0013-7227/90/1261-0325$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 126, No. 1 Printed in U.S.A.
Alterations of Human Growth Hormone Binding by Rat Liver Membranes during Hypo- and Hyperthyroidism ZEEV HOCHBERG, TOVA BICK, AND ZEEV HAREL Rappaport Family Institute for Research in the Medical Sciences and the Department of Pharmacology, Faculty of Medicine, Technion-Israel Institute of Technology, and the Department of Pediatrics, Rambam Medical Center, Haifa, Israel
Creatine kinase was also measured in homogenized livers. Liver membranes of the hypothyroid rats showed a significant decline in somatogenic and lactogenic binding of hGH. This was true for both the free unoccupied binding sites and total binding after dissociation of the endogenous ligand. Replacement of T4 for 2 weeks restored hGH binding to control values. Hyperthyroid rats had high somatogenic and lactogenic hGH binding. Creatine kinase activity decreased significantly in liver homogenates of hypothyroid rats, was restored by T4 replacement, and increased significantly in hyperthyroid rats. Thus, lactogenic and somatogenic receptors are directly related to the thyroid status in vivo. (Endocrinology 126: 325-329,1990)
ABSTRACT. Hypothyroid rats treated with human GH (hGH) were partially refractory to the latter's effects. The present study was undertaken to investigate the role of hypo- and hyperthyroidism on the GH receptor. Seven-week-old rats were rendered either hypothyroid, by methimazole, or hyperthyroid, by a daily overdose of T4, during weeks 7-14 of life. Livers were homogenized and overlaid on sucrose discontinuous density gradient. Removal of endogenous ligand from the receptor was performed by exposing the membranes to MgCl2. hGH was used with excess ovine PRL to characterize somatogenic specific binding. Lactogenic specific binding was calculated by subtracting somatogenic specific binding from the total specific binding.
T
Materials and Methods
HE GROWTH-attenuating effect of hypothyroidism is multifactorial. Chronically hypothyroid rats show depleted stores and decreased synthesis of GH (14). In addition, the growth-promoting effect of exogenously delivered GH is blunted in hypothyroid rats (5, 6). The growth stimulation of the two hormones has been shown to be higher than the expected additive effect, indicating potentiating synergism (7). A direct effect of the thyroid hormone on somatomedin secretion has also been suggested (8). We have recently shown that hypothyroid rats that were treated with human GH (hGH) were partially refractory to the latter's effects on epiphyseal growth plate cartilage and its adjacent bone (6). The possible mechanisms may include effects of hypothyroidism on the hGH receptors, postreceptor events, insulin-like growth factor-I synthesis, or insulin-like growth factor-I receptors and postreceptor events. The present study was undertaken to investigate the roles of hypo- and hyperthyroidism in modulation of the GH receptor as the first step in the link between GH secretion and its peripheral effect.
Experimental design Seven-week-old female Sprague-Dawley rats were maintained for 7 weeks (from 7-14 weeks of age) under controlled temperature and light/dark conditions. Food and water were available ad libitum. Animals were randomly divided into the following groups (four or five animals per group): 1) control; 2) hypothyroid, by maintaining the animals for 7 weeks on 75100 mg/kg BW-day methimazole; 3) hypothyroid, receiving T4 (15 Mg/kg BW, via daily sc injection) for the last 2 weeks of the study (12-14 weeks of life); and 4) hyperthyroid, receiving T4 (10 Mg/day, sc) during weeks 7-14 of life. At 14 weeks of age the animals were killed by decapitation, and cervical blood was withdrawn for thyroid hormone measurements. The anterior pituitaries were removed for rat GH (rGH) determination, and the livers were snap-frozen in liquid N2 for later homogenization. Preparation of rat liver membranes (9-11) Livers were excised and placed in ice-cold homogenization buffer consisting of 1 mM NaHCO3 and 0.5 mM CaCl2, pH 7.5. They were then minced finely by hand and homogenized in 5 vol/g wet wt homogenizing buffer using a glass-glass homogenizer, followed by filtration through two layers of cheesecloth. The homogenate was centrifuged at 1000 X g for 10 min at 4 C and the cloudy supernatant was removed. The loose pellet was resuspended in the homogenizing buffer and recentrifuged at 1000 X g for 10 min at 4 C. The supernatant was removed, and
Received June 8,1989. Address requests for reprints to: Dr. Zeev Hochberg, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, P.O.B. 9697, 31096 Haifa, Israel.
