Sex differences in binding of human growth hormone to isolated rat hepatocytes (hormone receptors/prolactin/estrogen effect)

Transcription

1 Proc. Nat. Acad. Sci USA Vol. 73, No. 3, pp , March 1976 Cell Biology Sex differences in binding of huan growth horone to isolated rat hepatocytes (horone receptors/prolactin/estrogen effect) MICHAEL B. RANKE, CHARLES A. STANLEY, DAVID RODBARD*, LESTER BAKER, ALFRED BONGIOVANNI, AND JOHN S. PARKS Departent of Endocrinology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 1914; and * Reproduction Research Branch, National Institute of Child Health and Huan Developent, National Institutes of Health, Bethesda, Maryland 214 Counicated by George B. Koelle, Deceber 19,1975 ABSTRACT Since liver is a target for growth horone action, binding of 125I-labeled huan growth horone to enyatically isolated rat hepatocytes was studied. Specific binding was shown with hepatocytes fro both ale and feale anials. There was a single class of receptors for huan growth horone on cells fro ales (affinity constant, Ka = 1.16 X 19 liters/ole; sites per cell, q = 62). In ales, bovine growth horone was alost as potent as huan growth horone in displacing bound 123I-labeled huan growth horone, while ovine prolactin was about 1 ties less potent. Cells fro feale rats bound ore 25I-labeled huan growth horone than cells fro ales. The cells fro feales contained at least two classes of receptors for huan growth horone. The receptor of highest affinity had the sae affinity for huan growth horone as the single receptor found in ales (Ka = 6 X 19 liters ole). However, there were three to four ties as any of these receptors per cell in feales (q = 21,). In feales, bovine growth horone and ovine prolactin were both about 2 ties less potent than huan growth horone. Treatent of ale rats with estrone produced cells that show the sae binding characteristics as feales. These results indicate that huan growth horone binds to a soatogenic receptor in hepatocytes fro ale rats. In feales and estrogen-treated ales, the receptors that bind huan growth horone recognie lactogenic as well as soatogenic properties. This suggests that the lactogenic and growth-prooting effects of huan growth horone in the rat are ediated by different receptors. The effects of growth horone on skeletal growth appear to be ediated by soatoedins (1). When 125I-labeled huan growth horone (hgh) is injected in rats in vivo, uch of the activity is localied in the liver (2). Soatoedin is produced when rat liver is perfused with growth horone (3, 4). These findings suggest that liver is a target organ for growth horone. The first step in the action of polypeptide horones is binding to specific receptors on the surface of target cells. Recently, binding of 125I-labeled hgh to cell ebranes prepared fro livers of rabbits, rats, and other species has been reported (5-8). Binding to rat liver ebranes could only be deonstrated using feale or estrogen-treated ale anials. In these ebrane preparations, ovine prolactin (oprl) was identical to hgh in displacing bound labeled hgh (8). It was therefore concluded that hgh binds to a lactogenic receptor in rat liver. The present study was carried out to exaine the binding characteristics of hgh to rat liver using enyatically isolated rat hepatocytes. Differences in ales and feales and ef- Abbreviations: hgh and bgh are, respectively, huan and bovine growth horone; oprl is ovine prolactin; ED5, dose at which displaceent is half-axial. fects of estrone treatent in ale anials on binding of 125I-labeled hgh were studied, leading to odels for growth horone receptors in the rat liver. MATERIALS AND METHODS Anials. Sprague-Dawley rats, obtained fro Charles River, Inc. and weighing g, were starved overnight prior to preparation of isolated hepatocytes. Estrone-treated ale rats were given 5.ug of estrone diluted in corn oil (8) by subcutaneous injection for 1 days. Hepatocytes. Suspensions of isolated hepatocytes were prepared by enyatic digestion of perfused