Interaction of Human Growth Hormone With Isolated Rat Liver Cells MARIE-CATHERINE POSTEL-VINAY1 AND BERNARD DESBUQUOIS Unite 30 INSERM, Hdpital des Enfants Malades, Paris, France ABSTRACT. The interaction of I25I-labeled human growth hormone (hGH) with isolated rat liver cells is a specific, time dependent and saturable process. In male rats, one cell binds a maximum of 2000 hormone molecules; the dissociation constant of the cell-hGH interaction is about 3 x 10"10M. Liver cells of female rats bind 5 to 10 times more hGH than do those of male rats at equivalent hormone concentrations. Binding of 125I-labeled hGH to liver cells is readily inhibited by native hGH; 50% inhibition occurs at about 2 x 10~9M hGH irrespective of sex. In male rats, bovine growth hormone (bGH) is almost as potent as hGH in inhibiting 125I-labeled hGH binding; no displacement occurs with ovine prolactin (oPRL) except at very high (>10~ 6 M) concentrations. In female rats, bGH competes less effectively, and oPRL, more effectively, than they do in males; in addition,
G
ROWTH hormone exerts various biological effects in rat liver. It stimulates amino acid uptake, DNA and RNA synthesis, and protein synthesis. It also modifies the activity of at least one liver enzyme, ornithine decarboxylase, and promotes the production of (a) factor(s) which stimulate(s) sulfate incorporation into cartilage. Sites that bind specifically human growth hormone have recently been identified in rat liver membranes (1-3). However, such sites showed specificity not only for human growth hormone but also for hormones possessing lactogenic activity. Only in rabbit liver membranes was the existence of specific "growth hormone" binding sites demonstrated. In the present studies, isolated hepatocytes have been used to characterize the cell -hGH interaction and to assess further Received March 31, 1976. Supported by the Institut National de la Sante et de la Recherche Medicale and by a grant from the Universite Rene Descartes (contract no. 3068). 1 To whom correspondence should be addressed.
oPRL demonstrates a higher apparent affinity for the hGH binding sites (4 x 10~ 9 M) than does bGH (1 x 10" 8 M). These findings suggest that in female rats hGH, unlike bGH, interacts with additional, "lactogenic" binding sites that are distinct from the "growth hormone" binding sites. The 125Ilabeled hGH eluted from liver cells as well as that which remains in the incubation medium retains full biological activity, as judged on its ability to bind specifically to liver membranes. Treatment of liver cells by phospholipase A causes a 5-fold increase in cell binding capacity. Liver cells bind about the same amount of hGH as do crude particulars fractions from these cells; this suggests that, in the intact cell, binding occurs at relatively accessible sites, presumably localized in the plasma membrane. (Endocrinology 100: 209, 1977)
the specificity of the hGH binding sites in rat liver. Materials and Methods hGH, purified from frozen pituitary glands using a modification of the method of Trygstad and Foss (4), was obtained from URIA, Institut Pasteur, Paris. Its activity, as tested by radioimmunoassay and the rat tibia test against, respectively, NIH-GH-HS 1634 A and bGH first international standard, was 2 IU/mg. The hGH used for iodination was further purified by ionexchange chromatography; this material was homogeneous on polyacrylamide gel electrophoresis. Bovine growth hormone (bGH) was a gift from Dr. Martin Sonenberg, Sloan-Kettering Institute for Cancer Research, New York. Ovine prolactin (oPRL) (NIH-P-S-11) was obtained from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institute of Health, Bethesda, Md. Collagenase (CLS) was purchased from Worthington. Phospholipases A (from Crotalus terrificus terrificus) and C (from Clostridium perfringens) were from Boehringer/ Mannheim and from Worthington, respectively. Bovine serum albumin (fraction V) was obtained from Sigma. Na125I (carrier free) was purchased 209
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Endo • 1977 Vol 100 • No 1
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[«l].hGH,M»10» FIG. 1. Specific binding of [125I]iodo-hGH to liver cells as a function of hGH concentration. Liver cells (2.3 x 106/ml) were incubated with [I25I]iodo-hGH at the indicated concentrations. Specific binding was determined as described in Materials and Methods.