325
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 21:48 For personal use only. No other uses without permission. . All rights reserved.
GH BINDING IN HYPO- AND HYPERTHYROIDISM
326
Endo • 1990 Vol 126 • No 1
the procedure was repeated twice more until the supernatant was fairly clear, and the pellet could be seen to consist of two distinct layers. The upper, tan layer was removed and suspended in 81% (wt/vol) sucrose (3.6 ml tan suspension to 10 ml sucrose). This suspension was overlaid with three sucrose solutions of different densities: 1.16,1.18, and 1.20. The discontinuous density gradient was centrifuged at 100,000 x g for 90 min at 4 C. The enriched plasma membrane fraction appeared as a band at the interface of densities 1.16 and 1.18 and was harvested. The suspension was diluted with the original buffer (~15 times) and centrifuged at 2,500 X g for 10 min at 4 C. This pellet was resuspended in 0.25 M sucrose and 49% (wt/vol) sucrose solution placed underneath via syringe and needle. After centrifugation for 4 h at 100,000 X g, the purified plasma membrane fraction was harvested from the interface. The membranes were diluted with 10 mM CaCl2 in 25 mM Tris-HCl, pH 7.4, and centrifuged at 100,000 X g for 60 min. The pellet was resuspended in the buffer, and the suspensions were frozen at —20 C until used. The membrane protein concentration was measured by the method of Lowry et al. (12), and the yield of membrane protein ranged from 1.5-2 mg/g liver.
Livers were homogenized in 50 mM Tris-HCl buffer, pH 6.8, with 5 mM MgSO4, 0.4 mM EDTA, 2.75 mM dithiothreitol, and 250 mM sucrose. After centrifugation at 38,000 X g for 10 min at 4 C, the supernatant was subjected to analysis of CK activity and Lowry protein determination (12). CK activity was measured by an enzyme kit (Sigma, St. Louis, MO).
Binding assay
Statistics
125
125
aspirated, and the radioactivity of the pellets was counted in a 7-counter. Nonspecific binding was defined as the binding of labeled hormone in the presence of 1 ^g unlabeled hormone. rGH RIA rGH was determined by a double antibody RIA. rGH for standard reference and iodination and monkey anti-rGH were provided by the National Hormone and Pituitary Program of the NIDDK. Precipitating antibodies against normal monkey serum were prepared in our laboratory and used in a 1:320 dilution. All samples were measured in duplicate. The sensitivity of the assay was 0.1 ng. Inter- and intraassay variabilities were less than 10% and 5%, respectively. Creatine kinase (CK) (18)
[ I]Human (h) GH for binding studies and [ I]rGH for RIA were prepared by the chloramine-T procedure of Greenwood et al. (13) using 0.5 mCi Na125I (Nuclear Research Center, Negev, Israel) and 5 ng authentic hGH (Biotechnology, General, Rehovot, Israel). The hormones were separated on a Sephadex G-50 column. Specific activities ranged from 50-70
The data were calculated from the means and variance in triplicate determinations of five rats per group (four in group 3). Unpaired Student's t test was used for statistical analysis. Statistical differences were considered significant at P < 0.05.
