Evidence for thyroid hormone-dependent and independent glucocorticoid actions in cultured cells. Studies on the induction of growth hormone and glutamine synthetase in GH1 cells and tyrosine aminotransferase in Reuber H-35 cells.

The induction of growth hormone synthesis and mRNA by thyroid hormone in cultured GH1 cells is mediated by the thyroid hormone nuclear receptor. In addition, the regulation of the growth hormone response by glucocorticoid is highly dependent on the action of thyroid hormone. To clarify whether thyroid hormone has a general influence on glucocorticoid action in GH1 cells, the glucocorticoid induction of growth hormone and glutamine synthetase was simultaneously examined. In contrast to the growth hormone response, the induction of glutamine synthetase by glucocorticoid was not influenced by thyroid hormone. Both responses appear to be modulated by the glucocorticoid receptor, and thyroid hormone had no influence on nuclear-associated glucocorticoid receptor levels. These results suggest that the thyroid hormone control of glucocorticoid induction of growth hormone may be a selective process, and the nuclear associated receptors for both thyroid and glucocorticoid hormones interrelate to control the growth hormone response.     

Inhibition of growth hormone synthesis by somatostatin in cultured pituitary of rainbow trout

Summary When the pituitary of rainbow trout (Oncorhynchus mykiss) was incubated in a serum-free medium, a high level of growth hormone release as well as an activation of growth hormone synthesis were observed, suggesting the existence of hypothalamic inhibitory factor(s) on growth hormone synthesis. Although an inhibitory effect of somatostatin on growth hormone release is well established in both mammals and teleosts, an effect on growth hormone synthesis has not been demonstrated. In this study, we examined the effect of somatostatin on growth hormone synthesis in organ-cultured trout pituitary using immunoprecipitation and Northern blot analysis. Somatostatin inhibited growth hormone release from the cultured pituitary within 10 min after addition without affecting prolactin release. Incubation of the pituitary with somatostatin also caused a significant reduction in newly-synthesized growth hormone in a dose-related manner, as assessed by incorporation of [³H]leucine into immunoprecipitable growth hormone. There were no changes in the level or molecular length of growth hormone mRNA after somatostatin treatment, as assessed by Northern slot blot and Northern gel blot analyses. Human growth hormone-releasing factor stimulated growth hormone release, although the spontaneous synthesis of growth hormone was not augmented. However, somatostatin-inhibited growth hormone synthesis was restored by growth hormone-releasing factor to the control level. The spontaneous increase in growth hormone synthesis observed in the organ-cultured trout pituitary may be caused, at least in part, by the removal of the inhibitory effect of hypothalamic somatostatin.Abbreviations GH growth hormone - GHRF GH-releasing factor - PRL prolactin - SDS sodium dodecyl sulphate - SRIF somatostatin (somatropin release-inhibiting factor)     

Immunological identity of human and porcine growth hormones. Application to determination of growth hormone in porcine serum.

In two radioimmunoassay systems with iodinated human growth hormone as tracer and anti-human growth hormone or anti-porcine growth hormone as binding site, the standard curves for human and for porcine growth hormone were parallel. In both systems the porcine growth hormone preparation had to be added in about the same excess as compared to the human hormone. It was concluded that the human and the porcine hormones behave in an identical way and that the values obtained in radioimmunoassay for human growth hormone represent the amount of immunologically active molecules in the porcine growth hormone preparation. As parallel standard curves were obtained with porcine serum, the concentration of growth hormone is porcine serum may be determined by radioimmunoassay for human growth hormone.     

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

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

Application of recombinant DNA technologies to studies on chicken growth hormone

Messenger RNA isolated from chicken pituitaries was used to construct a chicken pituitary cDNA library. A chicken growth hormone cDNA clone was isolated using 32P-labeled mammalian growth hormone cDNA probes. The amino acid sequence (derived from the DNA sequence) of the mature form of chicken growth hormone shows 77% homology with that of bovine growth hormone. The chicken growth hormone cDNA clone was used to generate a vector capable of producing chicken growth hormone in Escherichia coli. The recombinant E. coli-derived chicken growth hormone was similar to pituitary chicken growth hormone in several biochemical and immunological properties. The recombinant-derived hormone has been used to establish a sensitive radioimmunoassay for growth hormone determinations made from chicken sera. The chicken growth hormone gene has also been introduced into a retroviral vector capable of establishing productive infections of chicken cells both in in vitro and in vivo. The resulting infections are accompanied by the production of radioimmunoassay-detectable growth hormone. The concentrations of growth hormone in sera of Leghorn chickens infected with the recombinant retrovirus are three- to tenfold higher than in control animals.     

