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Endocrinology, doi:10.1210/en.2003-0526
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Endocrinology Vol. 144, No. 12 5537-5544
Copyright © 2003 by The Endocrine Society

Corticotropin-Releasing Hormone (CRH)-Induced Thyrotropin Release Is Directly Mediated through CRH Receptor Type 2 on Thyrotropes

Bert De Groef, Nesya Goris, Lutgarde Arckens, Eduard R. Kühn and Veerle M. Darras

Laboratories of Comparative Endocrinology (B.D.G., N.G., E.R.K., V.M.D.) and Neuroplasticity and Neuroproteomics (L.A.), Catholic University of Leuven, B3000 Leuven, Belgium

Address all correspondence and requests for reprints to: Bert De Groef, Naamsestraat 61, B3000 Leuven, Belgium. E-mail: bert.degroef{at}bio.kuleuven.ac.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRH is known as the main stimulator of ACTH release. In representatives of all nonmammalian vertebrates, CRH has also been shown to induce TSH secretion, acting directly at the level of the pituitary. We have investigated which cell types and receptors are involved in CRH-induced TSH release in the chicken (Gallus gallus). Because a lack of CRH type 1 receptors (CRH-R1) on the chicken thyrotropes has been previously reported, two hypotheses were tested using in situ hybridization and perifusion studies: 1) TSH secretion might be induced in a paracrine way involving melanocortins from the corticotropes; and 2) thyrotropes might express another type of CRH-R. For the latter, we have cloned a partial cDNA encoding the chicken CRH-R2. Neither {alpha}-melanotropin ({alpha}-MSH) nor its powerful analog Nle4,D-Phe7-MSH could mimic the in vitro TSH-releasing effect of ovine CRH. The nonselective melanocortin receptor blocker SHU91199 did not influence CRH- or TRH-induced TSH secretion. On the other hand, we have found that thyrotropes express CRH-R2 mRNA. The involvement of this CRH receptor in the response of thyrotropes to CRH was further confirmed by the fact that TSH release was stimulated by human urocortin III, a CRH-R2-specific agonist, whereas the TSH response to CRH was completely blocked by the CRH-R blocker astressin and the CRH-R2-specific antagonist antisauvagine-30. We conclude that CRH-induced TSH secretion is mediated by CRH-R2 expressed on thyrotropes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
NEXT TO ITS function as an ACTH-releasing factor, CRH has been shown to be a potent TSH-releasing factor in representatives of all nonmammalian vertebrates [fish (1), amphibians (2), reptiles (3), and birds (4)]. Our research group has been studying the TSH-releasing activity of CRH in the chicken. In vitro stimulation of chicken pituitaries clearly stimulated the release of TSH, indicating that CRH is able to exert its thyrotropic effect at the level of the pituitary (4, 5). However, it is not yet clear whether CRH acts directly at the level of the TSH-producing cells (thyrotropes) or through a paracrine pathway involving other hypophyseal cell types. The latter hypothesis has gained more credit because we were not able to demonstrate CRH type 1 receptor (CRH-R1) mRNA expression in thyrotropes, whereas ACTH-producing cells (corticotropes) expressed CRH-R1 mRNA abundantly (6).

