THE PRESENCE OF LACTOGEN BUT NOT GROWTH HORMONE BINDING SITES IN THE ISOLATED RAT HEPATOCYTE A. C. HERINGTON AND N. M. VEITH Medical Research Centre, Prince Henry's Hospital, St Kilda Road, Melbourne 3004, Victoria, Australia
(Received 7
December
1976)
SUMMARY
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]\g=a\\x=req-\ 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 \s=deg\C,binding reached a steady-state after 2\m=.\5h and had a half-life of dissociation of 2\p=n-\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\m=.\74\m=+-\3\m=.\73fmol/106 cells and a binding affinity of 1 \m=.\24\m=x\109 \m=+-\ 0\m=.\17\m=x\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.
The
=
INTRODUCTION
A number of studies (Posner, Kelly, Shiu «fe Friesen, 19746; Herington, Burger & Veith, 1976 ; Herington, Phillips & Daughaday, 1976 ; Herington, Veith «fe Burger, 1976c) have clearly demonstrated specific binding sites for lactogenic hormones in membrane preparations from rat liver. These sites bind prolactins and human growth hormone (hGH) but do not bind bovine growth hormone (bGH). They are characterized by unusually slow kinetics, by a marked dependence on the sex steroids and an intact pituitary gland, and by their regulation by prolactin itself. Since the liver is regarded as a target organ for GH, distinct binding sites for GH should be demonstrable in addition to those for prolactin and hGH. This is true for membrane preparations from rabbit liver (Tsushima «fe Friesen, 1973 ; Herington, Jacobs «fe Daughaday, 1974), but distinct GH sites have been reported by only one group in prepara¬ tions of rat liver membranes (Etzrodt, Musch, Schleyer & Pfeiffer, 1976). Recently, however, it has been reported that specific GH sites are present in preparations of isolated rat hepatocytes (Ranke, Stanley, Rodbard, Baker, Bongiovanni & Parks, 1976). Two classes of site were apparent in these studies. One class, present in male and female rats, has a high affinity for hGH, and competition for these sites is shown by bGH but not
a GH site). The second class, present only in female rats, has a lower affinity for hGH, and competition for these sites is shown by prolactin but not bGH (i.e. a lactogen site). The lactogen sites, but not the GH sites, were influenced by oestrogens in a similar manner to those present in membrane preparations and the time required for a binding equilibrium to be reached was much shorter. Although specific-binding sites for hGH and/or prolactin have been observed using both liver membranes and hepatocytes, there is no direct evidence that they are true hormone receptors, i.e. that they are involved in the expression of biological effects of GH or prolactin. The isolated hepatocyte would appear to be an ideal system to study this possible relationship since the full metabolic integrity of the cell (e.g. gluconeogenesis, protein synthesis, RNA metabolism) and responsiveness to at least some hormones (e.g. insulin, glucagon) are retained (Ingebretsen, Moxley, Allen «fe Wagle, 1972; Schreiber & Schreiber, 1973; Krebs, Cornell, Lund & Hems, 1974a; Claus, Pilkis & Park, 1975; Clark, Filsell & Jarrett,
prolactin (i.e.
1976).
This report therefore describes studies aimed at : (a) confirming the presence of both lactogen and GH sites in isolated rat hepatocytes ; (b) studying the characteristics and regula¬ tion of the sites more fully than previously described, and (c) determining whether the isolated hepatocyte would be suitable for an investigation of the possible physiological role of the binding sites in GH and/or prolactin action. MATERIALS AND METHODS
Materials 11.1) was obtained from the Commonwealth Serum Labora¬ and was purified by gel filtration on Sephadex G-100 (medium tories, Melbourne, Australia, 75 2-8 cm column). Bovine growth hormone (NIHSweden, grade: Pharmacia, Uppsala, B1003A for iodination, NIH-GH-B-18 as the standard) was a gift from the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland, U.S.A. Pork insulin (S836175) was obtained from Novo Industries, Denmark. Chloramine was obtained from Merck, A.G., Darmstadt, Germany, and Na125I (carrier free), 2-amino[l-14C]isobutyric acid and L-[U-14C]leucine were obtained from the Radiochemical Centre, Amersham. Human growth hormone (CSL
collagenase (127-154 units/mg) was purchased from Worthington Biochemical Corporation, New Jersey, U.S.A. and unlabelled amino acids were obtained from the Crude
Commonwealth Serum Laboratories, Melbourne, Australia. Oestradiol benzoate in oil (Oestramine) was obtained from Knoll Laboratories, Sydney, Australia, and testosterone propionate in oil (Testaviron) from Schering A.G., Berlin, Germany. Cycloheximide was obtained from Sigma, St Louis, U.S.A.
Preparation of isolated hepatocytes binding studies were prepared from female or male Spraguehepatocytes rats Dawley (120-200 g), by perfusion with calcium-free Krebs-Henseleit buffer containing collagenase (20 mg/100 ml) and glucose (2 mg/ml). The method was as described by Krebs, Cornell, Lund «fe Hems (19746), modified by the omission of hyaluronidase from the
The
used for all
perfusion medium. For binding studies with growth hormone, the cells were resuspended in 25 mM-Tris-HCl (pH 7-4) containing 10 mM-CaCl2. For binding studies with insulin, the CaCl2 was omitted. The cells were counted in a Neubauer haemocytometer and protein concentrations were measured by the method of Lowry, Rosebrough, Farr & Randall (1951). The cell yield was variable but represented 25 ± 2 (s.e.m.) % wet weight/wet weight of liver tissue (n 19). Viability of the cells was assessed by trypan blue exclusion and averaged 59 ± 4 (s.e.m.) % =
( 20). Phase-contrast microscopy indicated that the cells retained a normal gross morphological appearance with relatively little contamination by cell fragments. Hormones were iodinated as previously described using chloramine (Herington et al. 1976c) giving specific activities of 80-90/¿Ci//ig for hGH and 50-60µ€\/µ% for bGH. 125I-Labelled hGH, bGH and insulin were purified before use by chromatography on Sephadex G-100 (medium grade, 21 1-5 cm column). For some experiments 125I-labelled bGH was purified by a technique involving receptor purification on binding sites present in female rabbit liver membranes which are specific for growth hormones and not lactogenic hormones (Tsushima «fe Friesen, 1973). 125I-Labelled bGH was incubated with rabbit liver membranes at room temperature for 3 h. The bGH-receptor complex was separated from free bGH by centrifugation at 4 °C, washed and then resuspended in 1-5 ml 3 M-MgCl2. Preliminary studies had shown that 3 M-MgCl2, but not 2 M-NaCl or 25 mM-Tris-HCl (pH 7-4), would cause a rapid (2 h) dissociation of bound label. Approximately 30 % of bound tracer was recovered in this manner. Further extraction with 3 M-MgCl2 did not release any more bound bGH. The 'receptor purified' bGH was then desalted on Sephadex G-100 and routinely showed a single peak of radioactivity. The retention of the ability of this purified preparation to bind to GH receptors specifically was tested in a rabbit liver =
membrane system. All binding studies
were carried out in triplicate at 22 °C by conventional competitivebinding techniques as described previously (Herington et al. 1976 , b, c). Incubations were performed in 25 mM-Tris-HCl (pH 7-4) containing 10 mM-CaCl2, 0-1 % (w/v) bovine serum albumin and 0-02 % (w/v) sodium azide; bound and free hormone were separated by centri¬ fugation. Total binding was measured in the absence of unlabelled hormone and non-specific binding was taken as the 125I remaining bound in the presence of 1 µg unlabelled hormone. Specific binding was calculated as the total binding minus the non-specific binding. All statistical comparisons between the test groups and their respective controls were made by
Student's /-test.
