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Endocrinology Vol. 141, No. 7 2285-2293
Copyright © 2000 by The Endocrine Society

ARTICLES

Regulation of Corticotropin-Releasing Factor Receptor Type 2ß Messenger Ribonucleic Acid in the Rat Cardiovascular System by Urocortin, Glucocorticoids, and Cytokines1

Kazunori Kageyama, Georges E. Gaudriault, Margaret J. Bradbury and Wylie W. Vale2

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. E-mail: vale{at}salk.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CRF receptor type 2 (CRF R2) messenger RNA (mRNA) expression in the rodent heart is modulated by exposure to both the bacterial endotoxin lipopolysaccharide (LPS) and glucocorticoids. In this study we examined the roles of glucocorticoids, cytokines, and CRF R2ß ligands in the regulation of CRF R2ß expression in the cardiovascular system both in vivo and in vitro. Using ribonuclease protection assays, we found that, in addition to the injection of LPS or corticosterone, physical restraint caused a decrease in CRF R2ß mRNA levels in the rat heart and aorta. Adrenalectomy with corticosterone replacement at constant levels partially blocked LPS-induced decreases in CRF R2ß mRNA expression in the heart. Thus, elevations of endogenous circulating corticosterone could contribute to the down-regulation of CRF R2ß mRNA expression in heart. To identify other putative modulating factors, we examined CRF R2ß expression in the aorta- derived A7R5 cell line. Incubation with CRF R2 ligands or dexamethasone reduced CRF R2ß mRNA levels. In addition, incubation with a variety of cytokines, proteins released during immune challenge, also reduced CRF R2ß mRNA expression. The multifactorial regulation of CRF R2ß mRNA expression in the cardiovascular system may serve to limit the inotropic and chronotropic effects of CRF R2 agonists such as urocortin during prolonged physical or immune challenge.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TWO MAJOR SUBTYPES of receptors for the mammalian neuropeptide, CRF, have been identified; CRF receptor type 1 (CRF R1) (1, 2, 3) and CRF receptor type 2 (CRF R2) (4, 5, 6). Both types of CRF receptors are seven transmembrane-spanning G protein receptors positively coupled to adenylate cyclase. CRF R1 and CRF R2 share a 69% amino acid identify (5, 7), but have different tissue distributions and pharmacological properties with respect to ligands (8). CRF R1 is expressed in high concentrations in the pituitary and brain and in various peripheral tissues (9). CRF R2 is located within different areas of the brain than CRF R1 (10, 11) and is also abundant in the periphery (5, 7). CRF R2 has been reported to have at least three apparent splice variants: CRF R2{alpha} (4), CRF R2ß (5, 6, 7), and CRF R2{gamma} (12). In the rat, CRF R2{alpha} messenger RNA (mRNA) is found primarily in the brain, including sites in the hypothalamus, lateral septum, raphe nuclei of the midbrain, and olfactory bulb (13). By contrast, among other sites, rat CRF R2ß mRNA is predominantly expressed in the heart, gastrointestinal tract, arterioles, and muscles (7, 13). R2{gamma} was recently identified in human brain (12), but it has not yet been found in the rodent.

The natural ligand for CRF R2 is most likely not CRF. CRF has approximately 20-fold lower affinity for CRF R2 than it has for CRF R1 (14). In the rat brain, CRF immunoreactive fibers do not colocalize well with CRF R2-containing neurons. Instead, it is likely that urocortin (Ucn), a 40-amino acid, novel CRF family peptide discovered in mammals, is an endogenous CRF R2 ligand. Ucn has a 45% sequence identity to rat/human CRF and a 63% identity to the fish peptide, urotensin (14). Ucn has a 40-fold higher affinity for CRF R2 than does CRF. In rodent and human tissues, Ucn-like immunoreactivity and mRNA have been reported in the thymus, spleen, stomach, intestine, testis and liver (15, 16), lymphocytes (17), pituitary (18), placental and fetal membranes (19), as well as the brain (20, 21). The distribution of Ucn or urotensin-like immunoreactive fibers in the brain correlates with the distribution of CRF R2{alpha} rather than CRF R1 (14). Ucn mRNA and peptide were also found in rat heart and cardiac myocytes (16, 22). The administration of CRF or Ucn has positive inotropic effects on the heart in vivo and in vitro (23, 24). Ucn, however, has more potent coronary vasodilatory and cardiac inotropic effects in sheep than does CRF (25). In addition, CRF R2-deficient mice have a dramatically attenuated vascular response to Ucn injection (26). Together, these findings support the proposal that Ucn may be a natural ligand for CRF R2 in the periphery (8, 14).