CRH could release TSH indirectly via paracrine interactions between corticotropes and thyrotropes. Paracrine interactions within the pituitary gland of vertebrates are not uncommon (7), but relatively little is known about the paracrine influences to which thyrotropes are subject. Some cell types (gonadotropes, lactotropes) produce factors that stimulate mitosis in thyrotropes (7). A number of recent studies have focused on the paracrine actions of segments of proopiomelanocortin (POMC) on other pituitary cells. The POMC-derived peptide {gamma}3-melanotropin ({gamma}3-MSH) was demonstrated to be biologically active in the anterior pituitary of immature rats, showing a mitogenic action on lactotropes, somatotropes, and thyrotropes, and thus serving as a growth and/or differentiation factor within the pituitary (8, 9, 10). Porcine ACTH is able to stimulate corticosterone release in the chicken (11), but it has no effect on the in vitro TSH secretion by the chicken pituitary (4). Nevertheless, the porcine ACTH amino acid sequence is 77% identical to chicken ACTH, and when polarity similarities between amino acids are taken into account, both hormones are 95% similar. Therefore, ACTH can be discarded as being a paracrine factor involved in CRH-induced TSH release in the chicken. However, a variety of other candidates are released by chicken corticotropes. Cloning of chicken POMC cDNA has shown that POMC could be processed to give rise to all members of the melanocortin family (12, 13). To test the responsiveness of thyrotropes toward melanocortins, and hence the involvement of melanocortins in CRH-induced TSH secretion, we have set up some perifusion experiments to investigate whether MSHs can stimulate TSH release from pituitaries in vitro. We have also tried to alter the in vitro TSH response to CRH or TRH stimulation with the mammalian melanocortin receptor type 3 (MC3R) and 4 (MC4R) antagonist SHU9119.

An alternative possibility is the existence of CRH binding sites other than CRH-R1 on chicken thyrotropes. A number of CRH-Rs have been identified, and all of the identified CRH-like peptides bind to at least one of the CRH-Rs. In mammals, two types of CRH-Rs (CRH-R1 and CRH-R2) have been characterized (14, 15). Similar receptor types have also been cloned in Xenopus (16), and three different CRH-R subtypes were found in a catfish species (17). In the chicken, only a CRH-R1 cDNA has been identified so far (18). It is therefore highly probable that other CRH-Rs are yet to be found in the chicken. In our search for novel chicken CRH-Rs, we obtained a partial cDNA encoding the chicken CRH-R2 by an RT-PCR approach and examined the cellular localization of CRH-R1 and CRH-R2 mRNA in the pituitary gland. The results of this experiment were further investigated with perifusion experiments using the antagonists astressin (AS; blocker of both CRH-R1 and CRH-R2) and antisauvagine-30 (aSVG; CRH-R2-specific blocker), as well as the CRH-R2-specific agonist urocortin III.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All experiments described in this section were approved by the Ethical Committee for Animal Experiments of the Catholic University of Leuven.

Effect of {alpha}-MSH and a melanocortin receptor antagonist on in vitro {alpha}-subunit secretion
Pituitaries from 9- and 10-d-old Cobb broiler chicks (Avibel, Halle-Zoersel, Belgium) were dissected and placed individually in perifusion chambers (n = 3–4 per condition) at 37 C. For all perifusion experiments, M199 (Life Technologies, Gaithersburg, MD) was used as perifusion medium, and all reagents were prepared in this medium. After a 1.5-h equilibration period and a 30-min basal secretion measurement, SHU9119 (Neosystem, Strasbourg, France) was added to the medium (100 nM) 1 h before the stimulation period and until the end of the experiment. SHU9119 (Ac-Nle4-cyclo[Asp5, D-2-Nal7, Lys10]-{alpha}-MSH(4–10)-NH2) is a high-affinity nonselective antagonist at human MC3R and MC4R (19). Controls received plain medium during the same period. After a 30-min challenge with 100 nM ovine CRH (oCRH; Sigma, St. Louis, MO), 100 nM TRH (Sigma), or 100 nM {alpha}-MSH (Neosystem), the perifusion lasted for another 2 h. Flow rate was 12 ml/h. All samples were stored at -20 C before the measurement of {alpha}-subunit levels by RIA.

{alpha}-Subunit RIA was carried out according to Berghman et al. (20). Earlier studies have shown that {alpha}-subunit secretion is a good indication for TSH release in this type of studies (4, 5, 21, 22). The calculation of the stimulation factor (SF) and relative net increase (NI) were calculated for each individual chamber, slightly modified after Geris et al. (5). Briefly, the mean basal secretion (BS) of each single pituitary is calculated during the 30-min BS period. All obtained values are expressed as percentages relative to the mean BS (= 100%). The peak value (PV) obtained after the application of a test substance is determined. SF is calculated as the PV divided by the mean basal secretion (SF = PV/BS). SF gives an indication about the maximal amplitude relative to the mean BS. NI is the total increase above BS after application of the test substance. Relative NI is calculated as the sum of all values during which secretion is elevated in response to the test substance, expressed relatively to BS. NI is indicative for the magnitude and duration of the response. Statistical analysis of SFs and NIs was assessed by Student’s unpaired t test.

