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Endocrinology Vol. 139, No. 8 3416-3422
Copyright © 1998 by The Endocrine Society

ARTICLES

Thyrotropin-Releasing Hormone Gene Expression in the Anterior Pituitary. IV. Evidence for Paracrine and Autocrine Regulation

Thomas O. Bruhn, Jan M. M. Rondeel1 and Ivor M. D. Jackson

Division of Endocrinology, Department of Medicine, Brown University/Rhode Island Hospital, Providence, Rhode Island 02903; and Institut für Zellbiochemie, Universitätskrankenhaus Eppendorf, Universität Hamburg (T.O.B.), 22529 Hamburg, Germany

Address all correspondence and requests for reprints to: Ivor M. D. Jackson, M.D., Division of Endocrinology, Rhode Island Hospital, 593 Eddy Street, Providence, Rhode Island 02902. E-mail: ivor_jackson{at}brown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Disulfiram (Dis), an inhibitor of peptidyl-glycine {alpha}-amidating monooxygenase, the enzyme responsible for the production of {alpha}-amidated peptides from their immediate, glycine-extended precursors was used to investigate the paracrine effects of TRH on anterior pituitary (AP) hormone secretion. It reduces the production of TRH without directly affecting the classical pituitary hormones, none of which is amidated.

Dis (8 µM) decreased the accumulation of TRH accompanied by an equimolar increase in TRH-Gly levels, indicating that pro-TRH biosynthesis persisted. TRH and TSH release into the medium was significantly lowered, whereas other pituitary hormones were unaffected. In contrast, dexamethasone (10 nM), which up-regulates TRH gene expression in this system, increased TRH (+89.5%) and TSH (+61.3%) secretion. The combination of dexamethasone and Dis further diminished the release of TRH (-73%) and TSH (-40.3%) observed with Dis alone, indicating that TRH synthesized within the AP regulates TSH secretion.

Dis significantly elevated prepro-TRH (25–50) and pro-TRH messenger RNA levels, suggesting that reduced TRH formation leads to increased pro-TRH biosynthesis and that TRH regulates its own secretion. Thus, TRH synthesized by cultured AP cells not only stimulates TSH release through a paracrine effect, but has a negative feedback on its own biosynthesis by an autocrine mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRH (pGluHisProNH2) is derived from a 255 amino acid prohormone that yields 5 copies of the tripeptide amide when processed completely (1, 2). The immediate precursor to TRH is the C-terminal glycine-extended form pGluHisProGly, known as TRH-Gly (1, 2). TRH and TRH-Gly have been colocalized in a number of tissues, with the highest concentrations occurring in the hypothalamus and prostate, respectively (3). The conversion of TRH-Gly to TRH is accomplished by the enzyme peptidyl-glycine {alpha}-amidating monooxygenase (PAM), which has been found to be widely distributed throughout the body (4, 5, 6, 7, 8). PAM is a copper-, ascorbate-, and molecular oxygen-dependent enzyme that is responsible for the production of {alpha}-amidated peptides from their glycine-extended forms, a process generally essential for biological activity (4, 5, 9, 10). High levels of PAM have been reported in the anterior pituitary (AP) with varying levels of PAM messenger RNA (mRNA) found in all of the classical AP cell types (7). These studies suggest that AP cells are capable of synthesizing {alpha}-amidated peptides, a finding that is supported by reports of the localization of {alpha}-amidated peptides, classically associated with the hypothalamus, in the AP (11, 12, 13, 14).