liver with collagenase (Worthington, crude bacterial collagenase, Type I) using the ethod of Berry and Friend (9) odified by Krebs et al. (1). The yield of cells fro one 2-g rat was about 2 l of 1-15% (vol/vol) suspension or about 2 X 16 cells. The suspension consisted largely of single isolated cells with a few clups of 2 to 1 cells. Viability, as judged by exclusion of.4% Trypan blue, was 9-95%. For radioreceptor studies the cells were centrifuged and resuspended in Tris- HC1 buffer (ph 7.35), consisting of 25 M Tris-HCl, 12 M NaCl, 5 M KCl, 1.2 M MgSO4, 1 M EDTA, 1 M dextrose, 15 M Na-acetate, and.1% bovine seru albuin. Horones. Huan growth horone (NIH HS 1523D) was used for iodination. A preparation of hgh fro Kabi/ AS/Stockhol (lot no ) was used for unlabeled horone. This preparation was shown to be equal in potency to the NIH preparation by radioiunoassay as well as in a radioreceptor assay using cultured huan lyphoid cells (11). Other horones used were: bovine growth horone (bgh) (NIH GH-B ; 2 IU/g; lactogenic activity not specified); oprl (NIH PS 1; 25 IU/g; growth horone activity less than.1 IU/g); porcine insulin (Lilly, lot no ); porcine glucagon (Lilly, lot no B-167-1); and bovine parathorone (Dr. Howard Rasussen, PC ). All dilutions were ade in Tris- HCI buffer. lodination of hgh. 125I-Labeled hgh was prepared by a odification of the chloraine T ethod of Hunter and Greenwood (12). The reaction ixture consisted of 1 Ci of carrier-free Na'25I (Nuclear Chicago Corp.), 5,g of hgh,.1 l of 1. M Na2PO4 - buffer, ph 7.4, and 4,g of chloraine T in a total volue of.34 l. The reaction was stopped after 15 sec by addition of 8 ug of Na2S2O5 and.2 l of 5% bovine seru albuin solution. Labeled hgh was separated fro free 125I by gel filtration on Sephadex G-75 (colun sie:.5 X 12 c) using Tris-HCI buffer. The specific activity of the 125I-labeled hgh used was 12,Ci/,ug. Further purification of the labeled horone was achieved 847

2 848 Cell Biology: Ranke et al. Proc. Nat. Acad. Sci. USA 73 (1976) 2 6 x c 4 IL 2 w w e ' a r 1- : a w -j w 8- -L 6( - 4( - I B MINUTES Z 2' a-e a IT r 1 wlb ph x 1 O-6 CELLS/l FIG. 1. Effects of incubation conditions on binding of 1251-labeled hgh to isolated rat hepatocytes. (A) Effect of tie and teperature. The values depicted represent cobined data fro two experients with hepatocytes fro feale anials. Hepatocytes (2.2 and 5.3 X 16/l) were incubated with 125I-labeled hgh (.7 ng/l) with and without an excess of hgh (8,ug/l). The ordinate represents the aount of specifically bound labeled hgh as a percentage of the axial binding observed. (B) Effect of ph. Hepatocytes (2.2 X 16/l) fro a feale anial were incubated with 125I-labeled hgh (.7 ng/l) with and without an excess of hgh (8 Ag/l) for 12 in at 22. The ordinate represents the aount of specifically bound labeled hgh as a percentage of the axial binding observed. (C) Effect of cell concentration. Hepatocytes (2.3 to 11.8 X 16/l) of a ale anial were incubated with labeled hgh with and without an excess of hgh (8,g/l) for 12 in at 22. The ordinate represents the aount of labeled hgh specifically bound as a percentage of the total labeled hgh in the syste. by affinity chroatography. Aliquots of labeled horone (.5,ug) were applied to a sall colun (.3 X 2 c) containing anti-hgh antibody (Dr. M. McGillivray: R ) linked to Sepharose 4B. After 2 hr at roo teperature the colun was washed with 1 l of Tris.HCI buffer and the 125I-labeled hgh was eluted with 6 M guanidine- HGI,.2% bovine seru albuin, ph 3. Labeled horone in.5 l of this eluate was separated fro salts by gel filtration on Sephadex G-75, as above. Aliquots of the labeled horone purified weekly by this procedure allowed the use of aterial fro one iodination for 6 weeks. Incubation Procedure. Incubations were carried out in polypropylene tubes (12 X 75, Falcon no. 263). The incubation ixture consisted of.5 l of cell suspension,.5 l of 125I-labeled hgh containing approxiately.5 ng of hgh, and.5 l of unlabeled hgh, all