Effect of a melanocortin agonist on in vitro {alpha}-subunit secretion
Pituitaries from 10-d-old Cobb chicks were dissected and placed individually in perifusion chambers (n = 12) at 37 C. After an equilibration period of 1.5 h and a BS measurement of 90 min, NDP-MSH (Neosystem) was added to the medium (1 µM) for a 30-min stimulation period. NDP-MSH [(Nle4, D-Phe7)-{alpha}-MSH] is a synthetic {alpha}-MSH analog with high potency and long biological activity (23). The perifusion continued for another hour with plain M199. Flow rate was reduced to 6 ml/h during the whole perifusion experiment to concentrate the elution samples. This allowed us to detect smaller changes in {alpha}-subunit secretion that might be missed when using higher flow rates. Samples were stored at -20 C before the analysis of {alpha}-subunit levels by RIA.

Cloning of a partial cDNA encoding the chicken CRH-R2
RT-PCR.
mRNA was isolated from chicken brain (telencephalon), heart, and pituitary using the QuickPrep micro mRNA Purification kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) according to the manufacturer’s guidelines. Oligonucleotide primers were designed to highly conserved regions of previously cloned CRH-R2 sequences in rat (14), Xenopus laevis (16) and the catfish species Ameiurus nebulosus (17): 5'-ATAAACTACCTGGGGCACTG-3' and 5'-TTCCACAAACATCCAGAAGAA-3' (Invitrogen, San Diego, CA). Approximately 1 µg mRNA was heated for 5 min at 72 C with oligo(dT) primer, and then reverse-transcribed into cDNA in a volume of 20 µl containing reaction buffer [50 mM Tris-HCl (pH 8.3), 50 mM KCl, 10 mM MgCl2, 0.5 mM spermidine, 10 mM dithiothreitol], 1 mM of each deoxynucleoside triphosphate (dNTP), ribonuclease inhibitor (10 U) and avian myeloblastosis virus reverse transcriptase (2.5 U) (Roche Diagnostics, Basel, Switzerland). The mixture was incubated for 1 h at 42 C. A 5-µl aliquot of the cDNA product was amplified in a 20-µl PCR mixture containing PCR buffer [10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 0.01% stabilizer], 1 mM of each dNTP, 1 µM of each primer, and 2.5 U Super Taq DNA polymerase (HT Biotechnology, Cambridge, UK). The PCR cycles were performed in the GeneAmp PCR System 9700 (PerkinElmer, Wellesley, MA). After activation of the DNA polymerase by incubating at 94 C for 5 min, 35 reaction cycles including denaturation for 30 sec at 94 C and extension for 1 min at 60 C were performed, followed by a final extension at 72 C for 5 min. PCR products were analyzed on a 1.5% agarose gel containing ethidium bromide.

Subcloning and sequencing of the CRH-R2 PCR product.
The PCR product was excised from the agarose gel and extracted and purified using the QIAEX II Gel Extraction kit (QIAGEN, Hilden, Germany). The PCR product was subsequently inserted in a pCRII-TOPO plasmid using the TOPO TA Cloning kit (Invitrogen). Chemically competent Escherichia coli cells (TOP10) were transformed with vector containing the PCR product as insert and were spread on a culture plate containing Luria-Bertani medium with 50 µg/ml ampicillin and 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (Applichem, Darmstadt, Germany). A white colony was grown overnight in Luria-Bertani medium containing 50 µg/ml ampicillin, and plasmid was isolated using the High Pure Plasmid Isolation kit (Roche Diagnostics). The efficiency of transformation was checked by cutting out the insert of the vector with the restriction enzyme EcoRI (Roche Diagnostics) before agarose gel electrophoresis.

