0013-7227/90/1261-0317$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society

Vol. 126, No. 1 Printed in U.S.A.

The Paraventricular Nucleus of the Hypothalamus Has a Major Role in Thyroid Hormone Feedback Regulation of Thyrotropin Synthesis and Secretion T. TAYLOR, F. E. WONDISFORD, T. BLAINE AND B. D. WEINTRAUB Molecular, Cellular, and Nutritional Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; and Georgetown University Hospital, Washington, D.C. 20007

ABSTRACT. The role of the hypothalamic paraventricular nucleus (PVN) in thyroid hormone regulation of TSH synthesis during hypothyroidism was studied in adult male rats that were normal (n = 10), had primary hypothyroidism with sham lesions in the hypothalamus (n = 17), and had primary hypothyroidism with PVN lesions (n = 14). Two and 4 weeks after initiation of treatment, plasma levels of thyroid hormones (TSH, corticosterone and PRL) and pituitary content of TSH/3 and a-subunit mRNA were measured. TRH mRNA levels in the PVN were determined by in situ hybridization histochemistry. At 2 weeks, despite a decrease in plasma free T4 in both hypothyroid groups, plasma TSH levels increased, but to a lesser degree, in the hypothyroid PVN lesioned compared to hypothyroid sham-lesioned group (7.8 ± 1.3 vs. 20.5 ± 1.1 ng/dl; P < 0.05). Similarly, at 4 weeks, the hypothyroid PVN-lesioned group demonstrated a blunted TSH response compared to the hypothyroid sham-lesioned group (6.8 ± 0.7 us. 24.0 ± 1.3 ng/dl; P < 0.05). Plasma corticosterone and PRL did not significantly differ between sham-lesioned and PVN-lesioned groups. TSH/J mRNA levels markedly increased in hypothyroid

sham-lesioned rats compared to those in euthyroid controls at 2 weeks (476 ± 21% vs. 100 ± 39%; P < 0.05) and 4 weeks (1680 ± 270% vs. 100 ± 35%; P < 0.05). In contrast, TSH/3 mRNA levels did not increase with hypothyroidism in the PVN-lesioned group compared to those in euthyroid controls at 2 weeks (140 ± 16%, P = NS) and only partially increased at 4 weeks (507 ± 135; P < 0.05). a mRNA levels at 4 weeks markedly increased in hypothyroid sham-lesioned rats compared to those in euthyroid controls (1121 ± 226% vs. 100 ± 48%; P < 0.05), but did not increase in the hypothyroid PVN-lesioned rats (61 ± 15%; P = NS). TRH mRNA in the PVN increased in the hypothyroid sham-lesioned rats compared to those in euthyroid controls (16.6 ± 1.3 vs. 4.8 ± 1.2 arbitrary densitometric units; P < 0.05), and TRH mRNA was not detectable in the PVN of hypothyroidlesioned rats at 2 weeks. In summary, lesions in rat PVN prevented the full increase in plasma TSH, pituitary TSH/3 mRNA, and a mRNA levels in response to hypothyroidism. Thus, factors in the PVN are important in thyroid hormone feedback regulation of both TSH synthesis and secretion. {Endocrinology 126: 317-324, 1990)

T

administration decreased TSH subunit synthesis and secretion (3). In addition, thyroid hormone regulation of TSH subunit gene transcription appears to be proportional to T 3 nuclear receptor occupancy (9), and the hormone-receptor complex is believed to bind to the 5' flanking region of TSH subunit genes (10, 11). These in vitro models may not reflect in vivo physiological events, and these studies demonstrate the effects of increasing thyroid hormone levels on the thyrotrophs and not those events that may occur with decreasing thyroid hormone levels. The hypothalamus has been shown to be involved in thyroid hormone feedback regulation of TSH secretion. Hypothalamic lesions in the paraventricular nuclei (PVN) of rats resulted in decreased basal TSH secretion as well as TSH secretion in response to primary hypothyroidism (12-14). In addition, changes in thyroid hormone levels have been shown to inversely and specifically alter TRH mRNA levels in the PVN, as measured by in

HYROID hormone and TRH are important regulators of TSH secretion, but their relative contributions in stimulating TSH synthesis in response to hypothyroidism remain unclear. With hypothyroidism, TSH subunit transcription (1-8) and secretion (3) increase, and thyroid hormone administration reverses these effects in the rodent (4-8). The sites of thyroid hormone feedback may be solely at the level of the thyrotroph or may be in part through TRH or other hypothalamic factors. Thus, the relative contribution of the hypothalamus in thyroid hormone regulation of TSH synthesis and secretion was addressed in the current investigation. Direct thyroid hormone feedback at the thyrotroph has been suggested by in vitro studies of rat pituitary cell incubations which demonstrated that thyroid hormone Received June 20,1989. Address requests for reprints to: Dr. Terry Taylor, Building 10, Room 8D14, National Institutes of Health, Bethesda, Maryland 20892.

