The modulation by 3,5,3'-triiodothyronine (T3) of pituitary T3 nuclear receptors in hypothyroid rats is inhibited by cycloheximide

The density of T3 nuclear receptors is known to vary with tissues and physiopathological conditions, but the factors involved in their regulation are still unknown. We have previously shown in the anterior pituitary gland that T3 modulates its own receptors; the density of T3 receptors in hypothyroid rats is half that in normal rats, and one injection of T3 is able to restore normal density of T3 receptors within 1-3 h. To determine whether T3 has a direct action on the synthesis of its nuclear receptor, the effect of cycloheximide (Cy) on T3-induced nuclear receptor was studied. In addition, the relationship between the density of pituitary T3 receptors and the secretion of TSH in different thyroid states was examined. In normal rats one injection of Cy (0.5-8 mg/100 mg BW) induced within 3 h a dose-dependent reduction in the density of pituitary T3 receptors as well as an important decrease in plasma TSH, with no changes in T4, T3, or pituitary TSH content. In hypothyroid rats the 50% decrease in the density of pituitary T3 receptors was not further reduced by 1 mg Cy. However, when the same dose of Cy was given 30 min before T3 it completely inhibited the induction by T3 of its receptors. When Cy was given 30 min or 1 h after T3 the inhibition was only partial. An inverse correlation was found between the density of T3 receptors in the pituitary gland and plasma TSH (r = -0.8128) in all experimental groups except those treated with Cy; this drug had an inhibitory effect on both TSH secretion and the density of receptors. The present data, therefore, support the view that T3 in the pituitary gland may induce the synthesis of its own nuclear receptors and that the density of T3 receptors is also involved in the control of TSH secretion.     

The contribution of local tissue thyroxine monodeiodination to the nuclear 3,5,3'-triiodothyronine in pituitary, liver, and kidney of euthyroid rats.

The contributions of local T4 monodeiodination and plasma T3 to the nuclear T3 of anterior pituitary, liver, and kidney were measured in euthyroid rats. After injection of [125I]T4, there was a gradual increase in the quantity of plasma [125I]T3 in excess of injected contaminant, which peaked at approximately 12 h after injection and remained a constant fraction of plasma [125I]T4 (2.8 X 10(-3) after that time. In the nuclei of anterior pituitary tissue, there was also a slow increase in locally produced [125I]T3 (in excess of that which could be accounted for by plasma [125I]T3) which appeared to peak at about 16 h after [125I]T4 administration. The ratio of nuclear [125I]T3 generated intracellularly to plasma [125I]T4 was constant at 18 and 24 h after T4 injection and was 13 +/- 2 X 10(-3) in nuclei of pituitary, 2.0 +/- 0.4 x 10(-3) in liver, and 0.47 +/- 0.1 x 10(-3) in kidney (all values are mean +/- SD). This locally generated T3 resulted in a markedly higher nuclear to plasma (N:P) ratio for [125I]T3 than for injected [131I]T3 in the same animals. The N:P ratio for [125I]T3 at equilibrium after injected T4 was 2.4 +/- 0.6, 0.47 +/- 0.09, and 0.10 +/- 0.03 (nanograms of T3 (mg DNA)-1/ng T3 ml-1) in pituitary, liver, and kidney. Comparable values for [131I]T3 N:P ratios were 0.47 +/- 0.14 (pituitary), 0.18 +/- 0.01 (liver), and 0.036 +/- 0.008 (kidney). Using RIA values for plasma T4 and T3 concentrations in these rats and maximal nuclear T3-binding capacities estimated in parallel experiments, the gravimetric quantities of nuclear T3 derived from plasma T3 and from local T4 to T3 monodeiodination were estimated and expressed as the percentage of saturation of T3 receptors. Seventy-eight percent of nuclear T3 receptor sites in anterior pituitary were occupied with one-half of the nuclear T3 derived directly from plasma T3 and the other half from intrapituitary T4 monodeiodination. Local T4 monodeiodination provided only 28% and 14%, respectively, of the nuclear T3 in liver and kidney, and the nuclear receptors of these tissues were about 50% saturated. Since our previous studies have shown that the occupancy of the pituitary nuclear T3 receptors may regulate TSH release, these data provide a mechanism by which TSH secretion could be altered by changes in either plasma T3 or T4, whereas nuclear T3 in liver and kidney is predominantly a function of the plasma T3 concentration.     