Sequencing of the PCR product was done using the fluorescent dye-labeled dideoxynucleotide method with the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit and the automatic sequencer ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CA). Universal reverse and forward M13 primers (5'-CAGGAAACAGCTATGAC-3' and 5'-GTAAAACGACGGCCAG-3', respectively) were used in the sequencing reactions. The obtained sequences were analyzed using BLAST (24).

Cellular localization of CRH-R1 and CRH-R2 mRNA in the chicken pituitary
Animals and tissue processing.
Cellular localization studies were performed on female 6-wk-old Cobb chickens. Animals were killed by decapitation; pituitaries were removed and kept in 4% paraformaldehyde in PBS (pH 7.4) at 4 C. After 24 h, tissues were cryoprotected overnight at 4 C in the same solution containing 30% sucrose, and subsequently stored at -80 C until sectioning. Twenty-micrometer sections were cut with a cryostat. Sections were kept at -80 C in a cryoprotectant solution containing 30% (vol/vol) ethylene glycol and 30% (vol/vol) glycerol in 0.1 M phosphate buffer.

In situ hybridization.
Riboprobes were transcribed from 1 µg of linearized plasmids containing a cDNA insert of chicken CRH-R1 (18) or our own obtained chicken CRH-R2 fragment in the presence of 50 µCi [35S]UTP (PerkinElmer) and 20 U RNA polymerase (SP6 for antisense and T7 for sense probes) (Roche Diagnostics).

Free floating pituitary sections were treated at room temperature with 0.5% Triton X-100 in PBS (10 min), deproteinized with 0.2 N HCl (10 min), acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine buffer (pH 8.0) (10 min), postfixed in 4% paraformaldehyde at 4 C (10 min), and washed in PBS. Prehybridization was performed at 55 C for 3 h in a mixture of 50% formaldehyde, 0.62 M NaCl, 10% dextran sulfate, 5x Denhardt’s solution (0.1% BSA, 0.1% Ficoll 400, and 0.1% polyvinylpyrrolidone), 50 mM dithiothreitol, 0.01 M EDTA, 0.02 M piperazine-1,4-bis(2-ethanesulfonic acid) disodium salt (pH 6.8), 0.2% sodium dodecyl sulfate, 250 µg/ml herring sperm DNA, and 250 µg/ml yeast total RNA. Hybridization was performed in 0.5 ml of this solution overnight at 55 C, with 107 counts per minute of the [35S]-labeled riboprobes. Excess probe was washed away with 2x standard sodium citrate [SSC; 1x SSC is 0.15 M NaCl and 0.015 M sodium citrate (pH 7)] containing 10 mM ß-mercaptoethanol at room temperature for 30 min, followed by incubation with 4 µg/ml RNase A in 0.5 M NaCl, 0.05 M EDTA, and 0.05 M Tris-HCl (pH 7.5) at 37 C for 45 min. Stringency washes were performed in 0.5x SSC, 50% formamide, and 10 mM ß-mercaptoethanol at 55 C for 2 h, and then in 0.1x SSC plus 10 mM ß-mercaptoethanol at 68 C for 30 min.

Immunocytochemistry.
The in situ hybridization protocol was followed by an immunocytochemical staining to identify thyrotropes or corticotropes. The floating sections were incubated with a primary antibody overnight at 4 C. To identify thyrotropes, we used a 1:4000 dilution of anti-Tb3550, a polyclonal antiserum raised by injecting rabbits with a synthetic peptide corresponding to residues 35–50 of the ß-subunit of chicken TSH (25). Corticotropes were stained using a monoclonal antibody (1:2000) against N-terminal chicken POMC (26). The high hormone specificity of these antibodies was confirmed in the original papers. Detection was performed by use of a biotin-coupled goat antirabbit or antimouse IgG (1:200, 1 h, 4 C) (DAKO, Glostrup, Denmark), peroxidase-conjugated streptavidin (1:400, 1 h, 4 C) (Dako), and 3,3'-diaminobenzidine (Sigma) and hydrogen peroxide as color substrates.

