells that are refractory to the insulin-like effects of GH is indistinguishable from binding in responsive cells. Scatchard analysis of the data for both responsive and refractory cells gave linear plots consistent with a single class of about 20,000 receptors/cell, which become half-saturated at a concentration of approximately 20 ng/ml. This corresponds well with 30-50 ng/ml needed for half-maximal insulin-like responses and the 3-10 ng/ml ED50 for induction of refractoriness or lipolysis. It thus appears unlikely that there are appreciable spare receptors for insulin-like responses. These findings make it likely that refractoriness to the insulin-like effects of GH occurs at a postreceptor site.     

The presence of lactogen but not growth hormone binding sites in the isolated rat hepatocyte.

The binding of 125I-labelled human growth hormone (hGH) and bovine growth hormone (bGH) has been studied in hepatocytes isolated from female rats by perfusion with collagenase in situ. The cells appeared to retain normal membrane function, in that amino acid ([14C]alpha-aminoisobutyric acid) transport was both saturable and temperature-dependent. Amino acid ([14C]leucine) incorporation into protein was also linear over 3 h and was inhibited by cycloheximide. Binding of 125I-labelled hGH was dependent on time, temperature, hepatocyte concentration and hGH concentration. At 22 degrees C, binding reached a steady-state after 2-5 h and had a half-life of dissociation of 2-3 h. Hormone specificity studies indicated that binding was specific for hormones with prolactin-like activity (hGH, prolactins) and not for growth hormones themselves (bGH). Scatchard analysis revealed a single class of binding site with a binding capacity of 26-74+/-3-73 fmol/10(6) cells and a binding affinity of 1-24 X 10(9)+/-0-17 X 10(9) (S.E.M.) 1/mol (n=10). There was a significant sex difference in binding (female greater than male) and binding was subject to marked regulation by oestrogens (stimulation of binding) and by androgens (inhibition). The lactogen-binding sites, therefore, were comparable in many respects to those previously reported in rat liver membranes. No distinct GH binding sites were demonstrable as shown by the lack of specific binding by 125I-labelled bGH, purified either by Sephadex chromatography or by binding to and elution from GH receptors in rabbit liver membranes. The value of receptor purification of tracer for use in hormone binding studies was indicated by a substantial lowering of non-specific binding.     

Binding of bovine growth hormone to bovine liver membranes

The binding of [125I]bGH and [125I]hGH to bovine liver membranes is compared to characterize the somatotrophic hormone-receptor interaction. [125I]bGH binding exhibits higher nonspecific binding than [125I]hGH while the time-course of binding and displacement with unlabeled GH are similar. Divalent and monovalent cations enhance [125I]hGH binding with well-defined peaks of binding at specific cation concentrations. Monovalent cations do not enhance [125I]bGH binding at concentrations of 100 mM while divalent cations enhance binding over a range of cation concentration (4-80 mM). The binding of [125I]bGH is dependent upon the presence of divalent cations, with minimal effect of pH upon binding in the absence of calcium. Scatchard plots of bGH and hGH binding data indicate at least two binding sites. We conclude that somatotrophic GH exhibits unique and distinguishing characteristics of binding. The characteristics of hGH binding to the bovine liver membranes suggest that its binding may differ from bGH binding to its homologous receptor.     

Growth hormone receptors in cultured adipocytes: a model to study receptor regulation

Acutely isolated rat adipocytes have been maintained in primary culture for several days and the effects of culture on the kinetics of 125I-human growth hormone (hGH) binding to adipocytes have been determined. A marked increase (500-1000%) in specific binding of 125I-hGH was observed over the first 3 days of culture--acutely isolated adipocytes (5.5 +/- 1.4%, mean +/- SE, n = 47) compared to 3-day cultured adipocytes (48 +/- 7%, mean +/- SE, n = 8). Specific binding of 125I-hGH to both acutely isolated and cultured adipocytes was dependent on incubation time and temperature (equilibrium being reached in 1 h at 37 degrees C and 2 h at 22 degrees C). Binding was reversible (t1/2 approximately 1.5 h). Scatchard analysis revealed linear plots and showed that the increase in binding during culture was due to an increase in the number of receptors per cell (approximately 20 000 to approximately 170 000) with little or no change in binding affinity (Ka approximately 1 X 10(9) M-1). Cycloheximide inhibited the increase in binding sites during culture suggesting a requirement for de novo protein synthesis. Addition of unlabelled hGH to the culture medium resulted in a marked down-regulation of the GH receptor by 2 days. The GH-induced decrease in receptor number was to due to receptor occupancy by exogenously added GH. The studies to date indicate that the cultured rat adipocyte should provide a useful model for a comprehensive study of the cellular mechanisms and dynamics of GH receptor regulation.     

