Receptors and Hormone Action: Volume III

Index

1

Nuclear Receptors for Triiodothyronine: A Physiological Perspective

J.H. OPPENHEIMER and W.H. DILLMANN*

Publisher Summary

This chapter discusses the excess and deficiency of thyroid hormone, the dynamics of T3 bound to nuclear sites, physiological role of the nuclear T3 binding site, the possibility of other initiating sites, and speculations on molecular mechanisms. The excess or deficiency of thyroid hormone, which is termed as hypothyroidism and hyperthyroidism, results in a number of abnormalities. In particular, the changes in the enzyme activity are related with the alteration in thyroid state. Many enzymes enhances in activity with thyroid hormone administration, whereas others decrease. Among the enzymes that parallel thyroid state with an especially wide excursion are mitochondrial α-glycerophosphate dehydrogenase (a-GPD) and malic enzyme which is located in cytosol. In hypothyroidism, α-GPD and malic enzyme decline to almost undetectable levels. The function of these enzymes is poorly understood, although α-GPD is known to be linked to the respiratory chain, and malic enzyme is considered to be important in supplying NADPH for fatty acid synthesis.

I. Introduction: Thyroid Hormone Deficiency and Excess

II. Dynamics of T3 Bound to Nuclear Sites

III. Physiological Role of the Nuclear T3 Binding Site

IV. The Possibility of Other Initiating Sites

V. Speculations on Molecular Mechanisms

VI. Concluding Remarks

References

I INTRODUCTION: THYROID HORMONE DEFICIENCY AND EXCESS

The biological role of the thyroid hormones is currently understood largely in terms of a catalogue of apparently unrelated effects that are observed in the hypothyroid and hyperthyroid state. These are produced experimentally in animals or in the course of therapeutic manipulations or spontaneous disease in man. Thus, in the absence of a functioning thyroid gland, growth and development are severely retarded. In man, such retardation, especially in the growth and differentiation of the skeletal and central nervous systems, results in the syndrome of cretinism. In the tadpole, absence of thyroid hormone prevents progression to metamorphosis, a complex series of biochemical, physiological, and morphological events that characterizes the transition from the aquatic to the terrestrial state. In the adult form, the thyroprival state is associated yet with another series of abnormalities. Of these, the most generally recognized is the decrease in oxygen consumption, both in the whole animal and in certain excised tissues. Other changes that occur in hypothyroidism include slowing of the heart rate, accumulation of mucopolysaccharides in skin, alterations in lipid concentration in blood, and a decreased fractional metabolism of many metabolites and drugs. In fact, physiological and biochemical changes of one sort or another characterize almost every organ system. These are well described in standard texts. On the other hand, hyperthyroidism is associated with oppositely directed changes including increased oxygen consumption, both in the whole animal as well as in certain excised tissues, an accelerated heart rate, and an enhanced fractional turnover of metabolites and drugs.

Of particular interest are the changes in enzyme activity that are associated with alterations in thyroid state. Many enzymes increase in activity with thyroid hormone administration, whereas others decrease. Among the enzymes that parallel thyroid state with an especially wide excursion are mitochondrial α-glycerophosphate dehydrogenase (α-GPD) (Ruegamer et al., 1964) and malic enzyme, which is located in cytosol (Young, 1968). In hypothyroidism, α-GPD and malic enzyme decline to almost undetectable levels. Whether the residual activity is due to basal enzyme activity independent of thyroid hormone influence or reflects incomplete hypothyroidism has not been established. The function of these enzymes is poorly understood, although α-GPD is known to be linked to the respiratory chain (Ringler and Singer, 1959), and malic enzyme is considered to be important in supplying NADPH for fatty acid synthesis (Youngs et al., 1964).

The poorly defined interrelationships of these parameters pose an obstacle in any effort to define the mechanism of thyroid hormone action at a cellular level. It cannot even be assumed that there exists a single point of hormone initiation. Conceivably, multiple intracellular pathways could be involved in reaching separate end points of thyroid hormone action. Moreover, among the various thyroid hormone actions, only the suppression of TSH can be considered to be in any way specific. Unfortunately, inhibition of pituitary TSH appears to be an unusually complex process, since it is believed to occur as a result of the stimulation of an inhibitory protein (Lee et al., 1968). Under any circumstance, this process must be considered to be highly specialized and, thus, possibly unrepresentative of other manifestations of thyroid hormone action.

A fundamental problem also arises as to whether or not a given tissue is responsive to thyroid hormone. Of the various criteria that have been applied, perhaps the change in oxygen consumption with thyroid hormone administration and deprivation is generally regarded as the best index of response (Barker and Klitgaard, 1952). Although the association between thyroid hormones and oxygen consumption has been established for many years, it is not clear, however, that changes in oxygen consumption represent a sine qua non for thyroid hormone response. This presents an especially vexing problem in relationship to brain, a tissue which is apparently unresponsive by the criterion of oxygen consumption but nonetheless exhibits functional alterations in clinical states of thyroid excess and deficiency. It appears possible that the appropriate parameters for assessing thyroid hormone response in this tissue have not been defined.

