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Endocrinology Vol. 144, No. 4 1524-1532
Copyright © 2003 by The Endocrine Society

ARTICLE

Corticotropin-Releasing Factor Receptor Type 2 Messenger Ribonucleic Acid in Rat Pituitary: Localization and Regulation by Immune Challenge, Restraint Stress, and Glucocorticoids

Kazunori Kageyama1, Chien Li and Wylie W. Vale

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037

Address all correspondence and requests for reprints to: Wylie W. Vale, Ph.D., The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRF receptor 2 (CRF R2) has been identified in the rat pituitary. However, the cell types that express the receptor remained to be determined. In the present study, we localized CRF R2 mRNA in gonadotropes of the anterior pituitary. Ribonuclease protection assays of anterior pituitary mRNA further showed that the dominant receptor type is CRF R2{alpha}. We also demonstrated that the expression of CRF R2 in the pituitary is sensitive to alterations to the hypothalamic-pituitary-adrenal axis as CRF R2 mRNA levels in the anterior pituitary of male rats were significantly decreased 6 h after bacterial endotoxin lipopolysaccharide (LPS) injection or restraint stress. Subcutaneous corticosterone injections also resulted in significant suppression of CRF R2 mRNA levels in the pituitary, suggesting that glucocorticoids are involved in modulating CRF R2 mRNA levels in the pituitary under stress. LPS administration still caused a significant suppression of CRF R2 mRNA levels in the anterior pituitary of adrenalectomized rats. This suggests that one or more additional factors is involved in the regulation of CRF R2 expression in the anterior pituitary. Taken together, these data suggest that CRF R2{alpha} in the anterior pituitary might be involved in the regulation of gonadal functions under stress.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IN MAMMALS, CRF family peptides are involved in a variety of physiological functions including regulation of hypothalamo-pituitary-adrenal (HPA) axis activity due to stressors (1). The effects of the CRF/urocortin (Ucn)-related peptides are mediated by G protein-coupled seven transmembrane receptors (1). Two major subtypes of CRF receptors, CRF receptor type 1 (CRF R1; Refs. 2, 3, 4) and type 2 (CRF R2; Refs. 5, 6, 7), have been cloned and characterized. CRF R1, which has high affinity for CRF and Ucn (1), expresses predominately in the brain and the pituitary (8). In the pituitary, CRF R1 is found mainly in corticotropes and is responsible for mediating the effects of hypothalamic CRF on ACTH secretion in response to stress stimulation (9, 10).

CRF R2 shares 69% homology with CRF R1 on amino acid level, and yet it has different pharmacological properties from CRF R1 regarding ligand binding (6, 11). Whereas CRF has approximately 20-fold lower affinity for CRF R2 than for CRF R1, Ucn has a 40-fold higher affinity for CRF R2 than does CRF (12). In addition to Ucn, two additional Ucn-related peptides, Ucn II (also known as stresscopin-related peptide; Refs. 13 and 14) and Ucn III (as known as stresscopin; Refs. 14 and 15), have been found recently in mammals. Both Ucn II and Ucn III have very high affinity for CRF R2 with little or no binding affinity for CRF R1. Thus, the three Ucns may serve as natural ligands for CRF R2 in mammals. For example, it has been shown that both Ucn and CRF R2ß are expressed in the rat heart. Ucn is more potent in regulation of CRF R2 mRNA levels in aortic smooth muscles than CRF (16). Furthermore, Ucn is a more powerful inotrope and is also more potent to increase coronary blood flow and to reduce overall blood pressure than is CRF (17, 18). Thus, the natural ligand for CRF R2ß, at least in the cardiovascular system, is likely to be Ucn rather than CRF.

Several splice variants of CRF R2 have been reported. Two variants, CRF R2{alpha} and CRF R2ß, have been found in rat. CRF R2{alpha} and CRF R2ß differ in their N-terminal domain (5). CRF R2{alpha} is expressed primarily in several discrete brain regions including the hypothalamus, lateral septum, and raphe nuclei (19), whereas CRF R2ß is found predominately in peripheral tissues such as heart, gastrointestinal treat, the arterioles, and muscles (16, 19). CRF R2 mRNA has also been reported to be present in the rat pituitary (20), but neither the splice variant involved nor the function of the receptor has been defined in the rat pituitary.

