Saturable triiodothyronine-binding sites in the pituitary nuclei of salmonid teleost fish

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

77, 23-28 (1990)

Saturable Triiodothyronine-Binding Sites in the Pituitary Nuclei of Salmonid Teleost Fish 0. BRES' AND J. G. EALES Department

of Zoology,

University

of Manitoba,

Winnipeg,

Manitoba,

Canada

R3T

2N2

Accepted March 8, 1989 High-affinity, limited-capacity, 3,5,3’-triiodo-L-thyronine (T,)-binding sites were established by in vitro saturation analysis in cell nuclei of the pituitary gland of arctic charr. The sites were extracted from the purified nuclei using 0.4 M NaCl and incubated with [iz51]T, in the presence of 0.2 M NaCI. T, saturable binding attained equilibrium after 18-24 hr of incubation at 4”. The association constant ranged from 6.7 to 20.1 liters molF’ x 109, indicating a T, affinity greater than that for T,-binding sites in rainbow trout liver. The maximal binding capacity ranged from 0.93 to 2.05 lo-l3 mol . mg DNA-‘, representing a mean site abundance corresponding to 60% of that for nuclei from trout liver. Thyroxine (T4) completely displaced [‘251]T3 in the pituitary nuclei of arctic charr and T, completely displaced [‘*sI]T, in the pituitary nuclei of rainbow trout, suggesting that in salmonids both T, and T, bind to the same single class of sites. However, the site affinity for T, was approximately 20-50~ less than that for T,. The possible roles of these sites in pituitary function as well as their relationship to other nuclear T,-binding sites in salmonid fish are discussed. 0 1990 Academic Press, Inc.

Saturable T, (3,5,3’-triiodo-L-thyronine)-binding sites with properties resembling those of T, receptors found in the liver and other tissues have been described in the rat pituitary (Schadlow er al., 1972; Samuels and Tsai, 1973; Oppenheimer et al., 1974; Gordon and Spira, 1975). These sites are believed to reside predominantly in the thyrotrophs and somatotrophs. The former sites are probably involved in the negative feedback inhibition of thyrotroph activity in response to elevated plasma thyroid hormone levels (Bowers et al., 1967; Silva and Larsen, 1978). The latter sites, extensively studied in GH, cells in vitro, probably mediate the action of thyroid hormones in facilitating growth hormone synthesis and secretion (Bowers et al., 1967; Yaffe and Samuels, 1984). To date, thyroid hormone receptors in the pituitary gland ’ Present address: Anatomy, University 94720.

appear to have been described for mammals only. In teleost fish, negative feedback control of thyroid function by thyroid hormones acting at the level of the pituitary (Peter, 1972) suggests that T, receptors may also exist in the fish pituitary. In the present study we describe some properties of saturable thyroid hormone-binding sites (putative receptors), extracted from the cell nuclei of pituitaries from arctic charr, Salvelinus alpinus, and rainbow trout, Oncorhynthus mykiss. MATERIALS

AND METHODS

Pituitaries were removed from freshly killed arctic charr (three pools of approximately 300 pituitaries/ pool) and rainbow trout (one pool). The fish ranged in weight from 300 to 1000 g, and had been raised and held in running well water at 6.5” under a simulated natural photoperiod. They were not fed on the day of sampling. During collection (l-2 hr), pituitaries were held on ice in buffer 1 (0.32 M sucrose, 3 mM MgCI,, 3 mM dithiothreitol (DTT), 25 mM KCI, 2 mM ethylene glycol bis(O-aminoethyl ether)-N, N’-tetraacetic acid, 0.5 mM spermidine, 20 mM Tris-HCI, pH 7.2

Department of Physiologyof California, Berkeley, CA 23

0016~6480/90 $1.50 Copyright 0 1990 by Academic Pren. Inc. All rights of reproduction in any form reserved

