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Evidence to suggest the post synaptic α-adrenoceptor mediating contraction in the canine saphenous vein is not consistent with human α2a receptors Previous studies have suggested that the predominant functional α2 adrenoceptor in the canine saphenous vein (SV) is comparable to the human α2A receptor (MacLennan et al., 1997). The aim of this study was to confirm these findings by comparing the potency of standard adrenoceptor ligands tested in the canine SV with binding and functional data generated in a cell line expressing the human recombinant α2A receptor . Radioligand binding and β-lactamase reporter gene assays were carried out in CHO cells expressing human α2A receptors. For membrane binding studies, 3HUK-14,304 (NEN, specific activity 71 Ci/mmole) was incubated in the presence of test compound with 16 μg/well membrane protein for 120 min at room temperature as described previously (Turner et al., 1985). For the β-lactamase assay, cells and compounds were incubated for 3 h for the beta-lactamase to be synthesised (Kunapuli et al., 2003). For canine vascular studies; SV were removed from beagle dogs (either sex, 10-20kg), euthanised by pentobarbitone (0.5ml/kg iv, animals were used in several pharmacological studies, protocols were reviewed by Pfizer Ethics Committee). The endothelium was removed by gentle rubbing and vascular rings were mounted as described previously (Morris et al., 2005). All antagonists were pre-incubated for 30 min prior to noradrenaline challenge. Data are mean pIC50/pKb/pEC50± s.e.mean, n=number of experiments or dogs used. Data were compared using linear regression and Pearson’s correlation coefficient (r) Table 1. Mean binding affinity and functional potency values for adrenergic compounds.
Data represents mean ± s.e.mean, n=3-8 Overall, binding data confirmed high affinity for α2A compounds (Table 1), compared to selective α2B , α2C or α1 ligands. There was a good correlation between binding and functional cell based data at the human α2A receptor (r=0.93), with the exception of noradrenaline. In contrast, there were weak correlations between the canine SV and either α2A binding (r=0.53) or cell-based functional data (r=0.41). While, the high potency of WB4101, prazosin and doxazosin in the canine SV may suggest a role for a α1 receptor subtype, the high potency of certain α2 ligands (UK-14,304, oxymetazoline, yohimbine) does not support a role for a predominant α1 receptor mediated contraction in the canine SV. In contrast to previous findings (Maclennan et al., 1997), we conclude that the post-synaptic αadrenoceptor in the canine SV is not similar to the human α2A receptor and further studies to identify the predominant post-synaptic α-receptor mediating contraction in the canine SV are clearly warranted. Kunapuli, P. et al., (2003). Anal. Biochem., 314, 16-29. |
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