Sex steroids modulate the pituitary parameters involved in the regulation of TSH secretion in the rat

The sex-related differences observed in the regulation of TSH secretion was further investigated by determination of the densities of T3 nuclear and TRH membrane receptors as well as the activity of 5'-deiodinase (5'D) in the anterior pituitary gland of adult male and female rats. The respective modulatory roles of androgens and estrogens on these parameters were evaluated by similar determinations carried out in castrated and in estrogen-treated male rats. The density of pituitary T3 and TRH receptors and the activity of 5'D type II were significantly greater in the female than in the male rats. The E2-treated male rats disclosed a female profile, viz. also greater densities of T3 and TRH receptors when compared with control male rats (2.3 +/- 0.2 vs 1.8 +/- 0.2 fmol T3/mg gland and 9.4 +/- 0.8 vs 6.0 +/- 0.8 fmol TRH/mg gland, mean +/- SEM), whereas no changes were found in the castrated rats. The E2-treated rats and the castrated rats exhibited an increased pituitary activity of 5'D, type II (0.87 +/- 0.10 and 0.66 +/- 0.05, respectively, vs control 0.34 +/- 0.07 pmol rT3.h-1.(mg protein)-1), suggestive of a stimulatory effect of E2 and of an inhibitory effect of androgens on this parameter. In contrast, no differences in hepatic 5'D were found between all groups, illustrating the well-known tissue-specific regulation of 5'D. These results demonstrate that the sex difference in the density of pituitary T3 and TRH receptors and the activity of 5'D in the adult rat is mainly due to a modulatory effect of estrogens, which may be responsible for the sex-dependent regulation of TSH secretion.     

Modulation by oestrogen of thyroid hormone effects on thyrotrophin gene expression

The role of oestrogen in the regulation of TSH gene expression is unclear. We have examined the effect of administration of oestrogen in the rat on serum TSH, pituitary TSH content and pituitary cytoplasmic concentrations of mRNA encoding the TSH beta and alpha subunits, thus deriving measures of hormone release and synthesis. In addition, we have examined the effect of oestrogen on the binding of tri-iodothyronine (T3) to nuclear receptors in the anterior pituitary. Administration of oestrogen did not affect serum concentrations of TSH in euthyroid or untreated hypothyroid rats, but did augment the effects of T3 (1 and 2 micrograms) on serum TSH in hypothyroid animals 6 h after injection of T3. No influence of oestrogen or of thyroid status on pituitary content of TSH was seen. A marked increase in the concentrations of TSH beta and alpha mRNA in pituitary cytoplasm was found in hypothyroidism, compared with those in the euthyroid state. No effect of oestrogen on TSH mRNA was seen in euthyroid animals but concentrations of TSH beta and alpha mRNA were lower in hypothyroid animals than in vehicle-treated controls. A stimulatory influence of T3 on TSH mRNA was seen 6 h after injection of T3; this stimulation was absent in oestrogen-treated rats. No effect of oestrogen on the action of T3 was evident 72 h after beginning treatment with T3. In addition to effects on serum TSH and TSH mRNA, an increase in the number of pituitary nuclear receptors for T3 was seen after oestrogen treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thymus-hypophysis-thyroid interrelation in rats after administration of a heterologous extract of thymus gland

The Authors have investigated the effects of a thymus chromatographic fraction on TSH, T3, T4 serum values in thyroidectomized and normal controls rats before and after thymus treatment. The decrease in TSH values of thyroidectomized rats points out a possible inhibitory effect which the thymus extract may have at either the level of the adenohypophysis or hypothalamus. Moreover the TSH values in the euthyroid rats after thymus treatment showed a slight decrease, this changes are much more evident in the thyroidectomized rats after thymus treatment. In fact the plasma TSH of thyroidectomized rats drops from 97.5 +/- 4 microU/ml before thymus treatment, to 76.25 +/- 10 microU/ml after thymus treatment.     

