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Characterisation of alpha 2 adrenoceptor subtype involved in inhibition of neurally evoked dog ileum contractions α2 -adrenoceptor agonists are known to reduce nerve-evoked contractions in smooth muscle of the dog ileum (Boeckxstaens et al., 1993) however, the receptor subtype involved has not been defined. The aim of this study was to identify the α2-adrenoceptor subtype involved. All experiments were carried out in compliance with UK legislation. A 10cm section of dog ileum was removed from female and male Beagle dogs (10-20Kg) and strips of longitudinal muscle (2x2x10mm) dissected. Isometric tension recordings were performed from longitudinal ileum strips bathed in Krebs’ solution (37°C, 95% O2 / 5% CO2) containing propranolol (1μM), doxazosin (0.1μM) and naproxen (10μM). Strips were allowed to equilibrate (resting tension 3 g) for 1hr before electrical field stimulation (EFS, 0.2ms, 20Hz, 2 sec train, 30 volts) induced contractions were evoked at 5 minute intervals. The effects of α2-adrenoceptor agonists, in the absence and presence of antagonists, on EFS evoked contractions were investigated. Only a single agonist concentration response curve (CRC) was carried out on each strip. For calculation of antagonist affinities, agonist CRCs to UK-14304 were carried out in the absence and presence of 5 antagonist concentrations and pKBs calculated utilising the Angus analysis (Lew & Angus, 1995) . Data are reported as EC50 values (geometric mean) with 95% confidence intervals (CIs) n=4-6 or pKBs with 95% CIs n=4-6. The α2-adrenoceptor specific agonists UK-14304 and PGE-6201204 caused a concentration dependent inhibition of EFS evoked contractions with EC50 values of 15.3 (95% CIs of 12.9, 18.1) and 4.4 (2.6, 7.3) nM, respectively. The maximum inhibition of contraction with these agonists varied in the range of 90-100%. The imidazoline-1 (I1) receptor agonist moxonidine only inhibited EFS induced contractions at concentrations known to cause α2-adrenoceptor activation (EC50 value 418 (350, 499) nM). UK-14304 did not inhibit carbachol or KCl induced ileal longitudinal smooth muscle contraction. CRCs for UK-14304 were shifted to the right by the α2-adrenoceptor specific antagonists yohimbine, RX821002 and RS79948 (pKB 8.71 (8.57, 8.85), 8.53 (8.32, 8.74) and 9.19 (9.13, 9.26), respectively) and the α2A/D subtype preferring antagonist BRL44408 (pKB 7.68 (7.45, 7.91). However, the α2B and α2C preferring antagonists (Juvantia compound from patent WO 03-008387 and OPC28326 respectively (10, 30, 50, 100 and 300nM)) did not cause any significant shift in the UK-14304 CRC. These data are supportive of UK-14304-induced inhibition of nerve evoked contractions being mediated through presynaptic α2A -adrenoceptors.
Boeckxstaens GE. et al. (1993). Br J Pharmacol. 109(4) :1079-84. |
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