BINDING OF HUMAN GROWTH HORMONE TO HEPATIC LACTOGENIC BINDING SITES: REGULATION BY OESTROGENS AND ANDROGENS
A. C. HERINGTON, H. G. BURGER AND NOELLE M. VEITH Medical Research Centre, Prince Henry's Hospital, St Kilda Road, Melbourne, 3004, Victoria, Australia
(Received 22
December
1975)
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\p=n-\5\g=m\g/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\p=n-\200\g=m\g/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\p=n-\ovariectomyof female rats and castration of male rats. Scatchard analysis showed that, with the possible exception of adrenalectomy\p=n-\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. INTRODUCTION
(Herington, Phillips & Daughaday, 1976 ; Herington, Veith & Burger, 1976è; Posner, Kelly, Shiu & Friesen, 1974«) have demonstrated the presence of specific
Recent studies
binding sites for human growth hormone (HGH) in liver membranes prepared from rats. The binding of HGH to these sites appears to be due to its inherent 'lactogenic' activity and does not seem to be associated with the growth-promoting activity of HGH, although this has yet to be substantiated. The binding of HGH has been shown to depend on the physio¬ logical status of the rat from which the liver membranes were prepared (Kelly, Posner, Tsushima & Friesen, 1974; Herington et al. 1976 ). Thus, adult female rats show much greater binding than either adult male rats, prepubertal male or female rats, or hypophy¬ sectomized male or female rats. Pregnancy results in a two- to three-fold augmentation of binding as does the presence, in either sex, of a transplantable pituitary tumour which secretes massive amounts of GH and prolactin. *
Queen Elizabeth
II Fellow
Each of these situations reflects, among other things, a differing oestrogenic status and earlier experiments (Posner, Kelly & Friesen, 19746; Herington et al. 1976 ) had suggested that oestrogens probably played a role in the induction and maintenance of these binding sites. In the present study the basic phenomenon of oestrogen induction and the possible role of androgens in the regulation and turnover of these sites have been examined in greater detail. In particular, changes in HGH binding as a function of sex steroid dose and duration of treatment, and as a function of gonadectomy, have been investigated. MATERIALS AND METHODS
Materials Human growth hormone (CSL 11.1) was obtained from the Commonwealth Serum Labora¬ tories, Melbourne, Australia and was purified by gel filtration on Sephadex G-100 (medium grade: Pharmacia, Uppsala, Sweden, 75 2-8 cm column). Chloramine-T was obtained from Merck A.G., Darmstadt, Germany and Na 125I (carrier-free) was obtained from the Radio¬ chemical Centre, Amersham, Great Britain. Oestradiol benzoate in oil (Oestramine) was obtained from Knoll Laboratories, Sydney, Australia; oestradiol-17/? from Calbiochem, Sydney, Australia; and testosterone propionate in oil (Testoviron) from Schering A.G., Berlin, Germany. The reagent kit for measurement of 5'-nucleotidase activity was obtained from BDH Chemicals Ltd, Poole, England.
growth hormone binding studies the 15000-100000 studies, g membrane fraction was prepared as described binding & Friesen, 1973; previously (Tsushima Herington et al. 1976Ò) by serial centrifugation of Human
For all
homogenates of livers obtained from Sprague-Dawley rats. Because of earlier studies (Kelly et al. 1974) indicating that liver membranes prepared from immature rats exhibited neg¬ ligible specific binding of HGH, all rats used in this study were at least 10 weeks old and ranged in weight between 140 and 170 g. Iodination with 125I of HGH was performed as previously described (Herington et al. 19760) using chloramine-T. Specific activities of 80-90 µCi|µg were obtained routinely. Although the binding sites under study appear to be 'lactogenic' sites (Posner et al. 1974 ) the binding studies to be described were performed using 125I-labelled HGH rather than 125I-labelled prolactin for two main reasons. First, the initial observations which prompted the present investigation were made using HGH, and secondly, the characteristics of the binding of HGH are more fully understood (Herington et al. 1976 è). In addition, in two reports describing 125I-labelled ovine prolactin binding to rat liver membranes, the observations relating to oestrogen induction were essentially identi¬ cal to those seen when 125I-labelled HGH was used (Posner et al. 1974è; Gelato, Marshall, Boudreau, Bruni, Campbell & Meites, 1975). All binding studies were carried out in triplicate at 22 °C by conventional competitive binding techniques as described previously (Herington, Jacobs & Daughaday, 1974; Herington et al. 1976 , b). The reaction mixture (500/d) consisted of 100 µ membrane preparation (200 µ% membrane protein), 100/d 125I-labelled HGH (15000-25000 c.p.m.; 94-158 pg/tube), 100/tl unlabelled HGH as standard (0-5-1000 ng) and 0-025 M-Tris-HCl (pH 7-4) containing 10 mM-CaCl2, 0-1 % bovine serum albumin and 0-02 % sodium azide. Incubations were terminated after 16 h, the time required for full equilibrium to be reached in this system at 22 °C (Herington et al. 19766). Separation of bound and free hormone was achieved by centrifugation. Total binding was measured in the absence of unlabelled hor¬ mone and non-specific binding was taken as the 125I remaining bound in the presence of 1 µg of unlabelled hormone. Specific binding was calculated as the total binding minus non¬ specific binding.
