THE PRESENCE OF LACTOGEN BUT NOT GROWTH HORMONE BINDING SITES IN THE ISOLATED RAT HEPATOCYTE

1977 ◽  
Vol 74 (2) ◽  
pp. 323-334 ◽  
Author(s):  
A. C. HERINGTON ◽  
N. M. VEITH
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Binding Sites ◽  
Specific Binding ◽  
Female Rats ◽  
Scatchard Analysis ◽  
Hormone Binding ◽  
Binding Studies ◽  
Membrane Function ◽  
Liver Membranes

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]α-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 °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/106 cells and a binding affinity of 1·24 × 109 ± 0·17 × 109 (s.e.m.) l/mol (n = 10). There was a significant sex difference in binding (female > 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.

Pituitary regulation of human growth hormone binding sites in rat liver membranes

Metabolism ◽  
1976 ◽  
Vol 25 (3) ◽  
pp. 341-353 ◽  
Author(s):  
A.C. Herington ◽  
L.S. Phillips ◽  
W.H. Daughaday
Keyword(s):  
Growth Hormone ◽  
Rat Liver ◽  
Human Growth Hormone ◽  
Binding Sites ◽  
Human Growth ◽  
Hormone Binding ◽  
Liver Membranes

Growth hormone binding protein in patients with renal failure

1992 ◽  
Vol 127 (6) ◽  
pp. 485-488 ◽  
Author(s):  
Hiralal G Maheshwari ◽  
Ian Rifkin ◽  
Joan Butler ◽  
Michael Norman
Keyword(s):  
Growth Hormone ◽  
Renal Failure ◽  
Renal Disease ◽  
Binding Protein ◽  
Scatchard Analysis ◽  
Gel Chromatography ◽  
Hormone Binding ◽  
Binding Studies ◽  
Growth Hormone Binding Protein ◽  
Hormone Binding Protein

To investigate changes in the growth hormone binding protein (GH-BP) in renal disease, gel chromatography was used to separate free and bound hormone after incubation with 125I-GH, the results being expressed as a percentage of radioactive GH eluting in a high molecular weight (70–80 kD) peak. In 26 normal individuals, binding was 39.3±8.0%, while in 11 patients with renal disease who were off dialysis binding was reduced to 16.8±5.6%. Similarly, in 9 patients undergoing hemodialysis binding was reduced to 24.6±6.8%, in 8 patients undergoing chronic ambulatory peritoneal dialysis binding was reduced to 25.7±7.6%, and in 9 patients within three months of a renal transplant binding was reduced to 25.1±8.6%. Scatchard analysis showed that these changes were not a result of decreased affinity of GH-BP for GH, and receptor binding studies showed that uremic serum was not inhibiting binding. The decreased concentration of GH-BP may indicate decreased expression of the GH receptor in target tissues, and hence diminished responsiveness to GH in renal failure.

Comparison of growth hormone binding and metabolic response in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin

1985 ◽  
Vol 110 (1) ◽  
pp. 50-55 ◽  
Author(s):  
Stephen LaFranchi ◽  
Cheryl E. Hanna ◽  
Toni Torresani ◽  
Eugen Schoenle ◽  
Ruth Illig
Keyword(s):  
Growth Hormone ◽  
Binding Sites ◽  
Metabolic Response ◽  
Specific Binding ◽  
Human Growth ◽  
Scatchard Analysis ◽  
Rat Adipocytes ◽  
Significant Difference ◽  
Glucose Incorporation ◽  
Number Of Binding Sites

Abstract. We undertook a comparison of human growth hormone (hGH) binding and metabolic responses in rat adipocytes of epididymal, subcutaneous, and retroperitoneal origin to determine whether the site of fat depot biopsy might affect the response to hGH stimulation. The results showed highest specific binding in epididymal (3.6%), followed by subcutaneous (2.3%) and retroperitoneal adipocytes (1.5%); half-maximal binding was achieved at 14–18 ng/ml hGH for the three sites. Scatchard analysis of the binding data from each site was linear; there was no significant difference in binding affinities (2.1 to 3.3 × 109, m−1), but the number of binding sites was statiticially higher in epididymal (9.8 × 103) as compared to subcutaneous (7.5 × 103, P < 0.05) and retroperitoneal cells (3.3 × 103, P < 0.01). Stimulation with 5 to 2500 ng pituitary hGH produced a dose-related increase in glucose incorporation, with the largest increase in epididymal fat cells (31%, P <0.05) followed by subcutaneous cells (18%, P < 0.05); no significant increase was seen with retroperitoneal cells. Biosynthetic hGH produced a similar pattern of glucose incorporation in the three sites. Addition of hGH antibodies blocked the glucose incorporation in epididymal adipocytes using both pituitary-derived and biosynthetic hGH. It seems clear that this insulin-like effect is caused by hGH, not an insulin-like impurity. We conclude that the number of binding sites, perhaps related to adipose cell size, differs in adipose tissue from different locations and this influences the metabolic response to hGH stimulation.