In the pituitary, the changes in CRF R1 have been suggested to play a major role in modulating adaptive responses to stressors. It has been shown that CRF, vasopression, lipopolysaccharide (LPS), cytokines, and glucocorticoids can negatively modulate pituitary CRF R1 mRNA levels (21, 22, 23). The decrease in CRF receptor would contribute to a sustained reduction in the number of CRF binding sites and restricting subsequent effect of CRF to restore homeostasis. The existence of CRF R2 in the pituitary raised an important question as what function of pituitary CRF R2 may serve regarding the regulation of HPA axis activity. Our previous studies have demonstrated that both immune challenge and restraint stress can modulate CRF R2ß expression in the heart (16). It is conceivable that the expression of pituitary CRF R2 may be also sensitive to various stressors, which may suggest that CRF R2 in the pituitary is also involved in modulation of pituitary response to stressors.

To further understand the possible function and regulation of CRF R2 in the pituitary, we first examined the dominant receptor type and the localization of CRF R2 mRNA in the pituitary. We next determined whether the levels of CRF R2 mRNA in the anterior pituitary are modulated by HPA stimulation following immune or restraint stress.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague Dawley rats weighing 280–320 g were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). They were housed in a temperature-controlled room with controlled lighting (light 0600–1800 h) and were given free access to laboratory chow and tap water. All procedures were approved by The Salk Institute Animal Use and Care Committee.

Surgery
Jugular vein cannulation.
A jugular vein catheter (PE 50, Becton Dickinson and Co., Sparks, MD) was inserted into the right atrium of the rats under light halothane anesthesia 2 d before experiments. The catheter was filled with sterile heparinized saline, passed through an sc tunnel, and exteriorized at the back of the neck. After the cannulation, rats were housed in individual cages.

Subcutaneous cannulation.
In animals in which ACTH gel or corticosterone was injected, an sc catheter (PE 20, Becton Dickinson and Co.) was inserted into the back at the same time as jugular vein cannulation. The catheter was filled with saline, passed through an sc tunnel, and exteriorized at the back of the neck.

Adrenalectomy (Adx).
Halothane-anesthetized rats were bilaterally adrenalectomized via a dorsal approach, and sc implanted with a slow-release corticosterone pellet (35 mg, 21-d release; Innovative Research of America, Sarasota, FL; Adx + corticosterone). This regimen was chosen for its ability to retain basal CRF and vasopressin mRNA levels in the paraventricular nucleus (PVN) of the hypothalamus and proopiomelanocortin mRNA levels in the anterior pituitary after Adx (24). A control group of rats (sham) was anesthetized, received the same dorsal incision, and was implanted with a placebo pellet. After surgery, all rats were provided with water containing 0.9% NaCl. Five days later, sham and Adx + corticosterone rats participated in LPS experiments. In LPS-injected Adx + corticosterone rats, Adx was verified by the lack of change in plasma corticosterone. In saline-injected Adx + corticosterone rats, Adx was verified by the lack of circadian elevation in plasma corticosterone 8 h after lights-on.

Reagents
Lipopolysaccharide (LPS, Escherichia coli serotype O26: B6; code 3755, lot 37H4095) and corticosterone were purchased from Sigma (St. Louis, MO).

In vivo experimental procedure
On the day of the experiment, the rats were housed in opaque sampling cages, and the jugular vein catheter was connected to a sampling tube to allow for remote sequential blood sampling. After a period of 2–3 h, experiments were started at 0800–0900 h. The rats were killed by decapitation after final blood sampling. The anterior pituitaries were separated and frozen in liquid nitrogen.

Experiment 1: effects of iv injection of LPS [50 µg/kg body weight (BW)] on CRF R2 mRNA levels in the anterior pituitary
After blood sampling for measurement of basal plasma ACTH and corticosterone levels, vehicle (saline, 100 µl) or LPS at a dose of 50 µg/kg BW was injected iv at 0 min. Blood was drawn 30, 60, 120, 240, and 360 min later and stored for future measurements of plasma ACTH and corticosterone. After sampling blood at 6 h, some rats were decapitated, and organs were harvested. To examine time-dependent changes in CRF R2 mRNA levels, other rats were decapitated 2, 9, and 24 h after vehicle or LPS injection.