Inhibition of the response of thyrotrophin to thyrotrophin-releasing hormone by thyroxine in hypothyroid rats treated with iopanoic acid: evidence suggesting an intrinsic biological activity of thyroxine without prior conversion to 3,5,3'-tri-iodothyronine

The effects of a physiological replacement dose of thyroxine (T4) on the response of plasma TSH to TRH in hypothyroid rats were studied. Animals were treated with iopanoic acid (IOP; 5 mg/100 g body weight) or vehicle 24, 12 and 1.5 h before the experiment. Thereafter, 800 ng T4/100 g body weight were administered i.v. and 20 min later 1 microgram TRH/100 g body weight was injected i.v. In control rats the basal concentration of TSH was 1450 +/- 300 (S.E.M.) mu./1. The plasma concentration of TSH 10 min after injection of TRH increased to 167 +/- 14% of the mean basal value (P less than 0.001). The injection of T4 significantly (P less than 0.005) reduced the TSH response to TRH in both IOP-treated (118 +/- 15%) and vehicle-treated (108 +/- 15%) rats compared with untreated controls. Tri-iodothyronine (T3) was undetectable in the plasma of all rats, whereas the plasma concentration of T4 was 121 +/- 19 nmol/l in IOP-treated and 139 +/- 23 nmol/l in vehicle-treated rats 30 min after injection of 800 ng T4/100 g body weight. In pituitary cells of control rats, cytoplasmic radioactivity 30 min after injection of [125I]T4 was 1.28 +/- 0.13 X 10(-2)% (76 +/- 6% T4 and 17 +/- 3% T3), whereas that in nuclei reached 0.42 +/- 0.36 X 10(-2)% (51 +/- 4% T4 and 28 +/- 3% T3) of the injected dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of sex and age on T3 receptors and T3 concentration in the pituitary gland of the rat: consequences on TSH secretion

A reduced secretion of thyroid hormones with age has been documented in humans and animals with no substantial increase in TSH secretion, which may be indicative of an age-related impairment of the pituitary sensitivity to the negative control exerted by thyroid hormones. We have evaluated in rats the influence of sex and age on pituitary T3 nuclear receptors--known to be determinant in the regulation of TSH secretion--as well as on T3 concentration in the pituitary gland. As regards sex, the density of T3 receptors and the concentration of T3 in pituitary gland and plasma were greater in females than in males whereas pituitary and plasma TSH concentrations were less. As for age, the density of T3 receptors was greater in old male rats than in young ones with no changes in pituitary T3 and plasma TSH concentrations. In old female rats in contrast, there was no significant increase in T3 receptors but pituitary T3 was less and plasma TSH greater than in young female rats. In both sexes plasma thyroid hormones and pituitary TSH were reduced with age whereas TSH response to TRH was not altered. These results illustrate sex and age differences in pituitary T3 receptors and pituitary T3 concentration as well as in TSH secretion. In young animals of both sexes an inverse correlation is observed between the density of pituitary T3 receptors and plasma TSH. In contrast, in old animals the absence of this correlation is suggestive of an age-related impairment of T3 action on the thyrotrophs or of changes pertaining to other factors modulating TSH secretion.     

Reduced triiodothyronine content in liver but not pituitary of the uremic rat model: demonstration of changes compatible with thyroid hormone deficiency in liver only

Intracellular thyroid hormone concentration and action were examined in the liver and the pituitary of a nephrectomized rat model (Nx); the results were compared with those obtained from control (C), thyroidectomized (Tx), and nephrectomized-thyroidectomized (NxTx) littermates. Based on the severity of the uremia, Nx rats were subdivided into Nx1 and Nx2 groups; the former included rats with serum urea nitrogen of less than 100 mg/dl and the latter rats with serum urea nitrogen greater than 100 mg/dl. A group of rats pair-fed to the Nx rats was also included (PF). In the liver, nuclear T3 content (picograms per g liver) and T3-receptor binding capacity (Cmax, picograms T3 per mg DNA) were measured. The respective results from all groups of rats were as follows (asterisks denote values differing from C with a P value less than 0.05): C, 308 +/- (SE) 45 and 121 +/- 11; Nx1, 245 +/- 43 and 85 +/- 15; Nx2, 163 +/- 19 and 74 +/- 7; Tx, 43 +/- 11 and 91 +/- 10; NxTx, 33 +/- 10 and 54 +/- 6; and PF, 237 +/- 20 and 121 +/- 9. T3 receptor binding affinity (Ka), ranging from 3.62-5.28 X 10(9) M-1, was not significantly different among the six groups of rats. In the pituitary, T3 content (picograms per mg pituitary) was reduced only in the Tx rats, being 3.13 +/- 0.89 as compared to 7.04 +/- 1.48 in the C rats (P less than 0.05). In the Nx1, Nx2, and PF rats, pituitary T3 contents were 9.81 +/- 3.22, 13.01 +/- 3.60, and 7.83 +/- 1.08, respectively, and were not different from the C rats. Serum TSH was reciprocally elevated only in the Tx rats. The reduction in hepatic nuclear T3 content and T3-Cmax in the Nx2 rats is consistent with the presence of selective tissue deficiency of thyroid hormone. This is in agreement with the observation of reduced activity of two liver enzymes known to be under thyroid hormone regulation. The pituitary, however, had normal T3 content, suggesting a dissociation in thyroid hormone-dependent metabolic status between a peripheral tissue (liver) and the pituitary. This explains the failure to observe an increase in the serum TSH level, a manifestation of reduced intracellular rather than serum T3 concentration. Decreased food intake appeared not to be the cause of thyroid hormone abnormalities observed in uremia, as PF rats failed to manifest the changes found in Nx rats.     