Our laboratory has previously demonstrated the presence of pro-TRH-derived peptides in cultured AP cells (11), localized pro-TRH mRNA to a subpopulation of somatotrophs (12), and established that glucocorticoids and thyroid hormone regulate the TRH gene coordinately with the GH gene (15, 16). As AP cultures synthesize substantial amounts of TRH (11, 12, 15, 16), we investigated whether the peptide exerts paracrine effects within the AP. This could be achieved either by using antisense oligonucleotides complementary to prepro-TRH mRNA to inhibit the biosynthesis of the TRH prohormone and consequently TRH secretion or by employing TRH antiserum to absorb any released peptide. A third and novel approach would be to expose TRH-synthesizing cells to inhibitors of PAM; this treatment would reduce the amidation of TRH-Gly and, therefore, formation of bioactive TRH without directly affecting the biosynthesis of the prohormone. We chose the latter approach and used tetraethylthiuram disulfide [disulfiram (Dis)], a disulfide dimer of diethylthiocarbamate. Both substances are copper chelators that have been successfully used to reduce the formation of newly synthesized {alpha}-amidated peptides in vitro and in vivo (10, 17, 18, 19). We reasoned that Dis may provide a valuable tool by selectively reducing the accumulation of bioactive TRH within cultured AP cells without directly affecting the biosynthesis of classical pituitary hormones, none of which is amidated (7). The present studies were designed to 1) examine whether TRH in this location exerts a paracrine effect on the production of pituitary hormones and 2) address whether pituitary TRH regulates pro-TRH biosynthesis in an autocrine fashion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Lactating dams with litters (15-day-old males) were purchased from Charles River Laboratories (Wilmington, MA) with the pups serving as pituitary donors. Rats were housed at the Rhode Island Hospital animal facility, and animal care and euthanasia procedures were approved by the Rhode Island Hospital/Brown University Committee for Animal Welfare.

Tissue culture
AP cultures were obtained as previously described (11, 12). Briefly, AP tissue was separated from posterior/intermediate lobes, collected into sterile HBSS, enzymatically dispersed with neutral protease (1.5 U/AP; Sigma Chemical Co., St. Louis, MO), and plated in a monolayer on 16-mm wells (peptide or pituitary hormone measurements) or 35-mm wells (mRNA determination) at a density of 1000 cells/mm2. The cells were cultured for up to 16 days in a modified L-15/DMEM (Life Technologies, Grand Island, NY) medium (11, 12, 20) containing 10% FCS (Life Technologies).

Experimental procedures
Cultured AP cells were exposed to tetraethlythiuram disulfide, commonly referred to as Dis (Sigma) at a concentration of either 0.8 or 8.0 µM. Dis was dissolved in dimethylsulfoxide (DMSO); the final concentration of DMSO in culture medium was 0.01% for either dose. Control wells received an equal amount of DMSO. Cells were treated with Dis and/or 10 nM dexamethasone (Dex) for up to 12 days beginning after 2–3 days in culture. For the determination of TRH, TRH-Gly and prepro-TRH (25–50) (pYE27) by RIA, cells were extracted in 1 N acetic acid, boiled for 10 min, homogenized, and spun at 2000 x g, and the supernatants were lyophilized. TSH, PRL, and GH were measured from cells extracted in 0.1 M PO4 buffer containing 0.5% BSA. After homogenization and centrifugation, samples were directly subjected to RIAs at the appropriate dilution. Medium samples for TRH and TRH-Gly determinations were boiled for 10 min upon collection, whereas medium samples for TSH, PRL, and GH determinations were assayed directly. Samples for Northern blot analysis were extracted using the guanidine-cesium chloride method, with a recovery of 5–10 µg RNA/106 cells (12).

Unless stated otherwise, experiments were repeated twice. The data depicted in the figures are derived from one representative experiment.

RIAs
Lyophilized samples were reconstituted with RIA buffer and assayed for TRH and pYE27 immunoreactivities as previously described (11, 12, 21, 22, 23).

The TRH-Gly RIA was carried out as follows. TRH-Gly antiserum 632 was obtained from rabbits after immunization with synthetic TRH-Gly conjugated to bovine thyroglobulin as described previously (21, 23). Synthetic TRH-Gly was iodinated using the chloramine-T method, and the radioligand was purified by HPLC (21, 23). The RIA incubation volume was 500 µl and consisted of 100 µl TRH-Gly antiserum, 100 µl sample, and 200 µl RIA buffer (0.1 M PO4 with 0.5% BSA). The TRH-Gly antiserum was used at a final dilution of 1:2500. One hundred microliters of [125I]TRH-Gly (10,000 cpm) were added after a 24-h preincubation at 4°C. Tubes were then incubated for an additional 2 days at 4 C. Separation of bound and free [125I]TRH-Gly was accomplished by addition of 1.0 ml/tube of 0.1% activated charcoal. After centrifugation at 2,000 x g for 30 min, the supernatants were decanted and counted. The limit of detection (ED90) for the assay system was 12.6 ± 4.3 pg/assay tube; half-maximal displacement (ED50) was reached at 46.9 ± 13.8 pg/tube (n = 28). Intra- and interassay coefficients of variation were 7.5% and 10.9%, respectively. Cross-reactivity with related peptides including TRH and TRH-Gly-Lys were less than 0.001% and 0.1%, respectively. Cross-reactivity with unrelated peptides, including CRH, GH-releasing hormone, and somatostatin, was less than 0.001%, respectively. The specificity of the RIAs for TRH-Gly and TRH was further demonstrated by the absence of any TRH-Gly immunoreactivity in the area of TRH elution (and vice versa) on HPLC analysis (Fig. 2Go).