Activation of adenylate cyclase in renal medulla by bovine growth hormone. An artifact attributable to vasopressin.

The effect of bovine growth hormone on adenylate cyclase activity was studied in bovine and rat renal medulla. Highly purified growth hormone (lot B1003A) increased adenylate cyclase activity in plasma membranes from bovine renal medulla from 132+/-6 pmol cyclic AMP formed/mg protein per 10 min to 364+/-10 pmol cyclic AMP formed/mg protein per 10 min. Similar results were seen with homogenates of rat renal medulla. The minimum effective concentration of bovine growth hormone required to activate adenylate cyclase was 0.5 mug/ml and maximum activation was detected at 500 mug/ml. The amount of vasopressin determined by radioimmunoassay to contaminate the growth hormone caused an increase in adenylate cyclase activity comparable to that of the corresponding concentration of growth hormone that was tested. Dialysis of growth hormone and vasopressin resulted in parallel reductions in the effect of each hormone on adenylate cyclase activity. Similarly, both growth hormone and vasopressin produced increases in short circuit current in isolated toad bladders but these effects were not detectable after dialysis of the hormones. In contrast, the effect of growth hormone on the uptake of 35SO2-4 by cartilage from hypophysectomized rats was not decreased after dialysis. These results indicate that available preparations of growth hormone are contaminated by small but physiologically significant amounts of vasopressin and that the activation of adenylate cyclase activity in renal medulla in response to growth hormone can be explained by this contamination rather than by an effect of growth hormone per se.     

Differential regulation of pituitary hormone secretion and gene expression by thyrotropin-releasing hormone. A role for mitogen-activated protein kinase signaling cascade in rat pituitary GH3 cells

We examined the possible involvement of mitogen-activated protein (MAP) kinase activation in the secretory process and gene expression of prolactin and growth hormone. Thyrotropin-releasing hormone (TRH) rapidly stimulated the secretion of both prolactin and growth hormone from GH3 cells. Secretion induced by TRH was not inhibited by 50 microM PD098059, but was completely inhibited by 1 microM wortmannin and 10 microM KN93, suggesting that MAP kinase does not mediate the secretory process. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin, whereas expression of growth hormone mRNA was largely attenuated. The increase in prolactin mRNA stimulated by TRH was inhibited by addition of PD098059, and the decrease in growth hormone mRNA was also inhibited by PD098059. Transfection of the cells with a pFC-MEKK vector (a constitutively active MAP kinase kinase kinase), significantly increased the synthesis of prolactin and decreased the synthesis of growth hormone. These data taken together indicate that MAP kinase mediates TRH-induced regulation of prolactin and growth hormone gene expression. Reporter gene assays showed that prolactin promoter activity was increased by TRH and was completely inhibited by addition of PD098059, but that the promoter activity of growth hormone was unchanged by TRH. These results suggest that TRH stimulates both prolactin and growth hormone secretion, but that the gene expressions of prolactin and growth hormone are differentially regulated by TRH and are mediated by different mechanisms.     

Cycloheximide blocks insulin-like growth factor I but not somatostatin inhibition of growth hormone secretion