Removal of endogenous ligand from the receptor was performed by exposing the membranes to MgCl2 (14, 15). Purified plasma membranes containing 3.2 mg protein in 1.6 ml 25 mM Tris-HCl buffer, pH 7.4, were mixed with 6.4 ml Tris buffer alone or with buffer containing 3.8 M MgCl2 (3 M, final concentration). This amount of membrane was chosen because in earlier experiments treatment with MgCl2 was found to result in an average protein loss of 50%, leaving approximately 1.6 mg protein. After 5 min at 20 C, the mixture was diluted with 24 ml ice-cold Tris buffer containing 10 mM CaCl2 and 0.1% BSA, and the membrane was sedimented by centrifugation, washed in 24 ml buffer, and resedimented. The pellet was suspended by glass-glass homogenization in 1.6 ml Tris-CaCl2albumin buffer, and 200-/J aliquots were taken for binding studies. Binding studies were performed according to the method of Tsushima et al. (16), as previously reported by us (17). [125I] hGH was used with excess ovine PRL (oPRL; 1 /xg/tube) to characterize somatogenic specific binding. Lactogenic specific binding was calculated by subtracting somatogenic specific binding from total specific binding. Two hundred micrograms of membrane protein, suspended in 25 mM Tris buffer, were incubated with 1 ng [125I]hGH (1.14 X 10~10 M) with and without 1 ^g unlabeled hormone for 48 h at 4 C with constant agitation. The final incubation volume was 0.4 ml. Incubation was terminated by the addition of 2 ml cold Tris buffer and centrifugation at 4000 rpm for 30 min at 4 C. The supernatants were
Growth of the study animals was followed by monitoring their weight. Compared with control rats, the weight gain of hypothyroid rats was attenuated (Fig. 1). Replacement of T4 was followed by acceleration of growth from a mean of -3.5 ± 1 . 0 g/week to +9.0 ± 1.1 g/week. Hyperthyroid animals gained weight more slowly than the normal controls. Monitoring the efficacy of the drugs was carried out by taking measurements of thyroid hormones in the serum and of rectal temperature (Table 1). Serum T 4 and T 3 as well as body temperature were significantly decreased in the hypothyroid rats. In the T4 replacement animals, T4 was slightly elevated, and T 3 was reduced. In the hyperthyroid rats, serum T4 and T 3 were increased. To evaluate the effects of these thyroid states on the pituitary, rGH was determined in pituitary extracts. rGH was practically eliminated in the pituitaries of the hypothyroid animals and was partially restored over 2 weeks in the T4 replacement rats. Liver membranes of 45-day-old rats were subjected to displacement study of [125I]hGH binding by unlabeled hGH, oPRL, and bovine GH (bGH; Fig. 2). The lactogenic oPRL displaced about 50%, as did the somatogenic bGH. Scatchard analysis of unlabeled hGH displacement revealed two binding entities: a high affinity (Ka = 2 x
Results
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 21:48 For personal use only. No other uses without permission. . All rights reserved.
327
GH BINDING IN HYPO- AND HYPERTHYROIDISM
values. This was evident in both lactogenic and somatogenic binding, in MgCl2-treated and untreated membranes, and in the calculated occupancy of the lactogenic receptors. Hyperthyroid rats had higher somatogenic and lactogenic hGH binding. This was true for both total and MgCl2-dissociated binding. Occupancy was unaffected. As a measure of postreceptor events, CK activity was measured in liver extracts. CK activity decreased significantly in liver homogenates of hypothyroid rats, was restored by T4 replacement, and was increased significantly in hyperthyroid rats (Table 5).
Discussion
L L
i\ J
FIG. 1. Changes in body weight of female Sprague-Dawley rats from 7-14 weeks of age. * *, Control; +• • •+, hypothyroid; • - • - # , replacement therapy with T4 begun at 12 weeks of age; O- -O, hyperthyroid rats. Results are the mean ± SE (n = 4 or 5 in all groups).
