Characterization of human recombinant alpha sub(2A)-adrenoceptors expressed in Chinese hamster lung cells using intracellular Ca super(2+) changes: evidence for cross-talk between recombinant alpha sub(2A)- and native alpha sub(1)-adrenoceptors

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