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PVN AND REGULATION OF TSH

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situ hybridization histochemistry (15, 16), suggesting feedback effects of thyroid hormone on the synthesis of an important hypothalamic regulator of TSH. In vitro studies have shown that TRH can regulate TSH synthesis by increasing TSH/3 transcription (17-19), mRNA levels (20), and protein synthesis (21) in hypothyroid rat pituitary incubations, and TSH/? translation in normal rat pituitary incubations (20). In addition, we showed in vivo that anterior hypothalamic deafferentation decreased TSH subunit protein and carbohydrate synthesis, and TRH replacement in vitro resulted in marked increases in TSH synthesis (22). We have also shown that rats with PVN lesions secrete TSH with altered carbohydrate structures compared to euthyroid controls and rats with primary hypothyroidism (23); in vivo TRH administration to the PVN-lesioned rats were shown to normalize the altered carbohydrate structures (24). Therefore, hypothalamic factors such as TRH can regulate TSH secretion and glycosylation, but the full significance of the hypothalamus in response to hypothyroidism in vivo of TSH synthesis remains unclear. The present study investigated the contribution of PVN factors in the thyroid hormone feedback regulation of TSH synthesis and secretion during hypothyroidism. Rats with primary hypothyroidism received sham or PVN lesions, and plasma TSH and thyroid hormone, TSH subunit mRNA, as well as TRH mRNA levels were determined and compared to those in euthyroid controls. Our results demonstrate that PVN factors have a major role in thyroid hormone feedback regulation of both TSH synthesis and secretion. Materials and Methods Hypothalamic lesions Adult male Sprague-Dawley rats were anesthetized with 0.7 cc/kg BW Somnifer [1 g/ml sodium pentobarbital, 10% (vol/ vol) alcohol, 20% (vol/vol) propylene glycol, 2% (vol/vol) benzyl alcohol; Richmond Veterinary Supply Co., Richmond, VA] injected i.p. After placement in the stereotaxic apparatus with the incisor bar at -3.5 mm to auricular bars, electrodes with 0.5 mm exposed tips were inserted 0.5 mm bilateral to midline, 6.5 mm anterior to interaural line, with 7.5 mm depth from the dura. In the PVN-lesioned animals, 15 marap were applied for 20 sec through each electrode, and in the sham-lesioned animals no current was applied after electrode placement. Thirty thousand units of bicillin (penicillin-G benzathine and penicillin-G procaine; Wyeth, Philadelphia, PA) were administered ip after surgery. Localization of the lesions was determined in all rats, and only those with the appropriate placement were further studied. Propylthiouracil (PTU; 0.5%, wt/vol) was administered in the drinking water starting on the day of surgery. All rats were housed under a 12-h light, 12-h dark cycle.