Another obstacle to the analysis of thyroid hormone action has been the uncertain significance of many in vitro models for the study of thyroid hormone effects. Frequently, the concentrations of thyroid hormone required to produce changes have been many orders of magnitude above those required to achieve similar end points under physiological circumstances (Buchanan and Tapley, 1966; Gordon et al., 1973). Moreover, many of the in vitro interactions have been characterized by a rapid onset after the addition and a rapid cessation of effect after the withdrawal of hormone from the proposed initiating site. Thus, the increase in amino acid incorporation into mitochondrial proteins begins within minutes after addition of thyroxine (T4) and is quickly reduced when the concentration bound to the mitochondria is reduced. As will be detailed subsequently, these characteristics do not reflect the slow rates of initiation and dissipation of tissue responses to thyroid hormone in the intact animal.

In view of these problems, it is not surprising that, despite the venerable history of the thyroid hormones, our concepts of their mechanism of action at the cellular level remain both primitive and fragmentary. The recent description of what appear to be specific nuclear binding sites for triiodothyronine (T3) in various tissues of the rat (Oppenheimer et al., 1972a, 1974a; Samuels and Tsai, 1973; De Groot and Strausser, 1974; Latham et al., 1976) and in other species (Tsai and Samuels, 1974; Kistler et al., 1975) presents an unusual opportunity for the study of the mechanism of action of thyroid hormone at a molecular level. If it can be demonstrated that the nuclear binding sites are receptors instrumental in the initiation of thyroid hormone action, a biochemical intracellular reference point will be available, which should be helpful in defining the subsequent reactions which lead to hormone action.

Accordingly, we propose in this chapter to review our current state of knowledge with regard to the physiological properties of the thyroid hormone receptors in the intact animal. We shall consider first the quantitative relationships between hormone bound to the specific nuclear sites and hormone in cytosol and plasma. We shall discuss in vivo kinetic techniques, which have allowed definition of nuclear binding capacity, the mass of iodothyronine normally bound to the nucleus, and the affinity of these sites. We shall then consider the available evidence supporting the concept that these sites are true receptors involved in the initiation of thyroid hormone action. The possibility of extranuclear receptors will also be examined. An analysis of the quantitative relationship between nuclear hormonal occupancy and tissue response will follow. The temporal relationship between the hormone-nuclear interaction and the detection of tissue response will be discussed in relationship to the characteristic lag time exhibited by thyroid hormone. Lastly, we shall discuss the possible molecular basis for some of the physiological responses observed.

In this chapter, we shall assume that T3 rather than T4 is the primary thyroid hormone. Despite the fact that under most circumstances T4 appears to be the principal secretory product of the thyroid gland, current evidence suggests that T4 derives most of its hormonal potency from its conversion to T3 in the peripheral tissues. Thyroxine does exert thyroid hormone effects independent of its conversion to T3, but the intrinsic hormonal effects of T4 probably contribute only a small proportion of the overall thyroidal status of the target tissues. Moreover, as far as is known, T4 and T3 elicit qualitatively identical tissue responses.

II DYNAMICS OF T3 BOUND TO NUCLEAR SITES

In vivo techniques can be effectively used to determine the binding capacity of nuclear sites and the percentage of sites occupied by iodothyronine, as well as the rate of interchange of T3 between nuclei, cytoplasm, and plasma hormone pools. The original demonstration of nuclear receptors (Oppenheimer et al., 1972a, 1974a,b) was based on competitive studies performed in the intact rat using intravenously (i.v.) injected [¹²⁵I]T3 administered in tracer quantities, together with progressively increasing doses of unlabeled T3. Experiments were performed ½ hour after the injection of the dose. For the liver, this is the time when the specific activity of plasma T3 can be shown to be the same as nuclear T3. At ½ hour, only a small proportion of administered T3 has been metabolized, and the radioactivity bound to the purified nuclear preparation can be demonstrated chromatographically to be in the form of T3. The concentration of radioactive T3 bound to the nuclei (N) was expressed as a fraction of the radioactive T3 in plasma (P) to yield the N/P ratio. Injection of progressively larger doses of T3 resulted in decreasing values of N/P until baseline levels were achieved. These were considered to reflect nonspecific binding and were subtracted from the total binding ratios to yield the specific binding ratios. The product of the specific N/P ratio and plasma T3 as measured by radioimmunoassay provided an estimate of the mass of T3 bound to the nuclei at every level of T3 injected. The nuclear concentration of T3 in the physiological state, as well as the maximal binding capacity, thus, could be determined. Of interest in the context of current discussion about the possibility of extranuclear receptor sites was the finding that displacement of radioactive T3 could be demonstrated only in the nuclear fraction.

