GENERAL
AND
COMPARATIVE
Hepatic
ENDOCRINOLOGY
Receptors
81, 383-390 (1991)
for Homologous TETSUYA
Ocean
Research
Institute,
University
Growth
Hormone
in the Eel
HIRANO of Tokyo,
Nakano,
Tokyo
164, Japan
Accepted April 5, 1990 The specific binding of ‘2sI-labeled eel growth hormone (eGH) to liver membranes of the eel was examined. The specific binding to the lO,OOOgpellet was greater than that to the 600s pellet. The specific binding was linear up to about 100 mg fresh tissue, and was saturable with increasing amounts of membrane. The specific binding was pH-, temperature-, and time-dependent, with the optimum pH at 7.4, and greater specific binding was obtained at 15 and 25” than at 35”. Scatchard analysis of liver binding gave an association constant of 1.1 x lo9 M-’ and a capacity of 105 fmol/mg protein. The receptor preparation was highly specific for GHs. Natural and recombinant eel GHs as well as recombinant salmon GH competed equally with lz51-eGH for the receptor sites of the 10,OOOgliver membrane. Ovine GH was more potent in displacing the labeled eGH than the homologous eel hormone. Tilapia GH and ovine prolactin (PRL) were needed in greater amounts (40 times) than eGH to displace the labeled eGH. Salmon and tilapia PRLs were still less potent (500 times) than eGH. There was no displacement with eel PRL. No significant change in the specific binding was seen 1 week after hypophysectomy, whereas injection of eGH into the hypophysectomized eel caused a significant reduction after 24 hr. The binding to the membrane fractions from gills, kidney, muscle, intestine, and brain was low and exclusively nonspecific, indicating the presence of specific GH receptors predominantly in the liver. 0 1991 Academic Press, Inc.
It is well established that the mammalian with insulin, glucagon, prolactin (PRL), liver is a major target organ for growth hor- and EGF (Hayashi and Ooshiro, 1986). Remone (GH), some of whose effects are me- cently, Duan and Inui (1990) have demondiated by somatomedins produced mainly 1 strated the indirect action of GH through a in the liver. Receptors for GH have been somatomedin-like factor(s) on the synthesis detected on hepatocyte membranes from of mucopolysaccharides by the ceratobranseveral mammalian species and have chial cartilages of the eel. formed the bases for radioreceptor assay The basic properties of hepatic receptors (see Hughes et al., 1985; Nicoll et al., of lower vertebrates have been studied us1986). In contrast, studies on the hepatic ing mammalian GH as radioligand (see actions of GH in nonmammalian verteNicoll et al., 1986). However, interpretabrates are sparse, especially in teleost tion of data derived from heterologous sysfishes. McKeown et al., (1975) reported an terns is complex, owing to the overlapping increase in liver glycogen after injections of activities of GH and PRL (Nicoll, 1982; ovine GH in kokanee salmon. In the eel, Bern, 1983). With the use of a homologous Inui and Ishioka (1985) reported stimularadioreceptor assay one can study the regtory effects, both in vivo and in vitro, of ulation of GH-specific receptors in target ovine GH on incorporation of [14C]leucine organs in various physiological states. In into the liver protein. Growth hormone teleosts, Fryer (1979) was the first to deseems to be necessary to stimulate cell pro- scribe a radioreceptor assay for teleost GH, liferation of primary culture of the eel he- using a highly purified tilapia GH in a conpatocytes in serum-free medium, together specific assay system. The tilapia GH re383 0016~6480/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
384
TETSUYA
ceptors are highly hormone specific; they bind only tilapia GH and were inhibited only by large amounts of human, bovine, or ovine GHs and ovine PRL. Tarpey and Nicoll (1985) characterized hepatic GHbinding sites in a goby and a sturgeon using 1251-bovine GH, and suggested considerable evolutionary divergence among hepatic GH receptors in teleosts. Recently, a homologous radioreceptor assay for GH has also been reported in rainbow trout and coho salmon (Yao et al., 1990; Gray et al., 1990).
In our previous studies, two forms of GH have been isolated from the medium of organ-cultured eel pituitaries: eGH I is different from eGH II by three extra amino acids, Val-Glu-Pro, at the N terminal (Kishida et al., 1987; Yamaguchi et al., 1987). Recombinant eel GH (eGH I) has also become available (Saito et al., 1988). This study was undertaken to characterized the GH receptor in the eel liver using homologous eel GH. A preliminary account of parts of this work has been presented (Hirano and Kishida, 1989). MATERIALS
AND METHODS
Eels. Sexually immature cultured eels (Anguih juponica) were obtained from a commercial source. They were kept in a freshwater aquarium at 20” without food. Hypophysectomy was carried out as described by Inui er al. (1985). After anesthesia with triCaine methane sulfonate (Sigma), liver and other tissues (gills, kidney, muscle, intestine, and brain) were removed and frozen at - 80”. Hormones. Eel GH (eGH I) was purified from the culture medium of the pituitary of the Japanese eel as described by Kishida er al. (1987). Eel PRL was puritied from the eel pituitary in our laboratory (Suzuki er al., 1988). Recombinant eel and salmon GHs were supplied by the Tokyo Research Laboratories of Kyowa Hakko Kogyo Co. Ovine GH (NIAMDDK-oGH-S12) and PRL (NIAMDDK-oPRL-14) were supplied by the National Institutes of Health (Bethesda, MD). PuriIIed chum salmon PRL (Kawauchi et al., 1983) was provided by Professor H. Kawauchi (School of Fisheries Sciences, Kitasato University), and tilapia GH and PRL (Specker et al., 1985) was provided by Professor H. A. Bern (Department of Integrative Biology, University of California, Berkeley).