Hormone treatment Normal male rats (150 g) were treated with oestradiol benzoate (in oil), 25/¿g/day for 10 days, and normal female rats (150 g) were treated with testosterone propionate (in oil), 1 mg/day for 10 days. All rats were given a single subcutaneous injection (0-1 ml) each day. Isolation of hepatocytes by liver perfusion took place on the day after the last injection.
Measurement of amino acid transport and incorporation into protein The standard incubation medium consisted of 30 mM-Tris-HCl (pH 7-4) containing 100 mMNaCl, 5 mM-KCl, 0-3 mM-MgS04, 1 mM-Na2HP04,1 mM-CaCl2,1 mg glucose/ml and 0-1 % (w/v) bovine serum albumin. To study amino acid transport, a non-metabolizable amino acid [14C]a-aminoisobutyric acid (AIB) (0-4 mmol/1) was also added. To study amino acid incorporation into protein, [14C]leucine (0-13 mmol/1) was added together with essential amino acids and glutamine as previously described (Adamson, Herington & Bornstein, 1972). After incubation of the hepatocytes in a total volume of 1 ml buffer for varying times, the metabolic activity was stopped by adding 4 ml ice-cold isotonic saline and centrifuging (1600 g for 10 min) at 4 °C. The [14C]leucine incorporated into the protein fraction or the free intracellular [14C]AIB was separated and prepared for liquid scintillation counting using methods previously described for embryonic chick cartilage and muscle preparations (Adamson et al. 1972). The results were calculated as c.p.m./106 cells.
RESULTS
Amino acid transport and protein synthesis in isolated hepatocytes Although the isolated cells appeared to retain their gross morphological appearance after collagenase digestion and at least 50 % of the cells had intact membranes (as shown by their exclusion of trypan blue), a more definitive biochemical test of cellular integrity was required. For this purpose the transport of the non-metabolizable amino acid AIB into hepatocytes and the incorporation of leucine into protein were measured. As shown in Fig. 1 a, leucine incorporation into protein was linear for at least 3 h at 30 °C. Addition of cycloheximide (10/imol/l) inhibited the incorporation of leucine by as much as 66 %. Lower doses of cycloheximide were also effective. These results are consistent with similar but more detailed studies by Schreiber «fe Schreiber (1973). Figure 1 b shows that the transport of AIB through the cell membrane is a saturable process at 37 °C. The much higher intracellular concentration reached at 37 °C than at 4 °C indicates that the movement of AIB across the membrane is not due to non-specific leakage but to an intact active transport system.
Fig.
1.
(a) Time
course
of amino acid incorporation into protein by
hepatocytes (0-36 x 10e cells/
tube) isolated from a normal female rat. [14C]Leucine incorporation was measured as described under Methods in the absence (·) or presence (O) of 10 /tM-cycloheximide. (6) Time course of amino acid
transport by hepatocytes (1-88 x 10e cells/tube) isolated from a normal female rat. [14C]œ-Aminoisobutyric acid (AIB) transport was measured as described under Methods at 37 °C (·) or 4 °C (O). The values shown for 37 °C have been corrected for the 4 °C 'blank' values. All values are means + s.e.m. of triplicate determinations.
Association and dissociation rates of 12H-labelled hGH binding The binding of 125I-labelled hGH to isolated hepatocytes from normal female rats was both rapid and reversible (Fig. 2). Specific binding reached equilibrium in most experiments after 2-5 h at 22 °C the temperature at which all binding studies were subsequently performed. In a few experiments, binding was still increasing slowly after 3-4 h. Binding was marginally more rapid at 37 °C than at 22 °C, but was extremely slow at 4 °C. The maximum binding observed at 22 °C in all experiments was 10-3 ± 1-5 (s.e.m.) % (n 18) of the 125I-labelled hGH initially added per 10" cells. The reversibility of hGH binding is demonstrated (Fig. 2) by the rapid dissociation of -
=
Time (h)
Fig. 2. Time course for the association and dissociation of 125I-Iabelled human GH (hGH) binding to hepatocytes (1 x 10e cells/tube) isolated from normal female rats. Specific binding was determined as described under Methods. Values shown are means + s.e.m. of triplicate determinations and are expressed as a percentage of the total 125I-labelled hGH added per tube. Where no error bars are shown the s.e.m. was within the size of the point symbol. ·. Association curve; O, dissociation curve following addition of a 10000-fold excess (1 /¿g) of unlabelled hGH. The excess unlabelled hGH was added in only 10 /tl to prevent any dilution effects (arrow). bound 126I-labelled hGH following the addition of a 10000-fold excess (1 µg) of unlabelled hGH. Addition of excess hGH at 2 h (or at 1 h, not shown) led to dissociation with a halftime of 2-3 h at 22 °C.
Effects of hepatocyte concentration and 12hI-labelled hGH concentration 3 shows the effect of hepatocyte concentration on binding of hGH. Specific binding Figure was linear over the range of cell concentrations studied (0-4 IO6—1 -6 x 10e cells/ml). Although the cell yield from experiment to experiment varied it was within this linear portion of the dose-response curve. Binding as a function of increasing 125I-labelled hGH concentration is shown in Fig. 4. Specific binding was detected over the whole range of concentrations studied (1-4 1043-5 x 10e c.p.m.; 8-2100 pmol/1) and had reached saturation by approximately 1-5 x 10e c.p.m. or 900 pmol/1. Non-specific binding was linear over the entire concentration range and represented less than 4-7 % of the total c.p.m. added.
