Characterization of human recombinant alpha(2A)-adrenoceptors expressed in Chinese hamster lung cells using intracellular Ca(2+) changes: evidence for cross-talk between recombinant alpha(2A)- and native alpha(1)-adrenoceptors.
1. Human alpha(2A)-adrenoceptors expressed in Chinese hamster lung (CHL) fibroblasts have been pharmacologically characterized by measuring intracellular calcium (Ca(2+)(i)) changes using the Ca(2+)-sensitive dye Fluo3-AM, in conjunction with a fluorometric imaging plate reader (FLIPR). 2. Several alpha-adrenoceptor agonists were examined including the alpha(2)-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(2+)(i); A-54741 was a partial agonist relative to noradrenaline, achieving only 33% of the noradrenaline maximum. 3. Ca(2+)(i) 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 (1.0 nM). 4. Phenylephrine (100 microM) and UK-14304 (10 microM) alone were ineffective in causing Ca(2+)(i) increase. In the presence of a fixed concentration of UK-14304 (3.0 microM), phenylephrine induced concentration-dependent increases in Ca(2+)(i) (mean pEC(50)=5.33). In the presence of phenylephrine (30.0 microM) UK-14304 induced Ca(2+)(i) 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 [(3)H]-prazosin (B(max)=24 fmol mg protein(-1); pK(D)=10. 24). 6. Collectively, these data suggest that alpha-adrenoceptor agonist-induced Ca(2+)(i) 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.
Reynen
P H
PH
Department of Molecular Pharmacology, Center for Biological Research, Neurobiology Unit, Roche Bioscience, 3401 Hillview Avenue, Palo Alto, California, CA 94304, USA.
Martin
G R
GR
Eglen
R M
RM
MacLennan
S J
SJ
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000814:Aniline Compounds; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D001667:Binding, Competitive; D000068438:Brimonidine Tartrate; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D020927:Dexmedetomidine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D017292:Doxazosin / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005453:Fluorescence; D015870:Gene Expression; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D009638:Norepinephrine / Q000494:pharmacology; D011224:Prazosin / Q000378:metabolism / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D011869:Radioligand Assay; D020239:Receptor Cross-Talk; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism; D018341:Receptors, Adrenergic, alpha-2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013764:Tetrahydronaphthalenes / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014966:Xanthenes; D015016:Yohimbine / Q000494:pharmacology
C547768:ADRA2A protein, human; D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D000814:Aniline Compounds; D001381:Azepines; D004963:Estrenes; D007093:Imidazoles; D011760:Pyrrolidinones; D011810:Quinoxalines; D018340:Receptors, Adrenergic, alpha-1; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D013764:Tetrahydronaphthalenes; D014966:Xanthenes; C056298:5,6-dihydroxy-1-(2-imidazolinyl)tetralin; C074854:U 73343; C059715:Fluo-3; D015016:Yohimbine; D000068438:Brimonidine Tartrate; D019284:Thapsigargin; D020927:Dexmedetomidine; C024763:talipexole; D017292:Doxazosin; D002118:Calcium; D009638:Norepinephrine; D011224:Prazosin