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mRNA preparation Two weeks after brain surgery and initiation of 0.05% PTU treatment, anterior pituitaries from PVN-lesioned (n = 6), sham-lesioned (n = 12), and euthyroid adult rats (n = 6) were removed. RNA was extracted by the cytoplasmic dot/blot method previously described (8, 25). Total RNA content was determined by spectrophotometer (Beckman Instruments, Fullerton, CA). RNA purity was assessed by Northern blot analysis and 260:280 ratio. Liver and kidney samples were similarly handled. Four weeks after brain surgery and initiation of 0.05% PTU, anterior pituitaries were removed from PVN-lesioned (n = 8), sham-lesioned (n = 5), and euthyroid adult rats (n = 4). RNA was extracted by the guanidinium thiocyanate, sodium citrate, sarcosyl method as previously described (26). Slot/blot and Northern analyses For each sample, 1-9 /ig total RNA were incubated in 6 X SSC (1 x SSC = 0.15 M NAC1, 0.015 M Na citrate), 4% (vol/ vol) formaldehyde final concentration at 65 C for 15 min. Serial dilutions were applied to a Genescreen filter with the use of a slot/blot apparatus. Filters were baked for 2 h at 80 C, prehybridized in hybridization buffer [50% (vol/vol) formamide, 5 X SSC, 10 x Denhardt's, 1% sodium dodecyl sulfate (SDS), 250 Mg/ml tRNA, 100 /ig/ml salmon sperm DNA, 10% dextran, and 2 mM monobasic sodium phosphate] for 2-3 h at 70 C. The mouse TSH/? (cDNA was kindly provided by William Chin) was uniformly labeled with [32P]UTP (New England Nuclear, Boston, MA) and SP6 RNA polymerase using manufacturer's specification (Promega, Madison, WI) (27). Labeled TSH/3 riboprobe at 3 X 10~6 cpm/ml hybridization buffer was added to the filter and incubated for 16 h at 70 C. Filters were washed in 2 X SSC for 5 min at room temperature twice, in 2 X SSC and 1% SDS for 30 min at 65 C twice, and in 0.1 X SSC for 30 min at room temperature once. The filters were exposed to film, and densitometric measurements were made. hCGa probe (cDNA was kindly provided by John Fiddes) was labeled and extracted as described for TSH/3 riboprobe. Labeled a riboprobe was hybridized to filters, and filters were washed as described above. Hybridization of TSH/3 riboprobe with liver and kidney homogenates revealed minimal nonspecific binding. Samples were electrophoresed on a 1.5% agarose formaldehyde gel. Ribosomal RNA was visualized with UV light and photographed, and the intensity of the 28S ribosomal band was quantitated from densitometric measurement of the negative. Samples were standardized to ribosomal RNA for the 4-week treatment groups. RNA was transferred to nylon membranes, then probed with labeled TSH/3 and a riboprobes as described above. A second Northern gel was run with sample volumes corrected to 28S ribosomal concentration. /3-Actin mRNA was not used as a control because TRH has previously been shown to increase /?-actin mRNA levels (28). In situ hybridization histochemistry for TRH mRNA Brains were frozen on dry ice and stored at —70 C at the time of death. Tissue was sliced 12 /xm thick through the region

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PVN AND REGULATION OF TSH of the hypothalamus and placed on gelatin-coated slides. TRH cDNA probe previously characterized (15) was labeled with [3SS]dATP (New England Nuclear) with terminal deoxyribonucleotidyltransferase (Bethesda Research Laboratories, Bethesda, MD). Brain slices were fixed, dehydrated, incubated with labeled probe, and washed as previously described (15). Slides were exposed to Kodak X-Omat AR film (Eastman Kodak, Rochester, NY). Hybridization signal on the film in the region of the PVN was analyzed using an image analysis system (Loats Associates, Westminister, MD), background was subtracted, and measurements of four to six brains slices per animal were averaged. Other methods Plasma TSH, free T4, corticosterone, and PRL levels were measured by RIA (Hazelton Laboratories, Bethesda, MD) (29). Statistical significance of differences was determined by analysis of variance at the P < 0.05 level.

Results Plasma hormone levels PTU treatment in sham-lesioned animals compared to euthyroid controls significantly decreased plasma free T4 at 2 weeks (0.1 ±0.1 us. 1.8 ± 0.1 ng/dl) and 4 weeks (0.1 ± 0.1 us. 1.6 ± 0.2 ng/dl), and significantly increased plasma TSH at 2 weeks (20.5 ± 1.1 us. 2.6 ± 0.3 ng/ml) and 4 weeks (24.0 ± 1.3 us. 1.6 ± 0.2 ng/ml). In contrast, PTU treatment in PVN-lesioned animals compared to euthyroid controls lowered free T4 at both 2 and 4 weeks, but increased plasma TSH only partially at 2 and 4 weeks of hypothyroidism to levels approximately onethird of those in the sham-lesioned rats treated with PTU (7.8 ± 1.3, 6.8 ± 0.7 ng/ml, respectively; see Table 1). Serum corticosterone decreased in both sham-lesioned and PVN-lesioned rats compared to euthyroid rats at 2