HIRANO
Receptor preparation. Frozen or fresh livers were minced and then homogenized in 10 Vol (w/v) 40 mM Tris-HCl buffer (pH 7.4), containing 10 mM CaClr, 1 n&f phenylmethylsulfonyl fluoride (PMSF), and 10 ug/ml Pepstatin A (Sigma), using a Polytron homogenizer at a setting of 5 for two periods of 10 set each with an interval of 10 set for cooling. The tissue flasks were kept in an ice bath during all procedures. The homogenates were subsequently centrifuged at 6OOg for 10 min at 4”. The supematants were collected and centrifuged at 10,OOOgfor 20 min at 4”. The resulting pellets were washed twice with 5 Vol Tris buffer and suspended with a Teflon-glass homogenizer at 1 g starting tissue weight per milliliter of buffer. The suspensions were stored at -80”. Just before use, the frozen suspensions were thawed and diluted with the extraction buffer to appropriate concentrations. An aliquot was taken to determine protein content by the Bio-Rad protein assay, using bovine serum albumin (BSA, Fraction V, Sigma) as a standard. Gill tilaments, kidney, dorsal muscle, intestine, and brain were collected from freshwater-adapted eels and treated similarly. Binding experiments. The determination of GH receptors was carried out essentially following the procedures developed for gonadotropin receptors in lower vertebrates by Kubokawa and Ishii (1987). All solutions were made with 40 mM Tris buffer containing 10 mM CaCI, and 0.5% BSA. Eel GH (eGH I or recombinant eGH) was iodinated by the lactoperoxidase method as described previously (Kishida and Hirano, 1988). Tris-HCl buffer, instead of phosphate buffer, was used in separating the labeled hormone from free iodine. Labeled hormones were stored at - 20”. They were thawed and appropriately diluted with Tris-HCl buffer containing 0.5% BSA just before use. In Scatchard plot analysis, the specific radioactivity of the labeled eGH was estimated from data of the competition and saturation experiments with the same radioligand batch, with the aid of a microcomputer using a program of Ishii and Kubokawa (unpublished). Specific binding was estimated by incubation of the receptor preparation derived from 30 to 50 mg of the original tissue in 100 ~1 buffer with 10,000 to 20,000 cpm (about 0.5-l ng) of ‘*‘I-eGH in 50 pl buffer, in the presence or absence of an excess amount (1 ug in 50 ~1 buffer) of unlabeled recombinant eGH. The total volume of the reaction mixture was 200 ~1. Plastic sample tubes were used for incubation. They were tightly capped, placed almost horizontally in a water bath, and incubated with shaking (120 strokes/min) for 18-20 hr at 1s”. Incubation was terminated by the addition of 1 ml ice-cold buffer followed by centrifugation at lO,OOOgfor 2 mitt at 4”. The supematant was aspirated and the pellet was washed once with 1 ml chilled buffer. The radioactivity of the resultant pellet was counted in an automatic gamma counter.
HEPATIC
Statistics.
the Duncan’s
GH
RECEPTORS
Statistical significance was examined by new multiple range test (Duncan, 1955).
RESULTS
Optimal
Conditions
for the Binding Assay
As shown in Fig. 1, the specific binding of 1251-eGH to 10,OOOg liver membranes was greater than that to the 600g pellet. The specific binding to 10,OOOg membranes increased linearly up to about 100 mg fresh tissue, and increased further only slightly when membranes from 200 mg tissue were added. The receptor preparations were incubated at 25” for 4 hr in this experiment. When membranes from 50 mg tissue were used, the specific binding was 13% of total radioactivity and nonspecific binding was 2%. Further experiments utilized the 10,OOOg membranes derived from 30 to 50 mg fresh tissue (about 250 to 300 kg protein). 30 1
10,000
g Pellet
IN THE
385
EEL
The specific binding was dependent on both duration and temperature of incubation (Fig. 2). The specific binding at IS” increased over time from 2% after 1 hr to 11% after 12 hr, where it plateaued until 20 hr. The binding at 25” increased from 5% after 1 hr to 12% after 6 hr, and then reached a steady state. At 35”, the specific binding increased linearly to 5% during the first 2 hr, but declined gradually thereafter. Thus, the membranes were incubated at 15” for 18 to 20 hr in subsequent experiments. The specific binding was dependent also on the pH of the incubation medium (Fig. 3). It increased from 0 at pH 5.0 to a maximum of 14% at pH 7.4, and then decreased gradually at higher pH. In subsequent experiments, the membranes were incubated at pH 7.5. Receptor
Specificity
The binding of ‘*‘I-eGH to specific organs was examined using receptor preparations of various tissues (30 mg) from freshwater-adapted eels (Fig. 4). As compared with the binding to the liver membranes, the binding to the membranes from gills, kidney, muscle, and intestine was low and exclusively nonspecific. The membrane fractions of brain showed binding as high as in the liver, but no significant specific binding was detected.
20 t
”
600
g Petlet
50 Tissue
100 concentration
150 (mg1100
200 pl)
FIG. 1. Specific and nonspecific binding of ‘*‘I-eGH to membranes of freshwater eel liver (30 mg fresh tissue/tube) prepared by centrifugation at 10,OOOg or to membranes centrifuged at 600s. The receptor preparations were incubated at 25” for 4 hr. Each point is the mean of duplicate determinations.
10
5 Time
15
PO
(h)
FIG. 2. Effects of temperature and time of incubation on specific binding of **%eGH on hepatic recep tors of the freshwater eel (10,OOOg membranes derived from 30 mg fresh tissue/tube).
386
TETSUYA
HIRANO
4
I
0.4
old. 5
6
a2
25
100
400
nQ honnoneIMe
a
7
1s
9
PH
FIG. 3. The influence of pH on specific binding of ‘*‘I-eGH. The receptor preparations from 30 mg freshwater eel liver were incubated at 1s” for 20 hr. The buffers used were 40 mM acetate, pH 5.0-6.0; 40 mM phosphate, pH 6.5; 40 m&f Tris, pH 7.0-9.0. Each buffer contained 10 mM CaCl, and 0.5% BSA. One milliliter ice-cold buffer of the same pH was added to terminate the incubation.