109 M"1), low capacity (Bmax = 27.7 fmol/mg protein) lactogenic binder and a low affinity (Ka = 1.03 X 108 M"1), high capacity (Bmax = 139.5 fmol/mg protein) somatogenic binder. Scatchard analysis after lactogenic saturation revealed the somatogenic binder to have similar affinity (Ka = 1.17 X 10s M"1 and Bmax = 108.3 fmol/ mg protein). Liver membranes of the hypothyroid rats showed a significant decline in somatogenic, lactogenic, and total binding of hGH. This was true for both the free unoccupied binding sites (Table 2) and total binding after dissociation of the endogenous ligand with 3 M MgCl2 (Table 3). Subtraction of the MgCl2-untreated binding from the MgCl2-treated binding allowed us to calculate the occupancy of the hGH-binding sites (Table 4). In hypothyroid animals, somatogenic occupancy decreased significantly. Percent occupancy, however, remained unchanged. Lactogenic binding was not affected. Replacement by T4 for 2 weeks restored hGH binding to control
hGH binds to both lactogenic and somatogenic binding sites in female rat liver membranes (19-23). This is consistent with the lactogenic and growth-promoting actions of hGH in the rat (24). The binding of hGH to liver membranes of female rats was, therefore, suitable for evaluating the regulation of both receptor types. Careful attention was given to purification of plasma membrane fractions. Somatogenic binding was measured after the addition of excess cold oPRL. Treatment of the membrane fractions with MgCl2 enabled us to measure separately free, occupied, and total binding capacity. In the present study, hGH binding correlated positively with the thyroid status of the rats. Both lactogenic and somatogenic binding were significantly decreased in hypothyroid rats, restored by thyroid hormone replacement, and increased in hyperthyroid rats. This represents true changes in receptor capacity, rather than changes in occupancy, as seen from the persistence of the correlation after MgCl2 treatment, which dissociates receptor-ligand binding (14, 15). The lower number of the occupied somatogenic GH-binding sites in the T4treated hypothyroid rats may indicate that 2 weeks of replacement therapy was too short for complete restoration. The same can be said for the incomplete restoration of pituitary GH content. The decreased number of lactogenic sites in hypothyroid female rats extends previous reports. Binding of oPRL decreased in both in vivo and in vitro experiments (25-28). We now show that this effect is sustained after
TABLE 1. Thyroid hormone levels, body temperature, and pituitary GH content in the four studied groups
Total T4 (/ig/dl) Total T3 (ng/dl) Body temperature (C) Pituitary GH (jig/gland)
Group 1 (control)
Group 2 (hypothyroid)
Group 3 (hypothyroid + T4)
Group 4 (hyperthyroid)
3.1 ± 0.2 86 ± 6 37.2 ± 0.1 78 ±27
Vol. 126, No. 1 Printed in U.S.A.
Alterations of Human Growth Hormone Binding by Rat Liver Membranes during Hypo- and Hyperthyroidism ZEEV HOCHBERG, TOVA BICK, AND ZEEV HAREL Rappaport Family Institute for Research in the Medical Sciences and the Department of Pharmacology, Faculty of Medicine, Technion-Israel Institute of Technology, and the Department of Pediatrics, Rambam Medical Center, Haifa, Israel
Creatine kinase was also measured in homogenized livers. Liver membranes of the hypothyroid rats showed a significant decline in somatogenic and lactogenic binding of hGH. This was true for both the free unoccupied binding sites and total binding after dissociation of the endogenous ligand. Replacement of T4 for 2 weeks restored hGH binding to control values. Hyperthyroid rats had high somatogenic and lactogenic hGH binding. Creatine kinase activity decreased significantly in liver homogenates of hypothyroid rats, was restored by T4 replacement, and increased significantly in hyperthyroid rats. Thus, lactogenic and somatogenic receptors are directly related to the thyroid status in vivo. (Endocrinology 126: 325-329,1990)
ABSTRACT. Hypothyroid rats treated with human GH (hGH) were partially refractory to the latter's effects. The present study was undertaken to investigate the role of hypo- and hyperthyroidism on the GH receptor. Seven-week-old rats were rendered either hypothyroid, by methimazole, or hyperthyroid, by a daily overdose of T4, during weeks 7-14 of life. Livers were homogenized and overlaid on sucrose discontinuous density gradient. Removal of endogenous ligand from the receptor was performed by exposing the membranes to MgCl2. hGH was used with excess ovine PRL to characterize somatogenic specific binding. Lactogenic specific binding was calculated by subtracting somatogenic specific binding from the total specific binding.
