Search

Objective: Type 2 deiodinase gene (DIO2) polymorphisms have been associated with changes in pituitary-thyroid axis homeostasis. The -258A/G (SNP rs12885300) polymorphism has been associated with increased enzymatic activity, but data are conflicting. To characterize the effects of -258A/G polymorphism on intrathyroidal thyroxine (T(4)) to triiodothyronine (T(3)) conversion and thyroid hormone (TH) secretion pattern, we studied the effects of acute, TRH-mediated, TSH stimulation of the thyroid gland., Design: Retrospective analysis., Methods: The TH secretion in response to 500  μg i.v. TRH injection was studied in 45 healthy volunteers., Results: Twenty-six subjects (16 females and ten males, 32.8 ± 10.4 years) were homozygous for the ancestral (-258A/A) allele and 19 (11 females and eight males, 31.1 ± 10.9 years) were carriers of the (-258G/x) variant. While no differences in the peak TSH and T(3) levels were observed, carriers of the -258G/x allele showed a blunted rise in free T(4) (FT(4); P<0.01). The -258G/x92Thr/Thr haplotype, compared with the other groups, had lower TSH values at 60  min (P<0.03). No differences were observed between genotypes in baseline TH levels., Conclusions: The -258G/x DIO2 polymorphism variant is associated with a decreased rate of acute TSH-stimulated FT(4) secretion with a normal T(3) release from the thyroid gland consistent with a shift in the reaction equilibrium toward the product. These data indicate that the -258G DIO2 polymorphism causes changes in the pattern of hormone secretion. These findings are a proof of concept that common polymorphisms in DIO2 can subtly affect the circulating levels of TH and might modulate the TH homeostasis.

TRH is known to stimulate the transcription of the TSH gene in pituitary cells. To examine TRH-responsive elements of the human TSH alpha-subunit gene, we have used transient transfection of GH3 rat pituitary tumor cells. Using this system, TRH treatment stimulated expression of a reporter gene containing 846 base pairs from the 5'-flanking region of the human glycoprotein hormone alpha-subunit gene linked to luciferase. Analysis of 5'-deletions of the alpha-subunit sequence revealed that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 are important for regulation by TRH. The more proximal region includes a previously defined cAMP-response element (CRE) while the more upstream region contains an element with sequence similarity to the binding site for the pituitary transcription factor, Pit-1. The TRH responsiveness of each individual region was tested by inserting fragments upstream of a thymidine kinase-luciferase reporter gene. The -151 to -100 region had basal enhancer activity and permitted a 3.4-fold response to TRH. The -223 to -168 region did not permit a TRH response, but possessed basal enhancer activity. The combination of both regions resulted in a 5-fold stimulation by TRH. To assess the contributions of different signal transduction pathways, various combinations of treatments were examined. Combined treatment with TRH and forskolin led to an additive activity. Treatment with TRH plus phorbol 12-myristate-13-acetate resulted in the same level of reporter gene activity as with either agent alone.(ABSTRACT TRUNCATED AT 250 WORDS)

The TSH response to TRH administration (7 micrograms/kg) was measured in 68 infants (22 premature) who had abnormal thyroid screening tests by the filter paper method and whose serum thyroid function tests were only mildly abnormal. Twenty-eight infants (12 premature) had peak TSH values of 35 mU/L or less and were considered normal (group I). Forty infants (10 premature) had peak TSH values above 35 mU/L and were considered hyperresponsive (group II). The mean age at testing, screening T4, TSH levels that prompted the testing, as well as baseline T4, T3, and free T4 at the time of TRH testing were not different between the groups. The mean (+/- SD) baseline TSH value was greater in group II (6.8 +/- 2.3 mU/L) than in group I (4.4 +/- 2.2 mU/L; P < 0.001). However, there was a great deal of overlap in the individual TSH values (group I, 0.9-10 mU/L; group II, 1.9-10.6 mU/L). Mean peak TSH levels were significantly different in the two groups (group I, 24 +/- 7.7 mU/L; group II, 60.3 +/- 26.1 mU/L; P < 0.001). During long term follow-up, all 25 group I infants available for evaluation have been confirmed as clinically and biochemically normal. No infant diagnosed as normal was later found to have evidence of hypothyroidism. Fourteen infants in group II have had evidence of thyroid dysfunction. We conclude that the TSH response to TRH stimulation is a useful tool for the evaluation of infants suspected of having primary hypothyroidism. Whether hyperresponsiveness to TRH represents a form of neonatal hypothyroidism requiring treatment remains to be determined.