A vexing problem in quantitating the subcellular distribution of any noncovalently bound ligand is the possibility of redistribution of label during the homogenization and separatory processes. This does not, however, appear to be a significant technical problem in the experiments cited since the rate of interchange of T3 among the subcellular fractions is extremely slow at 4°C, the temperature at which fractionation is carried out. This was demonstrated by the failure to observe alterations in the distribution in subcellular T3 as a result of diluting the liver homogenate with aqueous buffer. Rapid dissociation of T3 from the nuclear sites would have resulted in a progressive fall in the percentage of radioactivity associated with this fraction. Direct measurements of the rate of dissociation of T3 from nuclear sites at 4°C have also been carried out (Thomopoulos et al., 1974; Samuels et al., 1974b) and shown to slow (tl/2 = 2 days).

The binding capacity of nuclear T3 in hepatocytes has been estimated by this technique to be approximately 0.6 ng/mg DNA or 1.0 pmole/mg DNA. This value is in excellent agreement with subsequent estimates performed under in vitro conditions with isolated nuclei and solubilized sites. Both in vivo as well as in vitro techniques indicate that nearly one-half of the nuclear sites are occupied under physiological conditions (Oppenheimer et al., 1974a; Silva et al., 1977; Surks and Oppenheimer, 1977). This appears to represent a substantially higher percentage occupancy than characterizes other hormone receptor systems. The physiological implications of these findings will be discussed subsequently. It should be emphasized that only approximately 12% of the total hepatic T3 is specifically bound (Oppenheimer et al., 1974a). The remainder is distributed among the other subcellular fractions.

The nature of the receptor has now been identified as a nonhistone protein of probable molecular weight (MW) of 50,500 (Latham et al., 1976). The chemical and physical characteristics of these sites as well as their association with DNA is discussed elsewhere in these volumes. Techniques for measuring the binding capacity and affinity of nuclear sites using isolated nuclei have now been developed (Koerner et al., 1974; Samuels et al., 1974a; De Groot and Torresani, 1975). Previous failures to demonstrate specific binding sites for T3 using in vitro incubation techniques with isolated nuclei had failed largely because of the presence of nonspecific binding sites which served to obscure specific nuclear binding. Treatment of nuclei with the detergent Triton X-100 reduces the nonspecific binding that is probably associated largely with the outer nuclear membrane. Since Triton X-100 is not required for the demonstration of specific nuclear binding with in vivo studies, it would appear probable that additional nonspecific binding sites are generated or exposed during the fractionation procedure. Substantial evidence from a number of laboratories indicates that the binding sites demonstrated in isolated nuclei are identical to those demonstrated by in vivo kinetic techniques. More recently, Samuels et al. (1974a) have succeeded in demonstrating a specific interaction of T3 with solubilized nuclear binding sites.

In vivo kinetic experiments have also indicated that there is a rapid exchange of T3 bound to the nuclei and T3 situated in cytosol (Oppenheimer et al., 1974b). This was established by serial measurements of tracer T3 in plasma, hepatic cytosol, and hepatic nuclei following the i.v. injection of tracer doses of [¹²⁵I]T3 (Fig. 1). The disappearance curves of labeled T3 in plasma and cytosol were parallel from the earliest period of observation (5 minutes), a relationship that reflects the rapid interchange of T3 between plasma and cells (Oppenheimer et al., 1969). Nuclear radioactivity, however, did not achieve maximum values until approximately ½ hour after the injection, after which it declined in a fashion parallel to plasma and cytosol [¹²⁵I]T3. The fractional rate of removal of radioactive T3 from nuclei calculated from these data was shown greatly to exceed the net irreversible fractional removal rate of T3 from the total body. The bulk of radioactivity labeled T3 entering the nucleus, thus, is not metabolized but is returned chemically unchanged to the cytoplasm. Moreover, nuclei cannot metabolize radioactively labeled hormone despite the addition of a variety of cofactors to the incubation mixture (Surks et al., 1975). In vivo techniques have been used to estimate the fractional rate of dissociation of T3 from the nuclei (Oppenheimer et al., 1976). This was accomplished by injecting loading doses of T3 ½ hour after the administration of a dose of [¹²⁵I]T3. The loading dose of T3 blocks the reassociation of the tracer hormone following initial dissociation from the nuclear sites. Animals were killed at serial intervals after the injection of the loading dose of T3 and the nuclear concentration of [¹²⁵I]T3 measured. The t1/2 of dissociation from hepatic nuclear sites was estimated to be approximately 15 minutes. This is rapid in comparison to the 7 hours tl/2 of plasma T3.

Fig. 1 ). From Oppenheimer et al. (1974b). These data were used to estimate the rate of interchange of T3 among nuclei, cytosol, and plasma.