Experiment 2: effects of restraint or sc injection of corticosterone on CRF R2 mRNA levels in the anterior pituitary
After blood sampling for measurement of the basal plasma ACTH and corticosterone levels, vehicle (100 µl of saline for iv injection, and 200 µl of 11% ethanol-containing saline for sc injection), LPS (50 µg/kg BW, iv), a low dose of corticosterone (37.5 µg/rat, sc), or a high dose of corticosterone (125 µg/rat, sc) was injected. Both low and high doses of corticosterone were also administered at 30, 60, 120, and 180 min. Some rats that received both vehicle injections were wrapped in cloth towels and restrained by rubber bands and labeling tape for 1 h. Blood was drawn 30, 60, 180, and 360 min after injection or onset of restraint for plasma ACTH and corticosterone measurements. After sampling at 6 h, the rats were decapitated for tissue collection.

Experiment 3: effects of Adx with corticosterone replacement and LPS injection (50 µg/kg BW) on CRF R2 mRNA levels in the anterior pituitary
After blood sampling for measurement of the basal plasma ACTH and corticosterone levels, vehicle (saline, 100 µl) or LPS (50 µg/kg BW, iv) was injected at 0 min in sham or Adx + corticosterone rats. Blood was drawn at 60 and 240 min for ACTH and corticosterone measurements. After the final blood sampling, the rats were decapitated for tissue collection.

Corticosterone and ACTH measurement
Plasma corticosterone and ACTH were measured in duplicate from unextracted samples as described previously (25). Plasma corticosterone levels were measured with a commercial immunoradiometric assay kit produced by ICN Biomedicals, Inc. (Costa Mesa, CA). Plasma ACTH levels were measured with a commercial immunoradiometric assay kit produced by Nichols Institute Diagnostics (San Juan Capistrano, CA). Samples repeated from individual rats were analyzed within the same assay.

Ribonuclease (RNase) protection
Total RNA was extracted using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH). Rat CRF R2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were measured simultaneously by RNase protection assay, using rat GAPDH as an internal loading control. CRF R2 and R2{alpha} antisense riboprobes specific to the rat CRF R2 and CRF R2{alpha} mRNA were synthesized using T3 RNA polymerase, respectively (Fig. 1AGo). A 165-nucleotide (nt) GAPDH antisense riboprobe specific to the rat GAPDH mRNA was synthesized using T3 RNA polymerase. All riboprobes were synthesized in the presence of [{alpha}-32P] uridine triphosphate (UTP; 3000 Ci/mmol) and 20 µM UTP, as described (26). The fragments protected by the CRF R2 probe 1 and the GAPDH riboprobe are 463 nt and 135 nt, respectively (Fig. 1BGo). The CRF R2{alpha} and CRF R2ß fragments protected by CRF R2 probe 2 are 154 nt and 113 nt, respectively (Fig. 1CGo).



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  		Figure 1. Detection of CRF R2 mRNA in the rat anterior pituitary by RNase protection assay. A, Schematic map of rat CRF R2 cDNA illustrating the probes used to detect rat CRF R2 (probe 1) or CRF R2{alpha} (probe 2) mRNA, respectively. Exons 1–12 (E1–E12) are shown in this map. Vertical black lines show exon junctions. A nucleotide position from the first nucleotide of exon 2 is indicated as a number (-42, 12, 113, or 475) on the map. B, Representative autoradiogram showing the detection of CRF R2 mRNA by RNase protection assay with probe 1. The probe protects a 463-nt band in RNA samples from the anterior pituitary (AP) and the heart (Ht), but not tRNA, which served as a negative control. C, Representative autoradiogram showing the detection of CRF R2{alpha} mRNA by RNase protection assay with probe 2. The probe protects a 154-nt CRF R2{alpha} fragment and a smaller 113-nt CRF R2ß fragment. AP1, Eight micrograms total RNA from anterior pituitary; AP2, 15 µg total RNA from anterior pituitary; LS, lateral septum; VMH, ventromedial hypothalamus; R2{alpha}-cell, R2{alpha}-transfected cells.

