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Endocrinology Vol. 144, No. 6 2396-2403
Copyright © 2003 by The Endocrine Society

Role of Corticotropin-Releasing Factor Receptors Type 1 and 2 in Modulating the Rat Adrenocorticotropin Response to Stressors

Catherine L. Rivier, Dimitri E. Grigoriadis and Jean E. Rivier

The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037; and Neurocrine Biosciences Inc., San Diego, California 92121

Address all correspondence and requests for reprints to: Catherine Rivier, Ph.D., The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: crivier{at}salk.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated the contribution of corticotropin-releasing factor (CRF) receptors types 1 and 2 (CRF1 and CRF2) in mediating the ACTH response to shock, alcohol injection, or endotoxemia in the rat. Peptidic (Astressin B and Astressin2-B) and nonpeptidic (NBI 30775) CRF antagonists were injected iv before the stressors at doses previously shown to be effective in blocking the corresponding receptors. Because NBI 30775, which specifically blocks CRF1, penetrates the brain following systemic injection, we also compared its effect with that of Astressin B, which primarily, though not exclusively, targets CRF1 but does not cross the blood-brain barrier. Shocks, alcohol (4.5 g/kg, intragastrically) or lipopolysaccharide (LPS, 1 µg/kg, iv) all significantly released ACTH. Astressin B or NBI 30775 markedly decreased the effect of shocks or alcohol and also interfered, though less significantly so, with the influence of LPS. In contrast, specific blockade of CRF2 with Astressin2-B, although not significantly altering the overall ACTH response to shocks, alcohol, or LPS, slightly enhanced ACTH levels during the early phase of some of these responses. Interestingly, combined administration of NBI 30775 and Astressin2-B decreased ACTH levels more than NBI 30775 alone, although this difference did not reach statistical significance. Finally, blockade of CRF1 and/or CRF2 augmented LPS- induced TNF-{alpha} and IL-6 release. Collectively, there results confirm the critical role played by CRF1 in mediating the ACTH response to shocks, alcohol and LPS, whereas the influence of CRF2 remains subtle. Finally, we showed that peripheral endogenous CRF restrains the ability of LPS to release cytokines.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CORTICOTROPIN-RELEASING FACTOR (CRF) is recognized as a central mediator of the body’s ability to respond to stress. Initially isolated from ovine hypothalamus and characterized as a 41-amino acid peptide in 1981 (1), CRF was subsequently characterized from rat hypothalami (2) and the identical structure for human CRF was deduced on the basis of the cDNA sequence of the human CRF precursor gene (3). Presently, more than a dozen members of the CRF family (including sauvagine, urotensins, and urocortins) have been described. CRF plays a major role in the maintenance or restoration of homeostasis by stimulating the activity of the hypothalamic-pituitary-adrenal (HPA) axis (4). It also acts within the central nervous system to control immune (5), reproductive (6), gastrointestinal/ingestive (7, 8), and cardiovascular (9) functions, as well as catecholamine release (10), drug withdrawal (11), behavior (12), and mood and anxiety (13). CRF also acts through the release of adrenal corticosteroids to alter immune parameters (1) and to participate in the regulation of carbohydrate metabolism by enhancing the availability of glucose (reviewed in Dallman et al., Ref. 14). Finally, the broad central and peripheral distribution of the peptide and its two classes of seven-transmembrane-helix G protein-coupled CRF receptors (15, 16, 17, 18, 19, 20, 21) support the notion that CRF is also an important local neurotransmitter within the central nervous and immune systems, among others.