Finally, sections were mounted on slides, air-dried, dipped in Hypercoat LM-1 photographic emulsion (Amersham Pharmacia Biotech), and exposed for 3 wk at 4 C. Slides were developed with D19 (Eastman Kodak, Rochester, NY), fixed with Ilford Hylam Rapid Fixer (Ilford, Cheshire, UK), dehydrated, and coverslipped.

Quantification and statistical analysis.
Slides were analyzed using a Leitz DM RBE microscope equipped with a color video camera (Optronics Engineering, Goleta, CA) and attached to a computer-aided image analysis system (Bioquant, R and M Biometrics, Nashville, TN). The number of grains per area of a certain cell type (thyrotropes or corticotropes) was determined for 30 cell groups in three different animals as the number of overlaying pixels, with a brightness exceeding a predetermined threshold, as described by Arckens et al. (27).

Statistical analysis between the grain counts of thyrotropes and corticotropes was carried out by Student’s unpaired t test.

Effect of a nonselective CRH-R antagonist and a selective CRH-R2 antagonist on CRH-induced in vitro {alpha}-subunit secretion
Two perifusions were carried out on consecutive days as described in Effect of {alpha}-MSH and a melanocortin receptor antagonist on in vitro {alpha}-subunit secretion. One hour before the stimulation period during which the pituitaries received oCRH (100 nM), and until the end of the experiment, the nonselective CRH receptor antagonist AS (28) or the selective CRH-R2 antagonist aSVG (29) were added to the medium (1 µM). Both peptides were purchased from Neosystem. Controls received plain medium during the pre- and poststimulation period and 100 nM oCRH during the stimulation period. Flow rate was 6 ml/h. For each condition, four to six pituitaries from 8-d-old (AS) or 9-d-old (aSVG) chicks were used.

Effect of urocortin III on in vitro {alpha}-subunit secretion
A perifusion protocol as described in Effect of a melanocortin agonist on in vitro {alpha}-subunit secretion was carried out. During the stimulation period, six pituitaries received 100 nM of the selective CRH-R2 agonist human urocortin III [hUCN III (30)] (Sigma), whereas the remaining pituitaries (n = 6) received oCRH (100 nM). The flow rate was 6 ml/h.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of melanocortins on in vitro {alpha}-subunit secretion
SHU9119 was unable to alter the TSH-releasing action of oCRH or TRH at the level of the pituitary. There was no significant difference between the mean SFs and NIs of the control and test conditions (Table 1Go). The fact that the relative NI value of the MSH/SHU condition appears to be lower than that of the MSH condition is probably caused by the gradually increasing secretion of one pituitary in the MSH condition, whereas the other pituitaries did not change their basal secretion rate. In the concentration used (100 nM), {alpha}-MSH did not stimulate the release of {alpha}-subunit from the chicken pituitary. Figure 1Go shows the effect of an NDP-MSH challenge on pituitary {alpha}-subunit secretion, scaled to a curve obtained after in vitro oCRH (100 nM) stimulation. It is clear that NDP-MSH did not stimulate the secretion of {alpha}-subunit from the chicken pituitary.


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  		TABLE 1. Effect of 1 µM SHU9119 on the in vitro {alpha}-subunit release from 9- and 10-d-old chicken pituitaries stimulated with 100 nM {alpha}-MSH, TRH, or oCRH in a perifusion system (n = 3–4 pituitaries per condition)

 


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  		FIG. 1. Absence of an effect of 1 µM (Nle4, D-Phe7)-{alpha}-MSH (NDP-MSH) on the in vitro {alpha}-subunit secretion from 10-d-old chicken pituitaries (filled circles) compared with a typical response to 100 nM oCRH challenge (open circles). Data shown are means ± SEM (n = 12 for NDP-MSH, and n = 4 for oCRH) and are expressed as percentage relative to mean basal secretion. The line indicates the presence of the stimulators (oCRH or NDP-MSH).