Growth hormone secretion is controlled by the two hypothalamic hormones, growth hormone releasing factor (GRF) and somatostatin. In addition, the insulin-like growth factors (IGF or somatomedins) which are themselves growth hormone dependent, inhibit growth hormone release in vitro, therefore acting to close the negative feedback loop. The studies reported here examine some of the differences between inhibition of growth hormone secretion by somatostatin and IGF-I in vitro. The major finding is that cycloheximide, a protein synthesis inhibitor, blocks inhibition of GRF-stimulated growth hormone release caused by IGF-I, without changing the inhibition caused by somatostatin. The experiments were done by exposing mixed rat adenohypophysial cells to secretagogues with or without cycloheximide for 24 h in a short term culture. Somatostatin (0.6 nM) totally blocked rat GRF (1 nM) stimulated growth hormone release to values 48% of control (nonstimulated values), while IGF-I (27 nM) only reduced the GRF-stimulated growth hormone release by 27 +/- 3% (N = 5). Cycloheximide (15 micrograms/mL) totally blocked the effect of IGF-I but not somatostatin. A low concentration (0.12 nM) of somatostatin, which only partly inhibited growth hormone release, was also unaffected by cycloheximide. In purified rat somatotrophs, somatostatin (0.1 nM) inhibited GRF-stimulated cAMP levels slightly and reduced growth hormone release while IGF-I (40 nM) had no effect. We suggest that IGF-I inhibits only the secretion of newly synthesized growth hormone, while somatostatin inhibits both stored and newly synthesized growth hormone pools.     

Monoclonal antibodies to the pituitary growth-hormone receptor by the anti-idiotypic approach. Production and initial characterization.

We obtained 10/192 and 3/384 antibody-secreting hybrids after immunization of Balb/c mice with either human growth hormone or affinity-purified rabbit anti-(human growth hormone) respectively. Radiolabelled rabbit anti-(human growth hormone) antibodies, but not human growth hormone, were specifically bound by supernatants from the 13 hybrids. The binding was completely inhibited by human-growth-hormone serum binding protein. However, anti-(human growth hormone antibodies) were detected in the sera of all the mice immunized with human growth hormone. In an independent fusion, which was carried out after immunization with fewer doses of human growth hormone, anti-(human growth hormone) antibodies were also obtained. Five hybrids, where the starting antigen was human growth hormone, were selected for ascites production, and the corresponding monoclonal antibodies were partially purified and characterized with respect to their immunoglobulin isotype and their interaction with human-growth-hormone receptors. These antibodies were found to enhance the binding of radioiodinated human growth hormone to human-growth-hormone serum binding protein from human and rabbit plasma by 40%. Scatchard analysis of the effect of one of the monoclonal antibodies showed that this enhancement was due to an increased number of binding sites. All of the partially purified antibodies but one (F12) inhibited the binding of human growth hormone to rat but not rabbit, liver microsomes to various extents, as well as to H-4-II-E rat hepatoma cells. Monoclonal antibody F12 enhanced the binding of radiolabelled human growth hormone to rat liver microsomes and H-4-II-E hepatoma cells. This enhancement was found to be due to an increase in the number of binding sites.     

Immunological relatedness of human and porcine growth hormones.

The immunological relatedness of human and porcine growth hormones is examined by means of labelled human growth hormone and guinea pig antiserum. 1) Labelled human growth hormone is found in the precipitate after reaction with antiserum against porcine growth hormone. Parallel dilution curves are obtained with antisera against human and porcine growth hormones. 2) After addition of antiserum against porcine growth hormone, all the radioactivity is eluted from Sephadex G-100 with the void volume. 3) The addition of an excess of porcine hormone displaces labelled human growth hormone from antibodies against human growth hormone to the same extent as an excess of non-labelled human growth hormone does. 4) The standard radioimmunoprecipitation curves for porcine and human growth hormones obtained in the assay system for the human hormone are parallel in slope, provided that the human hormone and our preparation of the porcine hormone are introduced at a proportion of 1 to 560. 5) In a double diffusion test in agarose gel layers, with human and porcine growth hormones diffusing against guinea pig anti-porcine serum, cross reaction is observed. The conclusion is drawn that with guinea pig antisera, human and porcine growth hormones behave immunologically in a similar fashion. Labelled human growth hormone seems to have only such immunodeterminants as are also found in porcine growth hormone.     

Response of serine antiproteases to growth hormone therapy in growth hormone deficient children

Growth hormone regulates the hepatic mRNA levels of alpha 1-antitrypsin and two contrapsin-like mRNAs in the rat. To determine whether growth hormone regulates similar serine protease inhibitors in humans, we measured serum alpha 1-antitrypsin, alpha 1-antichymotrypsin, and antithrombin III by radioimmunodiffusion in 16 growth hormone deficient children before and after growth therapy. Of the 19 determinations made, 17/19 showed an increase in alpha 1-antitrypsin after administration of growth hormone, 198.6 +/- 39.1 mg/dl before growth hormone and 239.4 +/- 44 mg/dl after growth hormone (p = 0.005). Specificity of the response for alpha 1-antitrypsin was indicated by the fact that neither alpha 1-antichymotrypsin or antithrombin III values changed after growth hormone (p = 0.6 and 0.5, respectively). These data are compatible with the hypothesis that growth hormone regulates serine protease inhibitors in humans and suggests that investigation of other members of the serpin gene family might prove fruitful in defining additional growth hormone target genes.     