 
RNase protection analyses were carried out as previously described (26). RNA samples (10 µg for anterior pituitary, 50 µg for heart, 10 µg for lateral septum and ventromedial hypothalamus of the brain, and 1 µg for R2{alpha}-transfected cells) were hybridized in 24 µl deionized formamide plus 6 µl hybridization buffer containing 5 x 105 cpm of CRF R2 and 2 x 104 cpm GAPDH antisense riboprobes. After heating at 85 C for 5 min, the samples were hybridized at 42 C for 15 h and subsequently digested by RNase (200 µg/ml RNase A and 350 U/ml RNase T1) at 24 C for 60 min. The samples were resolved on 6% polyacrylamide urea gels. Quantitative analysis was performed using the PhosphorImager system (Molecular Dynamics, Inc., Sunnyvale, CA) and the ImageQuant 4.0 software package. The intensity of each of the protected fragments was corrected for differences in loaded RNA according to the intensity of the protected GAPDH fragment of the same sample, and results are expressed as corrected arbitrary units. The intensity of CRF R2 mRNA was in proportion to the intensity of GAPDH mRNA in the same sample when added in a range from 5–15 µg (not shown).

Perfusion and tissue sectioning
Animals were anesthetized with an overdose of choral hydrate (1 g/kg BW, ip) and perfused transcardially with 150 ml of saline followed by 350 ml of 4% paraformaldehyde in borate buffer (pH 9.5). The pituitary was postfixed in 25% sucrose at 4 C for 6 h. Coronal sections (20 µm) were cut on a cyrostat, thaw-mounted onto glass slides and collected in one-in-three series. The sections were stored at -80 C until use.

Immunohistochemistry procedures
Tissue sections from all animals were processed in one assay to ensure uniformity of immunostaining. All solutions were treated with diethylpyrocarbonate to prevent RNase contamination. Tissue sections were rinsed in 0.05 M potassium PBS (KPBS) followed by treatment with 1% NaBH4-KPBS solution (Sigma). Sections were incubated in following primary antibodies raised in rabbit: anti-GH, prolactin, TSH, LH, ACTH (1:5000, NIH, Bethesda, MD) or S-100 (1:5000; Chemicon, Temecula, CA) in KPBS with 0.4% Triton X-100 at room temperature for 1 h, followed by 4 C for 48 h. After the incubation, the tissues were rinsed in KPBS and incubated in biotinylated donkey antirabbit IgG (1:600, Vector Laboratories, Burlingame, CA) in KPBS with 0.4% Triton X-100 for 1 h at room temperature. This was followed by another 1 h incubation at room temperature in avidin-biotin complex solution (Vectastain ABC Elite Kit, Vector Laboratories). The antibody-peroxidase complex was visualized with a mixture of 3',3'-diaminobenzidine (DAB, 0.2 mg/ml) and 3% H2O2 (0.83 µl/ml) in 0.05 M Tris buffer-saline solution. Following the staining, tissue sections were processed for in situ hybridization.

In situ hybridization
CRF R2 cRNA probe was transcribed from the CRF R2 probe 1 in the presence of the 33P-labeled UTP (NEN Life Science Products, Boston, MA). The specific activity of the probe ranged from 1–5 x 105 cpm/µl of hybridization buffer. The pituitary sections were fixed in 4% paraformaldehyde, digested with proteinase K (5 µg/ml, Sigma), treated with a fresh solution containing 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0), followed by a rinse in 2x saline-sodium citrate buffer (SSC), dehydrated through a graded series of alcohol, delipidated in chloroform, dehydrated through a second series of alcohol, and then air dried. The slides were exposed to the CRF R2 cRNA probe overnight in moist chambers at 55 C. After the incubation, the slides were washed in SSC that increased in stringency, in RNase, in 0.1x SSC at 65 C, and dehydrated through graded series of alcohol and dried. Slides were dipped in NTB-2 emulsion (Eastman Kodak Co., Rochester, NY), exposed for 15 d at 4 C and developed.