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and 4 weeks. PRL levels were not significantly different between study groups (see Table 1). TSHf3 and a mRNA leuels TSH/3 mRNA levels, compared to those in euthyroid controls, markedly increased in sham-lesioned rats treated with PTU for 2 weeks (476 ± 21% us. 100 ± 39%; P < 0.05) and 4 weeks (1680 ± 270% us. 100 ± 35%; P < 0.05). In contrast, TSH/3 mRNA levels, compared to those euthyroid controls, did not increase in the PVNlesioned rats treated with PTU for 2 weeks (140 ± 16%; P = NS) and had a comparably modest increase after 4 weeks (507 ± 135%; P < 0.05). Thus, the increase in TSH/3 mRNA levels in the PVN-lesioned group was markedly blunted compared to that in the sham-lesioned group after 2 and 4 weeks of hypothyroidism (P < 0.05; Fig. 1). a mRNA levels, compared to those in euthyroid controls, markedly increased in sham-lesioned rats treated with PTU for 4 weeks (1121 ± 226% us. 100 ± 48%; P < 0.05). In contrast a. mRNA levels did not increase in the PVN-lesioned rats treated with PTU for 4 weeks compared to those in euthyroid controls (61 ± 15%; P = NS). Thus, similar to TSH/? at 2 weeks, a-subunit mRNA levels did not increase in the PVN-lesioned rats compared to those in the sham-lesioned group after treatment with PTU for 4 weeks (P < 0.05; Fig. 2). TRH mRNA levels In situ hybridization histochemistry showed marked increases in TRH mRNA in the PVN in the shamlesioned rats treated with PTU compared to those in euthyroid controls (16.6 ± 1.3 us. 4.8 ± 1.2 arbitrary densitometric units; P < 0.05). In contrast, in the PVNlesioned rats treated with PTU, no TRH mRNA was

TABLE 1. Plasma hormone levels

2 weeks Rx Normal (N = 6) Sham/PTU (N = 12) PVN/PTU (N = 6) 4 weeks Rx Normal (N = 4) Sham/PTU (N = 5) PVN/PTU (N = 8)

Free T4 (ng/dl)

TSH (ng/ml)

Corticosterone (/*g/dl)

PRL (ng/ml)

1.8 ±0.1°

2.6 ± 0.3

43 ±6°

14 ± 3

0.1 ± 0.1

20.5 ± 1.1°

8±3

7± 1

0.1 ± 0.1

7.8 ± 1.3

2±1

7±4

1.6 ± 0.2°

1.6 ± 0.2

31 ±6°

34 ±10

0.1 ± 0.1

24.0 ± 1.3°

10 ± 3

17 ± 3

0.1 ± 0.1

6.8 ± 0.7

5±3

14 ± 2

Plasma hormone levels in untreated rats (Normal), sham-lesioned rats treated with PTU (Sham/PTU), and PVN-lesioned rats treated with PTU (PVN/PTU) for 2 weeks (2 weeks Rx) and 4 weeks (4 weeks Rx). ° Significant difference from the other study groups at that treatment time, as determined by analysis of variance at the P < 0.05 level.

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PVN AND REGULATION OF TSH NORMAL

Endo • 1990 Vol 126-No 1

NORMAL SHAM LESION/PTU PVN LESION/PTU

1500T

SHAM LESION/PTU PVN LESION/PTU

1000

II

I? ^ §

HI O CD Z

1000

500-

TREATMENT TIME (WEEKS)

5

6

8

STUDY GROUP FIG. 2. a mRNA levels in normal rats, sham-lesioned rats treated with PTU, and PVN-lesioned rats treated with PTU for 4 weeks, a mRNA levels were normalized to individual 28S ribosomal RNA content (mean ± SEM) *, Significant difference from normal (P < 0.05), as determined by analysis of variance.

detected in the hypothalamic PVN regions, although TRH mRNA remained detectable in the nonlesioned regions such as the reticular nucleus (Fig. 3).

Discussion

FIG. 1. Upper panel, TSHfi mRNA levels of euthyroid controls, shamlesioned rats treated with PTU, and PVN-lesioned rats treated with PTU for 2 and 4 weeks. TSH/3 mRNA levels were normalized to individual ribosomal RNA content and are the mean ± SE of the replicates. **, Significant difference from normal (P < 0.05), as determined by analysis of variance. Lower panel, Representative sam-

These data indicate that the PVN of the hypothalamus has a major role in thyroid hormone feedback regulation of TSH synthesis. Lesions in rat paraventricular nuclei prevented the full increase in plasma TSH, TSH/3, and a mRNA levels in response to hypothyroidism. After 4 weeks of hypothyroidism, plasma TSH and pituitary TSH/? mRNA levels in the PVN-lesioned rats were approximately one third of values in the sham-lesioned rats. Thus, factors in the PVN are important in thyroid hormone feedback regulation of TSH synthesis and secretion. Changes in TSH0 mRNA levels in the current study may reflect altered transcription and translation of the /? gene, but they may also reflect changes in RNA stabilples by Northern analyses of TSH/3 mRNA in normal rats (lanes 1 and 2), sham-lesioned rats treated with PTU (lanes 3-5), and PVN-lesioned rats treated with PTU (lanes 6-9).