The abilities of various GHs and PRLs to compete with 1251-eGH for the receptor sites of the 10,OOOg liver membranes were examined. The receptor did not distinguish between natural and recombinant eGHs (Fig. 5). Exactly the same displacement curve was obtained when recombinant eGH was used as a labeled hormone (data not shown). As shown in Fig. 6, recombinant salmon GH competed equally with ‘*‘I-eGH. Ovine GH was more potent than the homologous eel hormones. Greater amounts (40 times
FIG. 5. Specificity of binding of ‘*‘I-eGH to freshwater eel liver membranes (30 mg fresh tissue/tube). reGH, recombinant eGH.
more than eGH) of tilapia GH and ovine PRL were needed to displace the labeled eGH. Salmon and tilapia PRLs were still less potent (by 500 times) than eGH. There was no displacement with eel PRL. Equilibrium Association Constants and Concentration of eGH Binding Sites in the Eel Liver The specific binding of labeled eGH to liver membranes was saturable with increasing amounts of label. The Scatchard plot analysis revealed a single class of highaffinity binding sites (Fig. 7). The equilibrium constant of association for specific binding was 1.07 _+0.051 x lo9 M-’ (mean
15
5
ng hormone 0
liver
gills
kiy
msde
tiestine
brain
FIG. 4. Total (clear bars) and nonspecific (hatched bars) binding of ‘*‘I-eGH to crude membrane fractions of various organs of the freshwater eel. The receptor preparations derived from 30 mg tissue were incubated at 15” for 20 hr.
I tube
FIG. 6. Specilicity of binding of “‘I-eGH to freshwater eel liver membranes (30 mg fresh tissue/tube). oGH and oPRL, ovine GH and PRL; rsGH, recombinant chum salmon GH; sPRL, chum salmon PRL; tGH and tPRL, tilapia GH and PRL (20 K); ePRL, eel PRL.
HEPATIC
P *Et
GH
RECEPTORS
IN
THE
387
EEL
3
b x
-2 .-e P a g ‘5 m ::
r
1
/C
2
4 Free
6 6 ( x lO~‘cpm)
10
12
x rL Intact + sahe
HY~
HYF”
sahe
r&-l
8. Effects of hypophysectomy on specific binding of “‘1-eGH to liver membranes of the eel. Hepatic receptors (30 mg fresh tissue/tube) were prepared from freshwater eels 1 week after hypophysectomy or from hypophysectomized eels which received two daily injections of recombinant eGH (reGH, 2 &g) and were sacrificed 24 hr after the last injection. Membranes were also prepared from intact eels of the same lot sacrificed at the same time. Vertical bars represent means 5 SEM (n = 5). *Significantly (P < 0.05) different from intact fish and also from hypophysectomized fish (Hypox) receiving saline. FIG.
0
I
\ 0.05 Bound
0.10
0.15
(nM)
FIG. 7. Effect of concentration cific binding to liver membranes (upper), and Scatchard plot of (lower). Straight line was fitted method.
of ‘*‘I-eGH on speof the freshwater eel the specific binding by the least-squares
+ SEM of four independent determinations). The capacity was 0.56 + 0.078 pmol/ g tissue or 105 * 6.5 fmol/mg protein. Effect of Hypophysectomy and Seawater Transfer on SpeciJic Binding to Hepatic Receptor When freshwater eels fasted for about 2 weeks were hypophysectomized, no significant change was seen in the specific binding after 1 week. On the other hand, hypophysectomized eels receiving daily injections of recombinant eGH (2 pg/g) for 2 days and sacrificed 24 hr after the last injection showed significantly (P < 0.05) less specific binding than intact eels or hypophysectomized eels receiving the vehicle injection (Fig. 8).
DISCUSSION
In the present study, eGH bound specifically to crude membrane fractions of the eel liver. The binding to the membrane fractions from gills, kidney, muscle, intestine, and brain was minimal and exclusively nonspecific, indicating the presence of specific GH receptors predominantly in the liver. The hepatic GH receptors have the general characteristics of peptide hormone membrane receptors. The specific binding was saturable with increasing amounts of membrane as well as of radioligand. As shown by Scatchard plot analysis, the hepatic binding sites are of high affinity and limited number. The values of equilibrium parameters were similar to those reported for the hepatic GH receptors of tilapia (Fryer, 1979), goby and sturgeon (Tarpey and Nicoll, 1985), and of other vertebrate species (see Nicoll et al., 1986).
388
TETSUYA
The specific binding was pH-, temperature-, and time-dependent, with the optimum pH at 7.4, and greater specific binding at 15 and 25” than at 35”. The decline in binding after 2 hr at 35” suggests that denaturation and/or degradation of the receptor protein had occurred. This is in accord with the fact that eels are cultured at 20-25”, but are difficult to maintain at above 30”. Tarpey and Nicoll (1985) also noted a decrease in the specific binding of bovine GH to the goby liver membranes 8 hr after incubation at 37”. Similarly, amphibian gonadal receptors for gonadotropins have optimum equilibrium at about IS” (Kubokawa and Ishii, 1984). High hormone specificity is also an important characteristic of hormone receptors. According to Fryer (1979), the tilapia hepatic GH receptors are narrowly hormone specific, binding only to tilapia GH. In contrast, Tarpey and Nicoll(1985) found that human and bovine GHs are equipotent in inhibiting the binding of labeled bovine GH to goby hepatic membranes. The hepatic GH receptors of the eel also exhibited high hormone specificity. Natural and recombinant eGHs as well as recombinant salmon GH competed equally with 12?eGH (either natural or recombinant) for the receptor sites. The fact that recombinant eGH was equipotent to pituitary eGH in inhibiting binding of 12’I-eGH suggests that the recombinant hormone is equivalent in biological potency. In hormone-receptor interactions, the affinity of receptor for growth hormone has generally correlated with the established biological potency of the hormone. Exceptions have occurred most often when labeled hormones and receptors were derived from different species, i.e., in heterologous assays (see Hughes et al., 1985). The present observation that ovine GH was more potent in displacing the labeled eGH than the homologous hormone is in accord with the fact that ovine GH was more potent than eGH in stimulating the incorpora-
HIRANO
tion of 35S-sulfate into branchial cartilages of the eel in vitro (Duan and Inui, unpublished observation). It is not known if ovine GH stimulates growth of the eel, because they do not feed under laboratory conditions (Kishida et al., 1987). However, it is inexplicable why tilapia GH was less potent than eGH, or why ovine PRL was as potent as tilapia GH. Ovine PRL has been shown to have GH-like activity in amphibian larvae, birds, and dwarf mice (see Nicoll, 1982; Bern, 1983). According to Nicoll et al. (1986), there is greater similarity among PRL receptors than among GH receptors but the converse is true for the hormones themselves. At any rate, there was no displacement of the labeled eGH with eel PRL, indicating the highly specific nature of the hepatic GH receptor for endogenous GH as compared with endogenous PRL. In mammals, concentrations of the specific receptors for GH on target cells are known to be affected by the hormone concentration. GH receptors acutely decrease (down-regulation) after short-term exposure to GH, whereas prolonged treatment with GH increases GH-binding sites (upregulation) in liver of various species (see Hughes et al., 1985; Maiter et al., 1988a, b). Posner et al. (1980) reported a decrease in hepatic GH receptors in rabbits and sheep 20-30 days after hypophysectomy. In the present study, no significant change was seen in hepatic GH receptors of the eel 1 week after hypophysectomy, whereas treatment of the hypophysectomized eel with two daily injections of eGH resulted in a significant reduction 24 hr after the last injection, suggesting the occurrence of down-regulation also in the eel. According to Maiter et al. (1988a, b), a single injection of GH in hypophysectomized rats induced a transient decrease in the GH receptors, whereas continuous administration increased the receptors. The time courses of changes in the receptors after various treatments should be examined in various physiological states, in order to clarify whether
HEPATIC
down- and up-regulation also occurs in eels.