Effects of oestradiol benzoate on the pituitary secretion and peripheral kinetics of thyrotrophin in the rat

Previous works from this laboratory have demonstrated that oestradiol benzoate (EB) in euthyroid male and female rats induced a significant decrease in the pituitary content of TSH while serum levels of this hormone remained normal. The present work studied the effects of EB (25 micrograms/100 g body weight, during 9 days) on the peripheral metabolism of [125I]rTSH and on the pituitary and plasma concentration of TSH in euthyroid and hypothyroid rats. No significant variations were observed in [125I]rTSH kinetics of EB-treated euthyroid rats vs untreated controls: fractional turnover rate 2.8 +/- 0.2 vs 3.0 +/- 0.3%/min, distribution space 6.5 +/- 0.4 vs 6.8 +/- 0.5 ml/100 g body weight, disposal rate 18.4 +/- 2.4 vs 18.1 +/- 1.9 microU/100 g/min and extrapituitary pool 645 +/- 42 vs 614 +/- 43 microU/100 g body weight. Similarly, in hypothyroid rats oestrogens induced no changes in TSH kinetics except for an increase in distribution space (P less than 0.025). However, oestrogens decreased the pituitary pool of TSH (P less than 0.001) in both euthyroid and hypothyroid rats and increased the plasma TSH in hypothyroid animals (P less than 0.01), all vs their respective controls. Neither hypothyroid group had detectable plasma levels of T4 and T3. In summary: 1) the marked decrease of pituitary TSH with normal plasma TSH induced by EB appears unrelated to the peripheral metabolism of TSH, 2) the results from hypothyroid rats suggest that EB stimulates the release of TSH from the pituitary gland.     

Effect of administration of growth hormone on plasma and intracellular levels of thyroxine and tri-iodothyronine in thyroidectomized thyroxine-treated rats.

We have studied the effects of the administration of GH on plasma levels and peripheral production of tri-iodothyronine (T3) from thyroxine (T4) in thyroidectomized male Wistar rats given a continuous i.v. infusion of T4 (1 microgram/100 g body weight per day) and GH (120 micrograms per day) for 3 weeks. Tracer doses of 131I-labelled T3 and 125I-labelled T4 were added to the infusion. At isotopic equilibrium (10 days after the addition of 125I-labelled T4) the rats were bled and perfused. The plasma appearance rate for T3 was higher (10.6 +/- 1.3 vs 8.4 +/- 2.8 pmol/h per 100 g body weight, P = 0.05) and plasma TSH was lower (246 +/- 24 vs 470 +/- 135 pmol/l, P less than 0.01) in GH-treated rats. The amount of T3 in liver (12.3 +/- 2.8 vs 5.5 +/- 1.7 pmol/g wet weight, P less than 0.01), kidney (11.5 +/- 1.4 vs 6.5 +/- 1.4 pmol/g wet weight, P less than 0.01) and pituitary (8.8 +/- 2.7 vs 4.8 +/- 0.5 pmol/g wet weight, P less than 0.01) was higher than in controls, mainly as a result of an increased local production of T3 from T4, but plasma-derived T3 was also higher in most organs. We found an increased intracellular T3 concentration in the pituitary which may be responsible for the lower plasma TSH concentration in the GH-treated rats. Since the increase in locally produced T3 is found particularly in liver, kidney and pituitary, typical organs that express 5'-deiodinase activity, we suggest that GH acts on thyroid hormone metabolism by stimulating type-I deiodinase activity.     

