| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
* The Sackler Institute of Pulmonary Pharmacology, King’s College London, Guy’s Campus, London, UK; and
The School of Pharmacy, University of London, London, UK
1Correspondence: The Sackler Institute of Pulmonary Pharmacology, King’s College London, 5th Floor Hodgkin Bldg., Guy’s Campus, London SE1 1UL, UK. E-mail: james.moffatt{at}kcl.ac.uk
SPECIFIC AIMS
Corticotropin-releasing factor (CRF) and related peptides (urocortins) are emerging neuropeptide hormones and can have opposite biological effects, depending on which of two receptors, CRF1 and CRF2, are present in tissues. In the present study we investigated the bronchomotor and anti-inflammatory effects of the CRF2-preferring ligand, urocortin III, in the airways.
PRINICPAL FINDINGS
1. CRF2 is expressed in the airways
Using commercial antibodies raised against CRF1 and CRF2, we used immunohistochemistry to probe the location of these receptors in the mouse trachea. Both antibodies revealed intense staining of the airway epithelium. Colocalization of CRF1 with smooth muscle actin was not observed in sections from any of four animals. However, faint CRF2 immunoreactivity was consistently observed in the smooth muscle layer. Adventitial fibroblast-like cells displayed both CRF1 and CRF2 immunoreactivity, as did chondrocytes near the point of insertion of smooth muscle.
2. CRF2 activation causes bronchorelaxation
Whereas CRF binds and activates both CRF1 and CRF2, it is more potent at the CRF1 subtype. The CRF2-preferring ligand urocortin III caused slowly developing relaxation of smooth muscle (Fig. 1
A), which represented an approximate 30% reversal of methacholine-induced tone. Similar to urocortin III, CRF caused relaxation of the mouse trachea (Fig. 1A
), although it was significantly less potent than urocortin III (pEC50 6.79±0.08 vs. 7.153±0.09; P=0.02).
|
The highly selective CRF2 receptor antagonist (>100-fold vs. CRF1) astressin 2B potently inhibited relaxations induced by urocortin III (Fig. 1B
). Although a full pharmacological analysis (to determine a pA2 value) was not possible owing to availability and solubility of urocortin III, the IC50 for astressin 2B appeared to be 
3 nM (Fig. 1B
), consistent with the nanomolar affinity of astressin 2B for CRF2.
As CRF1 and CRF2 staining were most pronounced on epithelial cells, we also examined the effects of CRF1-preferring (CRF) and CRF2-preferring (urocortin III) peptides on ion transport across the airway epithelium in voltage-clamped preparations. Surprisingly, neither CRF nor urocortin III caused significant changes in ion transport, although the effect of elevating intracellular cyclic nucleotide levels with the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX; 100 µM) produced robust responses.
3. CRF2-mediated bronchorelaxation involves cyclic nucleotides but not NO or cyclo-oxygenase products
Like ßbeta;-adrenoceptors, CRF2 has repeatedly been reported to couple to Gs and hence to elevate cAMP in cells. If similar coupling occurs in the mouse trachea, responses should be amplified by agents that inhibit phosphodiesterases. In agreement with this, IBMX (30 µM), a nonspecific phosphodiesterase inhibitor, significantly potentiated relaxation responses to urocortin III.
The strong CRF1 and CRF2 immunoreactivity in airway epithelial cells also prompted us to consider that the response to urocortin III might depend on an epithelium-derived relaxing factor such as NO or prostaglandin E2, as has been demonstrated for some ligands. However, the combination of inhibitors of cyclooxygenase and NOS or removal of the epithelium had no effect on the relaxation caused by urocortin III, suggesting that the effect of this peptide is directly on smooth muscle cells in the trachea. The function(s) of CRF receptors abundantly expressed by airway epithelial cells remains an exciting area for future research.
4. CRF2 activation inhibits airway inflammation
Finally, we tested the potential anti-inflammatory activity of urocortin III in a standard acute model of lung inflammation, induced by LPS, that elicits neutrophil influx into the airways of mice. Few to no neutrophils are found in naive BALB/c mice in our hands, whereas LPS elicits neutrophil influx within 3 h. Pretreatment of mice with as little as 150 µg/kg i.p. of urocortin III reduced the number of neutrophils found in bronch-alveolar lavage (BAL) 3 h after administration of LPS by more than half compared to saline (Fig. 2
A). This anti-inflammatory effect was suppressed when astressin 2B was administered (500 µg/kg i.p.) 1 h prior to urocortin III (Fig. 2A
), confirming the involvement of CRF2 receptors. The mechanism(s) by which urocortin III exerts this protective effect is not clear from these preliminary studies. A previous study demonstrated that systemic CRF2 activation down-regulates and up-regulates proinflammatory and anti-inflammatory cytokines, respectively, in the spleen and liver during bacterial infection in mice. It is also known that the recruitment of neutrophils to the lungs of mice after LPS administration is dependent on production of TNF-
in the first 3 h within the airways, presumably by alveolar macrophages. However, TNF- levels (undetectable in naive animals) determined in BAL samples were not different between the experimental groups (Fig. 2B
). Therefore, ongoing production of inflammatory cytokines in the lung does not appear to have been modified by urocortin III, and the reduction of neutrophil influx after LPS may be modulated systemically.
|
CONCLUSIONS AND SIGNIFICANCE
In summary, we have demonstrated for the first time that activation of the urocortin III-preferring CRF2 receptor subtype causes bronchorelaxation in vitro and inhibits inflammatory processes in the airway in vivo. Therefore, we suggest that CRF2 receptor activation might be an interesting therapeutic strategy for treatment of inflammatory airway diseases. Additional studies are required to determine the basis for the anti-inflammatory effect of urocortin III.
|
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5315fje
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