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  		Figure 2. Reverse phase HPLC of TRH and TRH-Gly extracted from cultured AP cells. Cells were treated with 8.0 µM Dis for 12 days to enhance TRH-Gly levels. Top panel, HPLC of AP cell extract. Bottom panel, HPLC of AP cell extract spiked with a similar amount of synthetic TRH and TRH-Gly. Arrows indicate the elution positions of synthetic TRH and TRH-Gly.

 
TSH, PRL, and GH were determined using materials provided by the NIDDK as outlined previously (11, 12).

Reverse phase HPLC
Pooled, lyophilized cell extracts were reconstituted with 0.1% trifluoroacetic acid (TFA), spun, and the supernatant was directly applied to a Waters 200-SW size-exclusion column (Waters Associates, Milford, MA; 30 x 1 cm; flow rate, 0.5 ml/min). Fractions containing TRH and TRH-Gly immunoreactivities were pooled and applied to a Vydac C18 reverse phase HPLC column (25 x 0.46 cm; 5-µm particles; 300-Å pore size). Buffer A consisted of 0.1% TFA; buffer B consisted of 60% CH3CN, 39.9% H2O, and 0.1% TFA. Thirty-second fractions (flow rate, 1 ml/min) were collected and lyophylized before RIA for TRH and TRH-Gly.

Northern blot analysis
Northern blots and hybridization protocols were carried out as previously described (12, 15, 16). Briefly, RNA was fractionated on a 1% agarose gel containing 2.2 M formaldehyde and electrophoretically transferred to a nylon membrane support. Membranes were hybridized with a 32P-labeled antisense RNA probe complementary to the entire coding sequence for pro-TRH (3, 12). After hybridization, the blots were washed under stringent conditions and exposed to Kodak XAR5 film (Eastman Kodak, Rochester, NY). Blots were stripped and reprobed with a 32P-labeled antisense ß-actin riboprobe (12) to allow for minor differences in recovery. Hybridized bands were analyzed by a computerized image analysis system using Image 1.40 (NIH); data were expressed as the ratio of integrated pro-TRH/ß-actin optical densities.

Statistical analysis
Data were subjected to ANOVA followed by the Tukey-Kramer test for multiple comparisons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The cellular contents of TRH, TRH-Gly, and pYE27 all increased significantly (P < 0.01) with time in cultured AP cells (Fig. 1Go). Dis (8.0 µM) significantly lowered TRH content while significantly enhancing TRH-Gly and pYE27 contents (day 3, P < 0.05; days 6 and 12, P < 0.01; for all three peptides). After a 12-day exposure to Dis (8.0 µM), TRH was lowered by 39.4%, whereas TRH-Gly and pYE27 increased by 192.8% and 54.9%, respectively, compared with their respective controls without Dis. Although the ratio of TRH to TRH-Gly was dramatically altered after the addition of Dis, the combined molar quantities of TRH and TRH-Gly at each time point (data from Fig. 1Go, not shown) were similar in the control and Dis-treated groups.



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  		Figure 1. Time course of TRH, TRH-Gly, and pYE27 accumulation in cultured AP cells in the presence and absence of Dis (8.0 µM). Each point represents the mean ± SEM of six wells. Dis treatment started on day 2 of culture and persisted until day 14 of culture (12 days of Dis exposure). *, P < 0.05; **, P < 0.01 (compared with time-matched controls).

 
Extracts of cultured AP cells, treated with Dis to increase TRH-Gly levels, were subjected to HPLC, and TRH and TRH-Gly immunoreactivities were found to coelute with synthetic TRH and TRH-Gly (Fig. 2Go, top). In addition, spiking of the cellular extract with synthetic TRH and TRH-Gly resulted in single homogeneous peaks (Fig. 2Go, bottom). The recovery of the synthetic peptides, as determined by RIA, before and after HPLC fractionation was 85–90%.