0-4
0-8 10" IO6 Number of cells/tube
1-6 xlO6
Fig. 3. Effect of hepatocyte number on binding of 125I-labelled human GH (hGH) to hepatocytes isolated from normal female rats. Specific binding was determined as described under Methods and values are means ± s.e.m. of triplicate determinations, and are expressed as a percentage of the total 125I-labelled hGH added per tube.
12 r
9
-
X
O 4=
0-5x10
300
lxlO6 2xl06 125I-labelled hGH added (c.p.m.) 600
1200
3x10"
1800
125I-labelled hGH (pmol/1) Fig. 4. Effect of 12SI-labelled human GH (hGH) concentration on binding to hepatocytes (0-9 x 10e cells/tube) isolated from normal female rats. Specific (·) and non-specific (O) binding were deter¬ mined as described under Methods and are expressed (mean ± s.e.m. of triplicate determinations) as absolute counts bound or fmol bound. Where no error bars are shown the s.e.m. was within the size of the point symbol. Specific activity of the 125I-labelled hGH used in this experiment was
90/tCi/yttg.
Hormonal specificity and Scatchard analysis Figure 5 shows a typical binding displacement curve for hGH-binding to female rat hepato¬ cytes. Of the hGH bound, 22 % was not displaceable by 1 µg unlabelled hGH and represented the non-specific binding. The binding of 125I-labelled hGH to the hepatocytes appeared to be specific for lactogenic hormones and not growth hormones (Fig. 5). Ovine and human prolactin caused substantial displacement of labelled hGH but bovine, ovine and porcine growth hormones had no effect.
Bovine and
100
porcine f
GH
Ovine X GH
90
80
-
O 70
ZZ 60 o
01) c
50
S 40 o 30
20
Human GH
-
10
-//-
_L
J_
10
10 Unlabelled hormone
100
1000
(ng/tube)
Fig. 5. Hormonal specificity of binding of 125I-labelled human GH (hGH) to hepatocytes (1-6 x 10e cells/tube) isolated from a normal female rat. Competition for binding of 125I-labelled hGH (expressed as a percentage of binding in the absence of unlabelled hormone) was measured in the presence of increasing amounts of unlabelled hGH (·) and of a single dose (1 /tg/tube) of ovine prolactin, ovine growth hormone, porcine growth hormone and bovine growth hormone. Human prolactin was tested at 500 ng/tube. Each point is the mean ± s.e.m. of triplicate determinations. Scatchard analysis (Scatchard, 1949) of such binding displacement curves showed a single class of binding site (Fig. 6) with a binding affinity of 1 -24 109 ± 0-17 109 (s.e.m.) litres/mol and a binding capacity of 26-74 ± 3-75 fmol/106 cells (n 10). The number of binding sites per cell calculated from the binding capacity was 16041 ± 2273. =
Dependence on sex and effect of sex steroids Previous studies (Herington et al. 1976 ) have clearly demonstrated that binding of hGH to rat liver membranes is highly dependent on the sex of the animal from which the liver membranes were prepared. This is also true of hGH binding to hepatocytes (Fig. 7). Specific binding to male hepatocytes ranged from 0 to 7 %/10e cells with a mean of 2-0 ±0-9 % (n 8). Binding was too low in most cases to carry out Scatchard analysis. This is compared with a mean binding of 10-3 ± 1-5 %/106 cells (n 8) for normal female rats (P < 0-005). Administration of oestradiol benzoate (25 /¿g/day for 10 days) to male rats caused a marked (12-fold) stimulation of hGH binding (P < 0-02). Administration of testosterone propionate =
=
10 Bound
20
30
40
(fmol/106 cells)
Fig. 6. Representative Scatchard plot of the binding of 125I-labelled human GH (hGH) to hepato¬ cytes (0-98 x 10e cells/tube) isolated from a normal female rat. Scatchard analysis (specific binding only) was carried out on data obtained from a competition experiment similar to that shown in Fig. 5. Incubation of hepatocytes with 125I-labelled hGH was carried out for 4 h at 22 °C to ensure binding equilibrium had been reached. The negative of the slope of the Scatchard plot is a measure of the hormone binding affinity and the ^-intercept is a measure of the hormone-binding capacity. 40
30
X
O 20
-
S 14
-
12
10
[
(18)
(4)
Female
Testosteronetreated female
(4) Male
Oestradioltreated male
Fig. 7. Effect of sex and sex steroid treatment in vivo on binding of 125I-labelled human GH (hGH) to isolated rat hepatocytes. Treatment of female rats with testosterone propionate or male rats with
oestradiol benzoate is described in the text. The bars show the mean + s.e.m. with the number of rats in each group shown in parentheses. Specific binding, calculated as a percentage of the total 125Ilabelled hGH added, is expressed per 10e cells used in each experiment. This approach was required to account for variations in the number of hepatocytes isolated from each rat.
(1 mg/day for 10 days) to female rats caused a marked diminution but this decrease was not significant (P < 0-1).
(63 %) of hGH binding,
Binding of 125I-labelled bovine growth hormone In an attempt to study binding sites for GH as opposed to the lactogen sites, binding of bGH to male and female hepatocytes was investigated. 125I-Labelled bGH was purified before use by Sephadex chromatography or by receptor purification (see Methods) on rabbit liver membranes. In neither case did bGH bind specifically to rat hepatocytes, either male or female, although the bGH did bind to membranes freshly prepared from rabbit liver (Fig. 8) and to rabbit hepatocytes (not shown), both preparations having been shown previously to possess GH-specific binding sites. Receptor purification of bGH resulted in a highly signifi¬ cant reduction in the amount of non-specific binding, indicating perhaps a much more suitable purification method for iodinated hormones used in binding studies.
Binding of125I-labelled insulin Insulin binding to hepatocytes has been reported previously (Olefsky, Johnson, Liu, Edwards & Baur, 1975) and was measured in the present studies for additional assurance that the (n-7) 10-.