scholarly journals An investigation of sites that bind human somatotropin (growth hormone) in the liver of the pregnant rabbit

1981 ◽  
Vol 198 (3) ◽  
pp. 605-614 ◽  
Author(s):  
H F Cadman ◽  
M Wallis
Keyword(s):  
Growth Hormone ◽  
Binding Sites ◽  
Specific Binding ◽  
Bovine Somatotropin ◽  
Scatchard Analysis ◽  
Single Class ◽  
Pregnant Rabbit ◽  
Membrane Bound ◽  
Ovine Prolactin ◽  
Triton X

The binding of 125I-labelled human somatotropin (growth hormone) to a crude membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction by Triton X-100, was dependent on time, temperature and receptor concentration. At 4 degrees C a steady state was reached after 20 h, and maximum specific binding (as a percentage of total tracer added) was approx. 50% for both membrane-bound and solubilized receptors. Solubilization did not significantly affect the binding properties of the receptor at low concentrations of Triton X-100 (less than 0.05%, v/v, in the assay tube). However, at higher concentrations (approx. 0.1%, v/v), the detergent lowered the ability of some hormones, for example ovine prolactin, to displace 125I-labelled human somatotropin, but did not affect other hormones such as bovine somatotropin. Some somatogenic hormones, such as bovine somatotropin, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled human somatotropin from membrane-bound and solubilized receptor preparations. Furthermore, 85% of 125I-labelled bovine somatotropin was displaced from membrane-bound receptors by ovine prolactin, and 125I-labelled ovine prolactin was almost completely displaced by bovine somatotropin. Scatchard analysis of the binding data for human somatotropin suggested a single class of binding sites in the membrane-bound receptor preparation, with an affinity (Ka) of 1.9 X 10(9) M-1 and a capacity of 1726 fmol/mg of protein; these values were slightly increased by solubilization (Ka = 3.2 X 10(9) M-1, capacity = 2103 fmol/mg of protein). Scatchard analysis of binding to membrane-bound receptors also indicated a single class of high-affinity binding sites for bovine somatotropin (Ka = 4.8 X 10(9) M-1, capacity = 769 fmol/mg) and for ovine prolactin (Ka = 6.1 X 10(9) M-1, capacity = 187 fmol/mg).

scholarly journals Characterization of the binding of human growth hormone to microsomal membranes from rat liver

1976 ◽  
Vol 158 (1) ◽  
pp. 61-69 ◽  
Author(s):  
A C Herington ◽  
N Veith ◽  
H G Burger
Keyword(s):  
Growth Hormone ◽  
Binding Site ◽  
Rat Liver ◽  
Human Growth Hormone ◽  
Human Growth ◽  
Female Rats ◽  
Scatchard Analysis ◽  
Normal Female ◽  
Female Rat ◽  
Hormone Binding

The binding of 125I-labelled human growth hormone to the 100000g microsomal membrane fraction prepared from the livers of normal female rats was dependent on time, temperature, pH, membrane concentration and concentration of 125I-labelled human growth hormone. At 22 degrees C binding reached a steady state after 16h, with the mean maximal specific binding being 20% of the tracer initially added. Dissociation of 125I-labelled human growth hormone from the membranes, after addition of excess of unlabelled hormone, was relatively slow with a half-time greater than 24h. Only minor degradation of the 125I-labelled human growth hormone was observed during incubation with membranes for 16 or 25h at 22 degrees C. Similarly, no significant change in the ability of membranes to bind human growth hormone was evident after preincubation of the membranes for 16 or 25h. Specificity studies showed that up to 90% of the 125I-labelled human growth hormone bound could be displaced by 1 mug of unlabelled hormone. Ovine prolactin also showed considerable competition for the binding site. Non-primate growth-hormone preparations (ovine, bovine, porcine and rat) and non-related hormones (insulin, thyrotropin, lutropin and follitropin) all showed negligible competition. Scatchard analysis of the binding data was consistent with two classes of binding site with binding affinities of 0.64 × 10(10) +/- 0.2 × 10(10)M-1 and 0.03 × 10(10) +/- 0.007 × 10(10)M-1 and corresponding binding capacities of 98.4 +/- 10 fmol/mg of protein and 314.6 +/- 46.3 fmol/mg of protein. These studies provide data which, in general, are consistent with the criteria required for hormone-receptor interaction. However, proof of the thesis that the human-growth-hormone-binding sites in female rat liver represent physiological receptors must await the demonstration of a correlation between hormone binding and a biological response.