24

BRES AND EALES

and 5% glycerol (v/v)), then immersed in liquid nitrogen and stored in buffer 1 at -70” until analysis several weeks later. A thawed pituitary pool was minced in 10 vol (w/v) of buffer 1 containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF), homogenized by Polytron for 3 set and then by four passes with a motor-driven Teflonglass homogenizer. The homogenate was filtered through four layers of cheesecloth, and then centrifuged at 18OOgfor 10 min to produce a crude nuclear pellet, which was washed once in the original volume of buffer 1 containing 0.25% Triton X-100. Nuclei were viewed by Nomarski microscopy to assess purity and integrity and counted using a hemacytometer. Nuclear proteins were extracted from the purified nuclei by the methods of Seelig et ul. (1981). The nuclear pellet was resuspended in buffer 2 (30 mM TrisHCI, pH 8.0, 2 mM EDTA, 5 mM mercaptoethanol, and 10% (v/v) glycerol) containing 0.4 M NaCl and 5 mM MgCl,, briefly homogenized using the Polytron, and then vortexed briefly at S-min intervals for 45 min at @4”. The suspension was centrifuged at 130,OOOg for 20 min and the supernatant was diluted to 0.2 M NaCl with buffer 2. We have previously shown that this method for trout liver, results in essentially 100% recovery of the nuclear TX-binding proteins (Bres and Eales, 1988). The protein content of the extract was measured by the Bradford (1976) method. The DNA content of the purified nuclear pellet was measured by the diphenylamine reaction (Burton, 1956). The thyroid hormone-binding assays were carried out by incubating the extract at a final protein concentration of 200-400 ug/ml with [“‘I]T, (sp act = 1200 mCi/mg, Amersham) at 4” in a final volume of 1 ml. Nonsaturable binding was measured in parallel tubes containing lo--’ M unlabeled T,. Bound and free [“‘IIT, were separated by adding 1 ml Dowex 1 )i 8-400 ion-exchange resin (160 mg/ml incubation buffer). The suspension was vortexed briefly at S-mm intervals for 20 min after which the resin was removed by centrifugation at 18OOgfor 10 min, and 1 ml of the supematant was counted. We have previously shown (Bres and Eales, 1988) that the resin adsorbs over 99% of the free [“‘I]T, and “‘I~, leaving the proteinbound [‘*‘I]T, in solution. The association constant (KU) and the maximal binding capacity (MBC) were estimated by saturation analysis using either “hot only” or “cold displacement” methods (Bres and Eales, 1986, 1988). In the hot only approach, the [ IZ51]T3concentration was varied. In the cold displacement approach, [iZ51]T, or [iZ51]T, was held constant and increasing quantities of the homologous or heterologous unlabeled ligand (T4 or T, as the case may be) were added to the incubation tubes. In both instances the binding parameters were calculated using the LIGAND computer program (Munson and Rodbard, 1980).

RESULTS

The time course of [lZ51]T3 association with solubilized nuclear sites from charr pituitaries indicated low nonsaturable binding (0.2%) and attainment of equilibrium for saturable and total binding by 18-24 hr at 4” (Fig. 1). Saturation analyses were carried out for 24 hr at 4”. Both Scatchard (Fig. 2) and LIGAND (Table 1) analyses showed a single class of T,-binding sites. The K, values determined by hot only analysis for charr pools 1 and 2 were respectively 7.4 and 6.7 x 10” liters . mall’, and 20.1 x 10’ liters . rnol~~ ! as determined by cold displacement for pool 3 (Table 1). The MBC for these three pools ranged from 0.93 to 2.05 x 10 ” mol . mg DNA - ’ Based on a count of the isolated nuclei using the hemacytometer and the assumption that each nucleus has the same number of sites, this is equivalent to 340 sites per nucleus. Unlabeled T, completely displaced [i2’1]T, (Fig. 3A) and the approximate afl5nity of T, was 4.6 x IO’ liters . mall ‘, or 2.2% of that for T, itself (Table I, pool 3). On account of the unavailability of charr, the ability of T, to compete with [iz51]T, for binding to potential T, sites was tested on solubilized nuclear receptors from rainbow

INCUBATION

TIME

(hrl

FIG. 1. Time course of [“‘IIT, association with extracted proteins from charr pituitary nuclei (Pool 1) at 4”. The initial free hormone concentration was approximately 0.1 x 10-l” M. Each point represents the mean of three determinations (t2 standard deviations). 0, total binding; A, saturable binding: 0, nonsaturable binding.

PITUITARY

T3

25

SITES

the [‘251]T,-binding site. Since unlabeled T, can completely displace [1251]T3 and unlabeled T, can completely displace [‘251]T,, it is concluded that both hormones crossreact with the same binding sites in the nucleus. DISCUSSION

[ “*

I I

T3

BOUND

(molltube

x 1Oe’4

1

FIG. 2. Scatchard plot of [‘251]T, binding to proteins extracted from char-r pituitary nuclei (Pool 1). Using a “hot only” procedure, [‘ZSI]T, in concentrations varying from 0. I to 5 x IO- ” M was added to the nuclear extract and incubated for 24 hr at 4”. Nonsaturable binding was determined in the presence of lo-’ M unlabeled T,. The protein concentration of the nuclear extract was 302 kg ml-‘. K, and MBC values are given in Table I.