(n 4) =
NS
8 h
I6 I
1
(fl-5)
I
o oo c
Tn:
ß .S
1 G-100
Membrane
Membrane
purified
purified
purified
Male and female
hepatocytes
rat
Female rabbit liver membranes
Fig. 8. Binding of 125I-labelled bovine GH (bGH) to hepatocytes isolated from normal female and male rats and to a microsomal membrane preparation from the liver of a normal female rabbit. 12eILabelled bGH was purified before use by chromatography on Sephadex G-100 or by receptor purification on female rabbit liver membranes (see Methods). The bars represent the mean ± s.e.m. with the number of rats in each group in parentheses. Total binding (open bars) and non-specific binding (hatched bars) are expressed as a percentage of the total '"I-labelled bGH added/10e cells used in each experiment. NS, > 005 ; *P < 0-02 : compared with appropriate total binding value.
Table 1.
Binding of 12hI-labelled insulin to hepatocytes isolatedfromfemale and male rats Binding of lasI-labelled insulin (%/10· cells) Total
Non-specific
Specific
3-2 Rat 1, female 5-8 2-6 16-5 Rat 2, female 31-9 15-4 2-7 24-1 Rat 3, female 26-8 12-6 16-8 4-2 Rat 4, male Specific binding of insulin was measured in an identical manner to that for human GH, as described under Methods, except that calcium was omitted from the incubation mixture. All binding studies were done at room temperature (22 °C) for 2 h, the time required for equilibrium to be reached with this system.
failure to detect binding sites for bGH was not due to generalized damage to the cell mem¬ brane. Specific binding of insulin reached equilibrium by 1-2 h and, as shown in Table 1, ranged from 3-2 to 24-1 %/10e cells for hepatocytes isolated from female rats. Specific binding in the only preparation of hepatocytes from a male rat was 12-6 %/10e cells suggesting that there was no sex difference in binding of insulin to isolated hepatocytes. The lack of sex difference has been observed previously for insulin binding to liver membrane preparations (Kelly, Posner, Tsushima «fe Friesen, 1974). DISCUSSION
These studies show that a class of lactogen binding site, previously described in cell membrane preparations from rat liver, is also present in the intact isolated liver cell. The designation of these binding sites reflects their specificity for prolactin-like hormones only (hGH, pro¬ lactins) (Fig. 5) and their failure to bind bGH (Fig. 8). Unlike Ranke et al. (1976), we found
distinct GH sites. The properties of the sites in the intact hepatocyte resemble those previously established for the lactogen sites of rat liver membranes (Posner, Kelly 24 h in the membrane system (Herington et al. 1976 c). The slow kinetics of the membrane system may be due to inverted vesiculation of the isolated membrane fragments (Herington et al. 1976 c) which results in impedance of the binding reaction. The intact liver cell may well show kinetic properties close to those operating no
physiologically.
In agreement with Kelly et al. (1974) working with rat liver membranes, Scatchard analysis revealed a single class of binding site for hGH in the hepatocyte. This is in marked contrast to Ranke et al. (1976) who found two classes of site (a GH site and a lactogen site) in hepato¬ cytes and is also in contrast to the two classes of lactogen site observed by Herington et al. (1976 c) for rat liver membranes. The affinity of binding to the lactogen sites in the present study is comparable to that reported by others for binding of hGH to liver lactogen sites in membranes or whole cells (Kelly et al. 19 ; Herington et al. 19766, c; Ranke et al. 1976)
and is entirely compatible with the known levels of GH and prolactin in the blood. The inability to detect specific GH sites in these preparations of hepatocytes and in liver membrane preparations suggests that the GH sites might be extremely labile. Therefore, slight differences in experimental conditions, for example different batches of collagenase, might explain the apparent discrepancies between this study and that of Ranke et al. (1976). The influence of iodinated hormone on the ability to detect hGH sites might also be import¬ ant. However, no differences in binding were detectable between tracer iodinated with either chloramine or lactoperoxidase (A. C. Herington & . M. Veith, unpublished results) and tracer purified by conventional Sephadex chromatography or by receptor purification on rabbit liver membranes. Although it has not been possible to detect GH sites themselves, the use of receptorpurified 125I-labelled bGH has proved valuable. Such purification procedures, whichtheoretically would separate out only those labelled molecules which are capable of binding to a specific GH binding site, should therefore provide a more satisfactory tracer for receptor studies. Similar procedures have been used for thyroid-stimulating hormone (Manley, Bourke «fe Hawker, 1974), follicle-stimulating hormone (Ketelslegers & Catt, 1974) and human chorionic gonadotrophin (Dufau, Catt «fe Tsuruhara, 1972) but have not been reported previously for GH. The applicability of this procedure to GH-receptor studies is emphasized by the dramatic reduction in non-specific binding observed when this method is compared with the usual method of purification by Sephadex chromatography (Fig. 8). The demonstration of lactogen-binding sites and insulin-binding sites in the preparations of hepatocytes used here indicate that the hormonal responsiveness of the cells has not been totally destroyed. In addition, the cells appear to have retained normal metabolic function in that leucine incorporation into trichloroacetic acid-precipitable protein was linear and inhibited by cycloheximide (Fig. 1 a) and transport of AIB into the hepatocytes also showed normal kinetics and saturability (Fig. 1 b). Metabolic tests of cell function were not reported by Ranke et al. (1976), although they do claim that 90-95 % of their cells excluded trypan blue. The presence of lactogen-binding sites and the retention of metabolic function by the iso¬ lated hepatocytes permits a direct study of the relationship between binding of lactogenic hormones and the biological response. In other words, a study of whether the binding sites are true biological receptors for the lactogenic hormones and are involved in the expression of metabolic effects. Prolactin does have metabolic actions in liver tissue in vivo, for example on RNA synthesis (Chen, Hamer, Heininger «fe Meier, 1972), on ornithine decarboxylase activity (Richards, 1975), and on somatomedin release (Francis «fe Hill, 1975). Therefore the hepatocyte should provide an ideal system for such a study. Unfortunately, the major aim of the present paper remained unfulfilled because of the failure to detect distinct GH sites. The experiments do emphasize, however, the potential value of purifying GH tracers by adsorption and elution from particulate binding sites before use of the tracer in receptor studies.
These studies were supported by a grant from the National Health and Medical Research Council. The authors express their thanks to Dr H. G. Burger for many helpful discussions. REFERENCES
Adamson, L. F., Herington, A. C. «ft Bomstein, J. (1972). Evidence for the selection of intracellular or extra¬ cellular amino acids for protein synthesis. Biochimica et Biophysica Acta 282, 352-365. Chen, H. W., Hamer, D. H., Heininger, H. «ft Meier, H. (1972). Stimulation of hepatic RNA synthesis in dwarf mice by ovine prolactin. Biochimica et Biophysica Acta 287, 90-97.