Growth hormone receptors in the liver and osmoregulatory organs of rainbow trout: characterization and dynamics during adaptation to seawater

1991 ◽  
Vol 130 (3) ◽  
pp. 425-433 ◽  
Author(s):  
T. Sakamoto ◽  
T. Hirano
Keyword(s):  
Growth Hormone ◽  
Rainbow Trout ◽  
Binding Sites ◽  
Chum Salmon ◽  
Hormone Receptors ◽  
Sea Water ◽  
Specific Binding ◽  
Head Kidney ◽  
Scatchard Analysis ◽  
Single Class

ABSTRACT Specific binding sites for chum salmon growth hormone (sGH) were identified in the membranes obtained from tissues of rainbow trout. Specific binding of 125I-labelled sGH (% per mg protein) was found in the liver (37%), ovary (6%), brain (6%), gill (4%), intestine (4%) and posterior body kidney (4%). Specific binding was not significant in head kidney, anterior body kidney, spleen, heart, skeletal muscle or skin. Scatchard analyses demonstrated the presence of a single class of high-affinity low-capacity receptors in the liver, gill, intestine and kidney. The association constants for the membranes from liver, gill, intestine and kidney were of the same order (1 litre/nmol). Chum salmon prolactin did not inhibit the binding of 125I-labelled sGH to receptors in the liver, gill, intestine and kidney. Transfer of rainbow trout from fresh water to 80% seawater evoked a rise in plasma concentration of GH and a significant decrease in the GH binding to the liver membranes after 1 day. Binding in the gill and kidney was not altered significantly. Membranes were treated with 4 mol MgCl2/l to remove bound GH from the receptors, and the results indicated that the reduction in binding in the liver after transfer to sea-water was probably due to receptor occupancy by increased endogenous GH. The occupancy of liver GH-binding sites was maximal 4 days after transfer. Total (MgCl2-treated) binding sites in the liver increased significantly 14 days after transfer. Scatchard analysis indicated that receptors were altered in capacity without changes in binding affinity. Although GH may also directly affect osmoregulatory organs through their GH receptors, the present results indicate the likelihood of at least partial mediation by the liver of the seawater-adapting action of GH in the rainbow trout. Journal of Endocrinology (1991) 130, 425–433

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

1993 ◽  
Vol 293 (2) ◽  
pp. 345-349 ◽  
Author(s):  
T Amit ◽  
Z Hochberg ◽  
R J Barkey
Keyword(s):  
Growth Hormone ◽  
Binding Protein ◽  
Rabbit Serum ◽  
Scatchard Analysis ◽  
Hormone Binding ◽  
Binding Studies ◽  
Type Iii ◽  
Wide Range ◽  
Human Sera ◽  
Serum Gh

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.

The somatotrophic axis in young steers: influence of nutritional status and oestradiol-17β on hepatic high-and low-affinity somatotrophic binding sites

1988 ◽  
Vol 116 (2) ◽  
pp. 169-177 ◽  
Author(s):  
B. H. Breier ◽  
P. D. Gluckman ◽  
J. J. Bass
Keyword(s):  
Body Weight ◽  
Nutritional Status ◽  
Binding Site ◽  
Binding Sites ◽  
Dry Matter ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Binding Studies ◽  
High Affinity ◽  
High Affinity Site

