Bronchorelaxant and anti-inflammatory effects of urocortin III in mice

James D. Moffatt1, Rebecca Lever2 and Clive P. Page1..1The Sackler Institute of Pulmonary Pharmacology, King's College London, London SE1 1UL. 2The School of Pharmacy, University of London, London WC1N 1AX.

Urocortins are members of the corticotropin-releasing factor (CRF) family of peptides that bind to two receptors, CRF1 and CRF2. While CRF1 is a high affinity receptor for CRF, urocortin III binds with much greater affinity to CRF2. In the present study we investigated the effect of CRF2 receptor activation with urocortin III on airway smooth muscle tone in vitro, and in an acute model of airway inflammation in mice

All studies were performed in accordance with The Animals (Scientific Procedures) Act, 1986. For in vitro studies of tracheal responsiveness mice were killed with an i.p. injection of urethane (20g.kg-1) and the tracheae dissected from surrounding tissues. Ring preparations of trachea were mounted in conventional organ baths and connected to isometric transducers for recording of tracheal muscle tension. Preparations were maximally contracted with methacholine, washed and then recontracted with titrated concentrations of methacholine until a half maximal contraction was achieved. Cumulative concentrations of urocortin III (1-300 nM) were then added to the baths. For in vivo studies of inflammation, all mice were administered 50 μL of a 0.05 mg/mL solution of LPS in saline via the intranasal route while lightly anaesthetised with 5% isofluorane. In one experiment urocortin III (100 μg.kg-1 i.p.) or saline was administered one hour prior to LPS administration. In another group the CRF 2 antagonist astressin 2B was given one hour prior to urocortin III. In all groups, the mice were killed three hours after LPS and bronchoalveolar lavage was performed. Total and differential cell counts of cells retrieved were used to determine the number of neutrophils in the samples. Tumour-necrosis factor-α (TNF- α) levels in BAL fluids were determined by ELISA.

Urocortin III caused relaxation of methacholine-induced contraction (30.6% at 0.3 μM; pEC50 = 7.27±0.2, n=4) of mouse tracheal segments. CRF caused similar relaxation (27.7% at 0.3 μM, n=4), but with reduced potency (pEC50=6.78±0.1) compared to urocortin III, consistent with the CRF2 receptor subtype. Relaxation induced by urocortin III was concentration-dependently inhibited by the CRF2 antagonist, astressin 2B with an estimated IC50 in the nanomolar range. These relaxations were potentiated by inhibition of phosphodiesterases with isobutylmethylxanthine (30 μM; 60.3 vs control 30.3% relaxation) but unaffected by combined inhibition of cyclooxygenase and nitric oxide synthase with indomethacin (3μM) and L-NAME (100 μM). The number of neutrophils retrieved by broncholaveolar lavage 3 hours after intranasal administration of bacterial lipopolysaccharide was reduced (5.5 vs control 17.5 x105 cells/mL, n=5 each) by prior intraperitoneal injection of urocortin III. This anti-inflammatory effect was also completely suppressed by astressin 2B (0.5 mg.kg-1 i.p., n=5), again implicating a role for CRF2 receptors. BAL fluid TNF- α levels were not different in any of the groups, suggesting a systemic mechanism of action.

CRF2 agonists appear to have both bronchorelaxant and anti-inflammatory activities and might represent an interesting therapeutic approach to the treatment of inflammatory lung diseases.