Serum levels of thyrotropin, thyroid hormones and their response to thyrotropin releasing hormone in infective febrile illnesses.

In 25 patients suffering from fever of infection, serum levels of thyrotropin (TSH), thyroxine (T4), triiodothyronine (T3), and thyroxine binding globulin (TBG) were estimated on two consecutive days during the febrile period and again 3 to 10 days after the fever had subsided. The serum TSH and T3 responses to 100 mug iv TRH were also studied during fever. Hormones were estimated by specific radioimmunoassays and TBG by radioligand binding assay. As compared with age and sex matched normal controls, patients with fever of infection had significantly lowered levels of total serum T3 and TBG. The serum TSH and total T4 concentrations were not significantly altered. During fever both % FT4 and absolute FT4 were significantly elevated, whereas only % FT3 was significantly increased and due to lowered serum total T3 levels the absolute FT3 were not significantly altered as compared to that in normal subjects. After the fever had subsided, the serum T3 levels returned to normal and the serum TBG levels increased. There was no correlation between basal serum levels of T3 and TSH during fever. Although in response to iv TRH the mean rise in serum TSH during fever was comparable to that in normal subjects, the overall TSH response showed an inverse correlation with serum TT3 levels. Following iv TRH there was a significant increase in serum T3 levels and the T3 response in fever was comparable to that in normal subjects. These data suggest that hormone secretion by the thyroid and its responsiveness to endogenous TSH are maintained during fever. The lowered T3 levels are not suggestive of a hypothyroid state, but perhaps could be due to decreased peripheral conversion of T4 to T3 and to decreased binding of T3 to serum proteins. The exact mechanism or significance of these alterations in thyroid function during febrile illness remains to be elucidated.     

Modulation of thyroid hormone nuclear receptor levels by L-triiodothyronine (T3) in the rat pituitary

The concentration of thyroid hormone nuclear receptors varies from one tissue to another, the anterior pituitary (AP) gland possessing the highest. Since 3,5,3',1-triiodothyronine (T3) controls within a narrow range the secretion of TSH from the pituitary gland, this study was carried out to establish whether T3 modulates its own pituitary nuclear receptors and if so, whether this modulation is correlated with the thyroidal status and TSH secretion. Salt-solubilized T3 nuclear receptors were measured in the AP gland of thyroidectomized and intact adult male rats as well as in thyroidectomized rats treated with T3. In intact male rats the maximum binding capacity of pituitary T3 nuclear receptors (MBC-T3nR), determined by Scatchard analysis, was 578 +/- 45 fmoles T3/mg protein or 27 +/- 3 fmoles T3/AP (mean +/- SEM, n = 19). 2 weeks after thyroidectomy there was a marked decrease in serum T3 and T4 concentrations as well as in the MBC-T3nR (231 +/- 26 fmoles T3/mg protein or 9.3 +/- 1.2 fmoles T3/AP, n = 7) which was still observed 8 and 16 weeks after thyroidectomy. The affinity constant (Ka) of T3 for its pituitary nuclear receptors was significantly greater in thyroidectomized rats than in intact rats (3.61 +/- 0.70 vs. 1.09 +/- 0.15 X 10(10) M-1, P less than 0.001). To test whether treatment with T3 would restore a normal MBC-T3nR, 2-week thyroidectomized rats were injected with T3(0.5 micrograms/100 g b.w.) and killed 10 min, 1, 3, 15 or 24 h after T3 injection. 10 min after T3 injection MBC-T3nR was not altered but it returned to normal values 1 h after injection (441 +/- 97 fmoles T3/mg protein) and was maintained so for at least 3 h. 15 h after T3 injection MBC-T3nR was again decreased in spite of serum T3 levels that were twice as high as in normal rats. In contrast, when T3 was injected at the dose of 1.0 micrograms/100 g b.w. the MBC-T3nR was maintained within the normal range as long as 24 h after the injection (428 +/- 125 fmoles T3/mg protein) with serum T3 concentrations that were twice the normal levels (1.27 +/- 0.06 vs. 0.67 +/- 0.01 ng/ml). These results support the hypothesis that T3 modulates the concentration of its own nuclear receptors in the rat pituitary gland. The absence of any effect of T3 10 min after injection is suggestive of an effect of T3 on the synthesis of its receptors rather than on an alteration of unoccupied receptors that would require T3 for adequate configuration and detection. This modulation of pituitary T3 receptors by T3 may provide an additional mechanism of regulation of TSH secretion in thyroid insufficiency.     