of GH receptors
ACKNOWLEDGMENTS I am greatly indebted to Professor Susumu Ishii and Dr. Kaoru Kubokawa, School of Education, Waseda University, for their invaluable suggestions and encouragement during the course of this study. I gratefully acknowledge the gifts of hormones from Professor Howard A. Bern, Professor Hiroshi Kawauchi, the NIADDK Hormone Distribution Program, and Tokyo Research Laboratories of Kyowa Hakko Kogyo Co., Ltd. Thanks are also due to Dr. Tsuyoshi Ogasawara, Mitsuyo Kishida, Sanae Hasegawa, and Tatsuya Sakamoto, Ocean Research Institute, University of Tokyo, for their help. I am also grateful to Professor Howard A. Bern for a critical reading of the manuscript. This work was supported in part by grants-in-aid from the Fisheries Agency and the Ministry of Education, Japan.
REFERENCES Bern, H. A. (1983). Functional evolution of prolactin and growth hormone in lower vertebrates. Amer. 2001. 23, 663-671. Duan, C., and Inui, Y. (1990). Evidence for the presence of a somatomedin-like plasma factor(s) in the Japanese eel, Anguilla japonica. Gen. Comp. Endocrinol,
79, 326-331.
Duncan, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, 142. Fryer, J. N. (1979). A radioreceptor assay for purified teleost growth hormone. Gen. Comp. Endocrinol. 39, 123-130. Gray, E. S., Young, G., and Bern, H. A. (1990). Radioreceptor assay for growth hormone in coho salmon (Oncorhynchus kisutch) and its application to the study of stunting. .Z. Exp. Zool., in press. Hayashi, S., and Ooshiro, Z. (1986). Primary culture of the eel hepatocytes in the serum-free medium. Bull. Japan. Sot. Sci. Fish. 52, 1641-1651. Hirano, T., and Kishida, M. (1989). Growth hormone receptors in the eel liver. Proc. Znt. Symp. Comp. Endocrinol., 11th p. 160. [Abstract] Hughes, J. P., Elsholtz, H. P., and Friesen, H. G. (1985). Growth hormone and prolactin receptors. In “Polypeptide Hormone Receptors” (B. I. Posner, Ed.). pp. 157-199. Dekker, New York. Inui, Y., and Ishioka, H. (1985). In vivo and in vitro effect of growth hormone on the incorporation of [i4C]leucine into protein of liver and muscle of the eel. Gen.
Camp.
Endocrinol.
59, 295-300.
389
GH RECEPTORS IN THE EEL
Inui, Y., Miwa, S., and Ishioka, H. (1985). Effect of mammalian growth hormone on amino nitrogen mobilization in the eel. Gen. Comp. Endocrinol. 59, 287-294. Kawauchi, H., Abe, K., Takahashi, A., Hirano, T., Hasegawa, S., Naito, N., and Nakai, Y. (1983). Isolation and properties of chum salmon prolactin. Gen. Comp. Endocrinol. 49, 446-458. Kishida, M., and Hirano, T. (1988). Development of radioimmunoassay for eel growth hormone. Nippon Suisan Gakkaishi 54, 1321-1327. Kishida, M., Hirano, T., Kubota, J., Hasegawa, S., Kawauchi, H., Yamaguchi, K., and Shirahata, K. (1987). Isolation of two forms of growth hormone secreted from eel pituitaries in vitro. Gen. Comp. Endocrinol.
65, 478-488.
Kubokawa, K., and Ishii, S. (1984). Adaptation of testicular follicle-stimulating hormone receptors to ambient temperatures in vertebrates: Equilibrium analysis. Gen. Comp. Endocrinol. 54, 277-282. Kubokawa, K., and Ishii, S. (1987). Receptors for native gonadotropins in amphibian liver. Gen. Comp.
Endocrinol.
68, 260-270.
McKeown, B. A., Leatherland, J. F., and John, T. M. (1975). The effect of growth hormone and prolactin on the mobilization of free fatty acids and glucose in the kokanee salmon, Oncorhynchus nerka.
Comp.
Physiol.
Biochem.
B 50, 425-430.