Statistical analysis
All values are expressed as the mean ± SEM. Statistical analyses of these data were performed using one-way ANOVA, or two-way ANOVA on repeated measures with time and treatment as the factors, followed by Scheffé’s F post hoc test. P < 0.05 was accepted as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CRF R2 mRNA in the rat pituitary
Expression of CRF R2 mRNA in the anterior pituitary was first determined by RNase protection assays using a rat CRF R2 probe (probe 1) that detects both CRF R2{alpha} and CRF R2ß (Fig. 1AGo). CRF R2 mRNA was expressed in rat anterior pituitary as well as the heart (Fig. 1BGo). To further determine the dominant subtype of CRF R2 mRNA in the pituitary, a specific probe for the N-terminal end of rat CRF R2{alpha} was used in the RNase protection assays to distinguish between CRF R2{alpha} and CRF R2ß because the first exon of CRF R2ß is different from that of CRF R2{alpha} due to an alternative splicing that led to an exon substitution (Fig. 1AGo). As shown in Fig. 1CGo, a protected RNA fragment with the size of 154 nt for CRF R2{alpha} was found in the RNA samples from the anterior pituitary, the lateral septum, the ventromedial hypothalamus and CRF R2{alpha} transfected cells. This result suggests that CRF R2{alpha} is the dominant receptor type in the anterior pituitary.

Localization of CRF R2 mRNA in the rat pituitary
By using in situ hybridization, CRF R2 mRNA was visualized in the anterior pituitary. CRF R2 mRNA positive signals were only found in the tissues probed with antisense probes (Fig. 2AGo). No signals were found in the sense control. Immunohistochemistry on various pituitary-produced hormones and S-100 protein combined with in situ hybridization for CRF R2 mRNA showed that the majority of CRF R2 positive signals colocalized with gonadotropes as identified by LH-positive staining (Fig. 2BGo). Only a few ACTH-positive cells expressed CRF R2 mRNA (Fig. 2CGo). Table 1AGo summarizes the percentage of each cell type identified in CRF R2 positive cells. The data demonstrate that roughly 84% of CRF R2 positive cells are LH-immunopositive, whereas 3%, 11%, 15%, 7%, and 4% of CRF R2 mRNA positive cells are ACTH, TSH, prolactin, GH, and S-100 immunopositive cells, respectively. Table 1BGo summarizes the percentage of CRF R2 positive cells identified in each cell type. These data demonstrated that 52% of gonadotropes are CRF R2 positive, whereas approximately 2%, 6%, 3%, 1%, and 2% of corticotropes, thyrotropes, lactotropes, somatotropes, and S-100 positive cells, respectively, are CRF R2 positive.



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  		Figure 2. Localization of CRF R2 mRNA in the rat anterior pituitary. Representative dark-field photomicrographs showing anterior pituitary sections probed with either antisense (A) or sense (B) probe for CRF R2 mRNA. Positive signals indicated by silver grain clusters were only found in the tissues probed with antisense probes. No signals were found in the sense control. C, Bright-field photomicrograph showing LH-immunoreactive cells in the rat anterior pituitary. Gonadotropes are visualized by the brown DAB precipitates representing LH-immunoreactive cells. D, Dark-field image of the same area of C showing CRF R2 mRNA-positive cells identified by silver grain clusters. Some of the cells that are double-labeled with LH immunostaining and R2 mRNA are highlighted by arrows. Inset, High-power magnification of boxed area in panel D to illustrate a LH-positive cell overlapped with CRF R2 mRNA positive signal (scattered black silver grains). E, Representative bright-field photomicrograph of the anterior pituitary showing ACTH immunoreactive cells revealed by the DAB brown precipitates. F, Dark-field image of the same area of E showing CRF R2 mRNA signals identified by silver grain clusters. Only a few ACTH-immunoreactive and CRF R2 mRNA double-labeled cells (arrow) were observed. Scale bar, 50 µm.

 

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  		Table 1. Percentage of each cell type identified in CRF R2-positive cells (A) and of CRF R2-positive cells identified in each cell type (B)

 
Effects of LPS injection on CRF R2 mRNA levels in the pituitary
Rats were iv injected with saline or LPS (50 µg/kg), and plasma ACTH and corticosterone levels were measured. As previously reported (25), plasma ACTH and corticosterone levels immediately before these treatments (ACTH, 8 ± 1 pg/ml; corticosterone, 28 ± 9 ng/ml) were typical of rats under non-stress conditions. Iv injection of LPS elicited time-dependent increases in plasma ACTH and corticosterone levels, with peak concentrations measured 1 h (mean ± SEM, 820 ± 116 pg/ml) and 2 h (mean ± SEM, 523 ± 137 ng/ml) after the injection, respectively.