1,25-Dihydroxyvitamin D3 enhances thyrotropin releasing hormone induced thyrotropin secretion in normal pituitary cells

The findings of specific binding of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in normal rat pituitary tissue and selective effects of 1,25-(OH)2D3 on gene expression in clonal pituitary tumour cells have suggested that vitamin D may regulate pituitary function. Therefore, the in vitro effect of 1,25-(OH)2D3 on normal pituitary cells was investigated. Primary anterior pituitary cell cultures prepared from female rats were maintained in experimental medium +/- 10(-8) M 1,25-(OH)2D3 for up to 24 h and then incubated with fresh experimental medium containing TRH (10(-10)-10(-8) M) or vehicle for 1 h. Pretreatment with 1,25-(OH)2D3 for 24 h led to increased TSH release at all TRH concentrations tested (P less than 0.0001), a decrease in the half-maximal stimulatory dose of TRH for TSH release from 2 X 10(-9) M to 0.4 X 10(-9) M, a 22% increase in maximal TSH release (P less than 0.01), and an 81% increase in TSH release at 10(-9) M TRH (P less than 0.001). 1 X 10(-9) M 1,25-(OH)2D3 increased TRH (10(-9) M)-induced TSH release by 20% (P less than 0.05) but 10(-7) M and 10(-6) M 25-hydroxyvitamin D3 (25-OH D3) had no effect. The effect of 1,25-(OH)2D3 on TRH (10(-9) M)-induced TSH release was evident within 8 h and was maximal by 16 h. There was no effect on basal TSH release, TSH accumulation in the medium in the preceding 24 h nor on cell-associated TSH. 1,25-(OH)2D3 pretreatment had no effect on TRH-induced PRL secretion, PRL accumulation in the medium nor on cell-associated PRL. We have shown that 1,25-(OH)2D3 acts selectively on the thyrotroph to enhance in vitro responsiveness to physiologically relevant concentrations of TRH. These findings are consistent with the reported autoradiographic localization of [3H]-1,25-(OH)2D3 in the thyrotroph and support a permissive or regulatory role of vitamin D in the normal pituitary gland.     

Modulation of prolactin release by altered levels of thyroid hormones

In an attempt to delineate the effect of thyroid hormones on prolactin release, serum concentrations of prolactin in response to synthetic thyrotropin-releasing hormone (TRH) administration in subjects with normal and altered levels of thyroid hormones were determined by a heterologous radioimmunoassay for human prolactin. In 16 male and 19 female control subjects, an intravenous administration of TRH elicited a definite elevation in prolactin levels having a peak at 15 min after injection. This response of prolactin was markedly attenuated in all of the ten untreated hyperthyroid patients but was restored in eight subjects after the treatment. In contrast, TRH-mediated prolactin release was enhanced in most cases of primary hypothyroidism. Replacement of thyroid hormones in these patients resulted in a decrease in prolactin response to TRH. Treatment of six euthyroid subjects with triiodothyronine, on the other hand, unequivocally suppressed the release of prolactin. These results suggest that thyroid hormones per se are responsible for the blunted response of prolactin secretion to TRH administration.     

Changes of pituitary thyrotropin releasing hormone (TRH) receptor level and prolactin response to TRH during the rat estrous cycle.

The plasma PRL and TSH responses to TRH injected iv at different stages of the estrous cycle in normal rats under Surital anesthesia were maximal during the afternoon of proestrus and morning of estrus and lowest on diestrus I. As calculated from the areas under the plasma response curves, a 10-fold difference was found between the maximal and minimal PRL responses while a 2-fold difference was measured for TSH. The plasma PRL and TSH responses to TRH showed a correlation with the binding of [3H]TRH to anterior pituitary gland, a 3-fold difference being observed between the minimal binding measured on the morning of diestrus II and the maximal value found on the evening of proestrus. Contrary to findings with LHRH and LH, repeated injections of a small dose (10 ng) of TRH in the afternoon of proestrus abolished PRL and TSH responses to subsequent injection of the neurohormone.     