 
Cloning of a partial cDNA encoding the chicken CRH-R2
A partial cDNA encoding the chicken CRH-R2 was obtained by RT-PCR approach (EMBL accession no. AJ557031). RT-PCR on brain, heart, and pituitary total RNA yielded a single band (Fig. 2Go). The subcloned PCR product showed high homology to known CRH-R2 sequences, especially to base pairs 360–620 of the Xenopus laevis CRH-R2 (82%; Fig. 3Go), containing a part of the N-terminal extracellular domain, three transmembrane (TM) regions (TM1 and TM2 completely, TM3 partially), the first intracellular domain, and the second extracellular domain. This 260-bp fragment was used in subsequent in situ hybridization experiments to localize chicken CRH-R2 expression in hypophyseal cells.



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  		FIG. 2. DNA fragments resulting from RT-PCR amplification using primers for CRH-R2 on total RNA isolated from chicken brain (telencephalon), heart, and pituitary. The molecular marker on the left is a 100-bp ladder.

 


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  		FIG. 3. Sequence alignment of the obtained partial chicken (c) CRH-R2 cDNA and Xenopus laevis (x) CRH-R2 cDNA. Asterisks indicate identical nucleotides. The amino acid sequence derived from the chicken cDNA is shown in the upper line.

 
Cellular localization of CRH-R1 and CRH-R2 mRNA in the chicken pituitary
Each riboprobe was tested on pituitary sections of at least five different animals; the expression patterns were similar for all pituitary sections investigated. The chicken pars distalis consists of two distinctive lobes, called the cephalic and caudal lobes, which differ in their cellular components. In agreement with earlier immunocytochemical studies, both TSH- and POMC-immunoreactive cells were restricted to the cephalic lobe of the chicken pituitary. Combination of in situ hybridization with the antisense CRH-R1 probe and immunostaining of thyrotropes or corticotropes confirmed our previous findings, showing that CRH-R1 expression is confined to corticotropes, whereas no signal was detected on thyrotropes (Fig. 4Go, A and B). For CRH-R2, the situation was exactly the opposite. In situ hybridization with the antisense CRH-R2 riboprobe resulted in a hybridization signal that appeared as clusters of silver grains located in between corticotropes, but hardly any signal was detected on corticotropic cells (Fig. 4CGo). When thyrotropes were visualized, it was confirmed that this cell type expresses CRH-R2 mRNA significantly (Fig. 4DGo). Statistical analysis of overlaying silver grain numbers confirmed these findings (Table 2Go). Comparison of silver grain density is only possible between different cell types treated with the same probe; comparison between the different riboprobes is not allowed because of possible differences in labeling and/or hybridization efficiency and the fact that threshold indices have to be set accordingly. Hybridization with either sense probe did not result in any specific signal (data not shown).



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  		FIG. 4. Result of in situ hybridization with the CRH-R1 (A and B) and CRH-R2 (C and D) antisense riboprobes, combined with immunostaining of either corticotropes (A and C) or thyrotropes (B and D). Scale bar, 0.05 mm.

 

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  		TABLE 2. Quantification of silver grains (in situ hybridization signal) over thyrotropes and corticotropes in the anterior pituitary of 6-wk-old Cobb chickens, expressed as pixels per 1000 µm2

 
Effect of urocortin III, AS, and aSVG on in vitro {alpha}-subunit secretion
Stimulation with 100 nM hUCN III caused a modest increase in {alpha}-subunit release (Fig. 5Go). The SF for hUCN III (2.54 ± 0.49) is significantly lower than that for oCRH (6.53 ± 1.11) (P < 0.01), but relative NIs (hUCN III, 3344.0 ± 942.7; oCRH, 5913.0 ± 1141.3) do not differ significantly (P = 0.113). Treatment with 1 µM AS or aSVG completely abolished the {alpha}-subunit response to an oCRH stimulus (Figs. 6Go and 7Go and Table 3Go).