ABSTRACT The binding of bovine GH (bGH) to hepatic membranes obtained from steers on either high (3% dry matter of body weight per day) or low (1% dry matter of body weight per day) planes of nutrition with or without an oestradiol-17β implant was studied (n = 5 per group). Binding studies were performed on both crude membrane homogenates and on 100 000 g microsomal membrane fractions; identical results were obtained using both preparations. In all four groups of animals, linear Scatchard plots were obtained, but following pretreatment of the membranes with MgCl2 to remove endogenously bound hormone, curvilinear plots were obtained in the groups on the high plane of nutrition. Analysis of these curves suggested the presence of a high- and low-affinity binding site, the high-affinity site being fully occupied in the absence of MgCl2 pretreatment. The specific binding of bGH in MgCl2-pretreated crude membranes was greater (P < 0·01) in well-fed steers (14·8 ± 1·6%) than in poorly fed steers (9·8 ± 0·9%). Scatchard analysis showed this to be due to the presence of a high-affinity site (dissociation constant (Kd) = 11·6 ± 3·3 pmol/l) in the well-fed animals only. In addition, there was an increase (P < 0·01) in the affinity, but not in the capacity, of the low-affinity site (Kd = 106·4 ± 22·8 pmol/l in well-fed steers and 197·0 ± 23·8 pmol/l in poorly fed steers). Oestradiol treatment was associated with an increase (P < 0·01) in specific binding at both planes of nutrition, but binding was higher (P < 0·01) in well-fed (24·8 ± 2·9%) than in poorly fed (15·6 ± 3·7%) steers. Scatchard analysis after MgCl2 pretreatment again showed a curvilinear plot at the high and a linear plot at the low nutritional plane. The effect of oestradiol was to increase (P < 0·001) the capacity of the high-affinity site from 1·87 ± 0·61 pmol/100 mg in the control well-fed group to 6·56 ± 1 ·2 pmol/100 mg. The capacity of the low-affinity site was increased (P < 0·01) from 20·1 ± 2·6 to 30·1 ± 3·2 pmol/100 mg in the well-fed group, with a similar change in the poorly fed group. Oestradiol had no effect on the apparent affinity of either binding site. These studies demonstrate a heterogeneity of somatotrophic binding sites of hepatic membranes in steers. The presence of a high-affinity site is determined by nutritional status, whereas oestradiol primarily affects receptor capacity. Thus nutrition and oestradiol have independent and qualitatively different effects on somatotrophic binding. As the rate of weight gain correlated (P < 0·01) with the capacity of the high-affinity site, it is suggested that somatotrophic receptor modulation is a primary factor in the regulation of somatic growth in the ruminant. J. Endocr. (1988) 116, 169–177

BINDING OF HUMAN GROWTH HORMONE TO HEPATIC LACTOGENIC BINDING SITES: REGULATION BY OESTROGENS AND ANDROGENS

1976 ◽  
Vol 70 (3) ◽  
pp. 473-484 ◽  
Author(s):  
A. C. HERINGTON ◽  
H. G. BURGER ◽  
NOELLE M. VEITH
Keyword(s):  
Growth Hormone ◽  
Effective Dose ◽  
Rat Liver ◽  
Sex Steroids ◽  
Binding Sites ◽  
Human Growth ◽  
Female Rats ◽  
Male Rats ◽  
Minimum Effective Dose ◽  
Liver Membranes

SUMMARY The binding of 125I-labelled human growth hormone (HGH) to the 'lactogenic' binding sites of rat liver membranes has been shown to be highly dependent on the oestrogen and androgen status of the animal from which the membranes were prepared. Oestradiol treatment of either male or female rats induced a highly significant rise in HGH binding. The minimum effective dose used was 2–5 μg/day and the rise in HGH binding was apparent after 4 days of treatment. Following cessation of oestradiol treatment of male rats HGH binding declined with a half-time of approximately 9 days. In contrast to the stimulatory effect of oestrogen, treatment of female rats with testosterone propionate (minimum effective dose 100–200 μg/day) led to a marked reduction in HGH binding. The influence of both oestrogens and androgens was confirmed following the removal of endogenous sex steroids by adrenalectomy–ovariectomy of female rats and castration of male rats. Scatchard analysis showed that, with the possible exception of adrenalectomy–ovariectomy, all pharmacologically and physiologically induced changes in HGH specific binding reflected changes in binding site capacity; there were no changes in binding affinity. While earlier studies have indicated that the oestrogen effect is primarily indirect and is mediated by the pituitary gland, the mode of action of the androgens is currently unknown. The relatively slow response of HGH binding to hormonal changes would support an indirect action for both the sex steroids. The stimulatory effect of oestrogens and the inhibitory effect of androgens may provide an explanation for the marked sex difference in HGH binding to rat liver membranes.

Sign in / Sign up

Export Citation Format

Share Document