We and others have provided ample evidence that in rats, immunoneutralization of endogenous CRF significantly blunted the ACTH response to most known exteroceptive stressors (also called neurogenic), which involve a distinct cognitive and/or affective component comprised of footshocks and restraint (22, 23), as well as to interoceptive (systemic) stressors, whose essential features are not consciously appreciated and which involve inflammatory and infectious insults (reviewed in Ref. 24). Understanding the mechanisms through which CRF influences the HPA axis, requires knowledge of the type of receptor(s) through which this peptide acts within this axis, and in particular how it stimulates ACTH release. Present consensus holds that type 1 CRF receptors (CRF1), which are found on pituitary corticotrophs (see Ref. 25), represent the primary receptors that mediate stress-induced increases in ACTH levels. This concept was first derived from the observation that mice lacking the CRF1 receptor showed a significantly blunted ACTH response to restraint (26, 27). On the other hand, mice lacking the CRF2 gene tended to exhibit a slightly exaggerated stress-induced ACTH release, at least in the early phase of this response (27, 28). Interestingly, however, restraint-induced ACTH secretion in mice lacking both receptors has been reported by some investigators (29), though not others (27), to be smaller than that of mice only lacking CRF1. Whether this is due to compensatory mechanisms due to the lack of both CRF receptor types, or to other causes, has not been determined. Another puzzling aspect of the role of CRF receptors in mediating ACTH release is our finding that CRF1-/- mice release very significant amounts of ACTH when exposed to interoceptive (i.e. immune) stressors (30), which stands in stark contrast to their inability to respond to exteroceptive stimuli (see above). While the elevated levels of IL-6 measured in CRF1-/- mice injected with LPS or turpentine (30) have been invoked as potentially responsible for releasing ACTH in the absence of CRF drive (at least through the CRF1 receptor), this issue has not been settled. On the other hand, the lack of CRF1 leads to a significant loss of ACTH response to LPS in rats (31), though this loss is of lesser magnitude than that observed following exposure to shocks or injection with alcohol (32). In this species, it was determined that proinflammatory cytokines did not mediate the ACTH response to the immune stressors (31).

The present work was carried out to investigate the specific role of CRF1 and CRF2 in mediating the rat HPA axis response to stressors. The experiments were performed in rats to examine the consequence of the specific blockade of CRF receptors in a model unencumbered by the possible influence of compensatory mechanisms present during embryonic development of genetically altered mice. We used two types of CRF antagonists, peptidic and nonpeptidic. Peptidic antagonists allowed us to specifically block pituitary CRF receptors without altering receptors within the brain, particularly in regions of the hypothalamus that participate in the ACTH response to stressors. The disadvantage of this approach is that, although we have a peptidic antagonist specific for CRF2 (Astressin2-B, Ref. 33), the antagonist used to block CRF1 (Astressin B, Refs. 34 and 35) also provides some blockade of CRF2. We therefore also investigated the effect of a nonpeptidic antagonist which is specific for CRF1 (see Ref. 36 , in which NBI 30775 is referred to as R121919). This reagent provided specific information regarding the role of CRF1, but because it penetrates the brain following its peripheral administration (36), it did not allow us to uncouple its influence on pituitary and hypothalamic CRF1. The two types of antagonists were tested in three models of stress: alcohol injection, exposure to inescapable footshocks and endotoxemia. We show here that, whereas pituitary CRF1 receptors are the primary mediators of the rat ACTH responses, CRF2 receptors also modulate HPA axis activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and protocols
Adult male Sprague Dawley (Harlan Sprague Dawley, San Diego, CA) rats (200–220 g) were kept under standard light (lights on 0630–1830 h) and feeding (rat chow and water ad libitum) regimens. Aseptic insertion of an intragastric (ig) cannulae was done under isoflurane anesthesia 9–10 d before the assay (37). A right jugular iv catheter was inserted 2–3 d before testing (37). All animals were singly housed to prevent chewing of the cannulae. In view of the limited blood volume that can be withdrawn from rats without hemorrhage-induced activation of the HPA axis, not all time-courses could be studied in the same groups of animals. Also, it must be noted that not all comparisons were carried out in the same assay because this would have resulted in experiments that were too complex. Thus, for example, depending on the number of rats required per experimental group, we sometimes compared the influence of Astressin B and Astressin2-B in one experiment, and that of NBI 30775 and Astressin B in another.

On the day of the experiment, the animals were moved to a sound-proof room, singly housed in opaque buckets, and left undisturbed for 3 h. The antagonists or their vehicle were injected iv 15–20 min before CRF or the stressor. Blood samples (0.3 ml) were taken through the iv cannula in undisturbed rats and immediately replaced with an equivalent volume of apyrogenic isotonic saline. They were drawn into tubes that contained EDTA (10 µl of a 60 mg/ml solution) and placed on ice. They were centrifuged at 4 C, and plasma was stored at -20 C until assayed.