The nuclear uptake of T3 does not require the mediation of a cytoplasmic carrier as has been postulated for steroid hormones. Cytosol proteins binding T3 exhibit a substantially higher capacity and lower affinity than do nuclear sites. These proteins also bind thyroid hormone analogues with a different rank order than do nuclear sites (Dillmann et al., 1974). Moreover, nuclear binding of T3 can be demonstrated in a strictly aqueous medium without the requirement of cytosol factors (Surks et al., 1975). Triiodothyronine, therefore, appears to reach the nucleus after a series of relatively nonspecific interactions with cytosol binding proteins. Since T3 dissociates from protein before entering the nucleus, free rather than bound T3 probably is responsible for the initiation of thyroid hormone action at the cellular level. Changes in the level of cytosol protein, therefore, can serve to retard the fractional rate of transcellular movement of T3, but under true steady-state conditions will not influence the absolute mass of T3 bound to the nucleus. In this respect, the function of cytosol proteins is analogous to that of the plasma hormone binding proteins. Moreover, since the transfer of T3 from cytosol to nuclei is not inhibited by a variety of metabolic poisons, transcellular movement of T3 probably appears not to be dependent on active metabolism (Surks et al., 1975).

The rapid equilibration of T3 between nucleus and cytoplasm permits calculation of what can be designated as an "in vivo association constant." These calculations depend upon knowledge of the free T3 concentration in plasma as determined by standard equilibrium dialysis (Oppenheimer et al., 1963). The equilibrium relationship between free hormone and nuclear binding sites can be approximated by the expression ka = [T3N]/[T3][N], where ka = equilibrium association constant, [T3N] = number of occupied nuclear sites, [T3] = concentration of free T3, and [N] = number of unoccupied nuclear sites. Since [N], the number of unoccupied nuclear sites is equal to M - T37V, where M is the maximal binding capacity, ka = [T3N]/[T3][M -(T3N)]. Since 47% of the available sites are occupied [T3N]/[M] = 0.47 and [T3N]/[M - (T3N)] = 0.89. [T3] is approximately 2 × 10−12 M and therefore, ka = 4.4 × 10¹¹ M-1.

The in vivo association constant thus allows us to measure the intensity of binding of T3 which characterizes the physiological state. This approach obviates the necessity for simulating the nuclear microenvironment under in vitro assay conditions. As pointed out by Charles et al. (1975) and as discussed subsequently, the affinity constant determined in vitro depends on the specific conditions employed. The high in vivo association constant found indicates that nuclear binding is one to two orders more intense than thyroid hormone binding by plasma proteins.

Of considerable interest from a physiological point of view is the nature and concentration of those iodothyronines associated with specific nuclear sites in the normal state. As will be pointed out in the next section, many iodothyronine congeners bind to the nuclear sites. Moreover, the available data suggest that for a comparable duration of occupancy, hormonal activity at the tissue level appears to be determined exclusively by the molar concentration of iodothyronine specifically bound. Thus, if it could be shown that all thyroid hormone action is initiated at the nuclear level, then the ratio of T4 to T3 bound to specific sites in a given tissue should serve as an index of the relative hormonal contributions by these iodothyronines. A number of methods have been used to assess the concentration of specifically bound iodothyronines: (1) the in vivo techniques in which the isotopic nuclear plasma ratio is determined as described above, (2) direct measurements of the iodothyronine content of ethanolic extracts of nuclei pretreated with detergent to remove nonspecific binding; (3) long-term isotopic equilibration of animals with ¹²⁵I. The results of these experiments (Surks and Oppenheimer, 1977b) are summarized in Fig. 2. Excellent agreement among the results of the three methods used provides an internal check of the techniques applied. Only 10–15% of the bound iodothyronine is T4, both in liver and kidney. These findings, therefore, suggest that 10–15% of hormonal activity in liver and kidney can be attributed to T4 itself, independent of its subsequent conversion to T3. Of particular interest was that the isotopic equilibration experiments showed no evidence of any iodothyronine bound to the nuclear sites other than T3 and T4.

Fig. 2 Nuclear content of T3 and T4 per mg DNA were evaluated by direct radioimmunoassay and by chromatography of nuclear extracts of ¹²⁵I-equilibrated animals. Results were compared to previously reported in vivo kinetic studies (Oppenheimer et al., 1974A). No significant quantity of iodothyronine other than T3 and T4 were found in nuclear extracts of the equilibrated animals. Based on data by Surks and Oppenheimer, 1977.

III PHYSIOLOGICAL ROLE OF THE NUCLEAR T3 BINDING SITE

Several lines of evidence suggest that nuclear T3 binding sites act as true receptors in the initiation of thyroid hormone action. Whereas, none of these alone can be considered conclusive, the aggregate data appear convincing. As pointed out in the preceding section, nuclear binding sites exhibit an exceedingly high affinity and low capacity for iodothyronines. Although these properties are generally associated with hormone receptors, it should be pointed out that they are neither sufficient nor logically necessary characteristics. Serum hormone binding proteins are examples of low-capacity, high-affinity sites that clearly are not points for the initiation of thyroid hormone action. The nuclear location of the putative receptors and, more particularly, their identification as nonhistone nucleoproteins, however, favor the concept that they exert a regulatory function.