Regulation of receptor concentration by homologous hormone. Effect of human growth hormone on its receptor in IM-9 lymphocytes.

When cultured human lymphocytes of the IM-9 line were exposed to human growth hormone (hGH) at 37 degrees, washed for 2 hours, and incubated with 125I-hGH, the binding of 125I-hGH was reduced. The magnitude of the reduction in binding was dependent on the concentration of growth hormone present as well as the duration of the exposure. As little as 2 X 10(-11) M (0.5 ng/ml) growth hormone had a discernible effect. Growth hormone at 2 X 10(-10) M (5.0 ng/ml), which is a low resting concentration of hormone in vivo and occupies about 20% of the receptors at steady state at 30 degrees, produced a 50% reduction in binding while 20 mg/ml, which occupies about 50% of the receptors under steady state conditions, produced an 80% loss of receptors. Further increases in growth hormone concentration produced little further effect on receptor loss. Thus, the loss of receptors at a given concentration of growth hormone (up to 20 ng/ml) in the preincubation at 37 degrees was greater than the occupancy produced by that concentration of growth hormone receptors under steady state conditions at 30 degrees. Analysis of the data indicated that the decrease in binding of 125I-hGH was due to a loss of receptors per cell without any change in affinity of receptor for hormone or in cell number. The concentration of insulin receptors on these cells was affected by the insulin concentration in the medium, and the concentration of growth hormone receptors was affected by growth hormone, but neither hormone had any effect on the heterologous receptors. Exposure of the cells to cycloheximide (0.1 mM) produced a progressive but smaller loss of growth hormone receptors, and the effect of cycloheximide was additive to the receptor loss induced by growth hormone, suggesting that cycloheximide inhibited synthesis of receptors while growth hormone accelerated loss of receptors. When growth hormone was removed from the medium, receptor concentrations were restored rapidly; half of the loss was restored by 6 to 8 hours and the full complement of receptors was restored by 24 hours following removal of the hormone. If the growth hormone was removed and replaced with cycloheximide, the return of the receptors was delayed until the cycloheximide was removed. Thus restoration of the receptors appeared to require the synthesis of new proteins. These data indicate that in the IM-9 lymphocytes the concentration of growth hormone receptors is very sensitive to regulation by growth hormone and also add further support to the suggestion that hormones in general actively regulate the concentration of their own receptors.     

Effects of human growth hormone on immune functions: in vitro studies on cells of normal and growth hormone-deficient children

We have studied the in vitro effects of human growth hormone on cell surface markers and mitogenic responses of peripheral blood lymphocytes (PBL) of normal and growth hormone-deficient children before, during and after treatment with growth hormone. Growth hormone resulted in a decrease in B cell expression but it did not affect expression of T cell subsets. Growth hormone depressed the proliferation of PBL of normal and untreated growth hormone-deficient children. The proliferative responses to phytohemagglutinin (PHA) versus PHA with growth hormone were not statistically different, though the responses of most normal and on treatment children were diminished by the addition of growth hormone. PBL derived from growth hormone-deficient children during treatment with human growth hormone exhibited significantly greater spontaneous proliferation then the PBL of normal children. Growth hormone further significantly enhanced their proliferation. PHA and PHA with growth hormone resulted in significantly greater proliferation of these patients' PBL when compared to those of normal children. We demonstrated that human growth hormone had substantial in vitro effects on immune functions. These effects, some of which depend on the treatment status of the children, may need to be considered in the clinical use of human growth hormone.     