Maiter, D., Underwood, L. E., Maes, M., and Ketelslegers, J. M. (1988a). Acute down-regulation of the somatogenic receptors in rat liver by a single injection of growth hormone. Endocrinology 122, 1291-1296. Maiter, D., Underwood, L. E., Maes, M., Davenport, L., and Ketelslegers, J. M. (1988b). Different effects of intermittent and continuous growth hormone (GH) administration on serum somatomedinC/insulin-like growth factor I and liver GH receptors in hypophysectomized rats. Endocrinology 123, 1053-1059. Nicoll, C. S. (1982). Prolactin and growth hormone: Specialists on one hand and mutual mimics on the other. Perspect. Biol. Med. 25, 369-381. Nicoll, C. S., Tarpey, J. F., Mayer, G. L., and Russel, S. M. (1986). Similarities and differences among prolactins and growth hormones and their receptors. Amer. Zool. 26, %5-983. Posner, B. I., Patel, B., Vezinhet, A., and Charrier, J. (1980). Pituitary-dependent growth hormone receptors in rabbit and sheep liver. Endocrinology 107, 1954-1958. Saito, A., Sekine, S., Komatsu, Y., Sato, M., Hirano, T., and Itoh, S. (1988). Molecular cloning of eel growth hormone cDNA and its expression in Escherichia
coli.
Gene
73, 545-55
I.
Specker, J. L., King, D. S., Nishioka, R. S., Shira-
390
TETSUYA
hata, K., Yamaguchi, K., and Bern, H. A. (1985). Isolation and partial characterization of a pair of prolactin released in vitro by the pituitary of a cichlid fish, Oreochromis mossambicus. Proc. Natl. Acad. Sci. USA 82, 7490-7494. Suzuki, R., Yasuda, A., Kawauchi, H., and Hirano, T. (1988). Isolation of eel prolactin. 2001. Sci. 5, 1297. Tarpey, J. F., and Nicoll, C. S. (1985). Characterization of hepatic growth hormone binding sites in two fish species, Gillichthys mirabilis (Teleostei)
HIRANO
and Acipenser transmontanus (Chondrostei). Gen. Comp. Endocrinol. 60, 39-50. Yamaguchi, K., Yasuda, A., Kishida, M., Hirano, T., Sano, H., and Kawauchi, H. (1987). Primary structure of eel (AnguiNa japonica) growth hormone. Gen. Comp. Endocrinol. 66, 447-453. Yao, K., Niu, P., Le Gac, F., and Le Bail, P.-Y. (1990). Presence of GH specific binding sites in rainbow trout (Oncorhynchus mykiss) tissue: Characterization of the hepatic receptor. Gen. Comp. Endocrinol., in press.
AND
COMPARATIVE
Hepatic
ENDOCRINOLOGY
Receptors
81, 383-390 (1991)
for Homologous TETSUYA
Ocean
Research
Institute,
University
Growth
Hormone
in the Eel
HIRANO of Tokyo,
Nakano,
Tokyo
164, Japan
Accepted April 5, 1990 The specific binding of ‘2sI-labeled eel growth hormone (eGH) to liver membranes of the eel was examined. The specific binding to the lO,OOOgpellet was greater than that to the 600s pellet. The specific binding was linear up to about 100 mg fresh tissue, and was saturable with increasing amounts of membrane. The specific binding was pH-, temperature-, and time-dependent, with the optimum pH at 7.4, and greater specific binding was obtained at 15 and 25” than at 35”. Scatchard analysis of liver binding gave an association constant of 1.1 x lo9 M-’ and a capacity of 105 fmol/mg protein. The receptor preparation was highly specific for GHs. Natural and recombinant eel GHs as well as recombinant salmon GH competed equally with lz51-eGH for the receptor sites of the 10,OOOgliver membrane. Ovine GH was more potent in displacing the labeled eGH than the homologous eel hormone. Tilapia GH and ovine prolactin (PRL) were needed in greater amounts (40 times) than eGH to displace the labeled eGH. Salmon and tilapia PRLs were still less potent (500 times) than eGH. There was no displacement with eel PRL. No significant change in the specific binding was seen 1 week after hypophysectomy, whereas injection of eGH into the hypophysectomized eel caused a significant reduction after 24 hr. The binding to the membrane fractions from gills, kidney, muscle, intestine, and brain was low and exclusively nonspecific, indicating the presence of specific GH receptors predominantly in the liver. 0 1991 Academic Press, Inc.
It is well established that the mammalian with insulin, glucagon, prolactin (PRL), liver is a major target organ for growth hor- and EGF (Hayashi and Ooshiro, 1986). Remone (GH), some of whose effects are me- cently, Duan and Inui (1990) have demondiated by somatomedins produced mainly 1 strated the indirect action of GH through a in the liver. Receptors for GH have been somatomedin-like factor(s) on the synthesis detected on hepatocyte membranes from of mucopolysaccharides by the ceratobranseveral mammalian species and have chial cartilages of the eel. formed the bases for radioreceptor assay The basic properties of hepatic receptors (see Hughes et al., 1985; Nicoll et al., of lower vertebrates have been studied us1986). In contrast, studies on the hepatic ing mammalian GH as radioligand (see actions of GH in nonmammalian verteNicoll et al., 1986). However, interpretabrates are sparse, especially in teleost tion of data derived from heterologous sysfishes. McKeown et al., (1975) reported an terns is complex, owing to the overlapping increase in liver glycogen after injections of activities of GH and PRL (Nicoll, 1982; ovine GH in kokanee salmon. In the eel, Bern, 1983). With the use of a homologous Inui and Ishioka (1985) reported stimularadioreceptor assay one can study the regtory effects, both in vivo and in vitro, of ulation of GH-specific receptors in target ovine GH on incorporation of [14C]leucine organs in various physiological states. In into the liver protein. Growth hormone teleosts, Fryer (1979) was the first to deseems to be necessary to stimulate cell pro- scribe a radioreceptor assay for teleost GH, liferation of primary culture of the eel he- using a highly purified tilapia GH in a conpatocytes in serum-free medium, together specific assay system. The tilapia GH re383 0016~6480/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
384
TETSUYA
ceptors are highly hormone specific; they bind only tilapia GH and were inhibited only by large amounts of human, bovine, or ovine GHs and ovine PRL. Tarpey and Nicoll (1985) characterized hepatic GHbinding sites in a goby and a sturgeon using 1251-bovine GH, and suggested considerable evolutionary divergence among hepatic GH receptors in teleosts. Recently, a homologous radioreceptor assay for GH has also been reported in rainbow trout and coho salmon (Yao et al., 1990; Gray et al., 1990).