All protocols were approved by The Salk Institute Institutional Animal Use and Care Committee.

Electrofootshocks
All animals were tested in the shockers, in which they were exposed to the shocks (shocked) or not (controls). Mild, inescapable footshocks (0.5 mA, 1 sec duration, 2 shocks/min) were delivered to the rats’ paws using a Coulbourn HO2–08 grid floor shocker controlled by a Macintosh computer (Coulbourn Instruments, Allentown, PA) (38). Because of the low voltage used, this procedure does not cause injury and is considered primarily an emotional stressor accompanied by a modest amount of physical discomfort.

Reagents
Rat/human (r/h) CRF and the peptidic CRF antagonists Astressin B (34) and Astressin2-B (33) were synthesized by solid phase methodology (33, 34). A stock solution was made in apyrogenic water, and subsequent dilutions were made in 0.04 M PBS containing 0.1% crystalline BSA (to prevent nonspecific binding to glassware) and 0.01% ascorbic acid (to prevent oxidation). The nonpeptidic pyrrolopyrimidine CRF1 antagonist NBI 30775 was synthesized as described in (36, 39), solubilized in water containing 5% Cremophor (Sigma, St. Louis, MO), sonicated and mixed with 0.1 N NaOH for pH adjustment. Astressin B blocks CRF1 and CRF2 (34), Astressin2-B is specific for CRF2 (33) and NBI 30775 specifically blocks CRF1 (36). These antagonists were injected iv 20–30 min before the stressors at doses (Astressin B and Astressin2-B, 30 µg/kg; NBI 30775, 4–6 mg/kg) and over a time frame previously shown to fully block ACTH release (33, 34, 35, 36). Alcohol was diluted with saline so that its final injected concentration was 18% (vol/vol). It was injected through the indwelling ig cannulae to otherwise undisturbed, unhandled rats. The dose chosen (4.5 g/kg, ig) induces a moderate degree of intoxication and significant as well as reliable increases in plasma ACTH levels (37). LPS from Escherichia coli (serotype 026:B6) was purchased from Sigma and diluted in apyrogenic saline. It was injected iv at a dose (1 µg/kg) that is comparatively much lower than that usually reported in the literature, and was designed to significantly release ACTH without inducing severe sickness or disrupting the blood-brain barrier (BBB; Ref. 40).

ACTH assay
Plasma ACTH levels were determined by a commercially available two-site immunoradiometric assay (Allegro kit, Nichols Institute, San Juan Capistrano, CA), which has been validated for the measurement of rat ACTH (41). Assay sensitivity was 5 pg/ml, and the intraassay and interassay coefficients of variation were 3.2 and 6.8%, respectively.

Cytokine assays
TNF-{alpha} and IL-6 levels were measured by ELISA (TNF-{alpha}: R&D Systems, Minneapolis, MN; IL-6: Endogen, Inc., Rockford, IL) that were validated in our laboratory by comparing standard curves with serial plasma dilutions (42).

Statistical analysis
Statistical analyses were performed by one- or two-way ANOVA for the factors of group and time. The least square means post hoc tests were used to make comparisons between groups at a particular time point, and between time points within a particular group. Differences were considered statistically significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Comparison between the effect of Astressin B, Astressin2-B, or NBI 30775 on the ACTH response to r/hCRF (Fig. 1Go)
These experiments were designed to compare the effect of the blockade of CRF receptors on the ACTH response to exogenous CRF (2 µg/kg, iv), and to ensure that CRF1 antagonists completely abolished this response. Astressin B (30 µg/kg) or NBI 30775 (6 mg/kg) virtually abolished CRF-induced ACTH release (Fig. 1Go). In contrast, Astressin2-B slightly reduced this response (similar results were observed in two out of three experiments).



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  		Figure 1. Comparison between the effect of Astressin B, Astressin2-B or NBI 30775 on the ACTH response to r/hCRF (2 µg/kg, iv). {oplus}, Vehicle or antagonists alone; {circ}, CRF; {triangleup}, CRF + Astressin B; {triangleright}, CRF/NBI 30775; ({blacksquare}), CRF + Astressin2-B. Each point represents the mean ± SEM of five to six rats. **, P < 0.01 vs. CRF alone.