Hypophysial prolactin (PRL) and thyrotropin (TSH) cells of Rana perezi were examined after treatment in vitro with synthetic thyrotropin-releasing hormone (TRH). Ultrastructural morphometry applied to PRL and TSH immunoidentified cells estimated the volume density (Vv) of the secretory granules (SG), rough endoplasmic reticulum (ER), and Golgi complex (GC) as well as the numerical density (Nv) of the granules. Hemipituitaries were cultured in a superfusion system with or without 100 ng TRH/ml for 4, 14, or 24 hr. PRL cells showed significant degranulation (42%) of medium size SG after 14 hr of stimulation, whereas the biosynthetic machinery (ER and GC) was significantly developed after 4 hr of TRH culture (increase of Vv of GC, 1.5-fold, and ER, 1.3-fold, in comparison to the control). Most of these changes remained after 24 hr of TRH treatment. Compared with controls, TRH-treated TSH cells differed only after 24 hr when SG showed degranulation (40%), mainly of the medium size ones, and Vv of GC (1.6-fold) and ER (1.3-fold) increased. These results suggest that TRH acts directly on amphibian PRL and TSH cells stimulating hormonal synthesis and release. The time courses of responses to TRH differed in that PRL cells gave an immediate response while TSH cells gave a more delayed reaction.

We evaluated the predictive value of the thyrotropin (TSH) response to thyrotropin-releasing hormone (TRH) in 32 depressed outpatients completing a double-blind placebo-controlled trial of s-adenosyl-l-methionine (SAMe), which failed to show any significant difference between SAMe and placebo. Treatment response was defined as the change in Hamilton Rating Scale for Depression (HRSD-24) score between baseline and the end of the six-week trial. Subjects with TSH response outside the normal range (7-25 uU/ml) had a significantly greater response than patients with a normal response. There was also a significant correlation between absolute deviations from the mean TSH response (16 uU/ml) and changes in HRSD-24 scores.

The effects of a single intraperitoneal injection of ethanol (3 g/kg b.wt.) on the hypothalamic-pituitary-thyroid system was explored as a possible explanation of the hypothermic effect of ethanol. Serum thyroid hormones were significantly reduced by ethanol injection, but ethanol did not affect the cold-induced increase in serum thyroid hormones or thyroid-stimulating hormone (TSH). Since cold-exposure stimulates serum levels of TSH and thyroid hormones by stimulating thyroid-releasing hormone (TRH) release from neurons of the PVN, these findings demonstrate that ethanol did not block pituitary response to TRH or thyroid response to TSH. Paradoxically, ethanol increased cellular levels of TRH mRNA in the paraventricular nucleus (PVN), and blocked the cold-induced increase in TRH mRNA, suggesting that ethanol uncouples the regulation of TRH gene expression from the regulation of TRH release specifically in neurons of the PVN. Measurements of the effects of ethanol on TRH mRNA in thalamus, and beta-actin, vasopressin, somatostatin and corticotropin-releasing hormone (CRH) mRNAs in the PVN in addition to TRH mRNA revealed very specific effects of ethanol on the TRH neuronal system.