Increasing evidence suggests that both CRF R1 and CRF R2 in the brain and pituitary play major roles in modulating adaptive responses to stresses (8). The levels of CRF R2 and CRF R1 mRNA expression are altered in the hypothalamus after exposure to stressors (27, 28, 29). The responses of peripheral CRF receptors to stress, however, are not completely understood. In a previous study expression levels of CRF R2 mRNA in the heart were decreased after exposure to the immune challenge of lipopolysaccharide (LPS) (30). After immune stimulation, tumor necrosis factor-{alpha} (TNF{alpha}), interleukin-1 (IL-1), and IL-6 are known to be elevated in the systemic circulation. These cytokines also increase the activity of the hypothalamus-pituitary-adrenal (HPA) axis, resulting in the release of additional ACTH and corticosterone (31, 32). Injection of deoxycorticosterone also reduces the expression levels of CRF R2ß mRNA (33). It is not known, however, whether cytokines themselves modulate CRF R2ß mRNA expression, nor is it known whether changes in endogenous corticosterone levels are capable of regulating CRF R2ß mRNA expression.

In the present study we extended observations on stress- or hormone-induced changes in CRF R2ß mRNA expression in the cardiovascular system and examined possible mediators for changes in CRF R2ß mRNA levels. In vivo, we tested the hypothesis that the expression levels of CRF R2ß mRNA in the heart would be altered by HPA stimulation after both immune and nonimmune stress. To assess potential factors responsible for the changes in CRF R2ß mRNA levels, we chose an in vitro approach. Currently, the cell line A7R5, an aortic smooth muscle cell line, is available. We determined the effects of CRF, Ucn, glucocorticoids, and cytokines on CRF R2ß mRNA expression levels in the A7R5 cell line.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male Sprague Dawley rats (280–320 g BW) were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN). They were housed in a temperature-controlled room with controlled lighting (lights on, 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. In all rats, a jugular vein catheter (PE 50, Becton Dickinson and Co., Sparks, MD) was inserted into the right atrium under light halothane anesthesia 2 days before experiments. The catheter was filled with sterile heparinized saline, passed through a 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, a 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 a sc tunnel, and exteriorized at the back of the neck.

Adrenalectomy (Adx). Halothane-anesthetized rats were bilaterally Adx via a dorsal approach and sc implanted with a slow release corticosterone pellet (35 mg, 21-day 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 of the hypothalamus and POMC mRNA levels in the anterior pituitary after Adx (34). 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
LPS (Escherichia coli serotype O26: B6; code 3755, lot 37H4095) and corticosterone were purchased from Sigma (St. Louis, MO). ACTH gel (H.P. Acthar gel) was purchased from Rhone-Poulenc Rorer Pharmaceuticals, Inc. (Collegeville, PA).

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 whole hearts and the aorta (arch of aorta and thoracic aorta) were removed and frozen in liquid nitrogen.

Exp 1: effects of iv injection of LPS (50 µg/kg BW) on CRF R2ß mRNA levels in the heart. 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 R2ß mRNA levels, other rats were decapitated 2, 9, and 24 h after vehicle or LPS injection.

Exp 2: effects of sc injection of ACTH gel (0.8 U/animal) on CRF R2ß mRNA levels in the heart. After blood sampling for measurement of basal plasma corticosterone levels (0 min), vehicle (saline, 100 µl), LPS (50 µg/kg BW, iv), or ACTH gel (0.8 U/rat, sc) was injected. ACTH was also administered at 30 and 60 min. This dose regimen was chosen to ensure a prolonged endogenous corticosterone release (35). Blood was drawn 30, 60, 120, 180, 240, 300, and 360 min after LPS injection or first ACTH injection for plasma corticosterone measurement. After sampling at 6 h, the rats were decapitated for tissue collection.

Exp 3: effects of Adx with corticosterone replacement and LPS injection (50 µg/kg BW) on CRF R2ß and Ucn mRNA levels in the heart. 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.