Northern blot analysis of cultured AP cells treated with Dis (8.0 µM for 12 days) revealed a 79.2% increase (P < 0.05) in pro-TRH mRNA after Dis treatment (Fig. 3Go).



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  		Figure 3. Northern blot analysis of pro-TRH mRNA extracted from cultured AP cells in the presence and absence of Dis (8.0 µM, 12 days of exposure). The top panel shows the Northern blot images of three control and three Dis-exposed AP cell extracts. The bottom panel shows the densitometric analysis relative to ß-actin. Bars represent the mean ± SEM of four wells per group. *, P < 0.05.

 
Dis exposure lowered TRH content in a dose-dependent manner, with 8.0 µM Dis reaching significance (P < 0.01; Fig. 4Go, top). In contrast, TRH-Gly content rose dramatically (P < 0.01; Fig 4Go, bottom) after Dis exposure. Treatment with 10 nM Dex alone significantly increased the content of both TRH by 811.7% (P < 0.01) and TRH-Gly by 877.1% (P < 0.01; Fig. 4Go, top and bottom right axes). The combination of Dex (10 nM) and Dis (8.0 µM) resulted in an exaggeration of the Dis effect and lowered TRH by 84.5% vs. 57.3% in non-Dex-treated and increased TRH-Gly by 544.2% vs. 410.5% in non-Dex-treated cultures (Fig. 4Go).



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  		Figure 4. Dose-dependent effect of Dis (0.8 and 8.0 µM, 12 days of exposure) on TRH and TRH-Gly accumulation in cultured AP cells (left axis). Effect of simultaneously treating AP cells with Dis (8.0 µM) and Dex (10 nM) for 12 days on TRH and TRH-Gly contents (right axis). Bars represent the mean ± SEM of five or six wells. **, P < 0.01 relative to control. {ddagger}{ddagger}, P < 0.01 relative to control-Dex. Note that the treatment with Dex alone (control-Dex; right axis) increased TRH and TRH-Gly contents significantly (both P < 0.01; see Results).

 
TRH and TSH release from cultured AP cells was lowered in a dose-responsive fashion after exposure to Dis (0.8 and 8.0 µM), with significance reached at 8.0 µM Dis (P < 0.05; Fig. 5Go). Simultaneous exposure to Dis (8.0 µM) and Dex (10 nM) enhanced this effect (Fig. 5Go). In this group, TRH release was lowered by 73.0% compared with 59.2% in the non-Dex-treated group, whereas TSH secretion was decreased by 40.3% vs. 37.6% in the non-Dex group. Exposure to Dex (10 nM) alone resulted in significant increases in TRH and TSH release by 89.5% and 61.3%, respectively (both P < 0.05; Fig. 5Go). TRH-Gly release could not be determined due to insufficient sensitivity of the RIA.



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  		Figure 5. Dose-dependent effect of Dis (0.8 and 8.0 µM, 12 days of exposure) on TRH and TSH release from cultured AP cells. Effect of simultaneously treating AP cells with Dis (8.0 µM) and Dex (10 nM) for 12 days on TRH and TSH release from cultured AP cells. Bars represent the mean ± SEM of five or six wells. *, P < 0.05 relative to control. {ddagger}{ddagger}, P < 0.01 relative to control-Dex. Note that treatment with Dex alone (control-Dex) increased TRH and TSH release significantly (both P < 0.01; see Results).

 
To demonstrate that Dis specifically affects TRH formation, the intracellular accumulation of the nonamidated pituitary hormones GH and PRL was determined. PRL release and content tended to decrease, although not significantly, after treatment with Dis (Table 1Go). GH content and secretion were not affected by Dis exposure; on the other hand, Dex treatment significantly enhanced GH accumulation (Table 1Go), in agreement with previously reported data (15). Additionally, Dex significantly reduced PRL content, but not secretion into the medium (Table 1Go).