trout. Since the maximum total binding of [‘25]T, was low (approximately 2% of total added radioactivity), the results were very variable (Fig. 3B) and could not be analyzed by the LIGAND program. The approximate affinities, estimated from the dose of cold hormone which caused a 50% reduction of saturable [‘251]T, binding, were 9.5 x lo9 liters . mol-’ for T, and 4.0 x lo* liters * mol-’ for Tq. These affinities are similar to those for

SUMMARY

Pool I 2 3 3

OF PROPERTIES

Labeled ligand T3 T3 T3 T3

OF THYROID NUCLEI

Displacing ligand T3 T3 T3 T4

While not indicating their location in any particular cell type, these data show that high-affinity, low-capacity T,-binding sites exist in the nuclei of the pituitary of at least two salmonid teleost fish, the only species other than mammals studied in this regard. Based on the complete displacement of bound T, with T, and the complete displacement of bound T, with T,, there probably exists a single class of thyroid hormone-binding sites which binds predominantly T,. The possibility cannot be excluded that different TX-binding sites are present in the various cell types of the pituitary. However, if this is the case, their binding affinities are sufficiently similar to escape detention by LIGAND analysis which discriminates binding sites based on affinity alone. The pituitary site affinity of 6.7-20.1 x 10” liters . mall’ is greater than that recorded, using similar methodology, for liver, gill, kidney, and brain of rainbow trout but is more comparable to that for the nuclei of red blood cells (Bres and Eales,

TABLE I HORMONE-BINDING OF ARCTIC CHARR

Method H” H Cb C

(liters

SITES IN PROTEINS PITUITARIES K, mall’ 7.4 6.7 20.1 0.46

t 2 -+ lr

X 10’)

EXTRACTED

-

0.7’ 2.5 2.8 0.05

0 “Hot only” method in which saturation of sites was achieved by adding constant specific activity. ’ “Cold displacement” method whereby [‘251]T, was displaced by increasing ’ Approximate standard error as calculated by the LIGAND program.

(IO-l3

mol 2.05 0.93 1.45 1.40

increasing amounts

amounts

FROM THE

MBC mg DNA-‘) t t t 2

0.01 0.19 0.09 0.09 of [“‘IIT,

of unlabeled

at

T, or T,.

26

s

BRES

100

AND

a

r

80 60 ,.

. :

20

:

400 FI.Ysd+ ,o-ll

,o-lo

Unlabeled

,o-9

Hormone

0 0

.I IO-

lo-710-6

(mol/l)

FIG. 3. (A) Displacement of unlabeled T, (0) or T, (0) of [‘2SI]T, from protein sites extracted from charr pituitary nuclei (Pool 3). After extraction in 0.4 M NaCl, the proteins were incubated with 4.4 x IO- ” [“‘I]T, alone or in the presence of increasing concentrations of unlabeled T, or T,. K, and MBC values are given in Table 1. (B) Displacement by unlabeled T, (0) or T, (0) of [‘2SI]T, from protein sites extracted from rainbow trout pituitary nuclei. After extraction in 0.4 M NaCl, the proteins were incubated with 8.3 X lo-” mol . liter-’ [‘251]T, alone or in the presence of increasing concentrations of unlabeled T, or T,. K, values of 9.5 x IO9 liters mol-’ for T, and 4.0 x 10s for T, were estimated from the amount of cold hormone required for 50% displacement.

1988). The pituitary site affinity for T, is also higher than the affinity of hepatic nuclear sites of lampreys (Lintlop and Youson, 1983), coho salmon (Darling et al., 1982), which is now included in the same genus as the rainbow trout, and the lake trout, Salvelinus namaycush (Weirich et al., 1987), which is in the same genus as the arctic charr. This suggests that in salmonid teleosts the affinity of the extracted sites for T, is greater in the pituitary nuclei than it is in the liver nuclei and in the nuclei of most of the other tissues examined to date. This contrasts with the rat where pituitary T,-binding sites share similar affinities with hepatic sites (Oppenheimer et al., 1974; Spira and Gordon, 1986). Possibly the charr