To determine whether incubation of mouse thyrotropic tissue with TRH in vitro influenced the oligosaccharide structure of TSH, thyrotropic tumor tissue or pituitary tissue was incubated in vitro with [3H]mannose or with [35S]sulfate and [3H]methionine, in the absence or presence of TRH for times up to 24 h. [3H]mannose-labeled oligosaccharides from intracellular TSH and free alpha-subunits were analyzed by paper chromatography, and were predominantly Man9GlcNAc and Man8GlcNAc units both in the absence and presence of TRH. The [35S]sulfate/[3H]methionine ratio in secreted molecules was greater for TSH than for free alpha-subunits; within TSH heterodimers the ratio was greater for beta-subunits than alpha-subunits. The [35S]/[3H] ratio was not altered in TSH or free alpha-subunits by TRH. Analyses of [3H]mannose-labeled charged oligosaccharides by HPLC anion-exchange chromatography revealed similar types of oligosaccharides present on TSH subunits and free alpha-subunits (having one or two sulfate residues, one or two sialic acid residues, or both a sulfate and a sialic acid residue). These charged oligosaccharides occurred in different proportions on TSH subunits compared to free alpha-subunits, and also differed depending on whether the tissue source was tumorous or nontumorous. The proportions of oligosaccharide unit types were not altered by TRH. Thus, while this study provided information concerning the high-mannose and complex oligosaccharides of mouse TSH, there was no evidence that short incubations of tissues with TRH in vitro caused modulation of TSH oligosaccharide structures.

Our previous studies demonstrated TRH stimulation of TSH beta gene expression in rat pituitary cell cultures and GH3 tumor cells in a transient expression assay. To begin to characterize the gene-proximal elements of the pathways involved in TRH stimulation of TSH beta gene transcription, we examined the effects of factors that increase intracellular calcium concentration, [Ca2+]i, or activate protein kinase C on TSH beta promoter activity in transfected GH3 cells. TPA, a tumor-promoting phorbol ester, stimulated a dose-dependent increase in TSH beta promoter activity at 8 h similar to TRH (2-3-fold). TPA did stimulate protein kinase C activation without [Ca2+] mobilization. The calcium ionophore ionomycin increased cytoplasmic free [Ca2+] by stimulating both calcium influx and release from internal stores without affecting protein kinase C. Ionomycin also stimulated a dose-dependent increase (2-fold) in TSH beta promoter activity at 8 h. However, the voltage-dependent Ca2+ channel agonist Bay K 8644, which increased influx of extracellular calcium, had little or no effect on TSH beta gene expression until 48 h (5-fold). Similar effects on prolactin/mRNA levels were observed in these cells. Effects of these factors were not additive, suggesting a common pathway(s) to stimulate gene expression. Inhibition of intracellular calcium mobilization by treatment with 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) inhibited ionomycin effects on gene expression without affecting phorbol ester activity, and, conversely, inhibition of protein kinase C activity by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (H-7) or TPA desensitization blocked TPA effects without affecting ionomycin activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Thirty female and male essential hypertensive patients and eighteen normotensive controls were submitted to the TRH-TSH conventional test (200 ug intravenously, bolus injection of TRH). Supramaximal doses of 400 and 600 ug were repeated with a week interval to each subject. Hypertensives showed a significant lower response to both conventional and supramaximal TRH doses. Hypothalamic-pituitary-thyroid axis abnormalities, secondary to TRH-receptor alterations, could account for this result.

The nuclear receptor affinity for L-triiodothyronine (L-T3), L-thyroxine (L-T4), L-triiodothyroacetic acid (triac), and D-triiodothyronine (D-T3) was compared to the potency of these thyroid hormone analogues in regulating thyrotropin (TSH) production and the number of membrane receptors for thyrotropin-releasing hormone (TRH) in mouse thyrotropic tumor cells in culture. L-T3 and triac were equally potent and D-T3 was one-sixth to one-fifth as potent in binding to the receptor and in regulating TSH production and TRH receptor number. L-T4 was the least potent analogue in each instance, but its relative receptor-binding affinity, measured after 3 h, was significantly less than its somewhat variable relative biological potency, measured after 48 h. The cells were shown to monodeiodinate L-[125I]T4 to L-[125I]T3 in a time-dependent manner, and the enhanced biological potency of L-T4 was ascribed to its conversion to L-T3. Thyroid hormones appear to regulate TSH production and the number of receptors for TRH in thyrotropic cells in culture through interaction with a nuclear receptor.