Mediation of growth hormone-enhanced expression of the cartilage phenotype in vitro by the availability of the essential amino acid valine

Without increasing cell number, ovine growth hormone was shown to stimulate the incorporation of ²⁵SO₄ by cultured chick embryo chondrocytes into chondroitin sulfate. Since the stimulation of sulfation by growth hormone was abolished when the amino acid concentrations in the medium were doubled, the relationship between amino acids and growth hormone in promoting the synthesis of acid mucopolysaccharides was investigated. Comparison of the incorporation of various labeled amino acids into trichloroacetic acid-soluble and insoluble material revealed that growth hormone promoted the incorporation of only valine into trichloroacetic acid-insoluble material. Furthermore, growth hormone stimulated valine incorporation into both extracellular and intracellular protein, rather than preferentially into extracellular chondromucoprotein. Growth hormone gave a 4-fold stimulation of valine incorporation into collagen without stimulating collagen synthesis. That growth hormone enhances sulfation by stimulating valine availability was further supported by the observations: (a) doubling only the valine concentration in the medium enhanced sulfation; (b) in medium with twice the normal valine concentration, sulfation failed to be further stimulated with the addition of growth hormone; and (c) in medium with all the other amino acids except valine at twice normal concentrations, growth hormone enhanced sulfation. In addition the temporal relationships and synthetic events occurring between growth hormonealtered valine availability and enhanced chondromucoprotein synthesis were studied. It was found that growth hormone-promoted valine incorporation into acid-insoluble material is a rapid effect that can be detected by 10 min after hormone addition and does not require RNA synthesis. Increased valine availability is rapidly reversed after growth hormone removal ( ). On the other hand, growth hormone- and valine-enhanced chondromucoprotein synthesis are slower responses, taking over 24 hr of treatment for a maximal stimulation, and are mediated by RNA synthesis, as indicated by actinomycin D sensitivity. Enhanced chondromucoprotein synthesis is also relatively stable after removal of growth hormone or valine ( ).The evidence suggests that the availability of a single amino acid, valine, plays a regulatory role in the synthesis of a specialized cellular product and that growth hormone acts at some level to alter the availability of this essential amino acid.     

Altered release of growth hormone from dispersed adenohypophysial cells of streptozotocin diabetic rats. I. Effects of growth hormone releasing factor and somatostatin

As growth hormone has been implicated in the "dawn phenomenon," an early morning rise in serum glucose, we have studied the control of growth hormone release in diabetes using an acutely dispersed system of adenohypophysial cells from normal or diabetic rats (65 mg/kg streptozotocin, 8 days before sacrifice; serum glucose, 490 +/- 17 mg/dL). Growth hormone release is normally controlled by the two hypothalamic hormones, growth hormone releasing factor and somatostatin. We have found cells of the diabetic rats exhibit changes in sensitivity that result in increased growth hormone release in static incubation. In normal cells, rat growth hormone releasing factor increases growth hormone release three- to four-fold with an EC50 of 151 +/- 27 pM (n = 7). In contrast, in cells from diabetic rats, there was a significant (twofold) increase in sensitivity to growth hormone releasing factor (EC50 = 75 +/- 15 pM, n = 7) which resulted in increased growth hormone release with lower but not maximal (10 nM) growth hormone releasing factor. Basal nonstimulated release was unchanged. Somatostatin inhibition of stimulated growth hormone release was reduced (n = 7); half-maximal inhibition occurred with 0.21 +/- 0.03 nM (normal) and 0.76 +/- 0.17 nM somatostatin (diabetic). In perifusion the peak secretion rate was significantly lower for diabetic cells stimulated by a maximal dose of growth hormone releasing factor. These studies suggest somatotrophs of diabetic rats have altered sensitivity in vitro to the controlling hormones growth hormone releasing factor and somatostatin.     

Isolation and properties of the electrophoretic components of human growth hormone by Sephadex-gel filtration and preparative polyacrylamide-gel electrophoresis