EALES

pituitary T,-binding sites are not similar to T,-binding sites in other tissues. T, binds to the charr pituitary sites with approximately 20-50x lower affinity than T,. In the liver of the coho salmon and rainbow trout, T, binds with 7-10 x lower affinity than T, to saturable nuclear sites (Darling et al., 1982; Bres and Eales, 1986, 1988). The considerably greater discrepancy between K, values for T, and T, in the pituitary than reported for other salmonid tissues adds weight to the suggestion that the pituitary T, sites are not similar to T7 sites elsewhere. The pituitary MBC values of 0.93-2.05 x lo- I3 mol . mg DNA- ’ show that of all the tissues examined to date by similar techniques in the rainbow trout (Bres and Eales, 1988), only the hepatocyte nuclei have a greater site abundance. Furthermore, T, sites may be present in only one or two pituitary cell types, leading to an underestimate of site density in those cell types (e.g., thyrotrophs) which may not be the most common and which probably possess the most sites. The relative high abundance of the T, sites, their high affinity for T,, and, in relation to T,, their high specificity, strongly imply a receptor role. In mammals the T, receptors in the thyrotrophs mediate the action of circulating thyroid hormones in the negative feedback inhibition of thyroid-stimulating hormone release from the pituitary and hence suppression of thyroid function (Spira and Gordon, 1986). Available evidence also suggests a direct influence of the thyroid hormones on fish pituitary cells. T, alters the histological appearance of thyrotrophs and gonadotrophs when added to cultured pituitaries of rainbow trout and Poecilia (Baker, 1965, 1969; Sage and Bromage. 1970). T, containing pellets implanted into normal and autotransplanted pituitaries inhibit the release of TSH activity, as measured by radioiodide uptake by the goldfish thyroid (Peter, 1971, 1972). Thyrotroph responses to T, may be explained by T, ac-

PITUITARY

tion on the thyroid hormone nuclear sites since T, at the high levels used in several of these earlier studies may have acted directly on the sites, as described in mammals (Gershengorn, 1978). However, due to their relatively low affinity for T,, the pituitary sites in viva are probably occupied predominantly by T, unless T, is preferentially concentrated with respect to T, in the nucleus. Pituitary cells may be directly responsive to T, if they have the capacity to take up T, and convert it to T,. In the rat pituitary, a type II deiodinase occurs which converts T, and T, which is then believed to bind to pituitary nuclear receptors (Leonard et al., 1984; Larsen et al., 1979). A T, 5’-monodeiodinase which accomplishes the same conversion has been described in the trout and charr liver (Shields and Eales, 1986; MacLatchy and Eales, 1988) and could also be present in the pituitary in these species. ACKNOWLEDGMENTS This study was supported by a NSERC research grant (A1965) to J.G.E. and a NSERC postgraduate scholarship to O.B. Fish were supplied by Mr. R. Olson, Rockwood Experimental Fish Hatchery, Manitoba, by permission of the Department of Fisheries and Oceans.

REFERENCES Baker, B. I. (1965). Direct effect of thyroxine on the trout pituitary in vitro. Nature (London) 208, 1234-1235. Baker, B. I. (1969). The response of the teleost pituitary thyrotrophs to thyroxine in vitro: A histological study. Cert. Comp. Endocrinol. 12, 421-437. Bowers, C. Y., Scally, A. V., Reynolds, G. A., and Hawley, W. D. (1967). Interaction of L-thyroxine or L-triiodothyronine and thyrotropin releasing factor on the release and synthesis of thyrotropin from the anterior pituitary gland of mice. Endocrinology 81, 741-747. Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72, 248-254. Bres, O., and Eales, J. G. (1986). Thyroid hormone binding to isolated trout (Salmo gairdneri) liver

T3 SITES

27

nuclei in vitro: Binding affinity, capacity and chemical specificity. Gen. Comp. Endocrinol. 61, 29-39. Bres, O., and Eales, J. G. (1988). High-affinity, limited-capacity triiodothyronine-binding sites in nuclei from various tissues of the rainbow trout (Salmo gairdneri). Gen. Comp. Endocrinol. 69, 71-79. Burton, K. (1956). A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. .l. 62, 315-323. Darling, D. S., Dickhoff, W. W., and Gorbman, A. (1982). Comparisons of thyroid hormone binding to hepatic nuclei of the rat and a teleost, Oncorhynchus kisutch. Endocrinology 111, 1936-1943. Gershengorn, M. C. (1978). Regulation of thyrotropin production by mouse pituitary thyrotropic tumor cells in vitro by physiological levels of thyroid hormones. Endocrinology 102, 1122-l 128. Gordon, A., and Spira, 0. (1975). Triiodothyronine binding in the rat anterior pituitary, posterior pituitary, median eminence and brain. Endocrinology 96, 1357-1365. Hervas, F., Morreale de Escobar, G., and Escobar del Rey, F. (1975). Rapid effects of single small doses of L-thyroxine and L-triiodothyronine on growth hormone as studied in the rat by radioimmunoassay. Endocrinology 91, 91-101. Larsen, P. R., Dick, T. E., Markovitz, M. M., Kaplan, M. M., and Gard, T. G. (1979). Inhibition of intrapituitary thyroxine to 3,5,3’-triiodothyronine conversion prevents the acute suppression of thyrotropin release by thyroxine in the hypothyroid rat. J. Clin. Invest. 64, 117-128. Leonard, J. L., Silva, J. E., Kaplan, M. M., Mellen, S. A., Visser, T. J., and Larsen, P. R. (1984). Acute post transcriptional regulation of cerebrocortical and pituitary iodothyronine 5’-deiodinases by thyroid hormone. Endocrinology 114, 9981004.