Exp 4: effects of sc injection of corticosterone or restraint stress on CRF R2ß and Ucn mRNA levels in the heart or the aorta. After blood sampling for measurement of the basal plasma ACTH and corticosterone levels, vehicle (100 µl saline for iv injection and 200 µl 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.

Corticosterone and ACTH measurements
Plasma corticosterone and ACTH were measured in duplicate from unextracted samples; these data have been reported previously (16). 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.

Cell culture
The aortic smooth muscle cell line, A7R5, was obtained from American Type Culture Collection (Manassas, VA). Rat Ucn and rat/human CRF were synthesized and provided by Dr. J. Rivier (The Salk Institute, La Jolla, CA). Recombinant rat IL-1ß, IL-6, and TNF{alpha} purchased from Endogen, Inc. (Woburn, MA). Water-soluble dexamethasone was purchased from Sigma. The A7R5 cells were incubated at 37 C in a humidified atmosphere of 5% CO2 and 95% air and in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 U/ml penicillin. Cells were initially plated at 104 cells/cm2 7 days before each experiment. The medium was changed every 48 h. On the sixth day, cells were washed and starved overnight with DMEM supplemented with 0.2% FBS. On the seventh day, the cells were treated for each experiment. All treatments were performed in triplicate and repeated three times.

Ribonuclease (RNase) protection
Total RNA was extracted using Tri-Reagent (Molecular Research Center, Inc., Cincinnati, OH).

Rat Ucn or CRF R2ß, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels were measured simultaneously by RNase protection, using rat GAPDH as an internal loading control. A 361-nucleotide (nt) Ucn antisense riboprobe specific to the rat Ucn mRNA and a 294-nt CRF R2ß antisense riboprobe specific to the rat CRF R2ß mRNA were synthesized using T7 and T3 RNA polymerase, respectively. A 165-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]UTP (3000 Ci/mmol) and 20 µM UTP, as previously described (36). The fragments protected by the Ucn, CRF R2ß, and GAPDH riboprobes were 307, 166, and 135, respectively (Fig. 1Go).



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  		Figure 1. Expression of CRF R2ß and Ucn mRNA in the rat cardiovascular system. The protected fragments were resolved on a 6% polyacrylamide urea gel. Ao, Aorta; Ht, heart; A7R5, aortic smooth muscle cells. A, A representative autoradiogram of RNase protection assay of CRF R2ß mRNA. Total RNA isolated from each tissue or cells listed was hybridized with the antisense probe specific to rat CRF R2ß (5 x 105 cpm) and rat GAPDH (2 x 104 cpm). B, A representative image of RNase protection assay of Ucn mRNA. Total RNA isolated from each tissue listed was hybridized with the antisense probe specific to rat Ucn (5 x 105 cpm) and rat GAPDH (2 x 104 cpm).

 
RNase protection analyses were carried out as previously described (36). RNA samples (50 µg for heart, 15 µg for aorta, and 40 µg for A7R5) were hybridized in 24 µl deionized formamide plus 6 µl hybridization buffer containing 5 x 105 cpm Ucn and 2 x 104 cpm GAPDH antisense riboprobes. After heating at 90 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 Ucn or CRF R2ß mRNA was in proportion to the intensity of GAPDH mRNA in the same sample when added in a range from 12.5–50 µg (not shown).

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 R2ß and Ucn mRNA in heart or aorta
RNase protection assays were performed to determine the distribution of rat CRF R2ß or Ucn mRNA (Fig. 1Go). CRF R2ß mRNA was expressed in rat heart, aorta, and A7R5 aortic smooth muscle cells. By contrast, Ucn mRNA was detected in the heart, and not in the aorta. Ucn mRNA was not found in the A7R5 cell line (not shown).

Effects of LPS on CRF R2ß mRNA levels in the heart
Rats were iv injected with saline or LPS (50 µg/kg), and plasma ACTH and corticosterone levels were measured. As previously reported (16), plasma ACTH and corticosterone levels immediately before treatment in both saline- and LPS-treated groups were typical of those in rats under nonstress conditions. Levels in saline-injected rats demonstrated the expected diurnal variations. Intravenous administration of LPS elicited time-dependent increases in plasma ACTH and corticosterone levels, with peak concentrations measured 1 h (mean ± SEM, 820.42 ± 115.73 pg/ml) and 2 h (mean ± SEM, 522.61 ± 137.34 ng/ml) after injection, respectively.