Iv injection of LPS caused a time-dependent reduction of CRF R2 mRNA levels in the anterior pituitary (ANOVA; P < 0.005; Fig. 3Go). CRF R2 mRNA levels decreased to less than half those of the control values by 6 h after LPS injection (P < 0.05). CRF R2 mRNA levels in the anterior pituitary remained reduced 9 h and 24 h after the injection but tended to return toward control levels at 24 h.



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  		Figure 3. Time-dependent changes in CRF R2 mRNA levels in the rat anterior pituitary after iv injection of LPS or saline in intact male rats. Vehicle or LPS was injected iv at 0 min in intact male rats. Rats were decapitated 2, 6, 9, and 24 h after vehicle or LPS injection, and the anterior pituitaries were harvested to examine CRF R2 mRNA levels. Results were expressed as relative changes in CRF R2 mRNA levels compared with saline controls (mean ± SEM) of six to eight animals per group. *, P < 0.01 compared with control.

 
Regulation of CRF R2 mRNA levels in the pituitary by restraint stress or sc injection of corticosterone
To determine whether elevation of plasma corticosterone associated with LPS injection may modulate CRF R2 mRNA expression in the pituitary, the effects of exogenous corticosterone on pituitary CRF R2 mRNA expression was first examined. Subcutaneous administration of corticosterone elevated the levels of plasma corticosterone in a dose-dependent manner (area under the curve of plasma corticosterone concentrations from 0–360 min: high dose: 18,371 ± 5,502 ng/ml 360 min, low dose: 8,981 ± 2,006 ng/ml 360 min). In addition, the levels of plasma corticosterone in high dose injected animals were very similar to that of LPS-injected rats (17,040 ± 3,841 ng/ml 360 min) and of restrained rats (24,604 ± 6,181 ng/ml 360 min).

There was a significant interaction among treatments on CRF R2 mRNA levels (ANOVA; P < 0.001; Fig. 4Go). Restraint stress and both high and low doses of corticosterone significantly decreased CRF R2 mRNA levels in the anterior pituitary compared with that of the control groups (ANOVA; P < 0.01), indicating that corticosterone can modulate the expression of CRF R2 in the pituitary.



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  		Figure 4. Effects of restraint stress or corticosterone on CRF R2 mRNA levels in the rat anterior pituitary. Either vehicle, LPS, or two different doses of corticosterone was injected at 0 min. Corticosterone was administered again at 30, 60, 120, and 180 min later. A separate set of rats was restrained for 1 h. The rats were decapitated for tissue collection 6 h after the treatments. Results were expressed as relative changes in CRF R2 mRNA levels compared with controls (mean ± SEM) of six to nine animals per group. *, P < 0.01 compared with control.

 
Effects of endogenous corticosterone on CRF R2 mRNA levels in the pituitary
To determine the effects of endogenous corticosterone following LPS on CRF R2 mRNA levels, Adx rats in which plasma corticosterone was clamped at approximately 50 ng/ml (Adx + corticosterone), or sham-operated intact rats were injected with saline or LPS. Corticosterone replacement in the Adx + corticosterone rats caused a significant increase in the plasma ACTH levels [mean ± SEM, 1510 ± 234 pg/ml (1 h)] after LPS injection compared with that of LPS-injected sham rats [mean ± SEM, 831 ± 66 pg/ml (1 h)]. This augmented ACTH release in the Adx + corticosterone rats was probably due to reduced negative feedback, a consequence of their inability to secrete endogenous corticosterone during stress.

There was a significant interaction between adrenal surgery and treatment on CRF R2 mRNA levels (ANOVA; P < 0.05; Fig. 5Go). Adx with corticosterone replacement tended to attenuate the effects of LPS injection on CRF R2 mRNA levels, but not significantly (P = 0.06), compared with that of LPS-injected sham rats.