Lintlop, S. P., and Youson, J. H. (1983). Binding of triiodothyronine to hepatocyte nuclei from sea lampreys, Perromyzon marinus L., at various stages of the life cycle. Gen. Comp. Endocrinol. 49, 428-436. MacLatchy, D. L., and Eales, J. G. (1988). Shortterm treatment with testosterone increases plasma 3,5,3’-triiodo-t-thyronine and hepatic Lthyroxine 5’-monodeiodinase levels in arctic charr, Salvelinus alpinus. Gen. Comp. Endocrinol. 70, 10-16. Munson, P. J., and Rodbard D. (1980). Ligand: A versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107, 220-239. Oppenheimer, J. H., Schwartz, H. L., and Surks,

28

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AND

M. I. (1974). Tissue differences in the concentration of triiodothyronine nuclear binding sites in the rat liver, kidney, pituitary, heart, brain, spleen and testis. Endocrinology 95, 897-903. Peter, R. E. (1971). Feedback effects of thyroxine on the hypothalamus and pituitary of goldfish, Curassius auratus. J. Endocrinol. 51, 31-39. Peter, R. E. (1972). Feedback effects of thyroxine in goldfish, Carassius auratus, with an autotransplanted pituitary. Neuroendocrinology 10, 273281. Sage, M., and Bromage, N. R. (1970). Interactions of the TSH and thyroid cells with gonadotropic ceils and gonads in poecilid fishes. Gen. Camp. Endocrino[. 14, 137-140. Samuels, H. H., and Tsai, J. S. (1973). Thyroid hormone action in cell culture: Demonstration of nuclear receptors in intact cells and isolated nuclei. Proc.

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Schadlow, A. R., Surks, M. I., Schwartz, H. L., and Oppenheimer. J. H. (1972). Specific triiodothyronine binding sites in the anterior pituitary of the rat. Science 176, 1252-1254. Seelig, S., Schwartz, H. L., and Oppenheimer, J. H. (1981). Limitations in the conventional analysis of the interaction of triiodothyronine with solubilized nuclear receptor sites: Inapparent binding of triiodothyronine to nonspecific binding sites. J. Biol. Chem. 256, 21542161.

EALES

Shields, C. A., and Eales, J. G. (1986). Thyroxine 5’-monodeiodinase activity in hepatocytes of rainbow trout, Salmo gairdneri: Distribution, effects of starvation, and exogenous inhibitors. Grn. Comp.

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63, 334-343.

Silva, J. E., and Larsen, P. R. (1978). Contributions of plasma triiodothyronine and local thyroxine monodeiodination to triiodothyronine and nuclear triiodothyronine receptor saturation in pituitary, liver and kidney of hypothyroid rats: Further evidence revealing saturation of pituitary nuclear triiodothyronine receptors and the acute inhibition of thyroid stimulating hormone release. J. C’lirr Invest. 61, 1247-1259. Spira, O., and Gordon, A. (1986). Thyroid hormone feedback effects on thyroid-stimulating hormone. In “Thyroid Hormone Metabolism” (G. Henneman, Ed.), pp. 535-578. Dekker. New York. Weirich. R. T., Schwartz, H. L., and Oppenheimer. J. H. ( 1987). An analysis of the interrelationship of nuclear and plasma triiodothyronine in the sea lamprey. lake trout, and rat: Evolutionary considerations. Endocrinology 120, 664677. Yaffe. B. M.. and Samuels, H. H. (1984). Hormonal regulation of the growth hormone gene: Relation\hip of the rate of transcription to the level of nuclear thyroid hormone receptor complexes. .I Biol.

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