Figure 2Go shows significant time-dependent changes in heart CRF R2ß mRNA levels after iv injection of LPS (P < 0.0001, by ANOVA). CRF R2ß mRNA levels were decreased to less than half those of the control values by 6 h after LPS injection (P < 0.0001). Subsequently, CRF R2ß mRNA levels in the heart were still significantly decreased 9 h (P < 0.01) after the injection, but tended to return toward control levels 24 h after the injection.



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  		Figure 2. Time-dependent changes in CRF R2ß mRNA levels in the rat heart after iv injection of LPS or saline in intact male rats. Vehicle (saline, 100 µl) or LPS (50 µg/kg BW) was injected iv at 0 min in intact male rats. After the final blood sample, rats ware decapitated 2, 6, 9, and 24 h after vehicle or LPS injection, and organs were harvested to examine CRF R2ß mRNA levels. Relative changes in CRF R2ß mRNA levels compared with those in saline controls (mean ± SEM) of six to eight animals per group are shown. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post-hoc test. *, P < 0.01 (compared with control).

 
Regulation of cardiac CRF R2ß mRNA levels by ACTH administration
To determine whether the decreases in CRF R2ß mRNA after LPS were mediated through changes in ACTH or glucocorticoid production, ACTH gel was injected sc. The sc injection of ACTH gel and LPS each significantly elevated plasma corticosterone [ACTH gel peak, 497.54 ± 30.79 ng/ml (1 h); LPS peak, 517.02 ± 43.92 ng/ml (1 h)] compared with the saline-injected group [37.11 ± 23.23 ng/ml (1 h)].

The levels of CRF R2ß mRNA in the heart were significantly decreased 6 h after ACTH injection as well as after LPS treatment to levels approximately half those measured in saline-injected rats (Fig. 3Go). The decrease in CRF R2ß mRNA levels seen after the injection of ACTH was not significantly different from that observed after LPS injection.



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  		Figure 3. Effects of sc injection of ACTH gel on CRF R2ß mRNA levels in the heart of intact male rats. Vehicle (saline, 100 µl), LPS (50 µg/kg BW, iv), or ACTH gel (0.8 U/rat, sc) was injected at 0 min. ACTH was also administered at 30 and 60 min later. The rats ware decapitated for collection of tissue 6 h after injection of vehicle, LPS, or ACTH gel. Relative changes in CRF R2ß mRNA levels compared with control values (mean ± SEM) of seven to nine animals per group are shown. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post-hoc test. *, P < 0.05; **, P < 0.01 (compared with control).

 
Effects of endogenous corticosterone on CRF R2ß and Ucn mRNA levels in the heart
To determine the effects of an increase in endogenous corticosterone after LPS on CRF R2ß or Ucn 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. Plasma ACTH and corticosterone levels increased 1 h after LPS injection in sham rats, as previously described (16). The plasma ACTH levels in these Adx + corticosterone rats demonstrated marked and significant increases [mean ± SE, 1510.37 ± 233.82 pg/ml (1 h)] after LPS injection, which were greater than those in LPS-injected sham rats [830.87 ± 66.09 pg/ml (1 h)]. The increased ACTH levels in the Adx + corticosterone rats were 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 R2ß mRNA levels (P < 0.05, by ANOVA; Fig. 4AGo). As in the experiments detailed above, CRF R2ß mRNA levels in the heart decreased by half after LPS injection in sham rats (P < 0.001). Adx + corticosterone attenuated the effects of LPS injection on CRF R2ß mRNA levels in the heart compared with those in LPS-injected sham rats (P < 0.05). This reversal, however, was not complete; CRF R2ß mRNA levels after LPS injection in Adx + corticosterone rats were less than those in control sham rats (P < 0.05). This suggests that other factors in addition to corticosterone may have been involved in the regulation of CRF R2ß mRNA expression after immune challenge. There was no significant effect of LPS injection or Adx on Ucn mRNA levels in the heart (Fig. 4BGo).