Release of luteinizing hormone releasing hormone and thyrotropin releasing hormone from a synaptosome-enriched fraction of hypothalamic homogenates.

A mitochondrial fraction prepared from homogenates of rat hypothalamic tissue was found by means of electron microscopy to be enriched with synaptosomes. The release of luteinizing hormone releasing hormone (LHRH) and thyrotropin releasing hormone (TRH) from this preparation was investigated. After incubation, the synaptosomes were re-isolated by ultrafiltration; and the concentration of LHRH and TRH in the ultrafiltrate was determined by radioimmunoassay. When the synaptosome-enriched preparation was incubated in 0.32M sucrose at 1 or 30 C, less than 10% of the total LHRH and TRH was recovered in the ultrafiltrate. The two hormones were released by depolarizing concentrations (60 mM) of K+ in a Ca++-dependent manner, and the stimulatory effect of K+ was essentially complete within 2 min. In the presence of 2 mM Ca++, the release of LHRH and TRH increased with increasing K+ concentrations in the range 30-120 mM. Prostaglandin E2 (PGE2), PGF2 alpha, and PGF2 beta had little if any effect on LHRH or TRH release. When the synaptosome-enriched fraction was incubated in Hanks' balanced salt solution, the release of LHRH and TRH was about 10 times greater than that seen in 0.32M sucrose. It is concluded that a synaptosome-enriched fraction from the hypothalamus contains readily releasable pools of LHRH and TRH which are mobilized rapidly by depolarizing concentrations of K+ in a Ca++-dependent manner.     

Unchanged thyrotropin and prolactin responses to thyrotropin releasing hormone after indomethacin treatment.

Experimental effects of prostaglandin synthetase inhibitors have been considered in the literature as a clue to the possible interactions of prostaglandins with the hypothalamic releasing hormones at the pituitary level. Some results of the administration to man of these drugs are apparently in contrast with the in vivo and in vitro animal data. The present investigation deals with the comparison between the thyrotropin releasing hormone (TRH) effect on prolactin and thyrotropin when the hormone was administered intravenously at doses of 50, 100 and 200 microgram respectively to three groups of six men (aged 22 to 30 years), before and on the sixth day of indomethacin administration (50 mg orally at 6-hour intervals). No significant change in the releasing hormone effect was observed either in the case of prolactin, where TRH caused a consistently similar release of the hormone at every dose employed, or in the case of thyrotropin, where a dose-dependent releasing effect was obtained before and after indomethacin treatment.     

Luteinizing hormone pulsatile secretion and pituitary response to gonadotropin releasing hormone and to thyrotropin releasing hormone in male epileptic subjects on chronic phenobarbital treatment

Endocrine changes have been reported in treated epileptic subjects, who often exhibit sexual dysfunctions, but the endocrine effects of single antiepileptic drugs have not been completely elucidated. In this study we have investigated the influence of phenobarbital (PB) on adenopituitary function and on peripheral sexual steroid pattern in 8 epileptic males. Chronic PB treatment does not modify luteinizing hormone (LH) pulsatile secretion. In the same subjects, LH and follicle stimulating hormone (FSH) response to Gonadotropin Releasing Hormone was blunted with respect to healthy controls both in terms of absolute values and of secretion areas. No difference was found in prolactin (PRL) response to Thyrotropin Releasing Hormone. In the epileptic group a significant increase in the levels of sex hormone binding globulin and a consequent decrease of the percent free testosterone have been observed. PB treatment also significantly lowers 17-beta-estradiol mean levels. These data suggest that PB independently affects both gonadotropin secretion and peripheral steroid pattern.     

Distribution of thyrotropin releasing hormone receptor in rats: an immunohistochemical study

Thyrotropin releasing hormone (TRH) receptor was identified immunohistochemically in the rat tissues using anti-peptide antiserum. Anti-TRH receptor antiserum was raised in New Zealand white rabbits immunized with a conjugate of synthetic TRH-receptor peptide (15-28) to bovine serum albumin. Immunohistochemical analysis was performed by the avidin-biotin complex method. TRH-receptor immunoreactivity was visualized in the central nervous system and anterior pituitary thus supporting previous investigations of TRH receptor distribution using in vitro autoradiographic ligand binding. Significant stain was detected in neural perikarya, axons and dendrites as well as in many cells of the retina, adrenal medulla, stomach mucosa, Auerbach's nervous branch and Mysner's nervous branch of the stomach, small intestine and colon. When using antiserum preincubated with synthetic TRH-receptor peptide (15-28) or rat anterior pituitary homogenate which contains TRH-receptor peptide, no significant stain of the anterior pituitary cells or neurons in the hypothalamus was detected. These findings suggest that TRH-receptor is widely distributed and that this method is valuable in studying the distribution of TRH-receptor in rats.     

