Cloning of the porcine α2B-adrenoceptor: Tissue distribution and pharmacological characterization☆

Abstract

Constriction of carotid arteriovenous anastomoses in the pig serves as a predictive model for assessing the antimigraine efficacy of α2-adrenoceptors based drugs. To gain further insight in to α2-adrenoceptors mediated mechanisms, we amplified, cloned and sequenced a full-length cDNA encoding porcine α2B-adrenoceptor, assessed its tissue distribution and pharmacological characteristics. Sequence analysis of the porcine cDNA clone (1341 bp) revealed an open reading frame of 446 amino acids encoding α2B-adrenoceptor, which showed 88% and 85% similarity with the human and guinea pig sequences, respectively. The porcine α2B-adrenoceptor showed 7 transmembrane domains and differed with mouse and rat, both of which have 5 amino acids extension at the N-terminal. The expression of α2B-adrenoceptor mRNA was demonstrated in renal, femoral, mesenteric, carotid, pulmonary and coronary arteries, external jugular and saphenous veins as well as in the brain cortex, cerebellum and trigeminal ganglion. In transient transfection system based on HEK293 cells, receptor binding with the endogenous ligand, noradrenaline showed pEC50 ≥ 7.67 ± 0.4 and with the antagonist, rauwolscine pKB 9.76 ± 0.6. Stimulation of transfected HEK293 as well as COS-7 cells with noradrenaline, in dual luciferase assay, we found significant (P ≤ 0.05) elevation of serum response element (SRE) and activator protein-1 (AP-1), while nuclear factor of κB cells (NFκB) and nuclear factor of activated T-cells (NFAT) remained unchanged. In conclusion, we have cloned a full-length cDNA and established the amino acid sequence, ligand binding profile and signalling pathway and have demonstrated the tissue distribution of the porcine α2B-adrenoceptor. This information may be useful in exploring the role of α2B-adrenoceptor in pathophysiological processes relevant for novel drug discovery in diseases.