Clark, M. G., Filsell, O. H.
(Received 7
December
1976)
SUMMARY
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]\g=a\\x=req-\ 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 \s=deg\C,binding reached a steady-state after 2\m=.\5h and had a half-life of dissociation of 2\p=n-\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\m=.\74\m=+-\3\m=.\73fmol/106 cells and a binding affinity of 1 \m=.\24\m=x\109 \m=+-\ 0\m=.\17\m=x\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.
The
=
INTRODUCTION
A number of studies (Posner, Kelly, Shiu «fe Friesen, 19746; Herington, Burger & Veith, 1976 ; Herington, Phillips & Daughaday, 1976 ; Herington, Veith «fe Burger, 1976c) have clearly demonstrated specific binding sites for lactogenic hormones in membrane preparations from rat liver. These sites bind prolactins and human growth hormone (hGH) but do not bind bovine growth hormone (bGH). They are characterized by unusually slow kinetics, by a marked dependence on the sex steroids and an intact pituitary gland, and by their regulation by prolactin itself. Since the liver is regarded as a target organ for GH, distinct binding sites for GH should be demonstrable in addition to those for prolactin and hGH. This is true for membrane preparations from rabbit liver (Tsushima «fe Friesen, 1973 ; Herington, Jacobs «fe Daughaday, 1974), but distinct GH sites have been reported by only one group in prepara¬ tions of rat liver membranes (Etzrodt, Musch, Schleyer & Pfeiffer, 1976). Recently, however, it has been reported that specific GH sites are present in preparations of isolated rat hepatocytes (Ranke, Stanley, Rodbard, Baker, Bongiovanni & Parks, 1976). Two classes of site were apparent in these studies. One class, present in male and female rats, has a high affinity for hGH, and competition for these sites is shown by bGH but not
a GH site). The second class, present only in female rats, has a lower affinity for hGH, and competition for these sites is shown by prolactin but not bGH (i.e. a lactogen site). The lactogen sites, but not the GH sites, were influenced by oestrogens in a similar manner to those present in membrane preparations and the time required for a binding equilibrium to be reached was much shorter. Although specific-binding sites for hGH and/or prolactin have been observed using both liver membranes and hepatocytes, there is no direct evidence that they are true hormone receptors, i.e. that they are involved in the expression of biological effects of GH or prolactin. The isolated hepatocyte would appear to be an ideal system to study this possible relationship since the full metabolic integrity of the cell (e.g. gluconeogenesis, protein synthesis, RNA metabolism) and responsiveness to at least some hormones (e.g. insulin, glucagon) are retained (Ingebretsen, Moxley, Allen «fe Wagle, 1972; Schreiber & Schreiber, 1973; Krebs, Cornell, Lund & Hems, 1974a; Claus, Pilkis & Park, 1975; Clark, Filsell & Jarrett,
prolactin (i.e.
1976).
This report therefore describes studies aimed at : (a) confirming the presence of both lactogen and GH sites in isolated rat hepatocytes ; (b) studying the characteristics and regula¬ tion of the sites more fully than previously described, and (c) determining whether the isolated hepatocyte would be suitable for an investigation of the possible physiological role of the binding sites in GH and/or prolactin action. MATERIALS AND METHODS
Materials 11.1) was obtained from the Commonwealth Serum Labora¬ and was purified by gel filtration on Sephadex G-100 (medium tories, Melbourne, Australia, 75 2-8 cm column). Bovine growth hormone (NIHSweden, grade: Pharmacia, Uppsala, B1003A for iodination, NIH-GH-B-18 as the standard) was a gift from the National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, Maryland, U.S.A. Pork insulin (S836175) was obtained from Novo Industries, Denmark. Chloramine was obtained from Merck, A.G., Darmstadt, Germany, and Na125I (carrier free), 2-amino[l-14C]isobutyric acid and L-[U-14C]leucine were obtained from the Radiochemical Centre, Amersham. Human growth hormone (CSL
collagenase (127-154 units/mg) was purchased from Worthington Biochemical Corporation, New Jersey, U.S.A. and unlabelled amino acids were obtained from the Crude
Commonwealth Serum Laboratories, Melbourne, Australia. Oestradiol benzoate in oil (Oestramine) was obtained from Knoll Laboratories, Sydney, Australia, and testosterone propionate in oil (Testaviron) from Schering A.G., Berlin, Germany. Cycloheximide was obtained from Sigma, St Louis, U.S.A.
Preparation of isolated hepatocytes binding studies were prepared from female or male Spraguehepatocytes rats Dawley (120-200 g), by perfusion with calcium-free Krebs-Henseleit buffer containing collagenase (20 mg/100 ml) and glucose (2 mg/ml). The method was as described by Krebs, Cornell, Lund «fe Hems (19746), modified by the omission of hyaluronidase from the
The
used for all
perfusion medium. For binding studies with growth hormone, the cells were resuspended in 25 mM-Tris-HCl (pH 7-4) containing 10 mM-CaCl2. For binding studies with insulin, the CaCl2 was omitted. The cells were counted in a Neubauer haemocytometer and protein concentrations were measured by the method of Lowry, Rosebrough, Farr & Randall (1951). The cell yield was variable but represented 25 ± 2 (s.e.m.) % wet weight/wet weight of liver tissue (n 19). Viability of the cells was assessed by trypan blue exclusion and averaged 59 ± 4 (s.e.m.) % =
( 20). Phase-contrast microscopy indicated that the cells retained a normal gross morphological appearance with relatively little contamination by cell fragments. Hormones were iodinated as previously described using chloramine (Herington et al. 1976c) giving specific activities of 80-90/¿Ci//ig for hGH and 50-60µ€\/µ% for bGH. 125I-Labelled hGH, bGH and insulin were purified before use by chromatography on Sephadex G-100 (medium grade, 21 1-5 cm column). For some experiments 125I-labelled bGH was purified by a technique involving receptor purification on binding sites present in female rabbit liver membranes which are specific for growth hormones and not lactogenic hormones (Tsushima «fe Friesen, 1973). 125I-Labelled bGH was incubated with rabbit liver membranes at room temperature for 3 h. The bGH-receptor complex was separated from free bGH by centrifugation at 4 °C, washed and then resuspended in 1-5 ml 3 M-MgCl2. Preliminary studies had shown that 3 M-MgCl2, but not 2 M-NaCl or 25 mM-Tris-HCl (pH 7-4), would cause a rapid (2 h) dissociation of bound label. Approximately 30 % of bound tracer was recovered in this manner. Further extraction with 3 M-MgCl2 did not release any more bound bGH. The 'receptor purified' bGH was then desalted on Sephadex G-100 and routinely showed a single peak of radioactivity. The retention of the ability of this purified preparation to bind to GH receptors specifically was tested in a rabbit liver =
membrane system. All binding studies
were carried out in triplicate at 22 °C by conventional competitivebinding techniques as described previously (Herington et al. 1976 , b, c). Incubations were performed in 25 mM-Tris-HCl (pH 7-4) containing 10 mM-CaCl2, 0-1 % (w/v) bovine serum albumin and 0-02 % (w/v) sodium azide; bound and free hormone were separated by centri¬ fugation. Total binding was measured in the absence of unlabelled hormone and non-specific binding was taken as the 125I remaining bound in the presence of 1 µg unlabelled hormone. Specific binding was calculated as the total binding minus the non-specific binding. All statistical comparisons between the test groups and their respective controls were made by
Student's /-test.