An influence of thyrotrophin-releasing hormone (TRH) on TSH heterogeneity in close association with de-novo biosynthesis was studied in rat anterior pituitary glands. Hemipituitary glands from adult male rats were incubated in Krebs-Henseleit-glucose media containing [3H]glucosamine and [14C]alanine for 3 and 6 h in the presence or absence of 10 ng TRH per ml. Fractions of TSH in the pituitary extracts were obtained using affinity chromatography coupled with an anti-rat TSH globulin. These TSH fractions were analysed by isoelectric focusing. The control pituitary glands were composed of four component peaks (isoelectric point (pI) 8.7, 7.8, 5.3 and 2.5) of [3H]glucosamine and [14C]alanine incorporated into TSH, and the amounts of radioactivity of these components were increased with the incubation time. Of these peaks, radioactive components of pI 8.7 coincided with the non-radioactive TSH components measured by radioimmunoassay. Addition of TRH increased incorporation of [14C]alanine into TSH in each of the components to a greater extent than that of [3H]glucosamine. In addition, new components with pI 7.2, 6.5 and 6.2, each component corresponding to each unlabelled TSH component, were demonstrated in the presence of TRH. Because addition of TRH did not change the amounts of [14C]alanine-labelled TSH in the media, the newly formed components were assumed to be connected with protein synthesis occurring in the anterior pituitary gland, which may be specific substances in response to TRH administration. These results indicate that TRH principally elicits an increase in protein synthesis in TSH at the anterior pituitary level, resulting in an alteration of TSH heterogeneity.

The effects of in vivo TRH administered either as bolus or continuous doses on TSH secretion, synthesis, and glycosylation were studied in normal and hypothyroid rats. Nine-week-old normal or 3-week postthyroidectomy rats were administered bolus doses of saline or TRH (0.5 mg/kg) twice daily or continuous saline or TRH (1 mg/kg/day) via an osmotic pump. After 5 days, pituitaries were removed and incubated with [35S]methionine (MET) and [3H]glucosamine (GLCN), with or without 10(-8) M TRH, for 6 and 24 h. Samples were precipitated with anti-TSH beta sera and then analyzed by gel electrophoresis. In normal rats, plasma TSH, T4 and T3 increased with continuous in vivo TRH but not with bolus TRH; in hypothyroid rats, plasma TSH, T4 and T3 were not altered by continuous or bolus doses of TRH. Additionally, in normal rats, continuous in vivo TRH increased incorporation of MET in secreted TSH (477 vs. 212 X 10(3) dpm/mg DNA; P less than 0.05) and intrapituitary TSH (5035 vs. 2124 X 10(3) dpm/mg DNA; P less than 0.05), and GLCN in secreted TSH (148 vs. 50 dpm/mg DNA; P less than 0.05) and intrapituitary TSH (2344 vs. 744 X 10(3) dpm/mg DNA; P less than 0.05). In contrast, in hypothyroid animals, continuous in vivo TRH did not alter MET or GLCN incorporation in TSH. Bolus TRH did not alter secreted or intrapituitary MET or GLCN incorporation into TSH in the normal rat. However, bolus TRH in the intrapituitary MET or GLCN incorporation into TSH in the normal rat.(ABSTRACT TRUNCATED AT 250 WORDS)