Effect of neonatal hyperthyroidism upon the regulation of TSH secretion in rats

The neo-T4 syndrome was induced in rats by administration of 30 micrograms T4/in 5 doses starting on the first day of life. In the first experiment (A), neo-T4 and saline-control rats were divided into two populations, one of which was thyroidectomized on day 25. All rats then received 5 micrograms T4/100 g b.w. on days 42, 43 and 44, and were sacrificed on day 45. In the second experiment (B), neo-T4 and saline-control rats were thyroidectomized at 25 days of age and were sacrificed 10, 20 and 40 days after the operation. In both experiments, pituitary and plasma TSH and pituitary GH were determined. T4 administration has the same effect on plasma and pituitary TSH regulation in neo-T4 and control rats, thyroidectomized or not. The increase in pituitary GH produced by T4 is smaller in the thyroidectomized neo-T4 animals. The comparison between T4-induced increases of pituitary TSH in thyroidectomized neo-T4 and saline control-rats, and the corresponding decreases in non-thyroidectomized animals suggests an alteration in TSH synthesis which has previously been compared with that found in rats with lesions of the hypothalamus. However, the changes in pituitary and plasma TSH levels in neo-T4 rats at different intervals after thyroidectomy do not coincide with those described for rats with hypothalamic lesions. The possible perturbation in pituitary TSH synthesis proposed for neo-T4 rats accords with the lack of response to TRH found in adult animals treated with thyroxine, an effect which remains even when plasma T4 levels decrease.     

Estrogen induction of growth hormone in the thyroidectomized rat

GH production in the rat is almost completely dependent upon T3. Estrogens also stimulate GH in some rat models, and androgens have well documented stimulatory effects. This study examined estrogen and androgen effects on pituitary GH in rats with differing thyroid status. Diethylstilbesterol (DES; a potent synthetic estrogen, 5 mg Silastic implant), estradiol benzoate (50 micrograms/kg.48 h), or testosterone propionate (10 mg/kg.48 h) were administered for 3 weeks to ovariectomized rats that were either thyroid-intact or thyroid-ectomized. In intact rats, DES produced a 40% decrease in pituitary GH, whereas estradiol (at a lower relative dose) had no effect; testosterone produced a 65% increase in pituitary GH. Thyroidectomy decreased pituitary GH to less than 0.5% of intact values. DES and estradiol produced 50- to 70-fold increases in pituitary GH in thyroidectomized rats--reaching 23-36% of intact levels. In contrast, testosterone had no effect in thyroidectomized rats. Tamoxifen (an antiestrogen; 1 mg/kg.24 h) increased GH by 15-fold in thyroidectomized rats and completely blocked further GH induction by estradiol. T3 (20 micrograms/kg.24 h) increased pituitary GH levels by 200-fold in thyroidectomized rats--totally reversing the decrease produced by thyroidectomy; tamoxifen inhibited GH induction by T3 by 63%. The results indicate that estrogens powerfully induce pituitary GH in thyroidectomized but not intact rats through an estrogen receptor-mediated process. The DNA-binding domains of estrogen and T3 receptors, as well as their hormone response elements, share structural similarities. The present results are consistent with the hypothesis that estrogens and estrogen receptors may induce GH through unoccupied T3 response elements of the GH gene in thyroidectomized rats.     

PLASMA AND PITUITARY THYROTROPHIN (TSH) IN SEVERE AND PROLONGED HYPOTHYROIDISM, AS STUDIED IN THE RAT

Thyroidectomized rats, kept on a low iodine diet, were killed at 60, 80 and 270 days after thyroidectomy and plasma and pituitary TSH levels measured. Pituitary TSH content was lower in the thyroidectomized rats than in the controls at 60 and 80 days, starting to increase between 60 and 80 days, and reaching higher values than those of the controls by 270 days. Plasma TSH was higher in the thyroidectomized rats than in the controls at all the times studied, but declined markedly between 60 days (17.53 ± 1.98 μg/ml) and 270 days (3.63 ± 0.49 μg/ml). This decrease in plasma TSH levels was accompanied by a decrease in plasma PBI: from 0.69 ± 0.08 μg/dl at 60 days to 0.06 ± 0.01 μg/dl at 270 days. The daily injection of 1.75 μg T4/100 g body weight for 12 days in either thyroidectomized rats or normal intact rats resulted in a decline of plasma TSH levels in both groups. Pituitary TSH content increased in the thyroidectomized rats and decreased in the controls after T4 treatment. Present results agree with previous observations indicating that severe and chronic thyroid hormone deficiency is not accompanied by a progressive increase in circulating TSH levels, though an elevation is always found even in mild or subclinical forms or primary hypothyroidism. They show that in the rat this cannot be accounted for by an impairment of TSH synthesis, as previously suggessted: an impairment of TSH secretion appears more probable.     

