Characterization of human recombinant α_2A-adrenoceptors expressed in Chinese hamster lung cells using intracellular Ca²⁺changes: evidence for cross-talk between recombinant α_2A- and native α₁-adrenoceptors

1 Human alpha(2A)-adrenoceptors expressed in Chinese hamster lung (CHL) fibroblasts have been pharmacologically characterized by measuring intracellular calcium (Ca-i(2+)) changes using the Ca2+ sensitive dye Fluo3-AM, in conjunction with a fluorometric imaging plate reader (FLIPR). 2 Several alpha-adrenoceptor agonists were examined including the alpha-adrenoceptor agonists UK-14304, B-HT 920, dexmedetomidine and A-54741, the selective alpha(1)-adrenoceptor agonist phenylephrine and the non-selective adrenergic agonist noradrenaline. Of these only noradrenaline (mean pEC(50) = 6.49) and A-54741 (6.90) evoked changes in Ca-i(2+), A-54741 was a partial agonist relative to noradrenaline, achieving only 33% of the noradrenaline maximum. 3 Ca-i(2+) changes induced by noradrenaline and A-54741 were antagonized by the alpha(2)-selective antagonist rauwolscine (10 nM) and by the alpha(1)-selective antagonists prazosin (0.1 nM) and doxazosin 4 Phenylephrine (100 mu M) and UK-14304 (10 mu M) alone were ineffective in causing Ca-i(2+) increase. In the presence of a fixed concentration of UK-14304 (3.0 mu M), phenylephrine induced concentration-dependent increases in Ca-i(2+) (mean pEC(50) = 5.33). In the presence of phenylephrine (30.0 mu M) UK-14304 induced Ca-i(2+) release (pEC(50) = 6.92). The effects of phenylephrine were abolished by prazosin (1.0 nM) or rauwolscine (100 nM). 5 In saturation radioligand binding experiments using membranes of parental (non-transfected) CHL cells there was a small, specific binding of [H-3]-prazosin (B-max = 24 fmol mg protein(-1); pK(D) = 10.24). 6 Collectively, these data suggest that oc-adrenoceptor agonist-induced Ca-i(2+) release in CHL fibroblasts transfected with the human alpha(2A)-adrenoceptor is dependent upon co-activation of the recombinant receptor and a native alpha(1)-adrenoceptor.