In 17 patients with compensated autonomous adenomas of the thyroid, iv thyrotropin releasing hormone (TRH) tests (200 microgram) and oral TRH tests (40 mg) were performed. In nine of these patients, thyroid-stimulating hormone (TSH) 30 min after iv TRH showed a normal (greater than 2.7 microU/ml) and in eight patients a subnormal (less than 2.7 microU/ml) or negative response. However, after prolonged oral stimulation with 40 mg TRH, after 120-180 min TSH was normal (greater than 2.7 microU/ml) in 15 and subnormal (less than 2.7 microU/ml) in two patients. In 20 patients with decompensated autonomous thyroid nodules. TSH was not detectable (less than 0.8 microU/ml) after iv or oral TRH stimulation. Therefore, the oral TRH stimulation test seemed to be superior to the iv TRH test in the discrimination of compensated and decompensated autonomous adenomas of the thyroid.

Mouse thyrotropic tumor cells grown in primary culture were shown to synthesize TSH and proteins, as determined by the incorporation of radioactive proline into immunoprecipitable TSH and trichloroacetic acid-precipitable proteins. The net TSH content of the cells and medium determined by RIA is also increased during 24 h of incubation, and newly formed hormone is detected in the medium within 1 h after the addition of proline tracer. To study the effect of T4 and T3 on TSH synthesis, cultures were pulse-labeled with [3H]proline after they had been exposed to either T3 or T4. After 48 but not 24 h, exposure to either T3 or T4 was followed by inhibition. When studied after 48 h of incubation, T4, (10(-13) M) or T3 (10(-11) M) at the lowest concentration tested, was inhibitory to TSH synthesis. At concentrations of T4 and T3 greater than 10(-9) M, the inhibitory effects on TSH synthesis were partially reversed, suggesting a biphasic response. Incubation in TRH (10(-7) M) for 24 h led to a significant increase in TSH synthesis, total protein, acid-precipitable protein, and total DNA. The effect of TRH on TSH biosynthesis was a function of the logarithm of its concentration over the range of 10(-11)-10(-7) M. The inhibitory action of 10(-6) M T3 on TSH synthesis was reversed by exposure to 10(-10) or 10(-7) M TRH.

We have previously demonstrated differences in several cellular responses to TRH in mouse thyrotropic pituitary (TtT) cells and in rat mammotropic pituitary (GH3) cells. In this report, we further explore the mechanism of TRH action in TtT cells by measuring its effects on phosphoinositides and on cytoplasmic free Ca2+ concentration [( Ca2+]i). We demonstrate that TRH stimulates rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by a phospholipase C and elevates [Ca2+]i. Furthermore, we present evidence that hydrolysis of PtdIns(4,5)P2 is not secondary to the elevation of [Ca2+]i. TRH caused a rapid decrease in the level of PtdIns(4,5)P2 to 57% of control and stimulated an increase in inositoltriphosphate, the unique product of phospholipase C-mediated hydrolysis of PtdIns(4,5)P2, to a peak of 280% of control. In control cells, resting [Ca2+]i was 106 +/- (SE) 27 nM, and TRH stimulated a rapid elevation to 700 +/- 210 nM. In experiments performed to determine whether PtdIns(4,5)P2 hydrolysis induced by TRH may have been caused by the elevation of [Ca2+]i, the following results were obtained: the effect of TRH to decrease the level of PtdIns(4,5)P2 was not reproduced by the calcium ionophore A23187 or by membrane depolarization with 50 mM K+; the calcium antagonist TMB-8 did not inhibit the TRH-induced decrease in PtdIns(4,5)P2; and, most importantly, inhibition by EGTA of the elevation of [Ca2+]i did not inhibit the TRH-induced decrease in PtdIns(4,5)P2. We suggest that phospholipase C-mediated hydrolysis of PtdIns(4,5)P2 to yield inositoltriphosphate may be the initial event in TRH action in TtT cells, as in GH3 cells, that leads to elevation of [Ca2+]i and to TSH secretion.