Characterization of hepatic growth hormone binding sites in two fish species, Gillichthys mirabilis (Teleostei) and Acipenser transmontanus (Chondrostei)

To obtain information on the presence of growth hormone (GH) receptors in liver of nonmammalian vertebrates the specific binding of 125I-bovine growth hormone (bGH) to liver membranes of seven species representing the major groups was studied by radioreceptor assay. A substantial degree of specific binding was detected with sturgeon (Acipenser transmontanus) liver membranes and a much lower amount was detected on hepatic membranes of Gillichthys mirabilis. No significant specific binding was detected on liver membranes of pigeon, turtle, bullfrog, tilapia, or leopard shark. Gillichthys and sturgeon liver membranes were further characterized and compared with hepatic membranes from male rabbits. The sturgeon and Gillichthys membranes showed binding that was dependent upon time, temperature, pH, and membrane concentration. Scatchard analysis of the binding of 125I-bGH to sturgeon and rabbit membranes revealed both high and low affinity binding sites. The high affinity sites had KA values of 3.1 X 10(11) and 1.0 X 10(11) M-1, and capacities of 12 and 50 fmol/mg protein, respectively. Membranes from Gillichthys liver contained only a single class of binding sites with a KA of 6.7 X 10(9) M-1 and a binding capacity of 49 fmol/mg. Hormonal specificity of the sturgeon and Gillichthys hepatic binding sites was studied using methionyl-human GH (met-hGH), ovine prolactin (oPRL), and a crude preparation of sturgeon (st)GH. The met-hGH and stGH inhibited the binding of 125I-bGH to sturgeon liver membranes while only met-hGH displaced labeled bGH from Gillichthys liver membranes. One microgram of oPRL did not significantly inhibit 125I-bGH binding in either membrane assay. Based on these studies, sturgeon hepatic GH receptors seem to be more like those of nonprimate mammals than those of teleosts. Our results, in conjunction with the data of J. N. Fryer (Gen. Comp. Endocrinol. 39, 123-130 (1979)), indicate that considerable evolutionary divergence has occurred among teleost hepatic GH receptors. Thus, vertebrate GH receptors seem to have undergone at least as much evolution as has the hormone itself.     

Growth hormone potentiates colony formation of epiphyseal chondrocytes in suspension culture

The effect of human GH (hGH) on colony formation of rat epiphyseal plate chondrocytes was studied in suspension culture. Chondrocytes were isolated enzymatically from epiphyseal plates of the proximal tibia of 28-day-old normal male rats, and were cultured in a suspension stabilized with 0.5% agarose. After approximately 7 days of culture in the presence of 10% newborn calf serum (NCS), chondrocyte colonies developed consisting of varying numbers of cells in matrix. No colonies developed in the absence of NCS, and the number of formed colonies was proportional to the concentration of NCS (5-20%) in the medium. hGH potentiated the formation of large size colonies (diameter greater than 90 microns) after a culture period of 10 or 14 days. The lowest effective concentration of hGH was 10 ng/ml, while 40 ng/ml hGH gave a maximal stimulatory effect (40-50%). Higher concentrations of hGH (80 and 160 ng/ml) showed reduced potentiation of colony formation. The stimulatory effect of hGH was expressed at 10-15% of NCS at 14 days of culture. There was a linear relation between the number of seeded cells and the number of colonies formed, both in the absence and presence of hGH. These results show that GH potentiates colony formation in chondrocytes of the epiphyseal growth plate, providing further support for the contention that GH exert a direct stimulatory effect on epiphyseal cartilage and thus stimulates longitudinal bone growth directly. The finding that GH preferentially potentiated the formation of large size colonies suggests that GH promoted the differentiation of early proliferative chondrocytes or stem cell chondrocytes with an inherent high capacity to proliferate.     