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  		Figure 5. Effects of Adx following LPS injection on CRF R2 mRNA levels in the rat anterior pituitary. Vehicle or LPS was injected at 0 min in sham or Adx + corticosterone rats. The rats were decapitated for tissue collection 4 h after the treatments. Data are the mean ± SEM of seven to nine animals per group. Statistical analyses were performed using two-way ANOVA, following by Scheffé’s F test. *, P < 0.05 (compared with Sham/Saline control). **, P < 0.01 (compared with Sham/Saline control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In present study, we demonstrated the expression of CRF R2 mRNA in the anterior pituitary by using in situ hybridization and RNase protection assay. This observation corroborates a prior study of CRF R2 mRNA expression in the pituitary (20). The results from RNase protection assays of anterior pituitary mRNA showed that the dominant receptor type is CRF R2{alpha} instead of CRF R2ß, the major CRF R2 subtype found in rat peripheral tissues (16, 19). Furthermore, combined immunohistochemistry and in situ hybridization demonstrated that CRF R2 mRNA colocalizes mainly with gonadotropes but not corticotropes. These data suggest that CRF R2{alpha} in the anterior pituitary might be involved in modulation of gonadotropin secretions and/or gonadal function.

Activation of stress systems could potentially influence reproduction at any level of the hypothalamo-pituitary-gonadal axis (27). The decreases in the LH/FSH secretions by stress would influence the gonadal functions such as sex steroidogenesis and sperm production (27, 28). It has been shown that central CRF injections can inhibit LH/FSH secretion (29). However, this effect of CRF probably reflects a central mechanism involving modulation of the activity of GnRH neurons in the hypothalamus (30). In addition, peripheral administration of CRF did not affect LH secretion (31, 32). Taken together, it is likely that CRF, a low affinity ligand for CRF R2, does not have a direct effect on gonadotropes and stimulation of CRF R1 in the pituitary by CRF is not involved in LH secretion. We are currently investigating whether CRF R2 selective ligands including Ucn II and Ucn III can modulate LH/FSH secretions from pituitary. Further, studies in progress in several groups with CRF R2-specific antagonists and CRF R2-deficient mice should provide more definitive insight into the physiological roles of CRF R2 in the pituitary (33).

In the present studies, we observed a significant decrease in anterior pituitary CRF R2 mRNA levels 6 h after the rats treated with LPS, which has been shown to also suppress LH and FSH release in rats (34). Restraint stress also caused a decrease in CRF R2 mRNA levels. Furthermore, direct injection of either ACTH (unpublished observation) or corticosterone also down-regulated CRF R2 mRNA levels. These results suggest that glucocorticoids could play a role in mitigating CRF R2 mRNA levels in the pituitary during immune challenge or restraint stress. A direct effect of corticosterone on the expression of CRF R2 mRNA in the anterior pituitary is consistent with our previous report of CRF R2ß in the cardiovascular system (16). CRF R2ß mRNA levels in the rat heart or aorta were decreased after sc injections of corticosterone in vivo (16). CRF R2ß mRNA expression in the A7r5 aortic smooth muscle cells were also inhibited by dexamethasone (16). Thus, both CRF R2{alpha} and CRF R2ß expression in the periphery can be modulated by glucocorticoids (35, 36).

Interestingly, we found that LPS treatment can still suppress CRF R2 mRNA levels in the anterior pituitary of Adx rats with basal corticosterone supplement, suggesting that additional factors such as CRF-related ligands and cytokines might be involved in the regulation of CRF R2 mRNA. In fact, we and other investigators (16, 37) have observed that proinflammatory cytokines, IL-1ß, TNF-{alpha}, or IL-6, each decreased CRF R2ß messenger levels in A7r5 cells or heart. Therefore, following immune challenges, increased circulating concentrations of some cytokines or endotoxin itself might regulate CRF R2{alpha} mRNA levels in the anterior pituitary. Furthermore, glucocorticoids have been shown to negatively modulate proinflammatory cytokine levels (38, 39). Thus, it is possible that immune stress induced elevation of proinflammatory factors would be exaggerated and might have a more pronounced effect on CRF R2 expression in the pituitary in Adx rats in the absence of glucocorticoids.

Our previous in vitro study showed that treatment of A7r5 cells with Ucn or CRF decreased CRF R2ß mRNA levels, consistent with the possibility that ligands for CRF R2ß may be involved in the regulation of the receptor expression (16). The lack of glucocorticoids elevation in Adx rats after stress results in a large increase in CRF and AVP levels in the portal circulation followed by an elevation in plasma ACTH (40, 41). It has been suggested that the elevated portal levels of CRF or AVP might contribute to the decrease in CRF R1 mRNA levels in the pituitary (21, 22). Thus, it is conceivable that the endogenous ligands for CRF R2 might be involved in the modulation of CRF R2 mRNA levels in the pituitary under these conditions, although the endogenous ligand(s) for CRF R2{alpha} in the anterior pituitary is still unclear.

