Characterization of human recombinant α2A‐adrenoceptors expressed in Chinese hamster lung cells using intracellular Ca2+ changes: evidence for cross‐talk between recombinant α2A‐ and native α1‐adrenoceptors

Human α2A-adrenoceptors expressed in Chinese hamster lung (CHL) fibroblasts have been pharmacologically characterized by measuring intracellular calcium (Ca2+i) changes using the Ca2+-sensitive dye Fluo3-AM, in conjunction with a fluorometric imaging plate reader (FLIPR). Several α-adrenoceptor agonists were examined including the α2-adrenoceptor agonists UK-14304, B-HT 920, dexmedetomidine and A-54741, the selective α1-adrenoceptor agonist phenylephrine and the non-selective adrenergic agonist noradrenaline. Of these only noradrenaline (mean pEC50=6.49) and A-54741 (6.90) evoked changes in Ca2+i; A-54741 was a partial agonist relative to noradrenaline, achieving only 33% of the noradrenaline maximum. Ca2+i changes induced by noradrenaline and A-54741 were antagonized by the α2-selective antagonist rauwolscine (10 nM) and by the α1-selective antagonists prazosin (0.1 nM) and doxazosin (1.0 nM). Phenylephrine (100 μM) and UK-14304 (10 μM) alone were ineffective in causing Ca2+i increase. In the presence of a fixed concentration of UK-14304 (3.0 μM), phenylephrine induced concentration-dependent increases in Ca2+i (mean pEC50=5.33). In the presence of phenylephrine (30.0 μM) UK-14304 induced Ca2+i release (pEC50=6.92). The effects of phenylephrine were abolished by prazosin (1.0 nM) or rauwolscine (100 nM). In saturation radioligand binding experiments using membranes of parental (non-transfected) CHL cells there was a small, specific binding of [3H]-prazosin (Bmax=24 fmol mg protein−1; pKD=10.24). Collectively, these data suggest that α-adrenoceptor agonist-induced Ca2+i release in CHL fibroblasts transfected with the human α2A-adrenoceptor is dependent upon co-activation of the recombinant receptor and a native α1-adrenoceptor. British Journal of Pharmacology (2000) 129, 1339–1346; doi:10.1038/sj.bjp.0703184