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PVN AND REGULATION OF TSH

321

A.

D.

FIG. 3. Upper panel, TRH mRNA hybridization signal in representative brain slices from euthyroid control rats (A), sham-lesioned rats treated with PTU (B), and PVN-lesioned rats treated with PTU (C), as determined by in situ hybridization histochemistry. Arrows point to the TRH mRNA signal in the PVN which was specifically regulated by thyroid hormone level. The adjacent reticular nuclei of the thalamus contains TRH mRNA which was not regulated by thyroid hormone. D, A representative brain slice through the region of the PVN from a lesioned animal illustrates the size of the bilateral PVN lesion. Lower panel, TRH mRNA levels in the PVN of euthyroid rats (n = 6), shamlesioned rats treated with PTU (n = 5), and PVN-lesioned rats treated with PTU (n = 4) for 2 weeks (mean ± SEM) • * * , Significant difference from normal at (P < 0.05), as determined by analysis of variance.

400-1

300-

u

200-

fc

O X Z

STUDY GROUP

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PVN AND REGULATION OF TSH

ity and/or degradation. In addition, mRNA levels may not describe translational efficiency or rates. Nevertheless, changes in plasma TSH matched those changes in TSH/3 mRNA levels, suggesting a direct relationship between mRNA and secreted protein levels. The regulatory factor in the PVN involved in thyroid hormone feedback is most likely TRH (15,16); however, one of the other neuropeptides in the PVN, such as CRH (30), somatostatin (31), or vasopressin (31), may also be involved in the regulatory changes described. TRH mRNA levels in the PVN were not demonstrable in the hypothryoid PVN-lesioned animals and were increased in the hypothyroid sham-lesioned rats compared to those in normal rats, clearly demonstrating changes in TRH among the study groups. Plasma corticosterone levels were decreased in both sham- and PVN-lesioned animals

compared to normal values, suggesting an effect of anesthesia and surgery, but the differences in TSH synthesis and secretion between the hypothyroid-lesioned and hypothyroid sham groups cannot be explained by their similarly low corticosterone levels. If PVN lesions decreased somatostatin, a known inhibitor of TSH secretion (32), one would have expected further increases, rather than a decrease, in TSH synthesis and secretion. Future studies of animals administered specific antibodies or lesioned animals replaced with specific hormones will be necessary to clearly identify all of the factors in the PVN involved in thyroid hormone feedback. In addition, future studies will be needed to determine the site of thyroid hormone feedback that is modulated by TRH. Thyroid hormone may act directly on the PVN, indirectly through other central nervous system factors such as catecholamines, which are thought to be involved in thyroid axis regulation (33-35), or on the thyrotroph where TRH may modulate thyroid hormone effects. Prior in vivo studies have clearly shown that changes in thyroid hormone levels altered TSH subunit synthesis (1-8). Hypothyroidism increased pituitary TSH/3 mRNA in a near-linear manner up to 10 weeks of hypothyroidism (8). However, these in vivo studies have not defined the sites of thyroid hormone feedback regulation. An in vitro study showed that thyroid hormone administration decreased pituitary TSH/3 mRNA levels in a dose-related manner (3), suggesting direct feedback of high thyroid hormone levels on the thyrotroph. In addition, thyroid hormone appears to bind to its receptor in the thyrotroph, and this complex may regulate TSH/3 gene expression (9, 36). However, these in vitro studies do not necessarily demonstrate thyrotroph response to decreased thyroid hormone levels, and they may not reflect the entire feedback mechanisms that are present in an intact animal. Several lines of evidence have supported the role of the hypothalamus, and in particular TRH, in regulating