Estradiol-17 beta stimulates basal and thyrotropin releasing hormone induced prolactin secretion by bovine pituitary cells in primary culture.

A series of experiments were conducted which demonstrate that estradiol-17β directly affects bovine pituitary cells in primary culture causing an increase in basal and thyrotropin releasing hormone (TRH)-induced prolactin secretion. Prolactin release by pituitary cells incubated with TRH at concentrations of 0.001, 0.01, 0.1 and 1 ng/ml increased linearly with increasing log concentrations. Exposure of pituitary cells to 5, 50 or 500 ng/ml estradiol for 4 h did not affect basal or TRH-induced prolactin release. However, when the period of exposure to estradiol was prolonged to 6, 12, or 24 h, 0.5, 5 or 50 ng estradiol/ml medium caused pituitary cells to release more prolactin and there was more total prolactin in the system (medium +cell content) than for comparable controls. These increases were linearly related to increasing log concentrations of estradiol used. To determine the chronic effect of estradiol on prolactin secretion, pituitary cells were incubated with estradiol-17β for 11 days during which medium was collected at 24 h intervals beginning on day 3. On day 3, prolactin accumulation in medium of control cultures averaged 2.5 ng/ml, and decreased gradually reaching relatively low levels by day 11 (100 ng/ml). Although prolactin secretion decreased during the culture period, stimulatory effects of estradiol were evident throughout. In addition, these cells still released prolactin in response to TRH (1 ng/ml) on day 11 and magnitude of TRH-induced prolactin release increased with increasing concentrations of estradiol-17β. We conclude that estradiol will increase basal and TRH-induced prolactin release by bovine lactotrophs. These results are consistent with the view that the increase in estradiol that occurs at the end of pregnancy in cattle, may participate in the prolactin surge that occurs at parturition in this species.     

Development of glycoprotein hormones and their alpha- and beta-subunits in bovine fetal pituitary glands. I. Quantitation of thyrotropin, follicle-stimulating hormone, and luteinizing hormone by radioligand receptor assays.

The development of TSH, FSH, and LH in bovine fetal pituitary glands was studied by the respective radioligand receptor assays. The content of pituitary TSH increased gradually in fetuses of both sexes throughout gestation, and no significant difference was observed for the pituitary concentration of TSH between male and female fetuses at different stages of gestation. The pituitary content of FSH increased gradually in male fetuses, but increased dramatically in female fetuses after midgestation. Similarly, the concentration of pituitary FSH was 2- to 3-fold higher in female than male fetuses after midgestation. The pituitary content of LH increased gradually and proportionally with the age of the fetus, and no significant difference was observed between sexes. However, the concentration of pituitary LH remained relatively constant for both sexes throughout gestation.     

Ontogeny of the subcellular compartmentalization of thyrotropin releasing hormone and luteinizing hormone releasing hormone in the rat hypothalamus.

The 900 x g supernatant fluid prepared from hypothalamic homogenates from male and female rats (ranging in age from -1 to 120 days) was fractionated by means of continuous sucrose density gradient centrifugation. Thyrotropin releasing hormone (TRH) and LH releasing hormone (LHRH) in the gradient fractions were quantified by radioimmunoassay. In adult hypothalamic homogenates, TRH and LHRH were associated with two populations of particles distinguishable by their sedimentation properties. Each peptide was in turn distributed in two subpopulations of parties differing in size but similar in density. The distribution of each peptide within its subpopulations of particles was found to be a function of age. In hypothalami of 22-day-old fetuses, TRH was associated almost entirely with the subpopulation of small particles. However, in the neonates, an age-dependent increase in the fractional amount of the TRH confined to the subpopulation of large particles was observed. By the 7th day of age, the peptide was equally distributed in the two subpopulations. The buoyant density of the 1-day-old neonatal particles and that of the adult small and large particles containing TRH was similar. The ontogeny of the subcellular compartmentalization of LHRH differed appreciably from that of TRH. LHRH was barely detectable in hypothalami of 22-day-old neonates. Nevertheless, at this age, the peptide was confined primarily to the subpopulation of large particles, and a similar compartmentalization was noted in hypothalami of 5- and 7-day-old neonates. However, in hypothalami of 14-day-old males and 21-day-old females, association of LHRH with the subpopulation of small particles was evident. It is concluded that 1) the nature of the hypothalamic subcellular compartmentalization of TRH and LHRH is age dependent, 2) the compartmentalization of each peptide in neonatal hypothalami differs from that in the adults, and 3) the development of the mature profile of subcellular compartmentalization of TRH and LHRH proceeds asynchronously.     