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  		Figure 4. Effects of Adx after LPS injection (50 µg/kg BW) on CRF R2ß (A) or Ucn (B) mRNA levels in the rat heart. Vehicle (saline, 100 µl) or LPS (50 µg/kg BW, iv) was injected at 0 min in sham or Adx + corticosterone rats. The rats ware decapitated for tissue collection 4 h after vehicle or LPS in sham or Adx + corticosterone rats. Data are the mean ± SEM of seven to nine animals per group. Statistical analyses were performed using two-way ANOVA, followed by Scheffé’s F post-hoc test. *, P < 0.05 (compared with sham/saline control); **, P < 0.01 (compared with sham/saline control); #, P < 0.05 (compared with sham/LPS); ##, P < 0.01 (compared with sham/LPS).

 
Regulation of CRF R2ß and Ucn mRNA levels in heart or aorta by sc injection of corticosterone or restraint stress
To determine whether CRF R2ß or Ucn mRNA expression responds directly to changes in plasma corticosterone in the absence of immune challenge, exogenous corticosterone (low dose, 37.5 µg/rat; high dose, 125 µg/rat) was administered to intact rats. As previously reported, the areas under the curve of plasma corticosterone concentrations from 0–360 min (AUCs) measured in high dose corticosterone- and LPS-injected rats were not different (AUC mean ± SE, 18,370.75 ± 5,501.91 and 17,039.76 ± 3,840.92 ng/ml at 360 min, respectively). Low doses of corticosterone injection resulted in an AUC of plasma corticosterone concentrations (8,981.37 ± 2,006.04 ng/ml 360 min) between those in saline- and high corticosterone dose-injected rats. Some saline/saline-injected rats were physically restrained for 1 h. The restraint stress produced elevations of plasma corticosterone levels similar in magnitude and AUC (24,603.99 ± 6,180.53 ng/ml 360 min) to those following high dose corticosterone and LPS injections.

Figure 5Go demonstrates the effects of LPS, corticosterone, and restraint on CRF R2ß and Ucn mRNA levels in the heart and aorta (P < 0.001, by ANOVA). CRF R2ß mRNA levels in the heart and aorta were significantly decreased after LPS injection, high dose corticosterone injection, and restraint. Ucn mRNA levels in the heart were not significantly changed after LPS injection, low dose corticosterone, or restraint. By contrast, Ucn mRNA levels doubled 6 h after the injection of the high dose of corticosterone (P < 0.05) compared with those in saline-injected rats.



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  		Figure 5. Regulation of CRF R2ß and Ucn mRNA levels in rat heart (Ht) or aorta (Ao) by sc injection of corticosterone or restraint stress. Vehicle (100 µl saline for iv injection and 200 µl 11% ethanolcontaining 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 at 0 min. Both low and high doses of corticosterone were also administered 30, 60, 120, and 180 min later. A separate set of rats was restrained for 1 h. The rats ware decapitated for collection of tissue 6 h after injection of vehicle, LPS, or corticosterone or onset of restraint. Relative changes in CRF R2ß or Ucn mRNA levels compared with controls (mean ± SEM) in six to nine animals per group are shown. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post-hoc test. *, P < 0.05; **, P < 0.01 (compared with control).

 
Regulation of CRF R2ß mRNA levels in A7R5 aortic smooth muscle cells by dexamethasone administration
To determine whether the decrease in CRF R2ß mRNA levels after corticosterone injection in vivo required other blood-borne factors or represented a mechanism contained within cardiac or vascular tissues, we examined the regulation of CRF R2ß mRNA levels by glucocorticoids in A7R5 cells in culture. These cells are derived from aortic smooth muscle, and we found that they contain CRF R2ß (Gaudriault, G. E., and W. W. Vale, unpublished observations). We administered glucocorticoids to the cells and later determined the CRF R2ß content of the cells by RNase protection assays. Figure 6Go shows significant time- and dose-dependent changes in CRF R2ß mRNA levels after dexamethasone administration (P < 0.001, by ANOVA). Incubation with 0.2 µM dexamethasone decreased CRF R2ß mRNA levels 6 and 24 h, but not 2 h, after treatment (Fig. 6Go, A and B, demonstrates the effects of increasing concentrations of dexamethasone administration for 6 h on CRFR2ß mRNA levels). Concentrations of dexamethasone ranging from 0.02–20 µM produced a significant dose-dependent decrease in CRF R2ß mRNA levels.