Stimulation of liver mitochondrial alpha-glycerophosphate dehydrogenase activity by L-thyroxine in thyroidectomized rats: comparison with the suppression of pituitary TSH secretion

The rate of liver mitochondrial alpha-glycerophosphate dehydrogenase (GPD) induction was compared to the suppression of pituitary thyrotropin (TSH) secretion in thyroidectomized rats submitted to prolonged administration of small amounts of L-thyroxine (T4). With both 350 and 530 ng T4/100 g bw/day, liver alpha-GPD activity remained at post-thyroidectomy level (mean +/- SE: 0.030 +/- 0.002 and 0.034 +/- 0.001 delta A/mg prot/min, respectively) throughout all experiment. A sharp increase in enzyme activity was observed after 3 weeks of treatment in rats receiving 715 ng T4 / 100 g bw / day (mean +/- SE: 0.086 +/- 0.003 delta A/mg prot/min). In contrast, serum TSH levels were lower than pretreatment values (199 +/- 8ng/dI) in rats receiving 350 ng T4/100 g bw/day (mean +/- SE: 104 +/- 15 ng/dI; t = 7.48, p less than 0.001), decreased progressively with increasing T4 doses (m +/- SE:530 ng T4/100 g/day = 36 +/- 7 ng/dI); after only 48 h of treatment and were not significantly modified thereafter. The data are in agreement with the hypothesis of a nonlinear relationship between the degree of thyroid hormone receptor occupancy and the rate of liver mitochondrial alpha-GPD induction.     

Clomiphene citrate induces pituitary GnRH receptors in ovariectomized rats: its possible role in induction of ovulation

Since our previous studies have shown that clomiphene citrate (clomiphene) acts directly on the pituitary gland and exerts a facilitatory role on oestradiol-17 beta (E2)-induced LH surge in chronically ovariectomized rats, the effect of clomiphene on pituitary GnRH receptors was investigated. A single ip injection of either 5 micrograms E2 or 200 micrograms clomiphene did not induce LH release in adult rats ovariectomized 1-2 weeks before the injection. However, a significant increase in serum LH was noted 24 h after a single injection of E2 in the ovariectomized rats, if clomiphene was pre-injected 48 h before the E2 injection. The content of pituitary GnRH receptors in the ovariectomized rats (62 +/- 9 fmol/pituitary) remained almost unchanged until 24 h after a single injection of clomiphene but significantly increased 48 h after the injection (105 +/- 13 fmol/pituitary) without any alterations in the affinity for GnRH. To determine steroid specificity for the increase in pituitary GnRH receptors, other classes of steroids were injected in the ovariectomized rats. A single dose of E2 increased GnRH receptors, but either progesterone or 5 alpha-dihydrotestosterone failed to show any effect on the level of GnRH receptors. These results suggest that clomiphene may augment oestrogen-induced pre-ovulatory LH surge in anovulatory women, at least in part by increasing the number of pituitary GnRH receptors.     

Influence of age on the control of thyrotropin secretion by thyrotropin-releasing hormone in the male rat

Alterations with age in the control of thyrotropin (TSH) secretion by thyrotropin-releasing hormone (TRH) were evaluated at the hypothalamic and pituitary levels in young (3-5 months) and old (22-24 months) male rats. In the hypothalamus, TRH was quantified in the median eminence and in the mediobasal hypothalamus; in the adenohypophysis the membrane receptors for TRH were evaluated as well as the accumulation of TRH in the gland. As for TSH, its concentration was determined in the anterior pituitary gland and in plasma. In the hypothalamus, the concentration of TRH did not differ between young and old rats in the whole mediobasal hypothalamus, but it was significantly less in the old rats at the level of the median eminence (29.9 +/- 2.8 vs. 52.2 +/- 4.3 ng/mg protein). In the adenohypophysis, the density of receptors for TRH was greater in the old than in the young rats (23.2 +/- 3.2 vs. 13.7 +/- 1.1 fmol MeTRH/mg gland)--with no change in the affinity constant--, and the amount of TRH detected was larger (10.8 +/- 1.8 vs. 2.8 +/- 0.6 pg/mg gland), illustrative of an age-related increase in TRH accumulation in the pituitary gland. The latter results are contrasting with the findings of unchanged pituitary and plasma concentrations of TSH as well as unmodified TSH response to TRH in old rats. The present data concerning TRH and the analogy with previous observations regarding dopamine in old rats are indicative of reduced neuronal activities with age at the hypothalamic level associated with impairments in the processing of the hypothalamic hormones at the pituitary level.     