from the Radiochemical Centre, Amersham. Lactoperoxidase was from Calbiochem. Male and female rats weighing 150-200 g were obtained from Charles River, France. Liver cells were prepared by a modification of the method described by Berry and Friend (5). The liver was perfused in situ through the portal vein at a rate of 15 to 20 ml per minute. About 40 ml of a calcium-free Gey's Balanced Salt Solution (Gey's BSS) containing 2 mM EDTA were first perfused, followed by 150 ml of the same solution containing 1 mM CaCl2 and 0.05% collagenase. All media were kept at 37 C and gassed with a mixture of 95% O2 and 5% CO2. At the end of the perfusion, the liver was carefully removed and gently shaken in 100 ml Gey's BSS (calcium-free) for about 20 min. The suspension was then filtered through silk (mesh screen 40 /u,m) and cells were centrifuged at 50 x g for 1 min; the supernatant was removed and cells were washed twice. Viability of cells was checked by their ability to exclude trypan blue. Cells were counted in a hemocytometer. [125I]Iodo-hGH was prepared by a modification of the method of Greenwood, Hunter and Glover, as described by Lesniak et al. (6,7); its specific activity ranged from 50 to 120 ixCVfxg. [125I]iodooPRL was prepared using lactoperoxidase as described by Thorell and Johansson (8); it was purified by filtration on a G 75 Sephadex column
(60 x 0.9 cm) equilibrated with 50 mM sodium phosphate, pH 7.4. The assay procedure for binding determination was as follows: [125I]iodo-hGH was incubated with liver cells (3 to 5 x 105 cells) in 200 or 250 fjd of 50 mM phosphate buffer containing 0.12M NaCl and 0.1% bovine serum albumin. Incubations, in duplicate or triplicate, were carried out in polypropylene tubes at 23 C with constant shaking for 120 min. Bound and free hormone were separated by centrifugation as follows: an aliquot (160 /xl) of each incubation mixture was added to 800 fx\ of cold phosphate buffer in conical plastic tubes. These were centrifuged for 3 min at 4 C; the supernatant was aspirated and the walls of the tubes were washed once without resuspension of the pellet. Tips of tubes were cut off and counted in a Packard Scintillation Spectrometer model 548. Parallel incubations were performed in the presence of excess unlabeled hGH (0.5 x 10" 6 M). Specific binding was taken as the difference between the total uptake of radioactivity by the cells and that uptake which was not displaced by an excess of native hormone. The integrity of the [125I]iodo-hGH eluted from liver cells and of that present in the incubation medium was assessed from its ability to bind specifically to liver microsomal membranes (9) of female rats, and from precipitations by 5% TCA. Except when stated, all experiments were performed using cells of male adult rats. Results The specific binding of hGH to liver cells is a saturable process with respect to [125I]iodo-hGH (Fig. 1). At saturation, 2000 molecules of hormone are specifically bound per cell. The dissociation constant of the hGH—cell interaction, calculated from the data shown in Fig. 1, is 3.0 x 10~10M. The Scatchard plot drawn from the experiment shown in Fig. 1 is linear, suggesting one order of binding sites. As shown earlier with liver membranes (2,3), liver cells of female rats bind 5 to 10 times more hGH than do those of male rats at equivalent hormone concentrations (Fig. 2). The association of [125I]iodo-hGH to liver cells and the dissociation of the hormone-
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211