 
Comparison between the effect of Astressin B or Astressin2-B on the ACTH response to shocks, LPS or alcohol (Figs. 2–4GoGoGo)
Astressin B.
These experiments were designed to compare the effect of Astressin B and Astressin2-B in modulating the ACTH response to shocks, LPS, alcohol, or their vehicle. As these peptidic antagonists do not cross the BBB, these results provided information regarding the specific role of pituitary CRF receptors, which could not be addressed with the nonpeptidic CRF1 antagonist NBI 30775. There were no significant (P > 0.05) differences between ACTH release of animals injected with the vehicle or the antagonists alone (Figs. 2–4GoGoGo). All stressors induced the expected rise in ACTH concentrations, and in all three cases these responses were significantly (P < 0.01) reduced by Astressin B (30 µg/kg; Figs. 2–4AGoGoGo). It should be noted, however, that the magnitude of the decrease in the ACTH response to LPS was not as large as that seen in the shocks or alcohol model, a phenomenon that we have previously attributed to the role of vasopressin (VP) in the latter phase of ACTH release during endotoxemia (43).



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  		Figure 2. Comparison between the effect of Astressin B or Astressin2-B on the ACTH response to shocks. Two different time-courses were used (A, 45 min; B, 20 min) to differentiate between early and later phases of the ACTH response. {oplus}, Vehicle or antagonists alone; {circ}, shocks; {blacktriangleright}, shocks + Astressin B; {blacksquare}, shocks + Astressin2-B. Each point represents the mean ± SEM of five to six rats. **, P < 0.01 vs. shocks alone.

 


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  		Figure 3. Comparison between the effect of Astressin B or Astressin2-B on the ACTH response to the ig injection of alcohol. Two different time-courses were used (A, 60 min; B, 30 min) to differentiate between early and later phases of the ACTH response. {oplus}, Vehicle or antagonists alone; {triangleup}, alcohol (4.5 g/kg, ig); {blacktriangleright}, alcohol + Astressin B; {blacksquare}, alcohol + Astressin2-B. Each point represents the mean ± SEM of five to six rats. **, P < 0.01 vs. alcohol alone.

 


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  		Figure 4. Comparison between the effect of Astressin B or Astressin2-B on the ACTH response to the iv injection of LPS. Two different time-courses were used (A, 120 min; B, 40 min) to differentiate between early and later phases of the ACTH response. {oplus}, Vehicle or antagonists alone; {square}, LPS (1 µg/kg, iv); {blacktriangleright}, LPS + Astressin B; {blacksquare}, LPS + Astressin2-B. Each point represents the mean ± SEM of five to six rats. **, P < 0.01 vs. LPS alone.

 
Astressin2-B.
When we examined ACTH responses over the time courses that we have used in the past for the shocks (illustrated in A panels of Figs. 2–4GoGoGo), LPS, and alcohol models, Astressin2-B (30 µg/kg) only significantly (P < 0.01) augmented ACTH release due to LPS (Fig. 4AGo), but not shocks (Fig. 2AGo) or alcohol (Fig. 3AGo). In view of the reported early increase in ACTH release observed in CRF2-/- mice exposed to restraint stress (27, 28), we thought that it would also be important to examine earlier parts of ACTH responses in our models (illustrated in B panels of Figs. 2–4GoGoGo). We therefore measured ACTH levels shortly after exposure to shocks or injection with LPS or alcohol, in rats injected with this antagonist. In these experiments, Astressin2-B indeed significantly (P < 0.01) enhanced the early ACTH response to shocks (Fig. 2BGo) or LPS (Fig. 4BGo), but not to alcohol (Fig. 3BGo).