In our previous studies, two forms of GH have been isolated from the medium of organ-cultured eel pituitaries: eGH I is different from eGH II by three extra amino acids, Val-Glu-Pro, at the N terminal (Kishida et al., 1987; Yamaguchi et al., 1987). Recombinant eel GH (eGH I) has also become available (Saito et al., 1988). This study was undertaken to characterized the GH receptor in the eel liver using homologous eel GH. A preliminary account of parts of this work has been presented (Hirano and Kishida, 1989). MATERIALS
AND METHODS
Eels. Sexually immature cultured eels (Anguih juponica) were obtained from a commercial source. They were kept in a freshwater aquarium at 20” without food. Hypophysectomy was carried out as described by Inui er al. (1985). After anesthesia with triCaine methane sulfonate (Sigma), liver and other tissues (gills, kidney, muscle, intestine, and brain) were removed and frozen at - 80”. Hormones. Eel GH (eGH I) was purified from the culture medium of the pituitary of the Japanese eel as described by Kishida er al. (1987). Eel PRL was puritied from the eel pituitary in our laboratory (Suzuki er al., 1988). Recombinant eel and salmon GHs were supplied by the Tokyo Research Laboratories of Kyowa Hakko Kogyo Co. Ovine GH (NIAMDDK-oGH-S12) and PRL (NIAMDDK-oPRL-14) were supplied by the National Institutes of Health (Bethesda, MD). PuriIIed chum salmon PRL (Kawauchi et al., 1983) was provided by Professor H. Kawauchi (School of Fisheries Sciences, Kitasato University), and tilapia GH and PRL (Specker et al., 1985) was provided by Professor H. A. Bern (Department of Integrative Biology, University of California, Berkeley).
HIRANO
Receptor preparation. Frozen or fresh livers were minced and then homogenized in 10 Vol (w/v) 40 mM Tris-HCl buffer (pH 7.4), containing 10 mM CaClr, 1 n&f phenylmethylsulfonyl fluoride (PMSF), and 10 ug/ml Pepstatin A (Sigma), using a Polytron homogenizer at a setting of 5 for two periods of 10 set each with an interval of 10 set for cooling. The tissue flasks were kept in an ice bath during all procedures. The homogenates were subsequently centrifuged at 6OOg for 10 min at 4”. The supematants were collected and centrifuged at 10,OOOgfor 20 min at 4”. The resulting pellets were washed twice with 5 Vol Tris buffer and suspended with a Teflon-glass homogenizer at 1 g starting tissue weight per milliliter of buffer. The suspensions were stored at -80”. Just before use, the frozen suspensions were thawed and diluted with the extraction buffer to appropriate concentrations. An aliquot was taken to determine protein content by the Bio-Rad protein assay, using bovine serum albumin (BSA, Fraction V, Sigma) as a standard. Gill tilaments, kidney, dorsal muscle, intestine, and brain were collected from freshwater-adapted eels and treated similarly. Binding experiments. The determination of GH receptors was carried out essentially following the procedures developed for gonadotropin receptors in lower vertebrates by Kubokawa and Ishii (1987). All solutions were made with 40 mM Tris buffer containing 10 mM CaCI, and 0.5% BSA. Eel GH (eGH I or recombinant eGH) was iodinated by the lactoperoxidase method as described previously (Kishida and Hirano, 1988). Tris-HCl buffer, instead of phosphate buffer, was used in separating the labeled hormone from free iodine. Labeled hormones were stored at - 20”. They were thawed and appropriately diluted with Tris-HCl buffer containing 0.5% BSA just before use. In Scatchard plot analysis, the specific radioactivity of the labeled eGH was estimated from data of the competition and saturation experiments with the same radioligand batch, with the aid of a microcomputer using a program of Ishii and Kubokawa (unpublished). Specific binding was estimated by incubation of the receptor preparation derived from 30 to 50 mg of the original tissue in 100 ~1 buffer with 10,000 to 20,000 cpm (about 0.5-l ng) of ‘*‘I-eGH in 50 pl buffer, in the presence or absence of an excess amount (1 ug in 50 ~1 buffer) of unlabeled recombinant eGH. The total volume of the reaction mixture was 200 ~1. Plastic sample tubes were used for incubation. They were tightly capped, placed almost horizontally in a water bath, and incubated with shaking (120 strokes/min) for 18-20 hr at 1s”. Incubation was terminated by the addition of 1 ml ice-cold buffer followed by centrifugation at lO,OOOgfor 2 mitt at 4”. The supematant was aspirated and the pellet was washed once with 1 ml chilled buffer. The radioactivity of the resultant pellet was counted in an automatic gamma counter.
HEPATIC
Statistics.
the Duncan’s
GH
RECEPTORS
Statistical significance was examined by new multiple range test (Duncan, 1955).
RESULTS
Optimal
Conditions
for the Binding Assay
As shown in Fig. 1, the specific binding of 1251-eGH to 10,OOOg liver membranes was greater than that to the 600g pellet. The specific binding to 10,OOOg membranes increased linearly up to about 100 mg fresh tissue, and increased further only slightly when membranes from 200 mg tissue were added. The receptor preparations were incubated at 25” for 4 hr in this experiment. When membranes from 50 mg tissue were used, the specific binding was 13% of total radioactivity and nonspecific binding was 2%. Further experiments utilized the 10,OOOg membranes derived from 30 to 50 mg fresh tissue (about 250 to 300 kg protein). 30 1
10,000
g Pellet
IN THE
385
EEL
The specific binding was dependent on both duration and temperature of incubation (Fig. 2). The specific binding at IS” increased over time from 2% after 1 hr to 11% after 12 hr, where it plateaued until 20 hr. The binding at 25” increased from 5% after 1 hr to 12% after 6 hr, and then reached a steady state. At 35”, the specific binding increased linearly to 5% during the first 2 hr, but declined gradually thereafter. Thus, the membranes were incubated at 15” for 18 to 20 hr in subsequent experiments. The specific binding was dependent also on the pH of the incubation medium (Fig. 3). It increased from 0 at pH 5.0 to a maximum of 14% at pH 7.4, and then decreased gradually at higher pH. In subsequent experiments, the membranes were incubated at pH 7.5. Receptor
Specificity
The binding of ‘*‘I-eGH to specific organs was examined using receptor preparations of various tissues (30 mg) from freshwater-adapted eels (Fig. 4). As compared with the binding to the liver membranes, the binding to the membranes from gills, kidney, muscle, and intestine was low and exclusively nonspecific. The membrane fractions of brain showed binding as high as in the liver, but no significant specific binding was detected.