The probable participation of nuclear mechanisms in thyroid hormone initiation was first proposed by Tata and co-workers in the mid-1960’s as a result of sequential biochemical studies carried out in thyroidectomized rats injected with a single dose of T3 (Tata and Widnell, 1966). The earliest changes observed were enhanced incorporation of [¹⁴C]orotic acid into nuclear RNA, followed by enhanced RNA polymerase I activity. Terminally, an increase in total protein synthesis was observed. Recent extensions of these data will be discussed below.

One of the major lines of evidence supporting the biological importance of the sites is the correlation between nuclear binding affinity for various thyroid hormone analogues and their thyromimetic potency as determined by standard bioassay procedures. Nuclear binding of thyroid hormone analogues has been determined in the intact rat (Oppenheimer et al., 1973; DeGroot and Strausser, 1974), in incubated isolated hepatic nuclei (Koerner et al., 1975; De Groot and Torresani, 1975), and, more recently, by the study of solubilized nuclear extracts (Samuels et al., 1974b; Spindler et al., 1975; De Groot and Strausser, 1974; Thomopoulos et al., 1974; Silva et al., 1977). In the whole-animal studies of Oppenheimer et al. (1973), tracer T3, together with graded doses of unlabeled T3 and analogues, are injected and the animals killed ½ hour later. The dose of unlabeled T3 and analogues required to displace one-half of tracer T3 bound to hepatic and cardiac nuclei is determined. In the various in vitro studies reported, similar competition experiments with unlabeled analogue and tracer T3 have been carried out both with isolated nuclei and chromatin extracts.

The results of these approaches have yielded generally comparable results (Table I). Moreover, with only isolated exceptions, there is general agreement between reports from different laboratories (Oppenheimer et al., 1976). The substituted diphenyl group appears to be essential for nuclear binding. Thus, both mono- and diiodotyrosine fail to displace labeled T3 from nuclear sites. For maximal potency, a single group substitution either in the 3′ or 5′ position in the phenolic ring is required. T4 exhibits approximately one-tenth of the activity of T3 and tetrac, the acetic acid analogue of T4, shows even less activity. Isopropyl T2, a compound with a single bulky isopropyl grouping in the 3′ position and two iodine substituents in the 3 and 5 positions is bound as avidly as T3. In general, the binding activity of these compounds correlates well with their reported thyromimetic potency (Oppenheimer et al., 1973). The relatively low nuclear affinity of T4 can be reconciled with its well established physiological potency since, as pointed out above, it is known that T4 is converted to T3 peripherally. A singularly disturbing exception, however, is triac, the acetic acid analogue of T3. This analogue has an especially high affinity for nuclear sites, equivalent to that of T3 itself. Nevertheless, biological potency studies in the rat suggest that this compound exhibits only one-sixth to one-third of the potency of T3 (Greenberg et al., 1963; Short and Ruegamer, 1966). Failure of triac to exert its expected hormonal potency, however, can be readily attributed to the more rapid fractional metabolism of this analogue (Oppenheimer et al., 1973). Most bioassay procedures are based on an analysis of the effects of pulse injections of hormone and analogues into a test animal, generally the rat. As will be indicated subsequently, a number of lines of evidence suggest the effect of T3 appears to be mediated by an unidentified long-lived intermediate generated as a result of the interaction of T3 with the nuclear sites. In pulse injection of analogues, the longer the period of exposure of the analogue to the nuclear sites, the more intermediate can be presumed to be generated, and the more marked the biological effect terminally observed. Since triac is metabolized more rapidly than T3, the duration of nuclear binding at any given level of nuclear occupancy will be substantially less after injection of an equimolar dose of triac. The shorter nuclear occupancy of triac has been demonstrated in vivo by serial measurements of nuclear displacement carried out in groups of animals injected with equimolar doses of T3 and triac (Oppenheimer et al., 1973). Other studies have been carried out in which the metabolism and distribution of radioactive T3 and triac have been compared by the use of differently labeled isotopes of iodine and the application of noncompartmental analytic techniques (Goslings et al., 1976).

TABLE I

Relative Binding Affinities of Triiodothyronine and Analogues for Rat Liver Nuclei and Solubilized Receptora

aAliquots of nuclear extract were incubated with 0.2 × 10−10 M [¹²⁵I]T3 and increasing concentrations of T3, T4, isopropyl-T2 (3′-isopropyl-L-3,5-diiodothyronine), and reverse T3 (3,3′,5′-triiodo-L-thyronine)for 18 hours at 0°C. Values are expressed as the ratio of the concentrations of analogue and T3 required to produce a 50% decrease in binding of [¹²⁵I]T3. Data for whole nuclei determined by in vivo methods were taken from Oppenheimer et al. (1973) and for whole nuclei determined by in vitro incubation techniques from Koerner et al. (1975).