Consequence of specifically blocking CRF1 and CRF2, separately or together, on the ACTH response to shocks, LPS or alcohol (Figs. 5–7GoGoGo)
The experiments described above sought to give information regarding blockade of pituitary, but not hypothalamic, CRF receptors on ACTH responses to stressors and were therefore conducted with peptidic antagonists that do not cross the BBB. However, it must be noted that Astressin B, which was used to block CRF1, also targets CRF2. Consequently, these studies did not allow us to specifically and separately address the role of each receptor type. The experiments described below address this point, though the ability of NBI 30775 to cross the BBB (36) means that we now cannot distinguish between the role of pituitary and hypothalamic CRF1. Nevertheless, they provided a comparison with results previously published in mutant mice lacking the gene for both receptors.



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  		Figure 5. Effect of the single or combined injection of NBI 30775 and Astressin2-B on the ACTH response to shocks. {square}, Vehicle; , NBI 30775; {blacksquare}, Astressin2-B; , NBI 30775 + Astressin2-B. Each bar (mean ± SEM of six to seven rats) represents cumulative ACTH levels measured over 45 min. **, P < 0.01 vs. corresponding vehicle.

 


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  		Figure 6. Effect of the single or combined injection of NBI 30775 and Astressin2-B on the ACTH response to the ig injection of alcohol. {square}, Vehicle; , NBI 30775; {blacksquare}, Astressin2-B; , NBI 30775 + Astressin2-B. Each bar (mean ± SEM of six to seven rats) represents cumulative ACTH levels measured over 45 min. **, P < 0.01 vs. corresponding vehicle.

 


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  		Figure 7. Effect of the single or combined injection of NBI 30775 and Astressin2-B on the ACTH response to the iv injection of LPS. {square}, Vehicle; , NBI 30775; {blacksquare}, Astressin2-B; , NBI 30775 + Astressin2-B. Each bar (mean ± SEM of six to seven rats) represents cumulative ACTH levels measured over 120 min. **, P < 0.01 vs. corresponding vehicle.

 
The rats displayed the expected ACTH response to shocks, LPS, or alcohol, and blockade of CRF1 by NBI 30775 (6 mg/kg) consistently decreased this response (Figs. 5–7GoGoGo). Astressin2-B (30 µg/kg) did not significantly alter the stimulatory effect of LPS or alcohol, but slightly augmented that of shocks (for an explanation of the discrepancy between these results and those illustrated in Fig. 4BGo, see Discussion). Combined blockade of CRF1 and CRF2 tended to lower plasma ACTH levels below those observed for treatment with NBI 30775 alone, though this difference did not usually reach statistical significance.

Consequence of specifically blocking CRF1 or CRF2 on the TNF-{alpha} and IL-6 response to LPS (Fig. 8Go)
We previously reported that removal of endogenous CRF significantly enhanced the IL-6 response to LPS (31), and that mice lacking the CRF1 gene exhibited an elevated IL-6 response to endotoxemia (30). The present experiments were designed to extend these studies to TNF-{alpha}, and investigate the role of CRF1 and CRF2 on TNF-{alpha} and IL-6 release in rats injected with LPS. LPS induced the expected rise in plasma TNF-{alpha} and IL-6 levels (Fig. 8Go). Both responses were significantly (P < 0.05–0.01) augmented by either NBI 30775 (6 mg/kg) or Astressin2-B (30 µg/kg), and blockade of both receptor types did not provide a further increase.



