In vitro investigation of the functional role of adenosine receptor agonists in canine isolated vasculature

Rachael Morris, James Root, Sidath Katugampola, & Carolyn Napier, Candidate Research Group, Pfizer, Sandwich, Kent, CT13 9NJ, UK.

Adenosine receptors are expressed in canine vasculature (Hargreaves et al., 1991) and A2A receptor agonists have been reported to mediate vasodilatation and decrease blood pressure in canine in vivo studies (Schindler et al., 2005). It was our objective to determine the functional effects of A2A agonists and to investigate adenosine receptor subtypes involved in vasodilatation of canine isolated coronary arteries (CA), carotid artery (CaA) and saphenous vein (SV) in vitro.

Left anterior descending coronary arteries (LADCA), right coronary arteries (RCA) and CaA and SV were obtained from beagle dogs (10-20 kg, of either sex). Animals were used in other pharmacological studies and Pfizer ethics committee ethically reviewed the protocols. The CA, CaA and SV were dissected into 4mm rings and the endothelium was denuded (-e) by gentle rubbing. After mounting the tissues in organ baths containing 95%O2/5%CO2, 37°C Krebs at isometric resting tension of 1g (CA and CaA) or 2g (SV), tissue and endothelial viability were assessed using phenylephrine (1µM) and acetylcholine (1µM) respectively. The effects of the non-selective adenosine receptor agonist, NECA, A1/A3 preferring CCPA, and A2A selective CGS21680, (0.1 nM-100 µM) (Fredholm et al., 2001), the nitric oxide donor SNAP (0.1 nM-100 µM) and DMSO vehicle (0.000001% to 1%) were investigated on 1 µM (EC70 response) PE pre-constricted tissues. Data are mean pEC50/Emax ± s.e.mean, n-values refer to the number of dogs. Data were compared using Student’s t-test, with significance set at P<0.05.

Table 1. Mean pEC50 and E max ± s.e.mean values for test compounds (+e=endothelium intact).

CGS21680 failed to produce any vasodilatation in canine LADCA (either ± e, n=3), RCA (n=3) or CaA (n=8). The lack of any functional effect in the canine RCA is in agreement with previous studies (Balwierczak et al., 1991). High efficacy, low potency (>1000 fold the binding affinity at human A2A, (Fredholm et al., 2001)), vasodilator responses were obtained for CGS21680 in the canine SV (Table 1). CCPA did not produce vasodilatation either in the CaA (n=4) or RCA (n=3). However, similar to CGS21680, CCPA produced a concentration dependent high efficacy, low potency vasodilatation in canine SV (Table 1). NECA, produced no vasodilatation in the CaA (n=4). Interestingly, consistent low efficacy vasodilator responses were observed in the RCA with potency values 10 fold weaker than the binding affinity for the cloned human A2B receptor (Fredholm et al., 2001). This may suggest regional differences in functional vascular responses to NECA within the arterial circulation. Similar potency values with full reversal of PE responses were obtained for NECA in the canine SV (Table 1). Compared to the adenosine receptor agonists, SNAP, was a potent vasodilator in the canine vasculature. In contrast to the reported coronary vasodilator effects in vivo, there is no evidence to indicate a direct vasodilator effect of A2A receptor agonists in canine coronary arteries and it is possible that the low potency responses to NECA, CGS21680 and CCPA in SV maybe mediated via A2B receptors. Further studies using selective A2B receptor agonists/antagonists will be required to confirm these findings.

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