Actinomycin D and cycloheximide were used to clarify the tight relationship between protein synthesis and heterogeneous carbohydrate synthesis of rat pituitary thyroid-stimulating hormone (TSH) under thyrotropin-releasing hormone (TRH) stimulation in vitro. Rat anterior pituitaries were incubated with [3H]glucosamine in the presence of TRH, cycloheximide and actinomycin D at 37 degrees C for 3 and 6 h. The TSH fraction of pituitary homogenates was obtained using affinity chromatography coupled with anti-TSH globulin, and was analyzed by isoelectric focusing. Anterior pituitary in the presence of TRH showed heterogeneous components of [3H]glucosamine-labeled TSH with 6 different isoelectric points (components I-VI). Radioactivities of the components increased with the incubation period. Cycloheximide changed these heterogeneous components. Actinomycin D also caused a striking change in the heterogeneity of pituitary [3H]glucosamine-labeled TSH: components I and II decreased and components III and IV increased as compared to the control group after a 6-hour incubation. The present study implies that actinomycin D affects the TRH-induced heterogeneous components of pituitary TSH glycosylation. The data indicate that messenger RNA is essential for the normal processing of carbohydrate synthesis of pituitary TSH.

A cytological and optical microscopic study of the anterior lobe of hypohysis in the Wistar adult rat 10, 30 or 60 minutes after intravenous injection of 50 mug of synthetic TRH discloses modifications of thyrotropic cells and prolactin cells. It's possible to recognize a quick degranulation of the prolactin cells (with a maximum at the 10th minute) and a slower degranulation of the thyrotropic cells (perceptible at the 10th minute and very pronounced at the 60th minute). The somatrotopic cells did not show any modification.

To investigate whether TRH regulates TSH production through a pre- or posttranslational mechanism, we determined the pituitary levels of mRNAs for alpha-subunit and TSH beta in male Sprague-Dawley rats given TRH in the presence or absence of thyroid hormones, with or without hypothalamic influence. In normal rats, serum TSH increased 6-fold after a single sc injection of TRH (7 micrograms/kg BW), but the levels of mRNA for both TSH subunits did not differ from the control values. Infusion of TRH, achieved by osmotic minipumps that were implanted sc, increased serum TSH for 3 days. Conversely, the pituitary content of TSH dropped to and remained 35% of that in the controls. In these normal rats, throughout the TRH infusion, the pituitary levels of mRNA for the TSH subunits did not differ from those in the controls. Thyroidectomy increased, by 27 and 75 times, the normal levels of mRNAs for alpha and TSH beta, respectively. TRH, given either as a single injection or a 3-day infusion, did not further elevate these levels. We then studied thyroidectomized animals whose pituitaries were transplanted under their renal capsules. These pituitaries responded to TRH infusion by releasing TSH. T4 injection inhibited this response significantly, but not completely. In spite of this evidence of normal responsiveness to TRH, infusion of TRH for a week did not increase the level of mRNAs for either TSH subunit in transplanted pituitaries. We conclude that in the presence or absence of thyroid hormones, with or without concurrent hypothalamic influence, TRH did not affect rat pituitary level of mRNA for either TSH subunit despite persistent high levels of serum TSH. Therefore, TRH does not regulate TSH production through a pretranslational mechanism. Although a translational regulation cannot be completely excluded, the present data, in conjunction with previous findings, support the hypothesis that TRH regulates TSH production primarily by stimulating both posttranslational carbohydrate processing and secretion of this hormone.