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  		Figure 8. Effect of blockade of CRF1 or CRF2 on the TNF-{alpha} and IL-6 response to LPS (1 µg/kg, iv). {square}, Vehicle; , NBI 30775; {blacksquare}, Astressin2-B; , NBI 30775 + Astressin2-B. Each bar (mean ± SEM of six to seven rats) represents cumulative cytokine levels measured over 120 min. *, P < 0.05 and **, P < 0.01 vs. corresponding vehicle.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using mice lacking the gene for specific CRF receptors as well as the injection of peptidic and nonpeptidic CRF antagonists to rats, a number of laboratories have suggested the critical importance of pituitary CRF1 receptors in mediating the HPA axis responses to exteroceptive stressors such as shocks and restraint (26, 27, 29, 32, 35, 44, 45). We confirmed and extended these results here by showing that both the peptidic Astressin B and the nonpeptidic antagonist NBI 30775 significantly blunted the ability of shocks, alcohol, or endotoxemia to release ACTH. As these antagonists targeted different compartments of the HPA axis (i.e. Astressin B only blocked pituitary CRF1 and CRF2, whereas NBI 30775 blocked pituitary and hypothalamic CRF1), our results could not uncover any differences between the importance of peripheral and central CRF1. It must be also noted that blockade of CRF1 receptors only partially reduced LPS-induced ACTH release, which stands in contrast to the higher effectiveness of Astressin B or NBI 30775 in the shock or alcohol paradigms. We had previously reported that, although mice lacking the CRF1 gene showed a significantly decreased ACTH response to restraint compared with wild-type animals (26, 27), their ACTH response to immune stimuli was only minimally impaired (30). Here we report that acute blockade of CRF1 in the rat, which does not involve some of the compensatory mechanisms present when the corresponding gene is absent during embryonic development, is not as detrimental to pituitary activity during endotoxemia as it is during exposure to shocks. The markedly enhanced IL-6 release measured in CRF1-/- mice injected with turpentine or LPS (30), coupled to the fact that mice lacking IL-6 show a decreased ACTH response to immune stressors (40), had suggested that in mice, cytokines might stimulate ACTH release independently of CRF. The situation appears different in rats because we showed that endogenous CRF immunoneutralization significantly decreased ACTH levels and increased IL-6 concentrations following LPS injection (31), an observation that is supported by the present work. Furthermore, exposing isolated rat pituitary cells to LPS for 6 h increased IL-6 concentrations in the medium but did not alter ACTH release (46). Collectively, these results argue against a critically important influence of these proinflammatory cytokines on ACTH release in rats, which is not mediated by CRF. Nevertheless, as we show here, CRF1 receptors are not as important for the rat ACTH response to LPS as they are upon exposure to shocks or alcohol. Whereas VP participates in this response (47, 48), its ability to release ACTH depends on the presence of endogenous CRF and will be decreased by blocking interactions with this peptide (49). Indeed, both shocks and alcohol stimulate ACTH secretion through mechanisms that also depend on VP (50, 51), but as we show here, their influence was significantly decreased by NBI 30775. As prostaglandins play a critical role in LPS-induced ACTH release (48) but do not interact with the stimulatory effect of CRF on the corticotrophs (52), the relative lack of effectiveness of NBI 30775 in the endotoxemic rat may be, at least in part, due to the contribution of eicosanoids at a suprapituitary level. Finally, we need to address the questions of whether the use of peptidic and nonpeptidic CRF antagonists allowed us to dissociate the influence of peripheral and central CRF receptors in regulating HPA axis activity. By comparing the influence of the systemic injection of peptidic CRF antagonists that do not cross the BBB with that of intracerebroventricular injection, we had proposed that, whereas pituitary CRF1 receptors were critical for the rat ACTH response to shocks and alcohol injection, hypothalamic CRF1 did not play a significant role (53). The similarity between the effect of Astressin B and NBI 30775, which we report here, provides further support for this hypothesis.

The role of CRF2 in mediating ACTH release was first demonstrated by studies conducted in CRF2-/- mice. Originally, the finding that these receptors were not present in corticotrophs (25, 54) had suggested that they were probably not essential for the ability of stressors to release ACTH. This concept has been somewhat challenged by the fact that mice lacking the CRF2 gene displayed slightly enhanced ACTH levels during parts of initial phase of this response and/or an early termination of ACTH secretion (27, 28, 55). In the present work, we provide data supporting the hypothesis that in the rat, blockade of CRF2 alone may also, at least in some experiments, result in a slight enhancement of the early ACTH response to shocks, alcohol, and LPS. The question of where CRF2 receptors act within the HPA to alter the ACTH response to stressors has been investigated in a number of studies. Some investigators have identified CRF2{alpha} receptors in the rat PVN (56, 57), but levels of these transcripts are very low, if detectable at all, and are not up-regulated by stressors (57, 58, 59, 60, 61, 62). CRF2{alpha} receptors are also found in limbic regions (54, 60) that tonically restrain PVN activity through GABA-dependent mechanisms (63). Their absence in mutant mice might therefore result in disinhibited PVN neuronal activity, and hence increased ACTH release. However, in our rat models, blockade of PVN and/or limbic CRF2 by the peripheral injection of Astressin2-B is highly unlikely because this peptidic antagonist is not expected to cross the BBB. We therefore examined the ability of Astressin2-B to alter CRF-induced ACTH release and surprisingly, found that in two out of three experiments, the response was slightly decreased. Whether this phenomenon plays a role in the decreased ACTH response of rats injected with both NBI 30775 and Astressin2-B, remains to be determined. At present, there does not seem to be much experimental ground to provide the basis for fruitful explanations for these somewhat conflicting observations. One possibility is that pre- and/or perinatal events, over which investigators have no control when they use commercially purchased rodents, may influence later HPA axis responses, and in particular the role of specific CRF receptors. There is of course much information regarding the influence of prenatal stress, maternal separation or handling shortly after birth, on the adult offspring CRF and ACTH release (see, for example, Refs. 64, 65, 66, 67). Whether any of these manipulations are capable of altering the role played by pituitary CRF2 in selected batches of rats, remains to be determined, but it might be wise to keep in mind that conditions outside our control may influence results in a seemingly unpredictable fashion.