GROWTH HORMONE BINDING TO LIVER CELLS cell complex are time-dependent processes (Fig. 3). At 23 C, a steady state is obtained in 120 min; the latter is maintained for at least another 90 min. The time required for 50% dissociation of the complex is about 75 min. The binding of [125I]iodo-hGH is linearly related to cell concentration over the range of concentrations that can be achieved (Fig. 4). Native hGH readily inhibits [125I]iodohGH binding to liver cells (Fig. 5). Under the particular conditions of the assay, halfmaximal inhibition occurs at about 2 x 10~9M hGH. bGH is almost as potent as hGH in inhibiting [125I]iodo-hGH binding to male rat liver cells; the concentration of bGH required for 50% inhibition is about 3.5 x 10"9M (Fig. 5A). oPRL competes for binding to male rat liver cells only weakly and at concentrations greater than 10~6M; the presence of GH as a contaminant in the oPRL preparation (
G
ROWTH hormone exerts various biological effects in rat liver. It stimulates amino acid uptake, DNA and RNA synthesis, and protein synthesis. It also modifies the activity of at least one liver enzyme, ornithine decarboxylase, and promotes the production of (a) factor(s) which stimulate(s) sulfate incorporation into cartilage. Sites that bind specifically human growth hormone have recently been identified in rat liver membranes (1-3). However, such sites showed specificity not only for human growth hormone but also for hormones possessing lactogenic activity. Only in rabbit liver membranes was the existence of specific "growth hormone" binding sites demonstrated. In the present studies, isolated hepatocytes have been used to characterize the cell -hGH interaction and to assess further Received March 31, 1976. Supported by the Institut National de la Sante et de la Recherche Medicale and by a grant from the Universite Rene Descartes (contract no. 3068). 1 To whom correspondence should be addressed.
oPRL demonstrates a higher apparent affinity for the hGH binding sites (4 x 10~ 9 M) than does bGH (1 x 10" 8 M). These findings suggest that in female rats hGH, unlike bGH, interacts with additional, "lactogenic" binding sites that are distinct from the "growth hormone" binding sites. The 125Ilabeled hGH eluted from liver cells as well as that which remains in the incubation medium retains full biological activity, as judged on its ability to bind specifically to liver membranes. Treatment of liver cells by phospholipase A causes a 5-fold increase in cell binding capacity. Liver cells bind about the same amount of hGH as do crude particulars fractions from these cells; this suggests that, in the intact cell, binding occurs at relatively accessible sites, presumably localized in the plasma membrane. (Endocrinology 100: 209, 1977)
the specificity of the hGH binding sites in rat liver. Materials and Methods hGH, purified from frozen pituitary glands using a modification of the method of Trygstad and Foss (4), was obtained from URIA, Institut Pasteur, Paris. Its activity, as tested by radioimmunoassay and the rat tibia test against, respectively, NIH-GH-HS 1634 A and bGH first international standard, was 2 IU/mg. The hGH used for iodination was further purified by ionexchange chromatography; this material was homogeneous on polyacrylamide gel electrophoresis. Bovine growth hormone (bGH) was a gift from Dr. Martin Sonenberg, Sloan-Kettering Institute for Cancer Research, New York. Ovine prolactin (oPRL) (NIH-P-S-11) was obtained from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institute of Health, Bethesda, Md. Collagenase (CLS) was purchased from Worthington. Phospholipases A (from Crotalus terrificus terrificus) and C (from Clostridium perfringens) were from Boehringer/ Mannheim and from Worthington, respectively. Bovine serum albumin (fraction V) was obtained from Sigma. Na125I (carrier free) was purchased 209
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Endo • 1977 Vol 100 • No 1
POSTEL-VINAY AND DESBUQUOIS
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[«l].hGH,M»10» FIG. 1. Specific binding of [125I]iodo-hGH to liver cells as a function of hGH concentration. Liver cells (2.3 x 106/ml) were incubated with [I25I]iodo-hGH at the indicated concentrations. Specific binding was determined as described in Materials and Methods.