Hormone treatment Normal male rats (150 g) were treated with oestradiol benzoate (in oil), 25/¿g/day for 10 days, and normal female rats (150 g) were treated with testosterone propionate (in oil), 1 mg/day for 10 days. All rats were given a single subcutaneous injection (0-1 ml) each day. Isolation of hepatocytes by liver perfusion took place on the day after the last injection.
Measurement of amino acid transport and incorporation into protein The standard incubation medium consisted of 30 mM-Tris-HCl (pH 7-4) containing 100 mMNaCl, 5 mM-KCl, 0-3 mM-MgS04, 1 mM-Na2HP04,1 mM-CaCl2,1 mg glucose/ml and 0-1 % (w/v) bovine serum albumin. To study amino acid transport, a non-metabolizable amino acid [14C]a-aminoisobutyric acid (AIB) (0-4 mmol/1) was also added. To study amino acid incorporation into protein, [14C]leucine (0-13 mmol/1) was added together with essential amino acids and glutamine as previously described (Adamson, Herington & Bornstein, 1972). After incubation of the hepatocytes in a total volume of 1 ml buffer for varying times, the metabolic activity was stopped by adding 4 ml ice-cold isotonic saline and centrifuging (1600 g for 10 min) at 4 °C. The [14C]leucine incorporated into the protein fraction or the free intracellular [14C]AIB was separated and prepared for liquid scintillation counting using methods previously described for embryonic chick cartilage and muscle preparations (Adamson et al. 1972). The results were calculated as c.p.m./106 cells.
RESULTS
Amino acid transport and protein synthesis in isolated hepatocytes Although the isolated cells appeared to retain their gross morphological appearance after collagenase digestion and at least 50 % of the cells had intact membranes (as shown by their exclusion of trypan blue), a more definitive biochemical test of cellular integrity was required. For this purpose the transport of the non-metabolizable amino acid AIB into hepatocytes and the incorporation of leucine into protein were measured. As shown in Fig. 1 a, leucine incorporation into protein was linear for at least 3 h at 30 °C. Addition of cycloheximide (10/imol/l) inhibited the incorporation of leucine by as much as 66 %. Lower doses of cycloheximide were also effective. These results are consistent with similar but more detailed studies by Schreiber «fe Schreiber (1973). Figure 1 b shows that the transport of AIB through the cell membrane is a saturable process at 37 °C. The much higher intracellular concentration reached at 37 °C than at 4 °C indicates that the movement of AIB across the membrane is not due to non-specific leakage but to an intact active transport system.
Fig.
1.
(a) Time
course
of amino acid incorporation into protein by
hepatocytes (0-36 x 10e cells/
tube) isolated from a normal female rat. [14C]Leucine incorporation was measured as described under Methods in the absence (·) or presence (O) of 10 /tM-cycloheximide. (6) Time course of amino acid
transport by hepatocytes (1-88 x 10e cells/tube) isolated from a normal female rat. [14C]œ-Aminoisobutyric acid (AIB) transport was measured as described under Methods at 37 °C (·) or 4 °C (O). The values shown for 37 °C have been corrected for the 4 °C 'blank' values. All values are means + s.e.m. of triplicate determinations.
Association and dissociation rates of 12H-labelled hGH binding The binding of 125I-labelled hGH to isolated hepatocytes from normal female rats was both rapid and reversible (Fig. 2). Specific binding reached equilibrium in most experiments after 2-5 h at 22 °C the temperature at which all binding studies were subsequently performed. In a few experiments, binding was still increasing slowly after 3-4 h. Binding was marginally more rapid at 37 °C than at 22 °C, but was extremely slow at 4 °C. The maximum binding observed at 22 °C in all experiments was 10-3 ± 1-5 (s.e.m.) % (n 18) of the 125I-labelled hGH initially added per 10" cells. The reversibility of hGH binding is demonstrated (Fig. 2) by the rapid dissociation of -
=
Time (h)
Fig. 2. Time course for the association and dissociation of 125I-Iabelled human GH (hGH) binding to hepatocytes (1 x 10e cells/tube) isolated from normal female rats. Specific binding was determined as described under Methods. Values shown are means + s.e.m. of triplicate determinations and are expressed as a percentage of the total 125I-labelled hGH added per tube. Where no error bars are shown the s.e.m. was within the size of the point symbol. ·. Association curve; O, dissociation curve following addition of a 10000-fold excess (1 /¿g) of unlabelled hGH. The excess unlabelled hGH was added in only 10 /tl to prevent any dilution effects (arrow). bound 126I-labelled hGH following the addition of a 10000-fold excess (1 µg) of unlabelled hGH. Addition of excess hGH at 2 h (or at 1 h, not shown) led to dissociation with a halftime of 2-3 h at 22 °C.
Effects of hepatocyte concentration and 12hI-labelled hGH concentration 3 shows the effect of hepatocyte concentration on binding of hGH. Specific binding Figure was linear over the range of cell concentrations studied (0-4 IO6—1 -6 x 10e cells/ml). Although the cell yield from experiment to experiment varied it was within this linear portion of the dose-response curve. Binding as a function of increasing 125I-labelled hGH concentration is shown in Fig. 4. Specific binding was detected over the whole range of concentrations studied (1-4 1043-5 x 10e c.p.m.; 8-2100 pmol/1) and had reached saturation by approximately 1-5 x 10e c.p.m. or 900 pmol/1. Non-specific binding was linear over the entire concentration range and represented less than 4-7 % of the total c.p.m. added.