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  		FIG. 5. Effect of 100 nM oCRH (filled circles) and 100 nM hUCN III (open circles) on the in vitro {alpha}-subunit secretion from 10-d-old chicken pituitaries in a perifusion system. Data shown are means ± SEM (n = 6 per condition) and are expressed as percentage relative to mean basal secretion. The line indicates the presence of the stimulators (oCRH or hUCN III).

 


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  		FIG. 6. Effect of 1 µM AS on the in vitro oCRH-induced {alpha}-subunit secretion from 8-d-old chicken pituitaries in a perifusion system. Data shown are means ± SEM (n = 5–6 per condition) and are expressed as percentage relative to mean basal secretion. In both conditions, an oCRH stimulus was given during the period indicated with the thick line. The dashed line indicates the presence of AS in the blocker condition (open circles); the control condition (filled circles) received plain medium during this period.

 


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  		FIG. 7. Effect of 1 µM aSVG on the in vitro oCRH-induced {alpha}-subunit secretion from 9-d-old chicken pituitaries in a perifusion system. Data shown are means ± SEM (n = 4–5 per condition) and are expressed as percentage relative to mean basal secretion. In both conditions, an oCRH stimulus was given during the period indicated with the thick line. The dashed line indicates the presence of aSVG in the blocker condition (open circles); the control condition (filled circles) received plain medium during this period.

 

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  		TABLE 3. Effect of 1 µM AS or aSVG on the in vitro oCRH-induced {alpha}-subunit release from pituitaries of 8- and 9-d-old chicks, respectively

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The fact that chicken thyrotropes express CRH-R2 mRNA, as shown by our in situ hybridization experiments, strongly suggests that CRH stimulates TSH release directly at the level of the thyrotropes. The detection of CRH-R2 mRNA in the chicken adenohypophysis was somewhat unexpected because CRH-R2 was not found in the hypophysis of adult Xenopus by RT-PCR (16) and its expression in the anterior pituitary of mouse and rat had been questioned by several authors (31, 32). CRH-R2 was found in the pars distalis of the rhesus monkey by use of autoradiography, but no cellular localization was performed (31). The authors assumed that CRH-R2 is expressed by corticotropes and therefore supposed a role for CRH-R2 in the control of the pituitary-adrenal response to stress in primates. Other researchers were able to demonstrate the presence of CRH-R2 in the adenohypophysis of the mouse by RT-PCR, but they could not confirm this result by in situ hybridization (33). In vitro and in vivo experiments performed by the same researchers argued against the hypothesis that pituitary CRH-R2 could play a role in CRH- or UCN-induced ACTH secretion in the mouse. Therefore, it was suggested that pituitary CRH-R2 expression is confined to blood vessels (33). However, our results clearly demonstrate the existence of noncorticotropic hypophysiotropic functions of CRH, which is also supported by the very recent finding of CRH-R2 on gonadotropes in rat pituitary (34). In the chicken pituitary, CRH-R2 was clearly expressed by thyrotropes, indicating that it plays a role in the transduction of the CRH stimulus for TSH release. This was further confirmed by our perifusion experiments. The nonselective CRH-R blocker AS abolished CRH-induced TSH secretion, suggesting the direct involvement of a CRH-R. This receptor is most likely CRH-R2, because the specific CRH-R2 blocker aSVG also inhibited the TSH response to CRH. The CRH-like peptide hUCN III elicited an increase in {alpha}-subunit release, although modest compared with an equimolar concentration of oCRH. In mammals, hUCN III is a specific agonist for CRH-R2, showing no affinity for CRH-R1 (30), and therefore even this small increase suggests the involvement of CRH-R2. The higher potency of oCRH to stimulate the chicken CRH-R2 should be confirmed by pharmacological studies once the complete coding sequence is known.