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TSH secretion. Early studies in rats with PVN lesions demonstrated decreases in basal plasma TSH levels and TSH response to hypothyroidism (13). Administration of TRH antibody to rats decreased both basal plasma TSH levels (37, 38) and the TSH response to cold exposure (39-41). TRH mRNA levels in the PVN, the thyrotropic region of the hypothalamus, have been shown by in situ hybridization histochemistry to inversely change in relation to thyroid hormone levels (15, 16), suggesting feedback effects of thyroid hormone on TRH synthesis. Thus, the PVN has been shown to be involved in controlling the secretion of TSH basally and in response to thyroid hormone levels. The role of hypothalamic factors in altering TSH synthesis in vivo has been less clearly defined. We have previously shown that anterior hypothalamic deafferentation decreased incorporation of labeled amino acid and sugar into TSH, reflecting decreased protein and carbohydrate synthesis (22). We have also shown that rats with PVN lesions secrete TSH with altered in vitro carbohydrate structure, as analyzed by Concanavalin-A chromatography, to forms with biantennary structures rather than the more complex forms seen in primary hypothyroidism (23). TRH administration in vivo to these PVN-lesioned rats normalized TSH structural changes (24). Humans with hypothalamic hypothyroidism have been shown to have decreased TSH bioactivity, as determined by TSH receptor binding and adenylate cyclase-stimulating activity, and these functions were corrected after in vivo TRH administration (42). In addition to in vivo studies, in vitro administration of TRH has been shown to regulate gene transcription at the 5' flanking region (17-19), increase TSH/3 mRNA levels (18, 20), and alter TSH carbohydrate structure (43, 44). The current study, with the use of an in vivo model, demonstrated that factors in the PVN are important in obtaining the full increase in the synthesis of TSH/3 and a-subunits as well as secretion in response to hypothyroidism. In studies of other hypothalamic axes, hypothalamic releasing factors appear to have a significant role in regulating pituitary hormone synthesis and secretion. For example, PVN lesions (30) or anterolateral hypothalamic deafferentation (45) decreased CRH and caused significant decreases in POMC II mRNA levels in rat pituitaries despite decreasing corticosterone levels. Pituitary stalk sectioning in ovariectomized ewes resulted in lowered gonadotropin mRNA levels compared to those ovariectomized controls (46), and GnRH from the medial basal hypothalamus has a major role in regulating gonadotropin synthesis and secretion (47). These studies support the important role of the hypothalmus in feedback regulation of pituitary hormones. In conclusion, hypothalamic factors in the PVN have

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PVN AND REGULATION OF TSH a significant role in the increase in TSH synthesis seen in hypothyroidism. Future studies addressing the role of TRH and other hypothalamic factors involved in thyroid hormone feedback and the mechanism by which these factors alter TSH synthesis will provide interesting insight into the neuroendocrine regulation of thyroid axis.