0-4
0-8 10" IO6 Number of cells/tube
1-6 xlO6
Fig. 3. Effect of hepatocyte number on binding of 125I-labelled human GH (hGH) to hepatocytes isolated from normal female rats. Specific binding was determined as described under Methods and values are means ± s.e.m. of triplicate determinations, and are expressed as a percentage of the total 125I-labelled hGH added per tube.
12 r
9
-
X
O 4=
0-5x10
300
lxlO6 2xl06 125I-labelled hGH added (c.p.m.) 600
1200
3x10"
1800
125I-labelled hGH (pmol/1) Fig. 4. Effect of 12SI-labelled human GH (hGH) concentration on binding to hepatocytes (0-9 x 10e cells/tube) isolated from normal female rats. Specific (·) and non-specific (O) binding were deter¬ mined as described under Methods and are expressed (mean ± s.e.m. of triplicate determinations) as absolute counts bound or fmol bound. Where no error bars are shown the s.e.m. was within the size of the point symbol. Specific activity of the 125I-labelled hGH used in this experiment was
90/tCi/yttg.
Hormonal specificity and Scatchard analysis Figure 5 shows a typical binding displacement curve for hGH-binding to female rat hepato¬ cytes. Of the hGH bound, 22 % was not displaceable by 1 µg unlabelled hGH and represented the non-specific binding. The binding of 125I-labelled hGH to the hepatocytes appeared to be specific for lactogenic hormones and not growth hormones (Fig. 5). Ovine and human prolactin caused substantial displacement of labelled hGH but bovine, ovine and porcine growth hormones had no effect.
Bovine and
100
porcine f
GH
Ovine X GH
90
80
-
O 70
ZZ 60 o
01) c
50
S 40 o 30
20
Human GH
-
10
-//-
_L
J_
10
10 Unlabelled hormone
100
1000
(ng/tube)
Fig. 5. Hormonal specificity of binding of 125I-labelled human GH (hGH) to hepatocytes (1-6 x 10e cells/tube) isolated from a normal female rat. Competition for binding of 125I-labelled hGH (expressed as a percentage of binding in the absence of unlabelled hormone) was measured in the presence of increasing amounts of unlabelled hGH (·) and of a single dose (1 /tg/tube) of ovine prolactin, ovine growth hormone, porcine growth hormone and bovine growth hormone. Human prolactin was tested at 500 ng/tube. Each point is the mean ± s.e.m. of triplicate determinations. Scatchard analysis (Scatchard, 1949) of such binding displacement curves showed a single class of binding site (Fig. 6) with a binding affinity of 1 -24 109 ± 0-17 109 (s.e.m.) litres/mol and a binding capacity of 26-74 ± 3-75 fmol/106 cells (n 10). The number of binding sites per cell calculated from the binding capacity was 16041 ± 2273. =
Dependence on sex and effect of sex steroids Previous studies (Herington et al. 1976 ) have clearly demonstrated that binding of hGH to rat liver membranes is highly dependent on the sex of the animal from which the liver membranes were prepared. This is also true of hGH binding to hepatocytes (Fig. 7). Specific binding to male hepatocytes ranged from 0 to 7 %/10e cells with a mean of 2-0 ±0-9 % (n 8). Binding was too low in most cases to carry out Scatchard analysis. This is compared with a mean binding of 10-3 ± 1-5 %/106 cells (n 8) for normal female rats (P < 0-005). Administration of oestradiol benzoate (25 /¿g/day for 10 days) to male rats caused a marked (12-fold) stimulation of hGH binding (P < 0-02). Administration of testosterone propionate =
=
10 Bound
20
30
40
(fmol/106 cells)
Fig. 6. Representative Scatchard plot of the binding of 125I-labelled human GH (hGH) to hepato¬ cytes (0-98 x 10e cells/tube) isolated from a normal female rat. Scatchard analysis (specific binding only) was carried out on data obtained from a competition experiment similar to that shown in Fig. 5. Incubation of hepatocytes with 125I-labelled hGH was carried out for 4 h at 22 °C to ensure binding equilibrium had been reached. The negative of the slope of the Scatchard plot is a measure of the hormone binding affinity and the ^-intercept is a measure of the hormone-binding capacity. 40
30
X
O 20
-
S 14
-
12
10
[
(18)
(4)
Female
Testosteronetreated female
(4) Male
Oestradioltreated male
Fig. 7. Effect of sex and sex steroid treatment in vivo on binding of 125I-labelled human GH (hGH) to isolated rat hepatocytes. Treatment of female rats with testosterone propionate or male rats with
oestradiol benzoate is described in the text. The bars show the mean + s.e.m. with the number of rats in each group shown in parentheses. Specific binding, calculated as a percentage of the total 125Ilabelled hGH added, is expressed per 10e cells used in each experiment. This approach was required to account for variations in the number of hepatocytes isolated from each rat.
(1 mg/day for 10 days) to female rats caused a marked diminution but this decrease was not significant (P < 0-1).
(63 %) of hGH binding,
Binding of 125I-labelled bovine growth hormone In an attempt to study binding sites for GH as opposed to the lactogen sites, binding of bGH to male and female hepatocytes was investigated. 125I-Labelled bGH was purified before use by Sephadex chromatography or by receptor purification (see Methods) on rabbit liver membranes. In neither case did bGH bind specifically to rat hepatocytes, either male or female, although the bGH did bind to membranes freshly prepared from rabbit liver (Fig. 8) and to rabbit hepatocytes (not shown), both preparations having been shown previously to possess GH-specific binding sites. Receptor purification of bGH resulted in a highly signifi¬ cant reduction in the amount of non-specific binding, indicating perhaps a much more suitable purification method for iodinated hormones used in binding studies.
Binding of125I-labelled insulin Insulin binding to hepatocytes has been reported previously (Olefsky, Johnson, Liu, Edwards & Baur, 1975) and was measured in the present studies for additional assurance that the (n-7) 10-.