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  		Table 1. Effect of exposure to disulfiram alone (0.8 µM and 8.0 µM) and simultaneous treatment with Dis (8.0 µM) and dexamethasone (10 nM) on TSH, PRL, and GH content and release from cultured anterior pituitary cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we provide evidence for paracrine actions of TRH synthesized by cultured AP cells. We used Dis, a selective inhibitor of the enzyme PAM, to lower intracellular TRH accumulation and consequently, release of the peptide into the culture medium. As predicted, exposure to Dis resulted in a dose-dependent decrease in the intracellular accumulation of TRH that was accompanied by an approximately equimolar increase in TRH-Gly levels, indicating that the biosynthesis of pro-TRH persisted. Processing of the rat TRH prohormone yields five copies of TRH-Gly, the immediate precursor of TRH, as predicted by its complementary DNA structure (22). Other processing products include several cryptic peptides, one of which is pYE27 (23, 24). We have previously determined the intracellular content of pYE27 in AP cultures and found that the molar ratio of TRH/pYE27 was similar to the theoretical value of 5:1 (11, 12, 22). The low levels of TRH-Gly in extracts of AP cultures determined in this study (TRH/TRH-Gly molar ratio of approximately 1:0.1) suggest that TRH-Gly is a transitional peptide that is immediately processed to TRH.

It is well established that the expression of PAM is regulated in a tissue-specific manner (6, 8, 25). Differences in PAM activity may be responsible for the TRH/TRH-Gly molar ratio ranging from 1:0.04 in hypothalamic tissue to 1:5.5 in testicular tissue (3). PAM activity in AP cells expressing the TRH gene was sufficient to maintain the TRH/TRH-Gly molar ratio unaltered even after a 10-fold up-regulation of TRH-biosynthesis after Dex treatment. Dex may up-regulate PAM in somatotrophs in which the pro-TRH gene has been located, although glucocorticoids have been reported to down-regulate enzyme expression in parallel with POMC mRNA levels in corticotrophs (26). Precedence for tissue-specific expression of PAM was reported previously (8).

As a consequence of reduced intracellular pools of TRH after treatment with the PAM inhibitor Dis, basal release of the peptide into medium was lowered. This was paralleled by a significant reduction in basal TSH secretion, suggesting that TRH secreted by AP cells in vitro exerts a tonic stimulatory effect on thyrotrophs. Based on previous evidence that Dex stimulates TRH gene expression in both hypothalamic and hypophysial cultures, we reasoned that glucocorticoid treatment would increase TRH release into the medium and thus would probably enhance any paracrine effects of the peptide (15, 27). We previously determined that TRH mRNA was localized in somatotrophs and that treatment with glucocorticoids raised TRH and GH levels in parallel (12, 15, 28). Consistent with these results we confirmed in the present study that Dex profoundly raised both the intracellular accumulation and the release of TRH as well as GH. It is of note that Dex significantly increased TSH secretion. The intracellular accumulation of TSH, on the other hand, increased only moderately, indicating that the elevation in TSH secretion was not merely a reflection of a possible direct effect of the glucocorticoid on TSH biosynthesis. The combination of Dis and Dex treatments caused a parallel and highly significant decrease in both TRH and TSH release into the medium. Compared with the profound effect of Dex on TRH accumulation (10-fold), TRH release into medium rose only approximately 2-fold. Although we did not investigate this apparent discrepancy between changes in cellular content and secretion, we suggest that TRH may be rather unstable in cell culture medium due to the fact that pituitary cells express the recently cloned TRH-degrading ectoenzyme at a high level (29).

The parallel up- and down-regulations of TRH and TSH release by Dex and Dis strongly support the concept that TRH generated in the AP stimulates the secretion of TSH from thyrotrophs in a paracrine manner. It is of note that none of the classic pituitary hormones is amidated, and therefore, AP hormone expression would not be affected directly by blockade of the amidating enzyme PAM via Dis treatment. The hormone-specific effects of Dex treatment were verified in this study; the synthetic glucocorticoid significantly stimulated GH while inhibiting PRL gene expression. Both effects have been previously described and characterized as direct effects of the ligand-activated glucocorticoid receptor at glucocorticoid-responsive elements that are located in the respective promoter regions of the GH and PRL genes (30, 31, 32, 33).