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  		Figure 6. Changes in CRF R2ß mRNA levels in A7R5 aortic smooth muscle cells after dexamethasone administration. A, Time-dependent changes in CRF R2ß mRNA levels after incubation with 0.2 µM dexamethasone. Cells were incubated with medium alone (control) or with medium containing 0.2 µM dexamethasone for 2, 6, or 24 h. Results are based on a representative experiment (n = 3) performed in triplicate. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post-hoc test. *, P < 0.01 (compared with control). B, Dose-dependent effects of an incubation with dexamethasone for 6 h on CRF R2ß mRNA levels. Cells were incubated with medium alone (control) or with medium containing 0.02, 0.2, 2, or 20 µM dexamethasone for 6 h. Results are based on a representative experiment performed in triplicate. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post-hoc test. *, P < 0.01 (compared with control).

 
Regulation of CRF R2ß mRNA levels in A7R5 aortic smooth muscle cells by Ucn or CRF administration
The incomplete blockade of LPS-induced decreases in CRF R2ß mRNA levels in the Adx + corticosterone rats (Fig. 4Go) suggested that factors other than corticosterone regulate CRF R2ß mRNA levels in vivo. We examined the regulation of CRF R2ß mRNA levels by CRF R2ß ligands and cytokines in A7R5 cells. As in shown Fig. 7AGo, the peak decrease in CRF R2ß mRNA levels occurred 6 h after incubation with either CRF (1 µM) or Ucn (10 nM). Subsequently, the decrease in CRF R2ß mRNA levels by Ucn administration was shown to be dose dependent, with a maximal effect reached at 10 nM (P < 0.0001; Fig. 7BGo). Ucn was more potent than CRF in decreasing CRF R2ß mRNA levels (Fig. 7BGo).



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  		Figure 7. Regulation of CRF R2ß mRNA levels in A7R5 aortic smooth muscle cells by Ucn or CRF administration. A, Time-dependent changes in CRF R2ß mRNA levels after incubation with Ucn or CRF. Cells were incubated with medium alone (control) or with medium containing 10 nM Ucn (solid bar) or 1 µM CRF (open bar) for 2, 6, or 24 h. Results are based on a representative experiment (n = 3) performed in triplicate. Statistical analyses were performed using two-way ANOVA, followed by Scheffé’s F post-hoc test. *, P < 0.05 (compared with control). B, Dose-dependent effects of incubation with Ucn or CRF for 6 h on CRF R2ß mRNA levels. Cells were incubated with medium alone (control) or with medium containing 0.1, 1, 10, 100, or 100 nM Ucn (solid circle) or 10, 100, or 1000 nM CRF (open circle) for 6 h. Results are based on a representative experiment performed in triplicate. Statistical analyses were performed using two-way ANOVA, followed by Scheffé’s F post-hoc test. *, P < 0.05; **, P < 0.01 (compared with control).

 
Effects of IL-1ß, IL-6, and TNF{alpha} on CRF R2ß mRNA levels in A7R5 aortic smooth muscle cells
IL-1ß, IL-6, and TNF{alpha} are well known as proinflammatory factors that are induced by endotoxin. These proteins also contribute to HPA axis activation after immune challenge. The decreases in CRF R2ß mRNA concentrations measured after LPS injection in vivo were not entirely dependent on elevations of circulating corticosterone; therefore, we hypothesized that cytokines might have a role in regulating CRF R2ß mRNA levels during immune challenge. The cells were cultured for 6 h in the presence of IL-1ß, IL-6, or TNF{alpha} (500 pM). As shown in Fig. 8Go, CRF R2ß mRNA levels were significantly decreased 6 h after each of these treatments (IL-1 and TNF{alpha}, P < 0.001; IL-6, P < 0.01). The administration of IL-1ß caused a dose-dependent decrease in CRF R2ß mRNA levels that achieved statistical significance at 5 pM (P = 0.0013). A considerable decrease of approximately 47% in CRF R2ß mRNA levels was observed at the maximal concentration of IL-1ß.