It is apparent, therefore, that metabolism and distribution as well as nuclear affinity contribute to the relative hormonal potency of a given analogue. A tentative mathematical model incorporating variables has recently been suggested (Goslings et al., 1976). Surprising in light of these considerations is the generally excellent correlation between the reported biological potency and the relative affinities of some 35 thyroid hormone analogues that have recently been tested using an in vitro binding assay with isolated hepatic cell nuclei (Koerner et al., 1975). In these studies it was possible to show the following: (1) The hydroxyl group was necessary for both biological activity and nuclear binding. (2) Compounds with disubstitution in the 3′,5′ position in the phenolic ring were invariably less active both with respect to nuclear binding and hormonal potency than compounds with a single substitution in a 3′ or 5′ position. (3) Substitution in the tyrosyl ring is important for maximal nuclear binding and thyromimetic activity. Compounds such as reverse triiodothyronine (3,3′,5′-triiodo-L-thyronine) show negligible or no activity. (4) Compounds constrained in the distal conformation are more potent by both criteria than compounds in the proximal conformation. (5) The oxygen-ether linkage can be replaced by a disulfide bond with preservation both of biological and nuclear binding activity. (6) All of the iodine substituents can be replaced by non-halogen-containing groups with preservation of slight but definite hormonal- and nuclear binding activity. These findings, therefore, indicate that halogens are not essential for thyroid hormone activity. Jorgensen et al. (1974) have recently used these primary data to interpret changes in affinity of individual analogues in the light of the calculated lipophilicity of individual molecules.

In essence, there is an almost perfect correlation between thyroid hormone effects and nuclear binding when account is taken of metabolism and distribution of the analogues in question. The available data, thus, suggest that hormonal effect is related to the molar concentration of the analogues bound to the nuclear sites and to the duration of such exposure. To date, there have been no convincing data indicating that any compound can occupy T3 receptor sites without bringing about the characteristic effects of thyroid hormone. No compounds comparable to the false neurotransmitters, which by occupying the receptor sites block the effects of T3, have been identified. Although reverse T3 has been shown to antagonize the effect of T4 when administered in large quantities (Pittman et al.to–T3 conversion.

A second line of evidence that has been used to support the biological relevance of the nuclear sites is the correlation between the responsivity of individual tissues to thyroid hormone and the concentration of putative nuclear receptor sites in that tissue. Whereas the oxygen consumption of most excised rat tissues varies with the thyroidal status of the animal, this appears not to be the case for brain, spleen, lung, testis, and ovary (Barker and Klitgaard, 1952). Other data also show that thyroid status does not influence the rate of protein synthesis (Gelber et al., 1964) or the level of a-GPD in brain (Lee and Lardy, 1965). From these observations, the inference has been drawn that these tissues are not responsive to thyroid hormone. As pointed out in Section I, however, since the appropriate criteria for biological response may not have been applied, definitive conclusions cannot be drawn.

In attempting to establish correlations between nuclear binding and tissue responsitivity, in vivo studies analogous to those described for liver have been carried out. Specific nuclear sites have been identified in all the tissues examined (Oppenheimer et al, 1974a). These studies have yielded values for the following parameters: the mass of T3 bound to the nuclei under physiological conditions, the nuclear binding capacity or the total number of binding sites, and the fraction of total cellular T3 bound to specific nuclear sites. The tissues examined included liver, kidney, heart, anterior pituitary, spleen, and testis. Results of these studies are summarized in Table II. The binding capacity per mg DNA varied widely from tissue to tissue. The most marked reduction in binding sites per mg DNA were encountered in testis, spleen, and brain. Calculations suggested the following number of sites per tissue nucleus: liver, 4000; testis, 16; spleen, 120; brain, 1760; pituitary 5200; kidney, 3480; heart, 2600. Thus, those tissues which do not respond with changes in oxygen consumption appear generally to have the lowest level of nuclear binding sites. It is not clear, however, whether the reduction in the number of binding sites is sufficiently marked in brain to account for the failure to observe alterations in oxygen consumption. In order to resolve this problem, the precise limits of detectability in changes in oxygen consumption would have to be established and related to the observed number of specific nuclear binding sites.

TABLE II

Characteristics of Nuclear T3 Binding in Various Rat Tissuesa,b

aEach tissue was studied at the predetermined equilibrium time. Entries represent the average values from two to eight separate experiments for the different tissues. Corrections were made for losses of DNA.

bData from Oppenheimer et al. (1974b).

cSpecifically bound.

The high concentration of nuclear binding sites/gram pituitary is of interest. This reflects both a high concentration of DNA in this tissue and a large number of sites/mg DNA. Since over 50% of rat pituitary cells produce growth hormone (Surks and DeFesí 1977), it seems probable that somatotropes, as well as thyrotropes, contain nuclear receptors for T3. This would fit with the observation that thyroid hormone administration stimulates the synthesis of radioimmunoassayable pituitary growth hormone (Hervas et al., 1975). The heterogeneity of cell types in individual tissue represents a general problem complicating the physiological analysis. For example, in brain functions responsive to thyroid hormone could be carried out by a relatively small number of cells with a high receptor content. The contribution of such cells might not be reflected in any analysis of the total tissue homogenates. With the development of methodology for separating cell types, future studies should facilitate the quantitation of nuclear sites in individual cell species. This approach should be useful in providing broader insights into the physiological role of thyroid hormones.