20 t
”
600
g Petlet
50 Tissue
100 concentration
150 (mg1100
200 pl)
FIG. 1. Specific and nonspecific binding of ‘*‘I-eGH to membranes of freshwater eel liver (30 mg fresh tissue/tube) prepared by centrifugation at 10,OOOg or to membranes centrifuged at 600s. The receptor preparations were incubated at 25” for 4 hr. Each point is the mean of duplicate determinations.
10
5 Time
15
PO
(h)
FIG. 2. Effects of temperature and time of incubation on specific binding of **%eGH on hepatic recep tors of the freshwater eel (10,OOOg membranes derived from 30 mg fresh tissue/tube).
386
TETSUYA
HIRANO
4
I
0.4
old. 5
6
a2
25
100
400
nQ honnoneIMe
a
7
1s
9
PH
FIG. 3. The influence of pH on specific binding of ‘*‘I-eGH. The receptor preparations from 30 mg freshwater eel liver were incubated at 1s” for 20 hr. The buffers used were 40 mM acetate, pH 5.0-6.0; 40 mM phosphate, pH 6.5; 40 m&f Tris, pH 7.0-9.0. Each buffer contained 10 mM CaCl, and 0.5% BSA. One milliliter ice-cold buffer of the same pH was added to terminate the incubation.
The abilities of various GHs and PRLs to compete with 1251-eGH for the receptor sites of the 10,OOOg liver membranes were examined. The receptor did not distinguish between natural and recombinant eGHs (Fig. 5). Exactly the same displacement curve was obtained when recombinant eGH was used as a labeled hormone (data not shown). As shown in Fig. 6, recombinant salmon GH competed equally with ‘*‘I-eGH. Ovine GH was more potent than the homologous eel hormones. Greater amounts (40 times
FIG. 5. Specificity of binding of ‘*‘I-eGH to freshwater eel liver membranes (30 mg fresh tissue/tube). reGH, recombinant eGH.
more than eGH) of tilapia GH and ovine PRL were needed to displace the labeled eGH. Salmon and tilapia PRLs were still less potent (by 500 times) than eGH. There was no displacement with eel PRL. Equilibrium Association Constants and Concentration of eGH Binding Sites in the Eel Liver The specific binding of labeled eGH to liver membranes was saturable with increasing amounts of label. The Scatchard plot analysis revealed a single class of highaffinity binding sites (Fig. 7). The equilibrium constant of association for specific binding was 1.07 _+0.051 x lo9 M-’ (mean
15
5
ng hormone 0
liver
gills
kiy
msde
tiestine
brain
FIG. 4. Total (clear bars) and nonspecific (hatched bars) binding of ‘*‘I-eGH to crude membrane fractions of various organs of the freshwater eel. The receptor preparations derived from 30 mg tissue were incubated at 15” for 20 hr.
I tube
FIG. 6. Specilicity of binding of “‘I-eGH to freshwater eel liver membranes (30 mg fresh tissue/tube). oGH and oPRL, ovine GH and PRL; rsGH, recombinant chum salmon GH; sPRL, chum salmon PRL; tGH and tPRL, tilapia GH and PRL (20 K); ePRL, eel PRL.
HEPATIC
P *Et
GH
RECEPTORS
IN
THE
387
EEL
3
b x
-2 .-e P a g ‘5 m ::
r
1
/C
2
4 Free
6 6 ( x lO~‘cpm)
10
12
x rL Intact + sahe
HY~
HYF”
sahe
r&-l
8. Effects of hypophysectomy on specific binding of “‘1-eGH to liver membranes of the eel. Hepatic receptors (30 mg fresh tissue/tube) were prepared from freshwater eels 1 week after hypophysectomy or from hypophysectomized eels which received two daily injections of recombinant eGH (reGH, 2 &g) and were sacrificed 24 hr after the last injection. Membranes were also prepared from intact eels of the same lot sacrificed at the same time. Vertical bars represent means 5 SEM (n = 5). *Significantly (P < 0.05) different from intact fish and also from hypophysectomized fish (Hypox) receiving saline. FIG.
0
I
\ 0.05 Bound
0.10
0.15
(nM)
FIG. 7. Effect of concentration cific binding to liver membranes (upper), and Scatchard plot of (lower). Straight line was fitted method.
of ‘*‘I-eGH on speof the freshwater eel the specific binding by the least-squares
+ SEM of four independent determinations). The capacity was 0.56 + 0.078 pmol/ g tissue or 105 * 6.5 fmol/mg protein. Effect of Hypophysectomy and Seawater Transfer on SpeciJic Binding to Hepatic Receptor When freshwater eels fasted for about 2 weeks were hypophysectomized, no significant change was seen in the specific binding after 1 week. On the other hand, hypophysectomized eels receiving daily injections of recombinant eGH (2 pg/g) for 2 days and sacrificed 24 hr after the last injection showed significantly (P < 0.05) less specific binding than intact eels or hypophysectomized eels receiving the vehicle injection (Fig. 8).
DISCUSSION
In the present study, eGH bound specifically to crude membrane fractions of the eel liver. The binding to the membrane fractions from gills, kidney, muscle, intestine, and brain was minimal and exclusively nonspecific, indicating the presence of specific GH receptors predominantly in the liver. The hepatic GH receptors have the general characteristics of peptide hormone membrane receptors. The specific binding was saturable with increasing amounts of membrane as well as of radioligand. As shown by Scatchard plot analysis, the hepatic binding sites are of high affinity and limited number. The values of equilibrium parameters were similar to those reported for the hepatic GH receptors of tilapia (Fryer, 1979), goby and sturgeon (Tarpey and Nicoll, 1985), and of other vertebrate species (see Nicoll et al., 1986).