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  		Figure 8. Effects of IL-1, IL-6, and TNF{alpha} on CRF R2ß mRNA levels in A7R5 aortic smooth muscle cells. A, Effects of IL-1ß, IL-6, and TNF{alpha} on CRF R2ß mRNA levels in A7R5 aortic smooth muscle cells. Cells were incubated with medium alone (control) or with medium containing 500 pM IL-1ß, IL-6, or TNF{alpha} for 6 h. Results are based on a representative experiment performed in triplicate. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post-hoc test. *, P < 0.01 (compared with control); #, P < 0.01 (compared with IL-1ß). B, Dose-dependent effects of incubation with IL-1ß for 6 h on CRF R2ß mRNA levels. Cells were incubated with medium alone (control) or with medium containing 5, 50, or 500 pM IL-1ß for 6 h. Results are based on a representative experiment performed in triplicate. Statistical analyses were performed using one-way ANOVA, followed by Scheffé’s F post-hoc test. *, P < 0.01 (compared with control).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we demonstrated that activation of the immune system by endotoxin (LPS) injection in conscious rats decreased CRF R2ß mRNA expression levels in the heart and aorta. This observation corroborates a prior study showing that CRF R2 mRNA expression in the heart was decreased after LPS injection in mice (30). Two of our findings suggest that the decreases in CRF R2ß mRNA expression levels in these vascular tissues were at least in part dependent on HPA axis activation. First, CRF R2ß mRNA expression levels in the heart and aorta were decreased after restraint stress or injections of either ACTH or high doses of corticosterone. Second, LPS-induced decreases in CRF R2ß mRNA expression in the heart were partially blocked by Adx + corticosterone replacement in rats. A direct effect of corticosterone on the expression of CRF R2 mRNA is consistent with a previously reported decrease in CRF R2 mRNA levels in the rat heart after sc injections of corticosterone in vivo (37). In further support of a direct effect of glucocorticoids on the regulation of CRF R2ß mRNA, we found that dexamethasone inhibited CRF R2ß mRNA levels in A7R5 aorta cells. Thus, glucocorticoids can directly modify the CRF R2ß mRNA levels in these vascular tissues.

It is also possible that decreased CRF R2ß mRNA expression after corticosterone or dexamethasone administration in vivo is partly due to changes in vascular tone. CRF R2 mRNA levels in the heart of spontaneously hypertensive rats were reported to be higher than those in normotensive rats (33), supporting the idea that vascular tone modulates CRF R2 mRNA expression. Glucocorticoids have been postulated to inhibit the production of nitric oxide, a potent vasodilator, in vascular smooth muscle cells (38). Glucocorticoids, therefore, could potentially modulate the vasodilatatory response to stress through changes in nitric oxide production both in vivo and in vitro. It does not appear, however, that acute changes in blood pressure alone are responsible for decreased CRF R2ß mRNA expression levels. LPS injection eventually decreases blood pressure (39), whereas restraint stress increases blood pressure (40). As both treatments reduce CRF R2ß mRNA levels, it is unlikely that changes in vascular tone alone account for changes in gene expression.

LPS injection in vivo increases circulating levels of several classes of cytokines (32). We observed that in A7R5 cells, the proinflammatory cytokines, IL-1ß, TNF{alpha}, and IL-6, each decreased CRF R2ß messenger levels, with IL-1ß being the most effective at the doses tested. Therefore, after immune challenges, increased circulating concentrations of some cytokines might regulate CRF R2ß mRNA levels in vivo (41). It is known that IL-1ß contributes to the down-regulation of CRF R1 in the rat pituitary (42), suggesting that similar upstream elements for this cytokine signaling in the promoter between CRF R1 and CRF R2 may regulate the expression of these CRF receptors (43). The fact that these three cytokines modulate CRF R2ß gene expression levels suggests that there is a profound redundancy via the proinflammatory signal transduction system, because both TNF and IL-1 activate cell signaling via the same nuclear factor-{kappa}B pathway (44), whereas IL-6 acts through the Janus kinase/signal transducer and activator of transcription/suppression of cytokine signaling pathways (45). In addition, LPS may act directly via CD14/TLR4/nuclear factor-{kappa}B pathway (46).

It is of interest to note that preventing the LPS-induced increase in circulating corticosterone concentrations via Adx + corticosterone replacement only partially inhibited the decrease in CRF R2ß mRNA expression in the heart. This observation coupled with the effects of cytokines on CRF R2ß mRNA expression in A7R5 cells suggests that each factor or LPS itself may be involved in the decrease in CRF R2ß mRNA expression after immune challenge.