The effects of hypothalamic deafferentation on TSH synthesis were studied by making cuts of 180 degrees arc in the anterior hypothalamus (n = 18) or sham cuts (n = 12) in rats. After 21 days, pituitaries were incubated with [35S]methionine (MET), [3H]glucosamine (GLCN), with or without 10(-8)M TRH for 24 h. TSH and free alpha-subunits were immunoprecipitated and analyzed by gel electrophoresis. In the deafferented group as compared to sham, MET incorporation into both subunits of secreted TSH was decreased (alpha, 96 +/- (SE) 9 X 10(3) vs. 180 +/- 20 X 10(3) dpm/mg protein; beta, 35 +/- 9 X 10(3) vs. 84 +/- 15 X 10(3) dpm/mg protein; P less than 0.05). Basal GLCN incorporation into both subunits of secreted TSH was also decreased in the deafferented group (alpha, 6.5 +/- 11 X 10(3) vs. 132 +/- 17 X 10(3) dpm/mg protein; beta, 36 +/- 8 X 10(3) vs. 101 +/- 29 X 10(3), P less than 0.05). In vitro TRH did not stimulate MET incorporation into secreted TSH in the sham controls but did in the deafferented group (alpha, 270% of basal; beta, 374% of basal; P less than 0.01). In vitro TRH increased GLCN incorporation in secreted TSH in both the sham (alpha, 253% of basal; beta, 245% of basal; P less than 0.02) and the deafferented group (alpha, 692% of basal; beta, 630% of basal; P less than 0.01). GLCN/MET ratio, reflecting relative glycosylation, did not differ for sham or deafferented groups but increased 2-fold with in vitro TRH in each group for both secreted subunits (P less than 0.01). Free alpha-synthesis and intrapituitary TSH were not altered by deafferentation or TRH. In summary, 1) anterior hypothalamic deafferentation decreases basal TSH protein and carbohydrate synthesis; 2) such deafferentation increases sensitivity to TRH stimulation of TSH synthesis, most notably apoprotein synthesis; 3) TRH increases relative glycosylation of secreted TSH in both deafferented and sham groups. These data suggest that TRH plays a significant role in regulating basal TSH protein and carbohydrate synthesis, glycosylation of TSH subunits, and subsequent bioactivity.

The regulation of TSH apoprotein and carbohydrate biosynthesis by TRH was studied by incubating pituitaries from normal and hypothyroid (3 weeks postthyroidectomy) rats in medium containing varying doses of TRH, [14C] alanine or [35S]methionine, and [3H]glucosamine. Samples were sequentially treated with anti-TSH beta to precipitate TSH and free TSH beta, anti-LH beta to remove LH and free LH beta, and anti-LH alpha to precipitate free alpha-subunits. Total proteins were acid precipitated. All precipitates were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In hypothyroid samples, acute TRH (6 h) stimulated [3H] glucosamine incorporation into secreted combined alpha-subunit to 204% and secreted combined beta-subunit to 227% of control values (P less than 0.01), and stimulated [14C]alanine incorporation into secreted combined alpha-subunit to 201% and secreted combined beta-subunit to 258% of control values (P less than 0.01); pituitary content was not altered by TRH. In hypothyroid incubates, the half-maximal response was 8 X 10(-10) M TRH for both labeled precursors. In contrast, in normal samples, acute TRH (6 H) did not stimulate TSH subunit carbohydrate and apoprotein synthesis, but after 24 h, TRH stimulated [3H]glucosamine incorporation into both subunits of TSH to 270% of control values (P less than 0.02), with no change in [14C]alanine incorporation. Free alpha-subunit synthesis was not altered by TRH in normal or hypothyroid incubates. The glucosamine to alanine ratio of total newly synthesized TSH, reflecting its relative glycosylation, was increased by TRH in both combined subunits in hypothyroid samples as early as 6 h (P less than 0.05) and in normal samples only at 24 h (P less than 0.01). In summary, 1) TRH in hypothyroid incubates stimulated apoprotein and carbohydrate synthesis in combined alpha- and beta-subunits, but not free alpha-subunits, at 6 and 24 h. 2) In normal pituitary incubates, TRH stimulated TSH subunit carbohydrate, but not apoprotein, synthesis only at 24 h. 3) TRH increased the relative glycosylation of TSH in hypothyroid and normal rat pituitary incubates. Such alterations in TSH glycosylation may be due to structural changes in the carbohydrate moiety and may be important for hormone release, biological activity, or clearance.