All statistical comparisons between the test groups and their made by Student's r-test.
respective controls
were
Hormone treatment Liver membranes were prepared in some instances from rats that had been pretreated with oestradiol-17ß or oestradiol benzoate (in oil), testosterone propionate (in oil) or peanut oil (as group controls). All test and control groups were sex and weight matched at the beginning of the treatment period. All rats were given a single subcutaneous injection (0-1 ml) each day. For all experiments, except that shown in Fig. 7, on the day following the last injection for any particular group, the rats were bled by aortic puncture under ether anaesthesia and the serum assayed for oestradiol and testosterone content. The livers were then removed and membranes were prepared as described above. Doses of hormone and duration of treatment for each group of rats are given in the text for each experiment. Animals in the test groups 'castrated male rats' and 'adrenalectomized-ovariectomized female rats' were operated on in our laboratory. Successful gonadectomy was confirmed by radioimmunoassay measurement of circulating sex steroids. Serum oestradiol was measured using an antibody raised against 6-keto oestradiol coupled with thyroglobulin. This anti¬ body has a cross-reactivity of less than 15 % with other oestrogens from which oestradiol is separated by chromatography (Dufau, Dulmanis, Catt & Hudson, 1970). Serum testosterone was measured by the radioimmunoassay described by Wang, Youatt, O'Connor, Dulmanis & Hudson (1974). 5'-Nucleotidase measurement A commercial assay kit (BDH Chemicals Ltd) based on the colorimetrie method of van der Slik, Persijn, Engelsman & Riethorst (1970) was used to determine the 5'-nucleotidase (EC 3.1.3.5) activity (mu./mg protein) of a number of freshly made liver membrane preparations. Membrane protein concentrations were measured by the method of Lowry, Rosebrough, Farr & Randall (1951). RESULTS
Effect of oestradiol and testosterone treatment To confirm earlier experiments and to ascertain the gross effects of oestrogens and particu¬ larly androgens, liver membranes were prepared from the following groups of ráts: (a) male and female controls (0-1 ml peanut oil/day for 12 days); (b) oestradiol-treated male and female rats (oestradiol benzoate, 50/tg/day for 12 days); (c) testosterone-treated female rats (testosterone propionate, 3 mg/day for 12 days). Figure 1 shows the specific binding (expressed as a percentage of the total c.p.m. of 125Ilabelled HGH added) for each of these groups of rats. The marked sex difference in HGH binding previously reported (Kelly et al. 1974; Herington et al. 1976 ) is illustrated by the two columns for the control female and control male groups. In these studies the prepar¬ ations from female rats showed four times greater binding than those from male rats. Oestradiol treatment of either male or female rats resulted in a significant two- to four-fold rise in HGH binding compared with control animals the specific binding for the treated female rats reaching a level of 48 %. In direct contrast to this apparent oestrogen induction of HGH binding, administration of testosterone to female rats led to a significant 50 % fall in specific binding. Although measurement of specific binding can give an indication of comparative binding ability, a more stringent method of assessing binding parameters is that of Scatchard analy¬ sis (Scatchard, 1949). Under the conditions used for these binding studies and with this particular system, Scatchard analysis routinely reveals two classes of binding site for HGH -
in liver membranes from adult female rats (Herington et al. 1976b). Table 1 lists the binding capacity and binding affinity, for the high affinity site only, resulting from Scatchard analysis of the binding studies shown in Fig. 1. It is clear that the sex differences and hormoneinduced changes in specific binding are reflected accurately by changes in the binding capacity. There were no significant changes in binding affinity. Although these experiments were carried out using pharmacological doses of steroid, the circulating serum levels in the treated animals were not significantly different from levels observed in groups of normal male or female rats (Table 2). These observations indicate, therefore, that both oestrogens and androgens are involved in the induction and maintenance of hepatic binding sites for HGH. One other possible
x
50
f
r
o X
(5)
40
-
30
(3)
20
ID
(5)
F*3 Í(3)J
TPtreated female
Oiltreated male
//
Oiltreated female
En¬
treated female