Because no effects were seen in the SHU9119 perifusion experiment, we can conclude that neither MC3R nor MC4R is involved in CRH-induced TSH release. Moreover, it seems very unlikely that melanocortins are involved in the TSH-releasing activity of CRH in the chicken at all, because: 1) it was shown earlier that porcine ACTH has no effect on the in vitro TSH secretion by the chicken pituitary (4); and 2) in the present study, neither {alpha}-MSH nor the powerful {alpha}-MSH analog NDP-MSH could mimic the TSH-releasing effect of CRH, not even when high concentrations were used. Chicken MCRs show a high degree of homology with their mammalian counterparts (35, 36), and {alpha}-MSH and NDP-MSH are known to bind the chicken MCRs (37 ; and Schiöth, H. B., personal communication). Recent research on the ringdove indicated that the melanocortin system in birds is similar to that in mammals (38), and reports on the melanocortin system in fish suggest a similar mode of action in all vertebrates (39, 40). We are well aware of the fact that the TSH-releasing activity of only one class of POMC-derived peptides was investigated in our experiments. Besides melanocortins, opioids are important cleavage products of POMC. To our knowledge, the effect of ß-endorphin and other opioid peptides on TSH secretion has only been investigated in mammals, and results are quite contradictory (41). The significance of a pituitary site of action of opioids requires further investigation.

In summary, to elucidate the mechanism underlying the thyrotropic activity of CRH in the chicken, we have tested two plausible scenarios. The possibility that CRH releases TSH by indirect effects mediated by paracrine interactions between corticotropes and thyrotropes has become unlikely, because we have shown that chicken thyrotropes express a type 2 CRH receptor and are directly responsive to CRH through this receptor. On the other hand, thyrotropes are not responsive to an important class of hormones released by CRH, the melanocortins. The present results suggest that the hypophysiotropic actions of CRH in the chicken are mediated through different CHR-Rs. Corticotropes are stimulated to release ACTH after binding to CRH-R1, whereas CRH-induced TSH secretion is mediated by CRH-R2 on thyrotropes. This mechanism allows for the fine-tuning of the hypophysiotropic actions of CRH; according to the changing needs, CRH can preferentially release ACTH, TSH, or both by differentially regulated receptors.


   Acknowledgments
 
We thank Dr. Serge Van der Geyten, Sylvia Grommen, Willy Van Ham, Francine Voets, Lut Noterdaeme, and Tina Everaert for their help with the animal experiments and {alpha}-subunit RIAs, and Sofie Van Soest (Laboratory of Developmental Physiology, Genomics and Proteomics, Catholic University of Leuven) for sequencing. The chicken CRH-R1 cDNA was kindly donated by Dr. Abdul B. Abou-Samra (Massachusetts General Hospital and Harvard Medical School, Boston, MA). Purified chicken LH for {alpha}-subunit RIAs was a generous gift of Dr. John A. Proudman (Germplasm and Gamete Physiology Laboratory, United States Department of Agriculture-Agricultural Research Service, Beltsville, MD). The authors are also grateful to Dr. Helgi B. Schiöth (Department of Neuroscience, Uppsala University, Uppsala, Sweden) and Dr. Sakae Takeuchi (Department of Biology, Okayama University, Okayama, Japan) for their helpful information on chicken melanocortin receptors.


   Footnotes
 
This research was financed by the Fund for Scientific Research-Flanders (Fonds voor Wetenschappelijk Onderzoeck-Vlaanderen) (Grant FWO .0360.00).

Abbreviations: aSVG, Antisauvagine-30; BS, basal secretion; CRH-R, CRH receptor; hUCN III, human urocortin III; MC3R, melanocortin receptor type 3; MSH, melanotropin; NI, net increase; oCRH, ovine CRH; POMC, proopiomelanocortin; PV, peak value; SF, stimulation factor; SSC, standard sodium citrate; TM, transmembrane.

Received April 25, 2003.

Accepted for publication September 2, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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