Characterisation of alpha 2 adrenoceptor subtype involved in inhibition of neurally evoked rat bladder contractions

Sheridan Piper-Brown & Gordon McMurray (Introduced by Wesley Miner). Discovery Biology, Pfizer Global Research and Development, Sandwich, Kent, CT13 9NJ.

α2-adrenoceptor agonists are known to reduce nerve-evoked contractions in urinary bladder smooth muscle (Ali et al., 1998), however, the receptor subtype involved has not been defined. The aim of this study was to identify the α2-adrenoceptor subtype present on the intramural parasympathetic nerves of the rat bladder. All experiments were carried out in compliance with UK legislation. Whole bladders were removed from euthanased, female, Sprague Dawley rats (200-250 g) and longitudinal bladder strips (2x2x5 mm) dissected from the bladder dome. Isometric tension recordings were performed from bladder strips bathed in Krebs’ solution (37 ° C, 95% O2 / 5% CO2) containing propranolol (1 μM), Doxazosin (0.1 μM) and naproxen (10 μM). Strips were allowed to equilibrate (resting tension 1 g) for 1hr before electrical field stimulation (EFS, 0.1 ms, 15 Hz, 4 sec train, 30 volts) induced contractions were evoked at 5 minute intervals. The effects of a 2 -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 method of analysis (Lew MJ. & Angus JA, 1995) . Data are reported as EC 50 values (geometric mean) with 95% confidence intervals (CIs) N=5-8 or pKBs with 95% CIs N=5-8. The α2-adrenoceptor specific agonists UK-14304, PGE-6201204, dexmedetomidine and clonidine caused a concentration dependent inhibition of EFS evoked contractions with EC50 values of 26.0 (95% CIs of 9.1, 74.7), 1.1 (0.5, 2.6), 12.7 (5.2, 31.1) and 14.7 (9.3, 23.4) nM, respectively. The endogenous agonist noradrenaline caused a concentration dependent inhibition with an EC50 value of 91.7 (71.1, 118.3) nM. The α2A/D subtype preferring agonists guanfacine, and oxymetazoline also inhibited nerve evoked contractions (EC50 values 10.5 (5.4, 20.3) and 7.3 (4.5, 11.7) nM, respectively). The maximum inhibition of contraction with these agonists varied in the range of 40-100%. In addition, the imidazoline-1 (I1) receptor agonist moxonidine only inhibited EFS induced contractions at concentrations known to cause α2-adrenoceptor activation (EC50 value 353.9 (199.9, 626.5) nM). None of the agonists inhibited carbachol or KCl induced bladder smooth muscle contraction. UK-14304 CRCs were shifted to the right by the α2-adrenoceptor specific antagonists yohimbine, RX821002 and RS79948 (pKB –7.2 (-6.9, -7.5), -8.4 (-8.2, -8.6) and –8.7 (-8.4, -9.0), respectively) and the a α2A/D subtype preferring antagonist BRL44408 (pKB –7.4 (-7.2, -7.6). However the α2B and a α2C preferring antagonists ARC239, Juvantia compound from patent WO 03-008387 , OPC28326 and prazosin (10, 30, 50 100 and 300 nM) did not cause any significant shift in the UK-14304 CRC. It is concluded that α2-adrenoceptors are present on excitatory parasympathetic intramural nerve terminals of the rat urinary bladder, and that α2agonist-induced inhibition of nerve-evoked contractions is mediated by the α2A/D -adrenoceptor subtype (species orthologue of α2A ) in this species.

Ali A. et al, (1998). Br. J. Pharmacol., 125/127-135.
Lew MJ. & Angus JA. (1995). TiPS. 16/328-337.

Patent WO 03/008387 30.01.2003 exemplified compound C.