Dis has been used previously as a tool to reduce peptide amidation and thus formation of bioactive peptides in several in vitro and in vivo systems (18, 19, 34, 35, 36). PAM activity is expressed in all pituitary hormone-producing cells of the AP, a finding consistent with the presence of a multitude of regulatory peptides, many of which are amidated, in the pituitary gland (16, 37, 38). To investigate whether TRH acts in a paracrine fashion within cultured AP cells, we chose Dis as a highly specific tool to reduce the formation of bioactive TRH, which we previously established to be expressed in this system (11, 12, 15, 16, 28). The efficacy of this treatment was verified by HPLC of cell extracts followed by specific RIAs for TRH and TRH-Gly, which clearly demonstrated that the reduction of intracellular TRH accumulation after Dis treatment was accompanied by an equimolar increase in TRH-Gly levels.

Passive immunization and the use of antisense oligonucleotides complementary to prepro-TRH mRNA represent two alternative methods to study paracrine effects of TRH within the AP. The former method lowers the availability of extracellular TRH, whereas the latter inhibits the biosynthesis of the TRH prohormone. Both methods have been used in a variety of studies, but technical problems, such as the toxicity of antisense oligonucleotides or the necessity of using large amounts of high affinity TRH antiserum that may cause nonspecific RIA effects, have limited the use of these methods (40). The PAM inhibitor Dis represents a unique tool that prevents formation of the biological end product intracellularly before exocytosis. At the same time, transcription and translation of the TRH gene continue, processes that would be disrupted by antisense oligonucleotides.

To monitor the effect of Dis on pro-TRH peptides other than TRH itself, we measured pYE27 by RIA and observed a consistent increase in pYE27 levels after Dis treatment. This suggests that Dis treatment, by preventing the formation of TRH, led to an increase in the biosynthesis of the TRH prohormone. Northern blot analysis revealed that Dis treatment significantly elevated pro-TRH mRNA levels, indicating that pro-TRH biosynthesis was indeed elevated and that the observed effect on pYE27 was not merely an effect of altered processing of the prohormone. The findings suggest that TRH regulates its own biosynthesis in an autocrine fashion. Other examples for autocrine regulation have been reported. Peterfreund and Vale, for example, demonstrated that somatostatin inhibits somatostatin production in cultured hypothalamic cells (41). Although TRH and somatostatin appear to inhibit the expression of their genes, the pituitary-specific transcription factor Pit-1 stimulates the transcription of its own gene (42). Pit-1 is essential for the genesis of three pituitary cell types, somatotrophs, lactotrophs, and thyrotrophs, and also mediates the transcriptional regulation of their genes (42, 43, 44, 45). Positive autocrine regulation of the Pit-1 gene may ensure that sufficient concentrations of this essential transcription factor are available at all times, whereas negative autocrine regulation of genes encoding peptide hormones may provide the necessary fine tuning to control expression levels of these genes (41, 42).

How might TRH regulate its own production in an autocrine fashion? TRH receptors have been reported to be expressed in somatotrophs, the cell type that we have previously identified as a site of TRH biosynthesis (12, 46). Although the phosphokinase C pathway has been identified as the second messenger system conveying TRH activity, it is not clear which mediators would be involved in the activation of the TRH gene (47). Activation of TSHß as well as PRL transcription by TRH are mediated by the pituitary-specific transcription factor Pit-1 (43, 44, 45). Whether Pit-1-binding sites exist in the promoter region of the TRH gene that could mediate inhibitory rather than stimulatory effects has not been previously investigated (2, 48). In addition to the possibility of autocrine regulation, we cannot exclude that Dis might enhance TRH biosynthesis by stabilizing pro-TRH mRNA.

In conclusion, we have demonstrated that TRH synthesized by cultured AP cells regulates TSH secretion in a paracrine fashion. We have employed Dis, an inhibitor of the {alpha}-amidating enzyme PAM, to achieve a reduction of TRH formation without blocking TRH biosynthesis. Lower TRH levels after Dis administration were accompanied by an elevation of TRH biosynthesis, suggesting that TRH regulates its own biosynthesis in an autocrine fashion.


   Acknowledgments
 
We thank Roberta Todd for her assistance with the RIAs.


   Footnotes
 
1 Current address: Department of Clinical Chemistry, Drechtsteden Ziekenhuis, Refaja, P.O. Box 444, 3300 AK Dordrecht, The Netherlands. Back

Received December 5, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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