The in vivo studies discussed above have also provided a method for estimating the quantity of T3 bound to nuclear sites in the various tissues analyzed under physiological conditions. From these values and the binding capacity/gram of tissue determined in the same experiments, it was possible to calculate the percentage of nuclear sites normally occupied (Oppenheimer et al., 1974b). Of interest was that the percentage of sites occupied (35–50) did not appear to vary greatly from tissue to tissue. The variation observed may well have been within the error of the methods employed. Since it is generally believed that T3 in the plasma is in equilibrium with tissue pools, and since T3 bound to the nucleus is in equilibrium with the remainder of cellular T3, these findings imply that the affinity of the nuclear sites in different tissues is the same and, thus, support the concept that the sites are identical. This conclusion is further substantiated by recent data indicating similar physical properties of solubilized nuclear sites derived from brain and liver (Schwartz et al., 1977).

The same series of in vivo studies have also yielded information about the percentage of total cellular T3 specifically bound to the nuclear sites. This appears to vary from 10 to 15% for individual tissues with a striking exception that nearly 50% of total cellular T3 in the pituitary is specifically bound. The high percentage in the pituitary reflects the high concentration of nuclear binding sites discussed above. This made it possible in early experiments to demonstrate specific T3 binding sites in whole pituitary without the necessity of preliminary subcellular fractionation (Schadlow et al., 1972).

Of some interest in connection with the problem of tissue responsivity is a recent report suggesting that patients with apparent clinical resistance to T3 may exhibit abnormalities in nuclear binding as determined by in vitro tests with nuclei isolated from their lymphocytes, (Bernal et al., 1976). Such abnormalities may be reflected either in decreased affinity or decreased number of binding sites.

Lastly, efforts have been made to demonstrate the relevance of nuclear binding by correlating nuclear occupancy with cellular response. Such studies have been carried out both in tissue culture systems and in the intact animal. Studies at both levels of biological organization have inherent advantages and disadvantages. Thus, the tissue culture system provides an opportunity more easily to achieve steady-state hormone levels, since metabolism of hormones may be negligible under the conditions of incubations. Moreover, the defined conditions of the cell population and the removal of extraneous metabolic influences from other cellular systems reduces biological variability. Samuels and co-workers have extensively used the tissue culture of GH1 cells derived from experimental pituitary tumors in rats for the purposes of correlating nuclear occupancy with the rate of growth hormone synthesis by these cells. Details of these investigations are discussed elsewhere in these volumes. Another tissue culture system that probably can also be used to advantage for similar studies is the chick hepatocyte culture responding to thyroid hormone with synthesis of malic enzyme (Goodridge and Adelman, 1976). The disadvantage of any tissue culture system obviously lies in the fact that the cells are functioning in a controlled but not necessarily in a physiological environment. Analysis of hormone response in relationship to nuclear occupancy in the whole animal clearly obviates this problem. On the other hand, the active metabolism of hormone during the experiment poses obvious difficulties, as do the effects of poorly definable extraneous factors which contribute to biologic variation.

Although the problem of random variability of results can be minimized simply by the use of a sufficiently large number of animals, the problem of rapidly changing levels of hormone during the experiment presents a challenge. Two approaches are possible. First, efforts can be made to stabilize the plasma concentrations by achieving a steady state. Hormone is continuously supplied at the same rate at which it is metabolized. Unfortunately, it is difficult technically to achieve this experimental goal, since it would require the constant infusion of hormone over a period of days without disturbing the physiological system under study. Alternatively, it is possible to accept the nonsteady state of a rapidly changing plasma hormone concentration after pulse injection and to attempt to calculate the relevant parameters mathematically. The latter approach has recently been used (Oppenheimer et al., 1976). When possible, it is desirable to study the same system, both in the intact animal and in a tissue culture system. As will be discussed subsequently, this has been done in the case of the pituitary growth hormone response to thyroid hormone.

The interrelationships among the variables of tissue response, nuclear occupancy, plasma hormone concentrations, and i.v. hormone dose can be approximated by application of the following analysis. It will be useful first to consider the situation when the sites are fully saturated. The more general relationships, when sites are less than fully occupied, can then be considered. Definition of these functions may be useful, not only in evaluating the role of nuclear binding sites in the initiation of hormone action, but also in providing better insights into the subsequent molecular and physiological processes.