References 1. Shupnik MA, Chin WW, Habener JF, Ridgway EC 1985 Transcriptional regulation of the thyrotropin subunit genes by thyroid hormone. J Biol Chem 260:2900 2. Gurr JA, Kourides IA 1985 Thyroid hormone regulation of thyrotropin a and /3 subunit gene transcription. DNA 4:301 3. Shupnik MA, Ridgway EC 1985 Triiodothyronine rapidly decreases transcription of the thyrotropin subunit genes in thyrotropic tumor explants. Endocrinology 117:1940 4. Franklyn JA, Lynam T, Docherty K, Ransden DB, Sheppard MC 1985 Effect of hypothyroidism on pituitary cytoplasmic concentrations of messenger RNA encoding thyrotropin /3 and a subunits, prolactin and growth hormone. J Endocrinol 108:43 5. Carr FE, Ridgway EC, Chin WW 1985 Rapid simultaneous measurement of rat a and thyrotropin (TSH) /? subunit messenger ribonucleic acids (mRNAs) by solution hybridization: regulation of subunit mRNAs by thyroid hormones. Endocrinology 117:1272 6. Gurr JA, Vrontakis ME, Athanasian EA, Wagner CR, Kourides IA 1986 Hormonal regulation of thyrotropin alpha and beta subunit mRNAs. Horm Metab Res 18:382 7. Chin WW, Shupnik MA, Ross DS, Habener JF, Ridgway EC 1985 Regulation of the a and thyrotropin /3 subunit messenger ribonucleic acids by thyroid hormones. Endocrinology 116:873 8. Franklyn JA, Wood DF, Balfour NJ, Ramsden DB, Docherty K, Chin WW, Sheppard MC 1987 Effect of hypothyroidism and thyroid hormone replacement in vivo on pituitary cytoplasmic concentrations of thyrotropin /? and a subunit messenger ribonucleic acids. Endocrinology 120:2279 9. Shupnik MA, Ardisson LJ, Meskell MJ, Bornstein J, Ridgway EC 1986 Triiodothyronine (T3) regulation of thyrotropin subunit gene transcription is proportional to T3 nuclear receptor occupancy. Endocrinology 118:367 10. Gurr JA, Wolf O, Kourides IA, Pretranslational control of thyroid stimulating hormone biosynthesis. 69th Annual Meeting of The Endocrine Society, Indianapolis IN, 1987, p 7 (Abstract) 11. Wight PA, Crew MD, Spindler SR 1987 Discrete positive and negative thyroid hormone-responsive transcription regulatory elements of the rat growth hormone gene. J Biol Chem 262:5659 12. Aizawa T, Green M 1981 Delineation of the hypothalamic area controlling thyrotropin secretion in the rat. Endocrinology 109:1731 13. Martin JB, Boshans RI, Reichlin S 1970 Feedback regulation of TSH secretion in rats with hypothalamic lesions. Endocrinology 87:1032 14. Greer MA 1952 Evidence of hypothalamic control of the pituitary release of thyrotropin. Proc Soc Exp Biol Med 77:603 15. Segerson TP, Kauer J, Wolfe HC, Mobtaker H, Wu P, Jackson IM, Lechan RM 1987 Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleus of the rat hypothalamus. Science 238:78 16. Koller KJ, Wolff RS, Warden MK, Zoeller RT 1987 Thyroid hormones regulate levels of thyrotropin-releasing hormone mRNA in the paraventricular nucleus. Proc Natl Acad Sci USA 84:7329 17. Weintraub BD, Wondisford FE, Farr EA, Steinvelder H, Radovick S, Gesundheit N, Gyves PW, Taylor T, DeCherney GS, Pretranslational and post-translational regulation of TSH synthesis. International Symposium on Glycoprotein Hormones, Newport Beach CA, 1989 (Abstract) 18. Shupnik MA, Greenspan SL, Ridgway EC 1986 Transcriptional regulation of thyrotropin subunit genes by thyrotropin-releasing hormone and dopamine in pituitary cell culture. J Biol Chem 261:12675