1. Human growth hormone was prepared from acetone-dried residues after extraction of gonadotrophins from pituitary glands. 2. Crude growth hormone was purified by gel filtration on Sephadex, resulting in a product that is soluble in water or 0.5% sodium chloride. It is painless on injection and shows a twofold increase in biological potency. Aggregation of growth hormone on Sephadex columns can be avoided by the addition of urea (6m) and EDTA (1mm) to the buffer. 3. Growth hormone appeared as a single component from Sephadex and ion-exchange columns and sedimented as a single boundary in the ultracentrifuge. In the circular disk electrophoresis, however, the growth hormone showed one faster and two slower-moving anionic components. 4. These components were isolated by preparative electrophoresis on polyacrylamide columns. The purified growth hormone and its three components sedimented as single boundaries with coefficients 2.62, 2.66, 2.66 and 2.83s respectively. 5. Amino acid analyses of the purified growth hormone and its components were closely related. End-group analysis of purified growth hormone and its components showed only phenylalanine at both N- and C-terminals. 6. The purified growth hormone and its components were essentially free of other pituitary hormones, but contained significant prolactin activity. The biochemical similarities among the electrophoretic components of human growth hormone and the presence of the same three components in the growth hormone prepared from a single human pituitary gland suggest polymorphism of a biologically active protein molecule.     

Disulfide bonds in native and recombinant fish growth hormones.

Disulfide linkages were characterized for the first time in a fish growth hormone. Trypsin digestion of chum salmon growth hormone, followed by mass spectrometry established that Cys-49 is linked to Cys-161, while Cys-178 is linked to Cys-186. This is analogous to the big loop, little loop pattern found in human growth hormone. Ninety-three percent of the primary structure of a recombinant rainbow trout growth hormone whose cDNA codes for the same amino acid sequence as chum salmon growth hormone was confirmed by mass spectrometric peptide mapping.     

Activation of adenylate cyclase in renal medulla by bovine growth hormone: An artifact attributable to vasopressin

The effect of bovine growth hormone on adenylate cyclase activity was studied in bovine and rat renal medulla. Highly purified growth hormone (lot B1003A) increased adenylate cyclase activity in plasma membranes from bovine renal medulla from 132 ± 6 pmol cyclic AMP formed/mg protein per 10 min to 364 ± 10 pmol cyclic AMP formed/mg protein per 10 min. Similar results were seen with homogenates of rat renal medulla. The minimum effective concentration of bovine growth hormone required to activate adenylate cyclase was 0.5 μg/ml and maximum activation was detected at 500 μg/ml. The amount of vasopressin determined by radioimmunoassay to contaminate the growth hormone caused an increase in adenylate cyclase activity comparable to that of the corresponding concentration of growth hormone that was tested. Dialysis of growth hormone and vasopressin resulted in parallel reductions in the effect of each hormone on adenylate cyclase activity. Similarly, both growth hormone and vasopressin produced increases in short circuit current in isolated toad bladders but these effects were not detectable after dialysis of the hormones. In contrast, the effect of growth hormone on the uptake of ³⁵SO₄²⁻ by cartilage from hypophysectomized rats was not decreased after dialysis. These results indicate that available preparations of growth hormone are contaminated by small but physiologically significant amounts of vasopressin and that the activation of adenylate cyclase activity in renal medulla in response to growth hormone can be explained by this contamination rather than by an effect of growth hormone per se.     

Glucocorticoid induction of growth hormone synthesis in a strain of rat pituitary cells

Conditions were determined for measuring growth hormone synthesis by a clonal strain of rat pituitary cells grown in suspension culture. Incubation of the cells with [3H]leucine in either continuous labeling or pulse-chase experiments showed that secretion of newly synthesized growth hormone commences only after a lag of about 15 min. The pulse-chase experiments also demonstrated that there is no detectable degradation by the cells of growth hormone. Thus growth hormone synthesis could be measured, in the absence of complications arising either from secretion or degradation of growth hormone, by incubating the cells with [3H]leucine for 10 min. Exposure of cells grown under the usual culture conditions to dexamethasone (a synthetic glucocorticoid) led to an average stimulation of specific growth hormone synthesis (growth hormone synthesis/total cytoplasmic protein synthesis) of only 2.6-fold. However, two other growth conditions were found in which dexamethasone routinely yielded a 5- to 15-fold stimulation of specific growth hormone synthesis. One of these conditions, involving substitution of 10% fetal calf serum for the normal serum supplement, was employed in subsequent experiments. A stimulation of specific growth hormone synthesis could be observed at 10(-9) M dexamethasone, and the maximum stimulation was observed at dexamethasone concentrations of about 10(-8) to 10(-7) M. There was a lag of about 6 h before a stimulation by dexamethasone of specific growth hormone synthesis was detected. Thereafter, the stimulation increased in a nearly linear fashion until maximum stimulation was reached at about 48 h.