from the Radiochemical Centre, Amersham. Lactoperoxidase was from Calbiochem. Male and female rats weighing 150-200 g were obtained from Charles River, France. Liver cells were prepared by a modification of the method described by Berry and Friend (5). The liver was perfused in situ through the portal vein at a rate of 15 to 20 ml per minute. About 40 ml of a calcium-free Gey's Balanced Salt Solution (Gey's BSS) containing 2 mM EDTA were first perfused, followed by 150 ml of the same solution containing 1 mM CaCl2 and 0.05% collagenase. All media were kept at 37 C and gassed with a mixture of 95% O2 and 5% CO2. At the end of the perfusion, the liver was carefully removed and gently shaken in 100 ml Gey's BSS (calcium-free) for about 20 min. The suspension was then filtered through silk (mesh screen 40 /u,m) and cells were centrifuged at 50 x g for 1 min; the supernatant was removed and cells were washed twice. Viability of cells was checked by their ability to exclude trypan blue. Cells were counted in a hemocytometer. [125I]Iodo-hGH was prepared by a modification of the method of Greenwood, Hunter and Glover, as described by Lesniak et al. (6,7); its specific activity ranged from 50 to 120 ixCVfxg. [125I]iodooPRL was prepared using lactoperoxidase as described by Thorell and Johansson (8); it was purified by filtration on a G 75 Sephadex column
(60 x 0.9 cm) equilibrated with 50 mM sodium phosphate, pH 7.4. The assay procedure for binding determination was as follows: [125I]iodo-hGH was incubated with liver cells (3 to 5 x 105 cells) in 200 or 250 fjd of 50 mM phosphate buffer containing 0.12M NaCl and 0.1% bovine serum albumin. Incubations, in duplicate or triplicate, were carried out in polypropylene tubes at 23 C with constant shaking for 120 min. Bound and free hormone were separated by centrifugation as follows: an aliquot (160 /xl) of each incubation mixture was added to 800 fx\ of cold phosphate buffer in conical plastic tubes. These were centrifuged for 3 min at 4 C; the supernatant was aspirated and the walls of the tubes were washed once without resuspension of the pellet. Tips of tubes were cut off and counted in a Packard Scintillation Spectrometer model 548. Parallel incubations were performed in the presence of excess unlabeled hGH (0.5 x 10" 6 M). Specific binding was taken as the difference between the total uptake of radioactivity by the cells and that uptake which was not displaced by an excess of native hormone. The integrity of the [125I]iodo-hGH eluted from liver cells and of that present in the incubation medium was assessed from its ability to bind specifically to liver microsomal membranes (9) of female rats, and from precipitations by 5% TCA. Except when stated, all experiments were performed using cells of male adult rats. Results The specific binding of hGH to liver cells is a saturable process with respect to [125I]iodo-hGH (Fig. 1). At saturation, 2000 molecules of hormone are specifically bound per cell. The dissociation constant of the hGH—cell interaction, calculated from the data shown in Fig. 1, is 3.0 x 10~10M. The Scatchard plot drawn from the experiment shown in Fig. 1 is linear, suggesting one order of binding sites. As shown earlier with liver membranes (2,3), liver cells of female rats bind 5 to 10 times more hGH than do those of male rats at equivalent hormone concentrations (Fig. 2). The association of [125I]iodo-hGH to liver cells and the dissociation of the hormone-
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211
GROWTH HORMONE BINDING TO LIVER CELLS cell complex are time-dependent processes (Fig. 3). At 23 C, a steady state is obtained in 120 min; the latter is maintained for at least another 90 min. The time required for 50% dissociation of the complex is about 75 min. The binding of [125I]iodo-hGH is linearly related to cell concentration over the range of concentrations that can be achieved (Fig. 4). Native hGH readily inhibits [125I]iodohGH binding to liver cells (Fig. 5). Under the particular conditions of the assay, halfmaximal inhibition occurs at about 2 x 10~9M hGH. bGH is almost as potent as hGH in inhibiting [125I]iodo-hGH binding to male rat liver cells; the concentration of bGH required for 50% inhibition is about 3.5 x 10"9M (Fig. 5A). oPRL competes for binding to male rat liver cells only weakly and at concentrations greater than 10~6M; the presence of GH as a contaminant in the oPRL preparation (