(control)
E2B
treated male
(control)
Fig. 1. Effect of oestradiol benzoate (E2B) and testosterone propionate (TP) on specific binding of mI-labelled HGH to rat liver membranes. Liver membranes were prepared from female rats (open bars) and male rats (hatched bars) treated with peanut oil, E2B or TP as described under Methods. Each membrane preparation was assayed at least twice using two different batches of 125I-labelled HGH. The bars show the mean± s.e.m. with the numbers of different preparations in each group shown in parentheses. Specific binding was calculated as described under Methods and is expressed as a percentage of the total counts per minute la5I-labeIled HGH added. * < 001 ; ** < 0001 (Student's /-test). Table 1.
Effect of oestradiol and testosterone on binding affinity and binding capacity of HGH binding sites in rat liver membranes
Membrane
preparation
Oil-treated female (3) Oestradiol benzoate-treated female (5) Testosterone propionate-treated female (5) Oil-treated male (3) Oestradiol benzoate-treated male (4)
Binding capacity (fmol/mg protein)
Binding affinity (litre/mol)
1030+11-2
1·00±0-21
2440 + 48-2 47-6± 101*
1-14 + 0-lOx 0-99 + 018 0-75 + 018 1 08±008
29-2+2-3** 95-9±20-It
1010 1010 1010 1010 1010
Human GH binding capacity (fmol/mg protein) and binding affinity (litres/mol 1010) were calculated by Scatchard analysis of data obtained from the experiments described in Fig. 1. Each membrane preparation was assayed and analysed at least twice. The data are expressed as the mean ± s.e.m. with the number of different preparations in each group shown in parentheses. *P
A. C. HERINGTON, H. G. BURGER AND NOELLE M. VEITH Medical Research Centre, Prince Henry's Hospital, St Kilda Road, Melbourne, 3004, Victoria, Australia
(Received 22
December
1975)
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\p=n-\5\g=m\g/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\p=n-\200\g=m\g/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\p=n-\ovariectomyof female rats and castration of male rats. Scatchard analysis showed that, with the possible exception of adrenalectomy\p=n-\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. INTRODUCTION
(Herington, Phillips & Daughaday, 1976 ; Herington, Veith & Burger, 1976è; Posner, Kelly, Shiu & Friesen, 1974«) have demonstrated the presence of specific
Recent studies
binding sites for human growth hormone (HGH) in liver membranes prepared from rats. The binding of HGH to these sites appears to be due to its inherent 'lactogenic' activity and does not seem to be associated with the growth-promoting activity of HGH, although this has yet to be substantiated. The binding of HGH has been shown to depend on the physio¬ logical status of the rat from which the liver membranes were prepared (Kelly, Posner, Tsushima & Friesen, 1974; Herington et al. 1976 ). Thus, adult female rats show much greater binding than either adult male rats, prepubertal male or female rats, or hypophy¬ sectomized male or female rats. Pregnancy results in a two- to three-fold augmentation of binding as does the presence, in either sex, of a transplantable pituitary tumour which secretes massive amounts of GH and prolactin. *
Queen Elizabeth
II Fellow
Each of these situations reflects, among other things, a differing oestrogenic status and earlier experiments (Posner, Kelly & Friesen, 19746; Herington et al. 1976 ) had suggested that oestrogens probably played a role in the induction and maintenance of these binding sites. In the present study the basic phenomenon of oestrogen induction and the possible role of androgens in the regulation and turnover of these sites have been examined in greater detail. In particular, changes in HGH binding as a function of sex steroid dose and duration of treatment, and as a function of gonadectomy, have been investigated. MATERIALS AND METHODS
Materials Human growth hormone (CSL 11.1) was obtained from the Commonwealth Serum Labora¬ tories, Melbourne, Australia and was purified by gel filtration on Sephadex G-100 (medium grade: Pharmacia, Uppsala, Sweden, 75 2-8 cm column). Chloramine-T was obtained from Merck A.G., Darmstadt, Germany and Na 125I (carrier-free) was obtained from the Radio¬ chemical Centre, Amersham, Great Britain. Oestradiol benzoate in oil (Oestramine) was obtained from Knoll Laboratories, Sydney, Australia; oestradiol-17/? from Calbiochem, Sydney, Australia; and testosterone propionate in oil (Testoviron) from Schering A.G., Berlin, Germany. The reagent kit for measurement of 5'-nucleotidase activity was obtained from BDH Chemicals Ltd, Poole, England.