Our in vitro study showed that Ucn or CRF administration to A7R5 cells decreased CRF R2ß mRNA levels, consistent with the possibility that a ligand for CRF R2ß may be involved in the regulation of CRF R2ß mRNA expression in vivo during stress either directly or through the production of additional factors. If this is the case, it is not clear which endogenous ligands activate CRF R2 in the heart, when local concentrations of ligands rise in the heart and what the source of those ligands might be. Both CRF and Ucn mRNA are found in cardiac tissues (16, 22, 47). Prior studies show that Ucn has far higher affinity for CRF R2 than does CRF (14); furthermore, Ucn has more potent cardiac inotropic effects than does CRF (25). In the present study Ucn was more potent than CRF at decreasing CRF R2ß mRNA levels, in keeping with the hypothesis that CRF R2ß stimulation mediates down-regulation of itself. It is unclear, however, whether any locally produced Ucn normally regulates CRF R2ß mRNA levels in the heart and aorta in vivo. It is also possible that CRF R2ß in the heart and aorta may be stimulated by circulating Ucn (17).

In the present study the levels of Ucn mRNA in the heart were significantly increased after high doses of corticosterone injection, but not low doses of corticosterone or LPS injection. These results are different from those in the thymus, in which both LPS and corticosterone increased Ucn mRNA levels (16). In both of these tissues, there are relatively high concentrations of glucocorticoid receptors (48); therefore, the expression levels of Ucn mRNA in the heart and thymus may be stimulated directly by glucocorticoids. The susceptibility to proinflammatory factors might be different among the tissues. Alternatively, the regulation of Ucn mRNA expression in the heart may be controlled by multiple factors, some of which may counter corticosterone-induced increases.

The biological significance of changes in CRF R2ß mRNA levels in the cardiovascular system remains to be elucidated. In the anterior pituitary, a ligand-induced reduction in CRF-binding sites (49, 50) is achieved through both internalization of receptors (51) and suppression of new receptor synthesis (52). It is possible that the decrease in CRF R2ß mRNA levels in the cardiovascular system after stress contributes to a reduction in the number of ligand-binding sites, restricting subsequent ligand-mediated activity.

Ucn stimulates potent coronary vasodilatory and cardiac inotropic actions via CRF R2ß (53). Our results, therefore, suggest that these decreases in CRF R2ß mRNA levels by Ucn, glucocorticoids, and cytokines could limit the time course of Ucn-induced cardiovascular effects during prolonged stress, thus contributing to the restoration of homeostasis. The redundant effects of glucocorticoids, receptor ligands, and cytokines suggest that the regulation of CRF R2ß parallels that of CRF R1. Combined CRF and vasopressin administration in rat anterior pituitary cultures decreases CRF R1 mRNA levels synergistically (54). Furthermore, some elements may influence the production of other regulators. For instance, in A7R5 cell culture, we have shown that Ucn induces IL-6 secretion (Gaudriault, G. E., and W. W. Vale, unpublished observations). Thus, decreases in CRF R2ß mRNA expression measured after Ucn administration may occur indirectly through the actions of induced cytokines. The nature of the pharmacological interactions of these separate regulators of CRF R2ß has yet to be defined in vitro in A7R5 cells.

In summary, our present data demonstrate that the aorta expresses high levels of CRF R2ß mRNA, and the heart expresses both CRF R2ß and Ucn mRNA. Increased concentrations of circulating glucocorticoids from either exogenous administration or after HPA activation appear to decrease CRF R2ß mRNA expression levels in the cardiovascular system. It is possible that increased Ucn secretion within the heart contributes to the regulation of CRF R2ß mRNA levels during stress. LPS itself or IL-1ß, IL-6, and TNF{alpha} produced after immune challenge may also collaborate to suppress CRF R2ß mRNA levels in this system. These results indicate that regulation of CRF R2ß mRNA levels by these factors could serve to limit the cardiovascular effects of Ucn during stress or immune challenge.


   Acknowledgments
 
We thank Dr. J. Rivier for generously providing synthetic peptides, and R. Kaiser for their synthesis. We also acknowledge A. Blount for technical assistance, and S. Guerra and D. Dalton for assistance with manuscript preparation.


   Footnotes
 
1 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.); the Kleberg Foundation (to K.K.); and the Adler Foundation (to K.K., M.B., and G.G.). Back

2 Foundation for Research Senior Investigator. Back

Received January 14, 2000.


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 Introduction
 Materials and Methods
 Results
 Discussion
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