388
TETSUYA
The specific binding was pH-, temperature-, and time-dependent, with the optimum pH at 7.4, and greater specific binding at 15 and 25” than at 35”. The decline in binding after 2 hr at 35” suggests that denaturation and/or degradation of the receptor protein had occurred. This is in accord with the fact that eels are cultured at 20-25”, but are difficult to maintain at above 30”. Tarpey and Nicoll (1985) also noted a decrease in the specific binding of bovine GH to the goby liver membranes 8 hr after incubation at 37”. Similarly, amphibian gonadal receptors for gonadotropins have optimum equilibrium at about IS” (Kubokawa and Ishii, 1984). High hormone specificity is also an important characteristic of hormone receptors. According to Fryer (1979), the tilapia hepatic GH receptors are narrowly hormone specific, binding only to tilapia GH. In contrast, Tarpey and Nicoll(1985) found that human and bovine GHs are equipotent in inhibiting the binding of labeled bovine GH to goby hepatic membranes. The hepatic GH receptors of the eel also exhibited high hormone specificity. Natural and recombinant eGHs as well as recombinant salmon GH competed equally with 12?eGH (either natural or recombinant) for the receptor sites. The fact that recombinant eGH was equipotent to pituitary eGH in inhibiting binding of 12’I-eGH suggests that the recombinant hormone is equivalent in biological potency. In hormone-receptor interactions, the affinity of receptor for growth hormone has generally correlated with the established biological potency of the hormone. Exceptions have occurred most often when labeled hormones and receptors were derived from different species, i.e., in heterologous assays (see Hughes et al., 1985). The present observation that ovine GH was more potent in displacing the labeled eGH than the homologous hormone is in accord with the fact that ovine GH was more potent than eGH in stimulating the incorpora-
HIRANO
tion of 35S-sulfate into branchial cartilages of the eel in vitro (Duan and Inui, unpublished observation). It is not known if ovine GH stimulates growth of the eel, because they do not feed under laboratory conditions (Kishida et al., 1987). However, it is inexplicable why tilapia GH was less potent than eGH, or why ovine PRL was as potent as tilapia GH. Ovine PRL has been shown to have GH-like activity in amphibian larvae, birds, and dwarf mice (see Nicoll, 1982; Bern, 1983). According to Nicoll et al. (1986), there is greater similarity among PRL receptors than among GH receptors but the converse is true for the hormones themselves. At any rate, there was no displacement of the labeled eGH with eel PRL, indicating the highly specific nature of the hepatic GH receptor for endogenous GH as compared with endogenous PRL. In mammals, concentrations of the specific receptors for GH on target cells are known to be affected by the hormone concentration. GH receptors acutely decrease (down-regulation) after short-term exposure to GH, whereas prolonged treatment with GH increases GH-binding sites (upregulation) in liver of various species (see Hughes et al., 1985; Maiter et al., 1988a, b). Posner et al. (1980) reported a decrease in hepatic GH receptors in rabbits and sheep 20-30 days after hypophysectomy. In the present study, no significant change was seen in hepatic GH receptors of the eel 1 week after hypophysectomy, whereas treatment of the hypophysectomized eel with two daily injections of eGH resulted in a significant reduction 24 hr after the last injection, suggesting the occurrence of down-regulation also in the eel. According to Maiter et al. (1988a, b), a single injection of GH in hypophysectomized rats induced a transient decrease in the GH receptors, whereas continuous administration increased the receptors. The time courses of changes in the receptors after various treatments should be examined in various physiological states, in order to clarify whether
HEPATIC
down- and up-regulation also occurs in eels.
of GH receptors
ACKNOWLEDGMENTS I am greatly indebted to Professor Susumu Ishii and Dr. Kaoru Kubokawa, School of Education, Waseda University, for their invaluable suggestions and encouragement during the course of this study. I gratefully acknowledge the gifts of hormones from Professor Howard A. Bern, Professor Hiroshi Kawauchi, the NIADDK Hormone Distribution Program, and Tokyo Research Laboratories of Kyowa Hakko Kogyo Co., Ltd. Thanks are also due to Dr. Tsuyoshi Ogasawara, Mitsuyo Kishida, Sanae Hasegawa, and Tatsuya Sakamoto, Ocean Research Institute, University of Tokyo, for their help. I am also grateful to Professor Howard A. Bern for a critical reading of the manuscript. This work was supported in part by grants-in-aid from the Fisheries Agency and the Ministry of Education, Japan.
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389
GH RECEPTORS IN THE EEL
Inui, Y., Miwa, S., and Ishioka, H. (1985). Effect of mammalian growth hormone on amino nitrogen mobilization in the eel. Gen. Comp. Endocrinol. 59, 287-294. Kawauchi, H., Abe, K., Takahashi, A., Hirano, T., Hasegawa, S., Naito, N., and Nakai, Y. (1983). Isolation and properties of chum salmon prolactin. Gen. Comp. Endocrinol. 49, 446-458. Kishida, M., and Hirano, T. (1988). Development of radioimmunoassay for eel growth hormone. Nippon Suisan Gakkaishi 54, 1321-1327. Kishida, M., Hirano, T., Kubota, J., Hasegawa, S., Kawauchi, H., Yamaguchi, K., and Shirahata, K. (1987). Isolation of two forms of growth hormone secreted from eel pituitaries in vitro. Gen. Comp. Endocrinol.
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hata, K., Yamaguchi, K., and Bern, H. A. (1985). Isolation and partial characterization of a pair of prolactin released in vitro by the pituitary of a cichlid fish, Oreochromis mossambicus. Proc. Natl. Acad. Sci. USA 82, 7490-7494. Suzuki, R., Yasuda, A., Kawauchi, H., and Hirano, T. (1988). Isolation of eel prolactin. 2001. Sci. 5, 1297. Tarpey, J. F., and Nicoll, C. S. (1985). Characterization of hepatic growth hormone binding sites in two fish species, Gillichthys mirabilis (Teleostei)
HIRANO
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