growth hormone binding studies the 15000-100000 studies, g membrane fraction was prepared as described binding & Friesen, 1973; previously (Tsushima Herington et al. 1976Ò) by serial centrifugation of Human
For all
homogenates of livers obtained from Sprague-Dawley rats. Because of earlier studies (Kelly et al. 1974) indicating that liver membranes prepared from immature rats exhibited neg¬ ligible specific binding of HGH, all rats used in this study were at least 10 weeks old and ranged in weight between 140 and 170 g. Iodination with 125I of HGH was performed as previously described (Herington et al. 19760) using chloramine-T. Specific activities of 80-90 µCi|µg were obtained routinely. Although the binding sites under study appear to be 'lactogenic' sites (Posner et al. 1974 ) the binding studies to be described were performed using 125I-labelled HGH rather than 125I-labelled prolactin for two main reasons. First, the initial observations which prompted the present investigation were made using HGH, and secondly, the characteristics of the binding of HGH are more fully understood (Herington et al. 1976 è). In addition, in two reports describing 125I-labelled ovine prolactin binding to rat liver membranes, the observations relating to oestrogen induction were essentially identi¬ cal to those seen when 125I-labelled HGH was used (Posner et al. 1974è; Gelato, Marshall, Boudreau, Bruni, Campbell & Meites, 1975). All binding studies were carried out in triplicate at 22 °C by conventional competitive binding techniques as described previously (Herington, Jacobs & Daughaday, 1974; Herington et al. 1976 , b). The reaction mixture (500/d) consisted of 100 µ membrane preparation (200 µ% membrane protein), 100/d 125I-labelled HGH (15000-25000 c.p.m.; 94-158 pg/tube), 100/tl unlabelled HGH as standard (0-5-1000 ng) and 0-025 M-Tris-HCl (pH 7-4) containing 10 mM-CaCl2, 0-1 % bovine serum albumin and 0-02 % sodium azide. Incubations were terminated after 16 h, the time required for full equilibrium to be reached in this system at 22 °C (Herington et al. 19766). Separation of bound and free hormone was achieved by centrifugation. Total binding was measured in the absence of unlabelled hor¬ mone and non-specific binding was taken as the 125I remaining bound in the presence of 1 µg of unlabelled hormone. Specific binding was calculated as the total binding minus non¬ specific binding.
All statistical comparisons between the test groups and their made by Student's r-test.