T
Materials and Methods
HE GROWTH-attenuating effect of hypothyroidism is multifactorial. Chronically hypothyroid rats show depleted stores and decreased synthesis of GH (14). In addition, the growth-promoting effect of exogenously delivered GH is blunted in hypothyroid rats (5, 6). The growth stimulation of the two hormones has been shown to be higher than the expected additive effect, indicating potentiating synergism (7). A direct effect of the thyroid hormone on somatomedin secretion has also been suggested (8). We have recently shown that hypothyroid rats that were treated with human GH (hGH) were partially refractory to the latter's effects on epiphyseal growth plate cartilage and its adjacent bone (6). The possible mechanisms may include effects of hypothyroidism on the hGH receptors, postreceptor events, insulin-like growth factor-I synthesis, or insulin-like growth factor-I receptors and postreceptor events. The present study was undertaken to investigate the roles of hypo- and hyperthyroidism in modulation of the GH receptor as the first step in the link between GH secretion and its peripheral effect.
Experimental design Seven-week-old female Sprague-Dawley rats were maintained for 7 weeks (from 7-14 weeks of age) under controlled temperature and light/dark conditions. Food and water were available ad libitum. Animals were randomly divided into the following groups (four or five animals per group): 1) control; 2) hypothyroid, by maintaining the animals for 7 weeks on 75100 mg/kg BW-day methimazole; 3) hypothyroid, receiving T4 (15 Mg/kg BW, via daily sc injection) for the last 2 weeks of the study (12-14 weeks of life); and 4) hyperthyroid, receiving T4 (10 Mg/day, sc) during weeks 7-14 of life. At 14 weeks of age the animals were killed by decapitation, and cervical blood was withdrawn for thyroid hormone measurements. The anterior pituitaries were removed for rat GH (rGH) determination, and the livers were snap-frozen in liquid N2 for later homogenization. Preparation of rat liver membranes (9-11) Livers were excised and placed in ice-cold homogenization buffer consisting of 1 mM NaHCO3 and 0.5 mM CaCl2, pH 7.5. They were then minced finely by hand and homogenized in 5 vol/g wet wt homogenizing buffer using a glass-glass homogenizer, followed by filtration through two layers of cheesecloth. The homogenate was centrifuged at 1000 X g for 10 min at 4 C and the cloudy supernatant was removed. The loose pellet was resuspended in the homogenizing buffer and recentrifuged at 1000 X g for 10 min at 4 C. The supernatant was removed, and
Received June 8,1989. Address requests for reprints to: Dr. Zeev Hochberg, Rappaport Family Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology, P.O.B. 9697, 31096 Haifa, Israel.
325
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 21:48 For personal use only. No other uses without permission. . All rights reserved.
GH BINDING IN HYPO- AND HYPERTHYROIDISM
326
Endo • 1990 Vol 126 • No 1
the procedure was repeated twice more until the supernatant was fairly clear, and the pellet could be seen to consist of two distinct layers. The upper, tan layer was removed and suspended in 81% (wt/vol) sucrose (3.6 ml tan suspension to 10 ml sucrose). This suspension was overlaid with three sucrose solutions of different densities: 1.16,1.18, and 1.20. The discontinuous density gradient was centrifuged at 100,000 x g for 90 min at 4 C. The enriched plasma membrane fraction appeared as a band at the interface of densities 1.16 and 1.18 and was harvested. The suspension was diluted with the original buffer (~15 times) and centrifuged at 2,500 X g for 10 min at 4 C. This pellet was resuspended in 0.25 M sucrose and 49% (wt/vol) sucrose solution placed underneath via syringe and needle. After centrifugation for 4 h at 100,000 X g, the purified plasma membrane fraction was harvested from the interface. The membranes were diluted with 10 mM CaCl2 in 25 mM Tris-HCl, pH 7.4, and centrifuged at 100,000 X g for 60 min. The pellet was resuspended in the buffer, and the suspensions were frozen at —20 C until used. The membrane protein concentration was measured by the method of Lowry et al. (12), and the yield of membrane protein ranged from 1.5-2 mg/g liver.
Livers were homogenized in 50 mM Tris-HCl buffer, pH 6.8, with 5 mM MgSO4, 0.4 mM EDTA, 2.75 mM dithiothreitol, and 250 mM sucrose. After centrifugation at 38,000 X g for 10 min at 4 C, the supernatant was subjected to analysis of CK activity and Lowry protein determination (12). CK activity was measured by an enzyme kit (Sigma, St. Louis, MO).