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19. Carr FE, Shupnik MA, Burnside J, Chin WW 1989 Thyrotropinreleasing hormone stimulates the activity of the rat thyrotropin /3subunit gene promoter transfected into pituitary cells. Mol Endocrinol 3:717 20. Franklyn JA, Wilson M, Davis JR, Ramsden DB, Docherty K, Sheppard MC 1986 Demonstration of thyrotropin /3-subunit messenger RNA in rat pituitary cells in primary culture—evidence for regulation by thyrotropin-releasing hormone and forskolin. J Endocrinol 111:R1 21. Taylor T, Weintraub BD 1985 Thyrotropin (TSH)-releasing hormone regulation of TSH subunit biosynthesis and glycosylation in normal and hypothyroid rat pituitaries. Endocrinology 116:1968 22. Taylor T, Scouten CW, Jacobowitz DM, Weintraub BD 1986 The effects of anterior hypothalamic deafferentation on thyrotropin (TSH) biosynthesis and response to TSH-releasing hormone. Endocrinology 118:2417 23. Taylor T, Gesundheit N, Gyves PW, Jacobowitz DM, Weintraub BD 1988 Hypothalamic hypothyroidism caused by lesions in rat paraventricular nuclei alters the carbohydrate structure of secreted thyrotropin. Endocrinology 122:283 24. Taylor T, Weintraub BD 1987 Altered TSH carbohydrate structures in hypothalamic hypothyroidism created by paraventricular nuclear lesions are corrected by in vivo thyrotropin-releasing hormone administration. Clin Res 35.-402A (Abstract) 25. White BA, Bancroft FC 1982 Cytoplasmic dot hybridization. J Biol Chem 257:8569 26. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156 27. Butler ETI, Chamberlin MJ 1982 Bacteriophage SP6-specific RNA polymerase. J Biol Chem 257:5772 28. Weisman AS, Tixier-Vidal A, Gourdji D 1987 Thyrotropin-releasing hormone increases the levels of c-fos and /?-actin mRNA in GH3/B6 pituitary tumor cells. In Vitro Cell Dev Biol 23:585 29. Keiffer JD, Weintraub BD, Baigelman W, Leeman S, Maloof F 1974 Homologous radioimmunoassay of thyrotropin in rat plasma. Acta Endocrinol (Copenh) 76:495 30. Bruhn TO, Sutton RE, Rivier CL, Vale WW 1984 Corticotropinreleasing factor regulates proopiomelanocortin messenger ribonucleic acid levels in vivo. Neuroendocrinology 39:170 31. Swanson LW, Sawchenko PE 1980 Paraventricular nucleus: a site for the integration of neuroendocrine and autonomic mechanisms. Neuroendocrinology 31:410 32. Siler TM, Yen SSC, Vale W, Guillemin R 1974 Inhibition by somatostatin on the release of TSH induced in man by thyrotropinreleasing factor. J Clin Endocrinol Metab 38:742 33. Krulich L, Mayfield MA, Steele MK, McMillen BA, McCann SM, Koenig JI 1982 Differential effects of pharmacological manipulations of central ax and «2-adrenergic receptors on the secretion of thyrotropin and growth hormone in male rats. Endocrinology 110:796 34. Andersson K, Eneroth P 1985 Regression analysis of catecholamine utilization in discrete hypothalamic and forebrain regions of the male rat: effects of thyroidectomy. Acta Physiol Scand 123:105 35. Andersson K, Eneroth P 1987 Thyroidectomy and central catecholamine neurons of the male rat. Neuroendocrinology 45:14 36. Brent GA, Larsen PR, Harney JW, Koenig RJ, Moore DD 1989 Functional characterization of the rat growth hormone promoter elements required for induction by thyroid hormone with and without a co-transfected beta type thyroid hormone receptor. J Biol Chem 264:178 37. Koch Y, Goldhaber GL, Fireman I, Zor U, Shani J, Tal E 1977 Suppression of prolactin and thyrotropin secretion in the rat by antiserum to thyrotropin-releasing hormone. Endocrinology 100:1476 38. Szabo M, Kovathana N, Gordon K, Frohm LA 1978 Effect of passive immunization with an antiserum to thyrotropin (TSH)releasing hormone on plasma TSH levels in thyroidectomized rats. Endocrinology 102:799 39. Harris ARC, Christianson D, Smith MS, Fang SL, Braverman LE, Vagenakis AG 1978 The physiological role of thyrotropin-releasing hormone in the regulation of thyroid-stimulating hormone and

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prolactin secretion in the rat. J Clin Invest 61:441 40. Ishikawa K, Kakegawa T, Suzuki M 1984 Role of the hypothalamic paraventricular nucleus in the secretion of thyrotropin under adrenergic and cold-stimulated conditions in the rat. Endocrinology 114:352 41. Szabo M, Frohman LA 1977 Suppression of cold-stimulated thyrotropin secretion by antiserum to thyrotropin-releasing hormone. Endocrinology 101:1023 42. Beck-Peccoz P, Amr S, Menezes-Ferreira MM, Faglis G, Weintraub BD 1985 Decreased receptor binding of biologically inactive thyrotropin in central hypothyroidism. N Engl M Med 312:1085 43. Gesundheit N, Fink DL, Silverman LA, Weintraub BD 1987 Effect of thyrotropin-releasing hormone on the carbohydrate structure of secreted mouse thyrotropin. J Biol Chem 262:5197

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44. Gyves PW, Gesundheit N, Taylor T, Butler JB, Weintraub BD 1987 Changes in thyrotropin (TSH) carbohydrate structure and response to TSH-releasing hormone during postnatal ontogeny: analysis by Concanavalin-A chromatography. Endocrinology 121:133 45. Dallman MF, Makara GB, Roberts JL, Levin N, Blum M 1985 Corticotrope response to removal of releasing factors and corticosteroids in vivo. Endocrinology 117:2190 46. Hamernik DL, Nett TM 1988 Gonadotropin-releasing hormone increases the amount of messenger ribonucleic acid for gonadotropins in ovariectomized ewes after hypothalamic-pituitary disconnection. Endocrinology 122:959 47. Huckle WR, Conn PM 1988 Molecular mechanism of gonadotropin-releasing hormone action. II. The effector system. Endocr Rev 9:387

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The paraventricular nucleus of the hypothalamus has a major role in thyroid hormone feedback regulation of thyrotropin synthesis and secretion.

The role of the hypothalamic paraventricular nucleus (PVN) in thyroid hormone regulation of TSH synthesis during hypothyroidism was studied in adult m...
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