Homologous IM-9 lymphocyte radioreceptor and receptor modulation assays for human serum growth hormone

Radioreceptor assays for human GH (hGH) have been developed using the IM-9 cultured human lymphoid cell receptor. Varying degrees of nonspecific interference with [125I] hGH binding to these cells occurs in the presence of serum. We have modified the traditional IM-9 competitive binding assay for hGH and abolished the nonspecific serum effects. The modified competitive assay is sensitive to as little as 2 ng/ml hGH, and it has been validated by the quantitative recovery of purified pituitary hGH in hypopituitary serum. Sera from stimulated normals, acromegalics, and patients with severe growth retardation were assayed. The RIA to radioreceptor assay ratios for these groups were 0.98 +/- 0.10, 0.97 +/- 0.18, and 2.43 +/- 0.54, respectively. The assay has potential usefulness in screening and predicting growth-retarded patients most likely to respond to exogenous hGH therapy. In addition, a sensitivity receptor modulation assay, which uses the ability of hGH to regulate its homologous IM-9 receptors, is described. This is 8- to 10-fold more sensitive than the nonregulatory assays and has been applied to the measurement of hGH in sera from unstimulated normals and acromegalics. The ratios of RIA to receptor modulation assay for the two groups were 1.17 +/-0.68 ad 1.07 +/- 0.26, respectively. These sensitive receptor assays offer a powerful technique for the measurement of biologically active and inactive GH peptide in serum, and may be particularly useful in the evaluation of statural growth disorders.     

Growth hormone maintains its own receptors in rat adipocytes

Hypophysectomy decreased the capacity of adipocytes isolated from epididymal fat to bind [125I]human GH [( 125I]hGH) specifically without changing the apparent affinity for hGH. Specific binding of hGH by adipocytes of both normal and hypophysectomized rats appeared saturated when incubated with 75-80 ng/ml or higher concentrations of GH regardless of whether binding was studied for 2 h at 37 C or for 16 h at 0 C. Maximum binding of hGH by normal adipocytes was approximately 0.45 ng/10(6) cells, and that by adipocytes of hypophysectomized rats ranged from 0.15-0.25 ng/10(6) cells. In cells of both normal and hypophysectomized rats, only 25-30% of the hormone specifically bound at 37 was removed by digestion with trypsin, and about 75% was displaced by incubation with 5 M magnesium chloride, suggesting that these adipocytes internalized a significant fraction of bound hormone and that hypophysectomy did not alter the extent of internalization. Previously bound hormone was lost from normal adipocytes with a half-time of about 32 min and from adipocytes of hypophysectomized rats with a half-time of about 45 min, suggesting that hypophysectomy slowed the rate of processing bound hormone. To determine which pituitary hormone(s) might be required to maintain GH binding, we measured the binding of [125I]hGH at 3 or 30 ng/ml by fat cells prepared from hypophysectomized rats after various treatment regimens. Administration of bovine GH ip at a dose of 10 micrograms/rat every 4 h for 24 h doubled the binding of [125I]hGH by adipocytes prepared 4 h after the last injection. Similar results were obtained in fat cells examined 4 h after only one injection of 60 micrograms bovine GH to rats hypophysectomized 2-4 weeks previously. When binding was measured 16-24 h after GH administration, there was no apparent effect on restoration of binding even after treatment with 100 micrograms GH/day for up to 6 days, suggesting that the effects of GH in maintaining receptor number are transient. In accord with the apparently short-lived ability of GH to maintain its receptors on fat cells, GH binding was significantly reduced in adipocytes obtained form both hypophysectomized and sham-operated rats as early as 4 h after surgery, and by 8 h after surgery, declined to a level as low as that in adipocytes of chronically hypophysectomized rats. Twenty-four hours after surgery, GH binding by cells of sham-operated animals returned to normal. Fasting for 24 h also reduced GH binding by adipocytes of normal rats to a level comparable to that in adipocytes of fed hypophysectomized animals.(ABSTRACT TRUNCATED AT 400 WORDS)