Binding assay
Statistics
125
125
aspirated, and the radioactivity of the pellets was counted in a 7-counter. Nonspecific binding was defined as the binding of labeled hormone in the presence of 1 ^g unlabeled hormone. rGH RIA rGH was determined by a double antibody RIA. rGH for standard reference and iodination and monkey anti-rGH were provided by the National Hormone and Pituitary Program of the NIDDK. Precipitating antibodies against normal monkey serum were prepared in our laboratory and used in a 1:320 dilution. All samples were measured in duplicate. The sensitivity of the assay was 0.1 ng. Inter- and intraassay variabilities were less than 10% and 5%, respectively. Creatine kinase (CK) (18)
[ I]Human (h) GH for binding studies and [ I]rGH for RIA were prepared by the chloramine-T procedure of Greenwood et al. (13) using 0.5 mCi Na125I (Nuclear Research Center, Negev, Israel) and 5 ng authentic hGH (Biotechnology, General, Rehovot, Israel). The hormones were separated on a Sephadex G-50 column. Specific activities ranged from 50-70
The data were calculated from the means and variance in triplicate determinations of five rats per group (four in group 3). Unpaired Student's t test was used for statistical analysis. Statistical differences were considered significant at P < 0.05.
Removal of endogenous ligand from the receptor was performed by exposing the membranes to MgCl2 (14, 15). Purified plasma membranes containing 3.2 mg protein in 1.6 ml 25 mM Tris-HCl buffer, pH 7.4, were mixed with 6.4 ml Tris buffer alone or with buffer containing 3.8 M MgCl2 (3 M, final concentration). This amount of membrane was chosen because in earlier experiments treatment with MgCl2 was found to result in an average protein loss of 50%, leaving approximately 1.6 mg protein. After 5 min at 20 C, the mixture was diluted with 24 ml ice-cold Tris buffer containing 10 mM CaCl2 and 0.1% BSA, and the membrane was sedimented by centrifugation, washed in 24 ml buffer, and resedimented. The pellet was suspended by glass-glass homogenization in 1.6 ml Tris-CaCl2albumin buffer, and 200-/J aliquots were taken for binding studies. Binding studies were performed according to the method of Tsushima et al. (16), as previously reported by us (17). [125I] hGH was used with excess ovine PRL (oPRL; 1 /xg/tube) to characterize somatogenic specific binding. Lactogenic specific binding was calculated by subtracting somatogenic specific binding from total specific binding. Two hundred micrograms of membrane protein, suspended in 25 mM Tris buffer, were incubated with 1 ng [125I]hGH (1.14 X 10~10 M) with and without 1 ^g unlabeled hormone for 48 h at 4 C with constant agitation. The final incubation volume was 0.4 ml. Incubation was terminated by the addition of 2 ml cold Tris buffer and centrifugation at 4000 rpm for 30 min at 4 C. The supernatants were
Growth of the study animals was followed by monitoring their weight. Compared with control rats, the weight gain of hypothyroid rats was attenuated (Fig. 1). Replacement of T4 was followed by acceleration of growth from a mean of -3.5 ± 1 . 0 g/week to +9.0 ± 1.1 g/week. Hyperthyroid animals gained weight more slowly than the normal controls. Monitoring the efficacy of the drugs was carried out by taking measurements of thyroid hormones in the serum and of rectal temperature (Table 1). Serum T 4 and T 3 as well as body temperature were significantly decreased in the hypothyroid rats. In the T4 replacement animals, T4 was slightly elevated, and T 3 was reduced. In the hyperthyroid rats, serum T4 and T 3 were increased. To evaluate the effects of these thyroid states on the pituitary, rGH was determined in pituitary extracts. rGH was practically eliminated in the pituitaries of the hypothyroid animals and was partially restored over 2 weeks in the T4 replacement rats. Liver membranes of 45-day-old rats were subjected to displacement study of [125I]hGH binding by unlabeled hGH, oPRL, and bovine GH (bGH; Fig. 2). The lactogenic oPRL displaced about 50%, as did the somatogenic bGH. Scatchard analysis of unlabeled hGH displacement revealed two binding entities: a high affinity (Ka = 2 x
Results
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 21:48 For personal use only. No other uses without permission. . All rights reserved.