Changes in the multiple components of rat pituitary TSH and TSH beta subunit following thyroidectomy

Heterogeneity of pituitary TSH was investigated in rats following thyroidectomy. Adult male rats were sacrificed at varying periods (2-28 days) after thyroidectomy. In another experiment, thyroidectomized rats were injected daily with various doses of L-T4 (0.3-7.5 micrograms/100 g body weight, ip) and sacrificed 2 weeks later. The homogenate of the pituitaries was applied on an isoelectric focusing column or a Sephacryl S-200 column. The normal rat pituitary contained 5 major components of immunoreactive (IR) TSH in isoelectric focusing, in which the isoelectric point (pI) ranged from 6.6 to 8.8. The multiple components of IR-TSH beta were observed almost in the same areas as those of IR-TSH. Following thyroidectomy IR-TSH components with more acidic pI, associated with IR-TSH beta, were evident. A large amount of IR-TSH beta in the pituitaries of thyroidectomized rats appeared near the void volume in gel filtration, suggesting the presence of big TSH beta. Supplement of L-T4 minimized these thyroidectomy-induced changes in isoelectric focusing and gel filtration. Furthermore, big IR-TSH beta was little affected by ultracentrifugation and was relatively stable after treatment with 6 M guanidine hydrochloride. We demonstrated that the rat pituitary gland contained multiple components of IR-TSH and IR-TSH beta, both of which became variegated after thyroidectomy. It is suggested that a discernible degree of heterogeneity of TSH, particularly of TSH beta, is dependent upon the increased rate of TSH biosynthesis at the pituitary level.     

Effect of thyroid status and paraventricular area lesions on the release of thyrotropin-releasing hormone and catecholamines into hypophysial portal blood

TRH is a potent stimulator of pituitary TSH release, but its function in the physiological regulation of thyroid activity is still controversial. The purpose of the present study was to investigate TRH and catecholamine secretion into hypophysial portal blood of hypothyroid and hyperthyroid rats, and in rats bearing paraventricular area lesions. Male rats were made hypothyroid with methimazole (0.05% in drinking water) or hyperthyroid by daily injections with T4 (10 micrograms/100 g BW). Untreated male rats served as euthyroid controls. On day 8 of treatment they were anesthetized to collect peripheral and hypophysial stalk blood. In euthyroid, hypothyroid and hyperthyroid rats plasma T3 was 1.21 +/- 0.04, 0.60 +/- 0.04, and 7.54 +/- 0.33 nmol/liter, plasma T4 50 +/- 3, 16 +/- 2, and 609 +/- 74 nmol/liter, and plasma TSH 1.58 +/- 0.29, 8.79 +/- 1.30, and 0.44 +/- 0.03 ng RP-2/ml, respectively. Compared with controls, hyperthyroidism reduced hypothalamic TRH release (0.8 +/- 0.1 vs. 1.5 +/- 0.2 ng/h) but was without effect on catecholamine release. Hypothyroidism did not alter TRH release, but the release of dopamine increased 2-fold and that of noradrenaline decreased by 20%. Hypothalamic TRH content was not affected by the thyroid status, but dopamine content in the hypothalamus decreased by 25% in hypothyroid rats. Twelve days after placement of bilateral electrolytic lesions in the paraventricular area plasma thyroid hormones and TSH levels were lower than in control rats (T3: 0.82 +/- 0.05 vs. 1.49 +/- 0.07 nmol/liter; T4: 32 +/- 4 vs. 66 +/- 3 nmol/liter; TSH: 1.08 +/- 0.17 vs. 3.31 +/- 0.82 ng/ml). TRH release in stalk blood in rats with lesions was 15% of that of controls, whereas dopamine and adrenaline release had increased by 50% and 40%, respectively. These results suggest that part of the feedback action of thyroid hormones is exerted at the level of the hypothalamus. Furthermore, TRH seems an important drive for normal TSH secretion by the anterior pituitary gland, and thyroid hormones seem to affect the hypothalamic release of catecholamines.     