in Tris-HCI buffer. The ixture was shaken for 15 in every 3 in. At the end of the incubation period, 3 l of cold Tris.HCI buffer were added and the cells were iediately sediented by centrifugation (4) at 15 X g for 4 in. After the supernatant was decanted, the tip of the tubes containing the cell pellet was cut with a raor blade; radioactivity bound to the pellet was easured in a gaa counter (Searle, odel 1185). Nonspecific binding was defined as counts bound in the C 32 oi 28 F 24 LL C 2 ui. I 16 a g hgh IN INCUBATION (ng/l) FIG. 2. Displaceent of 125I-labeled hgh fro isolated rat hepatocytes in a representative feale (), ale (), and estronetreated ale (X) anial. Hepatocytes (5 X 16/l in the feale; 8.3 X 16/l in the ale; and 4 X 16/l in the estrone-treated ale) were incubated with 1251-labeled hgh (.7 ng/l) for 12 in at 22. The ordinate represents the radioactivity bound to the hepatocytes as a percentage of the total in the syste. presence of excess hgh (5 jig per tube). Dose response curves were established in each experient using quadruplicate saples at each dose level of unlabeled horone. The values were corrected for nonspecific binding. Results were calculated using the "Radioiunoassay Data Processing Package" of Rodbard and Faden (13). RESULTS Optiiation of assay conditions As shown in Fig. 1, binding of 125I-labeled hgh to isolated rat hepatocytes was tie and teperature dependent. Maxiu specific binding at 15, 22, and 37 was reached by 12 in and reained stable for an additional 12 in. Specific binding was greatest at 22. Little variation in binding was noted between ph 7.1 and 7.6. Specific binding of labeled hgh was directly proportional to the cell concentration over a range of 2 to 12 X 16 cells per l. To test degradation of labeled horone during incubation with hepatocytes, identical aounts of 125I-labeled hgh were incubated with 5 X 16 hepatocytes and with equal volues of buffer alone for 12 in at 22. Aliquots of the supernatants were used for binding to fresh hepatocytes and an excess of anti-hgh antibody. After preincubation with hepatocytes, binding to fresh cells was 96% of the control and binding to anti-hgh antibody was 98% of the control. Displaceent of 125I-labeled hgh fro hepatocytes by unlabeled horones Hepatocytes fro ales, feales, and estrone-treated ales deonstrated specific binding of 1251-labeled hgh. As shown in Fig. 2, the initial binding was siilar in feales and estrone-treated ales, but higher than in ales. There was significant displaceent of bound '25I-labeled hgh by 2 ng/l (1.47 X 1-1 M) of unlabeled hgh. Porcine insulin, porcine glucagon, and bovine parathorone, at concentrations up to 1.7 jig/l, failed to displace bound labeled hgh.

3 Cell Biology: Ranke et al. Proc. Nat. Acad. Sci. USA 73 (1976) lo, HORMONE IN INCUBATION (ng/l) FIG. 3. Specificity of binding of 125I-labeled hgh to hepatocytes fro ale rats. The abscissa denotes the concentration of horone-hgh (), bgh (X), and oprl ()-in the incubation ixture. The ordinate represents the ratio of the aount of labeled hgh bound at a given horone concentration to the aount of labeled horone bound in absence of nonlabeled horone. The points for hgh represent ean values of six experients; for bgh and oprl the eans of two experients. The dose-response curves for hgh, bgh, and oprl using hepatocytes fro ales, feales, and estrogen-treated ales are shown in Figs. 3, 4, and 5, respectively. Both bgh (3 Ag/l) and oprl (177,ug/l) would copletely displace specifically bound 125I-labeled hgh in all three groups of anials. Coputer analysis of these results using a logit-logarith transforation showed that the curves for bgh and hgh were parallel in ales, but were not parallel in feales and estrogen-treated ales. The curves for oprl were not parallel to those for hgh in any of the three groups. This invalidated the use of parallel line potency estiates. Instead, potencies were expressed as dose levels resulting in halfaxial displaceent of bound 125I-labeled hgh (ED5o). As shown in Table 1, the ED5 for hgh was lower in ales (22 ng/l) than in feales (4 ng/l, P <.1). Males and feales also differed in the potencies of bgh (48 copared to 99 ng/l) and of oprl (32, copared to 75 1.'