respective controls
were
Hormone treatment Liver membranes were prepared in some instances from rats that had been pretreated with oestradiol-17ß or oestradiol benzoate (in oil), testosterone propionate (in oil) or peanut oil (as group controls). All test and control groups were sex and weight matched at the beginning of the treatment period. All rats were given a single subcutaneous injection (0-1 ml) each day. For all experiments, except that shown in Fig. 7, on the day following the last injection for any particular group, the rats were bled by aortic puncture under ether anaesthesia and the serum assayed for oestradiol and testosterone content. The livers were then removed and membranes were prepared as described above. Doses of hormone and duration of treatment for each group of rats are given in the text for each experiment. Animals in the test groups 'castrated male rats' and 'adrenalectomized-ovariectomized female rats' were operated on in our laboratory. Successful gonadectomy was confirmed by radioimmunoassay measurement of circulating sex steroids. Serum oestradiol was measured using an antibody raised against 6-keto oestradiol coupled with thyroglobulin. This anti¬ body has a cross-reactivity of less than 15 % with other oestrogens from which oestradiol is separated by chromatography (Dufau, Dulmanis, Catt & Hudson, 1970). Serum testosterone was measured by the radioimmunoassay described by Wang, Youatt, O'Connor, Dulmanis & Hudson (1974). 5'-Nucleotidase measurement A commercial assay kit (BDH Chemicals Ltd) based on the colorimetrie method of van der Slik, Persijn, Engelsman & Riethorst (1970) was used to determine the 5'-nucleotidase (EC 3.1.3.5) activity (mu./mg protein) of a number of freshly made liver membrane preparations. Membrane protein concentrations were measured by the method of Lowry, Rosebrough, Farr & Randall (1951). RESULTS
Effect of oestradiol and testosterone treatment To confirm earlier experiments and to ascertain the gross effects of oestrogens and particu¬ larly androgens, liver membranes were prepared from the following groups of ráts: (a) male and female controls (0-1 ml peanut oil/day for 12 days); (b) oestradiol-treated male and female rats (oestradiol benzoate, 50/tg/day for 12 days); (c) testosterone-treated female rats (testosterone propionate, 3 mg/day for 12 days). Figure 1 shows the specific binding (expressed as a percentage of the total c.p.m. of 125Ilabelled HGH added) for each of these groups of rats. The marked sex difference in HGH binding previously reported (Kelly et al. 1974; Herington et al. 1976 ) is illustrated by the two columns for the control female and control male groups. In these studies the prepar¬ ations from female rats showed four times greater binding than those from male rats. Oestradiol treatment of either male or female rats resulted in a significant two- to four-fold rise in HGH binding compared with control animals the specific binding for the treated female rats reaching a level of 48 %. In direct contrast to this apparent oestrogen induction of HGH binding, administration of testosterone to female rats led to a significant 50 % fall in specific binding. Although measurement of specific binding can give an indication of comparative binding ability, a more stringent method of assessing binding parameters is that of Scatchard analy¬ sis (Scatchard, 1949). Under the conditions used for these binding studies and with this particular system, Scatchard analysis routinely reveals two classes of binding site for HGH -
in liver membranes from adult female rats (Herington et al. 1976b). Table 1 lists the binding capacity and binding affinity, for the high affinity site only, resulting from Scatchard analysis of the binding studies shown in Fig. 1. It is clear that the sex differences and hormoneinduced changes in specific binding are reflected accurately by changes in the binding capacity. There were no significant changes in binding affinity. Although these experiments were carried out using pharmacological doses of steroid, the circulating serum levels in the treated animals were not significantly different from levels observed in groups of normal male or female rats (Table 2). These observations indicate, therefore, that both oestrogens and androgens are involved in the induction and maintenance of hepatic binding sites for HGH. One other possible
x
50
f
r
o X
(5)
40
-
30
(3)
20
ID
(5)
F*3 Í(3)J
TPtreated female
Oiltreated male
//
Oiltreated female
En¬
treated female
(control)
E2B
treated male
(control)
Fig. 1. Effect of oestradiol benzoate (E2B) and testosterone propionate (TP) on specific binding of mI-labelled HGH to rat liver membranes. Liver membranes were prepared from female rats (open bars) and male rats (hatched bars) treated with peanut oil, E2B or TP as described under Methods. Each membrane preparation was assayed at least twice using two different batches of 125I-labelled HGH. The bars show the mean± s.e.m. with the numbers of different preparations in each group shown in parentheses. Specific binding was calculated as described under Methods and is expressed as a percentage of the total counts per minute la5I-labeIled HGH added. * < 001 ; ** < 0001 (Student's /-test). Table 1.
Effect of oestradiol and testosterone on binding affinity and binding capacity of HGH binding sites in rat liver membranes
Membrane
preparation
Oil-treated female (3) Oestradiol benzoate-treated female (5) Testosterone propionate-treated female (5) Oil-treated male (3) Oestradiol benzoate-treated male (4)
Binding capacity (fmol/mg protein)
Binding affinity (litre/mol)
1030+11-2
1·00±0-21
2440 + 48-2 47-6± 101*
1-14 + 0-lOx 0-99 + 018 0-75 + 018 1 08±008
29-2+2-3** 95-9±20-It
1010 1010 1010 1010 1010
Human GH binding capacity (fmol/mg protein) and binding affinity (litres/mol 1010) were calculated by Scatchard analysis of data obtained from the experiments described in Fig. 1. Each membrane preparation was assayed and analysed at least twice. The data are expressed as the mean ± s.e.m. with the number of different preparations in each group shown in parentheses. *P