Failure of thyrotropin releasing hormone to increase 125-I uptake by the thyroid in rana temporaria

Synthetic TRH, even when directly applied to the pars distalis of the hypophysis, failed to increase ¹²⁵I uptake by the thyroid in Rana lemporaria.     

Control of pituitary thyroid-stimulating hormone synthesis and secretion by thyroid hormones during Xenopus metamorphosis

We used ex vivo and in vivo experiments with Xenopus laevis tadpoles to examine the hypothesis that the set-point for negative feedback on pituitary thyroid-stimulating hormone (TSH) synthesis and secretion by thyroid hormones (THs) increases as metamorphosis progresses to allow for the previously documented concomitant increase in serum TH concentrations and pituitary TSH mRNA expression during this transformative process. First, pituitaries from climactic tadpoles were cultured for up to 96 h to characterize the ability of pituitary explants to synthesize and secrete TSHβ in the absence of hypothalamic and circulating hormones. Next, pituitary explants from tadpoles NF stages 54-66 were exposed to physiologically-relevant concentrations of THs to determine whether stage-specific differences exist in pituitary sensitivity to negative feedback by THs. Finally, in vivo exposures of tadpoles to THs were conducted to confirm the results of the ex vivo experiments. When pituitaries from climactic tadpoles were removed from the influence of endogenous hormones, TSHβ mRNA expression increased late or not at all whereas the rate of TSHβ secreted into media increased dramatically, suggesting that TSH secretion, but not TSH mRNA expression, is under the negative regulation of an endogenous signal during the climactic stages of metamorphosis. Pituitaries from pre- and prometamorphic tadpoles were more sensitive to TH-induced inhibition of TSHβ mRNA expression and secretion than pituitaries from climactic tadpoles. The observed decrease in sensitivity of pituitary TSHβ mRNA expression to negative feedback by THs from premetamorphosis to metamorphic climax was confirmed by in vivo experiments in which tadpoles were reared in water containing THs. Based on the results of this study, a model is proposed to explain the seemingly paradoxical, concurrent rise in serum TH concentrations and pituitary TSH mRNA expression during metamorphosis in larval anurans.     

Modulation of adenovirus transformation by thyroid hormone.

We have examined the effect of triiodothyronine (T3) on de novo transformation of a cloned population of Fischer rat embryo fibroblasts (CREF) by a temperature-sensitive mutant (H5ts125) of type 5 adenovirus and on the expression of the transformed phenotype in these cells. When CREF cells were grown in medium lacking T3 before, during, and after infection with H5ts125, the yield of transformed foci was half that in the cultures supplemented with 1 nM T3. Selective addition or removal of T3 during various phases of the transformation process indicated that the hormone exerted its maximal effect within 72 hr after viral infection. T3 was also required for optimal growth in agar of two clones of CREF cells previously transformed by type 5 adenovirus, wt-3A and ts-7E. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate could substitute for T3 in enhancing growth in agar of wt-3A but not of ts-7E, suggesting that the promoter and T3 modify anchorage-independent growth by different mechanisms. Normal CREF cells and both of the transformed CREF clones grew equally well in monolayer culture in medium containing or lacking T3. Both of the transformed CREF clones contained a lower number of nuclear T3 receptors than did CREF cells and they bound somewhat lower levels of phorbol dibutyrate. These results indicate that thyroid hormone modulates an early stage involved in adenovirus transformation and that it also enhances the expression of the transformed state in previously transformed cells.