(n 4) =
NS
8 h
I6 I
1
(fl-5)
I
o oo c
Tn:
ß .S
1 G-100
Membrane
Membrane
purified
purified
purified
Male and female
hepatocytes
rat
Female rabbit liver membranes
Fig. 8. Binding of 125I-labelled bovine GH (bGH) to hepatocytes isolated from normal female and male rats and to a microsomal membrane preparation from the liver of a normal female rabbit. 12eILabelled bGH was purified before use by chromatography on Sephadex G-100 or by receptor purification on female rabbit liver membranes (see Methods). The bars represent the mean ± s.e.m. with the number of rats in each group in parentheses. Total binding (open bars) and non-specific binding (hatched bars) are expressed as a percentage of the total '"I-labelled bGH added/10e cells used in each experiment. NS, > 005 ; *P < 0-02 : compared with appropriate total binding value.
Table 1.
Binding of 12hI-labelled insulin to hepatocytes isolatedfromfemale and male rats Binding of lasI-labelled insulin (%/10· cells) Total
Non-specific
Specific
3-2 Rat 1, female 5-8 2-6 16-5 Rat 2, female 31-9 15-4 2-7 24-1 Rat 3, female 26-8 12-6 16-8 4-2 Rat 4, male Specific binding of insulin was measured in an identical manner to that for human GH, as described under Methods, except that calcium was omitted from the incubation mixture. All binding studies were done at room temperature (22 °C) for 2 h, the time required for equilibrium to be reached with this system.
failure to detect binding sites for bGH was not due to generalized damage to the cell mem¬ brane. Specific binding of insulin reached equilibrium by 1-2 h and, as shown in Table 1, ranged from 3-2 to 24-1 %/10e cells for hepatocytes isolated from female rats. Specific binding in the only preparation of hepatocytes from a male rat was 12-6 %/10e cells suggesting that there was no sex difference in binding of insulin to isolated hepatocytes. The lack of sex difference has been observed previously for insulin binding to liver membrane preparations (Kelly, Posner, Tsushima «fe Friesen, 1974). DISCUSSION
These studies show that a class of lactogen binding site, previously described in cell membrane preparations from rat liver, is also present in the intact isolated liver cell. The designation of these binding sites reflects their specificity for prolactin-like hormones only (hGH, pro¬ lactins) (Fig. 5) and their failure to bind bGH (Fig. 8). Unlike Ranke et al. (1976), we found
distinct GH sites. The properties of the sites in the intact hepatocyte resemble those previously established for the lactogen sites of rat liver membranes (Posner, Kelly 24 h in the membrane system (Herington et al. 1976 c). The slow kinetics of the membrane system may be due to inverted vesiculation of the isolated membrane fragments (Herington et al. 1976 c) which results in impedance of the binding reaction. The intact liver cell may well show kinetic properties close to those operating no
physiologically.
In agreement with Kelly et al. (1974) working with rat liver membranes, Scatchard analysis revealed a single class of binding site for hGH in the hepatocyte. This is in marked contrast to Ranke et al. (1976) who found two classes of site (a GH site and a lactogen site) in hepato¬ cytes and is also in contrast to the two classes of lactogen site observed by Herington et al. (1976 c) for rat liver membranes. The affinity of binding to the lactogen sites in the present study is comparable to that reported by others for binding of hGH to liver lactogen sites in membranes or whole cells (Kelly et al. 19 ; Herington et al. 19766, c; Ranke et al. 1976)
and is entirely compatible with the known levels of GH and prolactin in the blood. The inability to detect specific GH sites in these preparations of hepatocytes and in liver membrane preparations suggests that the GH sites might be extremely labile. Therefore, slight differences in experimental conditions, for example different batches of collagenase, might explain the apparent discrepancies between this study and that of Ranke et al. (1976). The influence of iodinated hormone on the ability to detect hGH sites might also be import¬ ant. However, no differences in binding were detectable between tracer iodinated with either chloramine or lactoperoxidase (A. C. Herington & . M. Veith, unpublished results) and tracer purified by conventional Sephadex chromatography or by receptor purification on rabbit liver membranes. Although it has not been possible to detect GH sites themselves, the use of receptorpurified 125I-labelled bGH has proved valuable. Such purification procedures, whichtheoretically would separate out only those labelled molecules which are capable of binding to a specific GH binding site, should therefore provide a more satisfactory tracer for receptor studies. Similar procedures have been used for thyroid-stimulating hormone (Manley, Bourke «fe Hawker, 1974), follicle-stimulating hormone (Ketelslegers & Catt, 1974) and human chorionic gonadotrophin (Dufau, Catt «fe Tsuruhara, 1972) but have not been reported previously for GH. The applicability of this procedure to GH-receptor studies is emphasized by the dramatic reduction in non-specific binding observed when this method is compared with the usual method of purification by Sephadex chromatography (Fig. 8). The demonstration of lactogen-binding sites and insulin-binding sites in the preparations of hepatocytes used here indicate that the hormonal responsiveness of the cells has not been totally destroyed. In addition, the cells appear to have retained normal metabolic function in that leucine incorporation into trichloroacetic acid-precipitable protein was linear and inhibited by cycloheximide (Fig. 1 a) and transport of AIB into the hepatocytes also showed normal kinetics and saturability (Fig. 1 b). Metabolic tests of cell function were not reported by Ranke et al. (1976), although they do claim that 90-95 % of their cells excluded trypan blue. The presence of lactogen-binding sites and the retention of metabolic function by the iso¬ lated hepatocytes permits a direct study of the relationship between binding of lactogenic hormones and the biological response. In other words, a study of whether the binding sites are true biological receptors for the lactogenic hormones and are involved in the expression of metabolic effects. Prolactin does have metabolic actions in liver tissue in vivo, for example on RNA synthesis (Chen, Hamer, Heininger «fe Meier, 1972), on ornithine decarboxylase activity (Richards, 1975), and on somatomedin release (Francis «fe Hill, 1975). Therefore the hepatocyte should provide an ideal system for such a study. Unfortunately, the major aim of the present paper remained unfulfilled because of the failure to detect distinct GH sites. The experiments do emphasize, however, the potential value of purifying GH tracers by adsorption and elution from particulate binding sites before use of the tracer in receptor studies.
These studies were supported by a grant from the National Health and Medical Research Council. The authors express their thanks to Dr H. G. Burger for many helpful discussions. REFERENCES
Adamson, L. F., Herington, A. C. «ft Bomstein, J. (1972). Evidence for the selection of intracellular or extra¬ cellular amino acids for protein synthesis. Biochimica et Biophysica Acta 282, 352-365. Chen, H. W., Hamer, D. H., Heininger, H. «ft Meier, H. (1972). Stimulation of hepatic RNA synthesis in dwarf mice by ovine prolactin. Biochimica et Biophysica Acta 287, 90-97.
Clark, M. G., Filsell, O. H.