Ucn mRNA is expressed in discrete regions of the brain including the supraoptic nucleus of hypothalamus (42, 43) and a number of peripheral tissues such as the thymus, spleen, heart, stomach, intestine, testis, liver (25), lymphocytes (44), and placental and fetal membranes (45). Thus, both supraoptic nucleus and peripheral tissues could potentially provide the source of Ucn for pituitary CRF R2. Several studies even showed that Ucn is expressed in the rat and human pituitary (42, 46). Limited studies have shown that in the brain Ucn II is expressed in the PVN (13), suggesting that Ucn II may reach the pituitary through the portal circulation to interact with pituitary CRF R2. Ucn III is expressed in discrete areas in the hypothalamus and amygdala (15). In addition, Ucn III immunoreactivity has been found in the internal zone of the median eminence (47). It hasn’t been excluded that one of the Ucns could reach the pituitary from the general circulation under some circumstances. Clearly, more studies are needed to further determine the endogenous ligand responsible for interacting with CRF R2 in the pituitary.

Similar to CRF R1 in the pituitary (21, 22, 23), CRF R2 mRNA levels in the pituitary are suppressed by LPS, restraint stress, and glucocorticoids. By contrast, in the PVN of the hypothalamus, the regulation of CRF R2{alpha} mRNA levels seems to be different from those of CRF R1 mRNA levels (48). CRF R2 mRNA levels in the PVN are not modulated by LPS or corticosterone injection, or Adx, whereas CRH R1 mRNA levels in the PVN are altered by these treatments (48). This differences between the regulation of receptors in the pituitary and the PVN might result from the altered susceptibility of these tissues to these factors.

The reduction in CRF-binding sites or number of CRF receptors is achieved through internalization of receptors induced by CRF in the pituitary (49, 50). In addition, the decrease in mRNA levels of the receptors might reflect the suppression of the receptor synthesis, consequently diminishing number of the receptors (21, 51, 52, 53). It is therefore possible that the decrease in CRF R2{alpha} mRNA levels in the anterior pituitary contributes to a sustained reduction in the number of CRF family peptide-binding sites, restricting subsequent ligand actions to restore homeostasis. Additional studies are needed to determine the biological role that occurs after down-regulation of CRF R2{alpha} in the pituitary.

In summary, the present study identified that CRF R2 mRNA is expressed in gonadotropes in the anterior pituitary. RNase protection assays of anterior pituitary mRNA show that the dominant receptor type is CRF R2{alpha}. The CRF R2 mRNA levels in the anterior pituitary are regulated, in part, by glucocorticoids, in response to an immune challenge or restraint stress. Other factors such as CRF-related ligands and cytokines might also be involved in the regulation of CRF R2 mRNA. Further studies on the role of the CRF family of ligands and their receptors would elucidate the novel regulation of gonadotropins under stress.


   Acknowledgments
 
We thank A. Blount for technical assistance, and S. Guerra and D. Dalton for assistance with manuscript preparation.


   Footnotes
 
This work was supported by NIH Program Project DK-26741, the Foundation for Research, The Third Department of Internal Medicine, Hirosaki University School of Medicine (to K.K.), and the Adler Foundation (to K.K.). K.K. is supported by a grant from the Ministry of Education, Science and Culture of Japan (No. 14770582). C.L. is supported by a National Research Service Award (MH-12654). W.W.V. is a Foundation for Research Senior Investigator.

1 Present address: The Third Department of Internal Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan. Back

Abbreviations: Adx, Adrenalectomized; BW, body weight; CRF R1, CRF receptor type 1; CRF R2, CRF receptor type 2; DAB, 3',3'-diaminobenzidine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPA, hypothalamo-pituitary-adrenal; KPBS, potassium PBS; LPS, lipopolysaccharide; nt, nucleotide; PVN, paraventricular nucleus; RNase, ribonuclease; Ucn, urocortin; UTP, uridine triphosphate.

Received October 9, 2002.

Accepted for publication December 17, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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