.8.7' HORMONE IN INCUBATION (ng/l) FIG. 4. Specificity of binding of 125I-labeled hgh to hepatocytes fro feale rats. The abscissa denotes the concentration of horone-hgh (-), bgh (X), and oprl ()-in the incubation ixture. The ordinate represents the ratio of the aount of labeled hgh bound at a given horone concentration to the aount of labeled horone bound in absence of nonlabeled horone. The points for hgh represent ean values of eight experients; for bgh and oprl the eans of four experients i HORMONEIN INCUBATION (ng/l) FIG. 5. Specificity of binding of 1251-labeled hgh to hepatocytes fro estrone-treated ale rats. The abscissa denotes the concentration of horone-hgh (-), bgh (X), and oprl ()-in the incubation ixture. The ordinate represents the ratio of the aount for labeled hgh bound at a given horone concentration to the aount for labeled horone bound in absence of nonlabeled horone. The points of hgh represent ean values of four experients; for bgh and oprl, the values fro one experient. ng/l). In estrone-treated ales, the potencies of the three horones were siilar to those found in feales. Scatchard plot analysis Shown in Fig. 6 are Scatchard plots (14) for hgh binding to hepatocytes fro a ale, a feale, and an estrone-treated ale rat. The plot is linear in the ale, indicating the presence of a single class of receptors. In the feales and estrone-treated ales, curvature of the plots indicates the presence of two or ore classes of receptors. The Scatchard plots fro each of the experients in six ales, eight feales, and four estrone-teated ales were calculated and analyed using the coputer progra previously described (13). This progra perits a choice, based on residual variance and objective F-test, aong three odels for the best fit of the data: a two-paraeter odel corresponding to a single class of noninteracting binding sites; a three-paraeter odel corresponding to one class of satura- Table 1. Relative potency of hgh, bgh, and oprl in copeting with 1251-labeled hgh for binding to isolated rat hepatocytes: Dose at which displaceent is half-axial (ED5)* hgh bgh oprl Male 22.35t , ( ) (4.5; 6.3) (26,; 4,) (7) (2) (2) Feale 39.73t ( ) (25-388) (17-325) (8) (4) (4) Estrone t treated ( ) ales (4) (1) (1) * Mean (ng/l); in parentheses, 95% liits and nuber of experients. Means and 95% confidence liits calculated fro logariths to approxiate a noral distribution. t For hgh: ale copared to feale, P <.1; ale copared to estrogen-treated ales, not significant; feale copared to estrogen-treated ales, not significant.

4 85 Cell Biology: Ranke et al.,-..12, TOA.OROEBON 1' OLLTR TOTAL HORMONE BOUND (i` MOL/LITER) FIG. 6. Scatchard plot of data fro dose response curves for hgh using hepatocytes fro a representative feale (), ale (), and estrone-treated ale (X) anial. The concentrations of hepatocytes in the incubation ixtures were 2.1 X 16/l in the feale, 8.2 X 16/l in the ale, and 6.2 X 16/l in the estronetreated ale anial. The ordinate depicts the ratio of bound to free horone, and the abscissa the total aount of horone bound to the hepatocytes. ble binding sites in the presence of a second class of binding sites with "infinite" capacity but infinitesial affinity; and a four-paraeter odel corresponding to the presence of two separate classes of saturable sites. The first odel fits a straight line Scatchard plot, while the second two fit curved lines. In all of the six experients using ales, the two-paraeter odel gave the best fit. In contrast, for seven of the eight feales and three of the four estrone-treated ales, a three-paraeter odel proved a significantly better fit (P <.5). This suggests the presence of a single class of binding sites for hgh in ales and ore than one class of binding sites in feales and estrone-treated ales. The affinity constants (K) and binding capacities (q) of the priary (high affinity) binding sites for hgh are shown in Table 2. The affinity constant of the single class of receptors in ales (1.2 X 19 liters/ole) was the sae as that of the priary class of receptors in feales (6 X 19 liters/ ole) and estrone-treated ales (1.4 X 19 liters/ole). The nuber of these receptors per cell was about four ties higher in feales (21,) or in estrone-treated ales (26,) than in ales (62). DISCUSSION The binding of 125I-labeled hgh to isolated hepatocytes is copatible with the presence of physiologically iportant horone receptors on the surface of liver cells. Dose-response curves for hgh in ales, feales, and estrone-treated ales show significant displaceent of '25I-labeled hgh at physiologic dose levels. The affinity constants and nuber of binding sites per cell in all three groups of anials are of Proc. Nat. Acad. Sci. USA 73 (1976) Table 2. Affinity constants (K) and nuber of sites* for binding of hgh to isolated rat hepatocytes No. of K x 1-9 expts. (liters/ol) Sites/cell Male t 62t ( ) (46-82) Feale 8 6t 21,t ( ) (1,-42,) Estrone-treated t 26,t ales ( ) (6-11,) * Mean and 95% liits are given. Mean and 95% confidence liits calculated fro logariths to approxiate a noral distribution. t Differences between groups, not significant. t Males copared to feales, P <.5; ales copared to estrone-treated ales, P <.5; feales copared to estronetreated ales, not significant. the sae order of agnitude found for other polypeptide horone receptors (11, 15). Hepatocytes prepared fro ale and feale rats show differences in binding of 125I-labeled hgh. Linearity of the Scatchard plots of hgh binding to ale heptocytes indicates the presence of a single class of receptors. In rodent bioassays, hgh has been shown to have both growth-prooting (soatogenic) (16) and lactogenic (17) effects. Bovine growth horone has soatogenic (17) but not lactogenic effects and oprl has only lactogenic effects (18). Binding of bgh to hepatocytes fro ale rats is nearly identical to binding of hgh, as indicated by siilar potency estiates and parallel dose-response curves for the two horones. In contrast, oprl is 1-fold less potent than hgh. Displaceent of 125I-labeled hgh by bgh but not oprl at physiologic concentrations suggests that ebrane receptors on hepatocytes fro ale rats recognie only the soatogenic properties of hgh. The interactions of hgh with hepatocytes fro feale rats are considerably ore coplex. Total binding of hgh is greater than to ale hepatocytes. Scatchard plots of hgh binding are nonlinear. This indicates either the presence of two or ore classes of receptors with different affinities for hgh or a negative cooperative interaction of the horone with its receptor, such as has been shown for insulin binding to lyphocytes (19). If the forer interpretation is correct, the higher affinity site has the sae affinity constant for hgh as the single site in ales. Estiates of the nuber of such receptors per cell are higher than for ales. The nuber and affinity of the secondary class of receptors for hgh cannot be estiated fro the present data. With hepatocytes fro feale rats, bgh and oprl are approxiately equipotent in displacing bound 125I-labeled hgh. In contrast to results using ale cells, the ED5 of bgh is 2-fold greater than that of hgh and the displaceent curves are not parallel. Ovine prolactin is uch ore potent in displacing 125I-labeled hgh fro feale than fro ale cells. However, the ED5 of ovine prolactin is also 2-fold greater than that of hgh and the displaceent curves are not parallel. Since physiologic concentrations of either bgh or oprl give partial displaceent of 125I-labeled hgh, the binding of hgh in feales appears to cobine both soatogenic and lactogenic properties. The characteristics of 125I-labeled hgh binding to hepatocytes fro feale rats suggest the presence of two distinct classes of binding sites with differing affinities and specifici-