327
GH BINDING IN HYPO- AND HYPERTHYROIDISM
values. This was evident in both lactogenic and somatogenic binding, in MgCl2-treated and untreated membranes, and in the calculated occupancy of the lactogenic receptors. Hyperthyroid rats had higher somatogenic and lactogenic hGH binding. This was true for both total and MgCl2-dissociated binding. Occupancy was unaffected. As a measure of postreceptor events, CK activity was measured in liver extracts. CK activity decreased significantly in liver homogenates of hypothyroid rats, was restored by T4 replacement, and was increased significantly in hyperthyroid rats (Table 5).
Discussion
L L
i\ J
FIG. 1. Changes in body weight of female Sprague-Dawley rats from 7-14 weeks of age. * *, Control; +• • •+, hypothyroid; • - • - # , replacement therapy with T4 begun at 12 weeks of age; O- -O, hyperthyroid rats. Results are the mean ± SE (n = 4 or 5 in all groups).
109 M"1), low capacity (Bmax = 27.7 fmol/mg protein) lactogenic binder and a low affinity (Ka = 1.03 X 108 M"1), high capacity (Bmax = 139.5 fmol/mg protein) somatogenic binder. Scatchard analysis after lactogenic saturation revealed the somatogenic binder to have similar affinity (Ka = 1.17 X 10s M"1 and Bmax = 108.3 fmol/ mg protein). Liver membranes of the hypothyroid rats showed a significant decline in somatogenic, lactogenic, and total binding of hGH. This was true for both the free unoccupied binding sites (Table 2) and total binding after dissociation of the endogenous ligand with 3 M MgCl2 (Table 3). Subtraction of the MgCl2-untreated binding from the MgCl2-treated binding allowed us to calculate the occupancy of the hGH-binding sites (Table 4). In hypothyroid animals, somatogenic occupancy decreased significantly. Percent occupancy, however, remained unchanged. Lactogenic binding was not affected. Replacement by T4 for 2 weeks restored hGH binding to control
hGH binds to both lactogenic and somatogenic binding sites in female rat liver membranes (19-23). This is consistent with the lactogenic and growth-promoting actions of hGH in the rat (24). The binding of hGH to liver membranes of female rats was, therefore, suitable for evaluating the regulation of both receptor types. Careful attention was given to purification of plasma membrane fractions. Somatogenic binding was measured after the addition of excess cold oPRL. Treatment of the membrane fractions with MgCl2 enabled us to measure separately free, occupied, and total binding capacity. In the present study, hGH binding correlated positively with the thyroid status of the rats. Both lactogenic and somatogenic binding were significantly decreased in hypothyroid rats, restored by thyroid hormone replacement, and increased in hyperthyroid rats. This represents true changes in receptor capacity, rather than changes in occupancy, as seen from the persistence of the correlation after MgCl2 treatment, which dissociates receptor-ligand binding (14, 15). The lower number of the occupied somatogenic GH-binding sites in the T4treated hypothyroid rats may indicate that 2 weeks of replacement therapy was too short for complete restoration. The same can be said for the incomplete restoration of pituitary GH content. The decreased number of lactogenic sites in hypothyroid female rats extends previous reports. Binding of oPRL decreased in both in vivo and in vitro experiments (25-28). We now show that this effect is sustained after
TABLE 1. Thyroid hormone levels, body temperature, and pituitary GH content in the four studied groups
Total T4 (/ig/dl) Total T3 (ng/dl) Body temperature (C) Pituitary GH (jig/gland)
Group 1 (control)
Group 2 (hypothyroid)
Group 3 (hypothyroid + T4)
Group 4 (hyperthyroid)
3.1 ± 0.2 86 ± 6 37.2 ± 0.1 78 ±27