Localization of 7B2, neuromedin B, and neuromedin U in specific cell types of rat, mouse, and human pituitary, in rat hypothalamus, and in 30 human pituitary and extrapituitary tumors

The distributions of three novel peptides, 7B2, neuromedin B, and neuromedin U, in rat, mouse, and human pituitaries, rat hypothalamus, and 30 human pituitary tumors were investigated with immunocytochemistry. Immunoreactivity for 7B2 was present in rat, mouse, and human gonadotropes, in intermediate lobe cells and posterior lobe nerve fibers in rats and mice, in rat hypothalamus (particularly in the median eminence), and in eight human pituitary gonadotropinomas. In gonadectomized rats, larger, more numerous LH beta- and 7B2-immunoreactive gonadotropes were seen than in controls. Extractable 7B2-like immunoreactivity was elevated but not significantly so in gonadectomized rat pituitaries [males: castrated, 37.4 +/- 4.3 (mean +/- SE); controls, 26.9 +/- 4.3; females: ovariectomized, 27.2 +/- 2.7; controls, 19.1 +/- 2.2 pmol/gland]. Neuromedin B immunoreactivity was found in normal rat and mouse thyrotropes and weakly in "thyroidectomy" cells in hypothyroid rats, in which extractable pituitary neuromedin B was significantly depleted (thyroidectomized, 87.0 +/- 14.0; methimazole-treated, 82.0 +/- 11.4; control, 230.7 +/- 25.6 fmol/gland). Hyperthyroid rat pituitaries showed increased TSH beta and neuromedin B immunoreactivities and neuromedin B content (TRH-treated, 385.2 +/- 30.2; T4-treated, 352.6 +/- 20.2; control, 230.7 +/- 25.6 fmol/gland). Neuromedin U immunoreactivity occurred in corticotropes of all species, in rat and mouse intermediate lobe, and throughout the rat hypothalamus, with immunoreactive cell bodies in the arcuate nucleus. Neuromedin U-immunoreactive cells were present in six of six human pituitary and five of six human extrapituitary corticotropinomas. In adrenalectomized rats, corticotropes were larger and more numerous than in controls, but extractable anterior pituitary neuromedin U-like immunoreactivity was not raised (adrenalectomized, 3.30 +/- 0.45; control, 3.32 +/- 0.27 pmol/gland). Our findings suggest that 7B2, neuromedin B, and neuromedin U may be involved in pituitary function.     

Regulatory effect of lithium on thyroxine metabolism in murine neural and anterior pituitary tissue

The conversion of T4 to T3 in the brain and anterior pituitary gland contributes significantly to the T3 content of these tissues and appears to be an important modulator of thyroid hormone action. In the present study, the antimanic agent lithium was demonstrated in cultured neural and pituitary tissue to have a significant inhibitory effect on the activity of low Km (type II) iodothyronine 5'-deiodinase (I5'D), the enzyme mediating T3 formation. At medium lithium concentrations of 3.3-5 mM, 15'D activity was decreased 44 +/- 3% (P less than 0.001) in the NB41A3 mouse neuroblastoma cell line and 48 +/- 2% (P less than 0.001) in the GH3 rat pituitary tumor cell line. This inhibitory effect was only observed in intact cells. Significant inhibition of this enzymatic process was also noted in the anterior pituitary gland of thyroidectomized rats injected 3-24 h earlier with either 4 or 10 mmol/kg BW LiCl. This decrease in low Km I5'D activity was accompanied by significant decreases in the serum T3 concentration and the pituitary nuclear T3 content. Renal high Km (type I) I5'D activity was unaffected by lithium administration. These studies demonstrate that lithium, an agent of proven therapeutic benefit in patients with manic-depressive illness, can affect changes in T4 metabolism and cellular T3 content in neural and anterior pituitary tissue. Given the prominent mood changes that occur in patients with disordered thyroid function, this finding suggests that the therapeutic benefits of lithium in affective illness may be derived in part from alterations in thyroid hormone economy in the brain.