5 Cell Biology: Ranke et al. ties. In this odel, the first site has a higher affinity for hgh, recognies the soatogenic properties of hgh, andl recognies the soatogenic horone, bgh. This site ay be identical to the receptor found in ales. The second site has a lower affinity for hgh, recognies the lactogenic properties of hgh, and recognies the lactogenic horone, oprl. This site is lacking in ales but can be induced by estrogen treatent. The fact that oprl at high concentrations copletely displaces '25I-labeled hgh in feales, as in ales, suggests soe weak interaction with the first site. This ay represent trace containation of the oprl or weak intrinsic activity of the oprl olecule. A siilar weak interaction of bgh with the second site is suggested by the fact that bgh at high concentrations also copletely displaces 125I-labeled hgh in feales. While the two-site odel is consistent with the experiental data, validation of the odel and full characteriation of the postulated second class of receptors will require experients using labeled oprl. The postulated second site for hgh binding to hepatocytes fro feale and estrogen-treated ale rats ay be identical to the "lactogenic" receptor reported by Posner et al. (8) for rat liver ebranes. However, liver ebranes fro untreated ale rats did not bind '25I-labeled hgh and there was no evidence for a separate site with soatogenic specificity in liver ebranes fro feale rats. The deonstration of "soatogenic" binding sites in rat hepatocytes is consistent with the hypothesis that the liver is a target organ for the growth prooting properties of growth horones and suggests that the lactogenic and soatogenic effects of hgh in rats are ediated by different receptors. We thank the National Institute of Arthritis, Metabolis, and Digestive Diseases for providing growth horones and prolactin; Kabi/AS/Stockhol for hgh; Dr. M. McGillivray, Buffalo, N.Y., for anti-hgh antibody; Dr. H. Rasussen, Philadelphia, Pa., for bovine parathorone; and Dr. J. Galloway, Eli Lilly Laboratories, for insulin and glucagon. The assistance of Martin Z. Weingarten and Dr. Charlotte H. Greene is greatly appreciated. This research was supported by the following grants: Deutsche Forschungsgeeinschaft Ra 232/1-2 (M.R.), USPHS Training Grant no. 259 (C.S.), Proc. Nat. Acad. Sci. USA 73 (1976) 851 RCDA 1K4-AM 5 (J.S.P.), AM 13518, HD 215, HD 371, and GM Van Wyk, J. J., Underwood, L. E., Hint, R. L., Cleons, D. R., Voina, S. J. & Weaver, R. R. (1974) Recent Prog. Hor. Res. 3, Mayberry, H. E., Van den Brande, J. L., Van Wyk, J. J. & Waddell, W. J. (1971) Endocrinology 88, McConaghey, P. & Sledge, C. B. (197) Nature 225, McConaghey, P. & Dehel, J. (1972) J. Endocrinol. 52, Tsushia, T. & Friesen, H. G. (1973) J. Clin. Endocrinol. Metab. 37, Posner, B. I., Kelly, P. A., Shiu, R. P. C. & Friesen, H. G. (1974) Endocrinology 95, Kelly, P. A., Posner, B. I., Tsushia, T. & Friesen, H. G. (1974) Endocrinology 96, Posner, B. I., Kelly, P. A., & Friesen, H. G. (1974) Proc. Nat. Acad. Sci. USA 71, Berry, M. N. & Friend, D. S. (1969) J. Cell Biol. 43, Krebs, H. A., Cornell, N. W., Lund, P. & Hes, R. (1974) in Alfred Benon Syposiu VI, eds., Lundquist, F. & Tygstrup, N. (Acadeic Press, New York), pp Lesniak, M. A., Gorden, P., Roth, J. & Gavin, J. R., III (1974) J. Biol. Che. 249, Hunter, W. M. & Greenwood, F. G. (1962) Nature 194, Rodbard, D. & Faden, V. B. (1975) Radioiunoassay Data Processing Report PB (National Technical Inforation Service, Springfield, Va.), 3rd ed. 14. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51, Gavin, J. R., III, Gorden, P., Roth, J., Archer, J. A. & Buell, D. N. (1973) J. Biol. Che. 248, Evans, H. M., Sipson, M. E., Marx, W. & Kibrick, E. (1943) Endocrinology 32, Kleinberg, D. L. & Frant, A. G. (1971) J. Clin. Invest. 5, Li, C. H. (1972) "Lactogenic horones," in Ciba Foundation Syposiu, eds. Wolstenhole, G. E. W. & Knight, J. (Churchhill Livingstone, London), p DeMeyts, P., Roth, J., Neville, D. M., Jr., Gavin, J. R., III & Lesniak, M. A. (1973) Bioche. Biophys. Res. Coun. 55,