A last comment pertains to the role of endogenous CRF in regulating the production of proinflammatory cytokines. We showed here that pretreatment with Astressin B, NBI 30775 or Astressin2-B augmented LPS-induced TNF-{alpha} and IL-6 release. Depending on the experimental model, peripheral CRF has been reported to augment or inhibit immune responses independently of glucocorticoids (68, 69, 70, 71, 72). For example, we found that the peripheral injection of CRF antibodies significantly decreased symptoms in arthritic rats (73). These results, which were recently confirmed with the use of a nonpeptidic CRF antagonist in arthritic Lewis rats (74), suggest a proinflammatory role of this peptide. However, we also observed that immunoneutralization of endogenous CRF augmented the TNF-{alpha} and IL-6 response to LPS (31), which supports the hypothesis that, in the rat, CRF acts peripherally to inhibit the release of these particular cytokines. The present data confirm and extend these latter findings, and further indicate that the influence of the peptide is likely exerted through both subtypes of CRF receptors (type 1 and 2). The observation that blockade of both receptor types did not augment cytokine levels above those measured in the presence of NBI 30775 or Astressin2-B alone further suggests that the contribution of these receptors is not additive. Interestingly, a recent report indicated that in mice, blockade of CRF1 suppressed LPS-induced TNF-{alpha} and IL-6 release (75). We therefore need to consider the possibility that the role of CRF on the immune system might be species specific.

In summary, our results confirm that pituitary CRF1 receptors are essential for the rat ACTH response to extero- as well as interoceptive stressors, even though they appear to be more critical in mediating the ACTH-releasing effectiveness of shocks and alcohol, compared with LPS. This observation may indicate a species difference in the role of these receptors because, in mice, the absence of CRF1 only marginally compromises the ACTH response to LPS (30). We also extended earlier results obtained in mutant mice and showed that CRF2 in areas not protected by the BBB also play a role in regulating corticotroph function. Finally, we showed that blockade of CRF1 and CRF2 enhances TNF-{alpha} and IL-6 release following LPS injection. This observation not only supports the hypothesis that peripheral endogenous CRF restrains the production of these cytokines, but also that TNF-{alpha} and IL-6 do not provide a significant drive to ACTH release in the absence of CRF. Collectively, these data extend our previous understanding of the role played by CRF receptors in regulating HPA axis activity in the rat.


   Acknowledgments
 
The authors are indebted to Dr. Jozsef Gulyas, Elaine Law, Melissa Herman, Yaira Haas, Dean Kirby, and Bill Low for excellent technical help.


   Footnotes
 
This research supported by NIH Grants AA-06420, AA-08924, DK-26741, and the Foundation for Research. J.R. is the Frederik Paulsen Chair in Neurosciences Professor.

Abbreviations: BBB, Blood-brain barrier; CRF, corticotropin-releasing factor; HPA, hypothalamic-pituitary-adrenal; ig, intragastric; LPS, lipopolysaccharide; r/h, rat/human; VP, vasopressin.

Received December 9, 2002.

Accepted for publication February 18, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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