If one assumes that thyroid hormone action originates at a common specific receptor site, then it is reasonable to expect that, when the receptor site is fully saturated for a given time t, any tissue response measured at t will be maximal, regardless of the dose injected. In order to test this proposition experimentally, it is first necessary to establish that the number of receptor sites is not altered as a result of the administration of hormone. This appears to be the case, since no significant changes in nuclear binding capacity were observed by in vivo kinetic analysis following the injection of large doses of hormone (Oppenheimer et al., 1975). Moreover, the number of hepatic nuclear binding sites in hypothyroid animals does not appear to differ significantly from the values measured in euthyroid rats (Oppenheimer et al., 1975). Similar conclusions have been drawn on the basis of in vitro measurements of the binding capacity of nuclei derived from hypothyroid and euthyroid animals (Surks et al., 1975; Spindler et al., 1975).

Figure 3 illustrates the results of a representative experiment designed to test the hypothesis that full occupation of receptor sites constrains hormonal response (Oppenheimer et al., 1977). The induction of the mitochondrial enzyme α-GPD and soluble malic enzyme was used to quantitate the tissue response to thyroid hormone administration. Previous studies by other investigators based on the use of inhibitors of protein synthesis had suggested that the increased activity of these enzymes brought about by thyroid hormone administration reflected de novo protein synthesis, rather than simple enzyme activation (Sellinger and Lee, 1964; Tarentino et al., 1966). Other experiments have been carried out with the use of specific antiserum to malic enzyme; these have established the fact that the increased enzyme level produced by thyroid hormone truly reflects increased enzyme mass (Li et al., 1975; Murphy and Walker, 1974).

Fig. 3 Response of mitochondrial α-glycerophosphate dehydrogenase (α-GPD) (upper panel) and malic enzyme (ME) (lower panel) in same group of animals treated with 200 µg and 5000 µg T3/100 g body weight. Each point represents the mean value of four animals ± SE. Duration of 95% nuclear occupancy is indicated by horizontal stippled bar with a time shift to the right to take into account the lag period for α-GPD, 13.4 hours and for ME, 8.2 hours. By analysis of variance, statistically significant differences in the level of enzyme activity between animals treated with 200 and 5000/µg/100 g body weight were observed for α-GPD on day 2 (p < 0.005) and days 3, 4, 5, and 7 (p < 0.001) and for ME, day 3 (p < 0.001), 4 (p < 0.001), and 7 (p < 0.05). From Oppenheimer et al. (1977).

The experimental protocol involved the i.v. injection of two doses of T3, 200 µg and 5000 µg/100 g of body weight. Groups of animals were killed at designated intervals after injection. Hepatic enzyme levels and the concentration of plasma T3 by radioimmunoassay were determined. The nuclear concentration of T3 can be approximated from the plasma T3 concentration on the basis of reference studies in which the level of nuclear T3 were related to the plasma T3 (Oppenheimer et al., 1975). Since the binding capacity of nuclear sites is known, it is possible to estimate the fraction of nuclear sites that are occupied at any time following injection of hormone. The data in Figure 3 suggest that during the period of saturation, when over 95% of the sites are occupied, the rate of accumulation of enzyme is maximal for both dose levels injected. As a consequence, until almost 36 hours, the level of enzyme attained in both groups appears the same. With the desaturation of the nuclear sites, accumulation of new enzyme ceases. This occurs in the animals treated with the 200 µg/100 g body weight at 36 hours and in the group treated with a larger dose at about 60 hours. With the larger dose, accumulation of enzyme continues from 36 to 60 hours resulting in marked differences between the enzyme levels achieved in both groups. The terminal rate of disappearance of enzyme proceeds with an approximate t, where Rmax is the maximal appearance rate of new enzyme and λ, the fractional disappearance rate.

Figure 4 illustrates some of the relationships implicit in the model. The receptors limiting response must be in rapid equilibrium with plasma hormone. If this were not the case it would have been impossible to relate the time of cessation of enzyme accumulation with a given plasma hormone concentration in these experiments. A saturating plasma concentration can be defined at which the receptor sites are fully occupied and the observed hormonal responses are maximal. It is obvious that, in order to saturate the sites for progressively longer periods of time with a single injection, the dose of T3 required increases exponentially. This is simply a function of the exponential decrease in the plasma T3 concentration. The higher doses of T3 and longer periods of occupation will result in the accumulation of larger amounts of enzyme. The terminal plasma concentration at which maximal levels are observed, however, should be the same, regardless of the length of occupancy or the quantity of enzyme accumulated. A study was, therefore, designed to test this prediction. (Oppenheimer et al., 1977). Dose-response studies were carried out in which animals were killed at 24 and 36 hours after injection. Activities of hepatic α-GPD and malic enzyme were assessed, as well as the terminal plasma concentration of T3. The results of these experiments are illustrated in Figs. 5A and B. Indicated are the enzyme levels achieved as a function of the terminal plasma T3 concentration. The plasma concentration at which maximal effects were observed, 12 ng/ml, was the same for experiments conducted at 24 and 36 hours. These findings thus serve to support the validity of the basic