Characterization of human recombinant alpha(2A)-adrenoceptors expressed in Chinese hamster lung cells using extracellular acidification rate changes.
1. Human alpha(2A)-adrenoceptors heterologously expressed in Chinese hamster lung (CHL) fibroblasts have been characterized pharmacologically using a cytosensor microphysiometer to measure ligand-induced extracellular acidification rate changes. 2. In untransfected CHL cells, noradrenaline had no effect at concentrations up to 100 microM. In alpha(2A)-adrenoceptor transfected cells the rank order of agonist potency was A-54741 (mean pEC(50)=8.96)>dexmedetomidine (8.88)>UK-14304 (8.42)>B-HT 920 (7.05)>noradrenaline (6.92). A-54741, UK-14304 and noradrenaline had the same maximum response while dexmedetomidine and B-HT 920 behaved as partial agonists. 3. The selective alpha(2)-adrenoceptor ligand rauwolscine antagonized acidification rate changes with an affinity independent of the agonist used; the affinity (mean pK(B)) against noradrenaline was 8.43. 4. The selective alpha(1)-adrenoceptor ligands prazosin and doxazosin (each 3 microM) had no effect on noradrenaline responses. 5. Acidification rate changes induced by each agonist were abolished by pre-treatment of cells with pertussis toxin. 6. These data suggest that agonist-induced acidification rate responses in CHL cells transfected with the human alpha(2A)-adrenoceptor are mediated exclusively by the recombinant protein, via pertussis toxin sensitive G(i/o) proteins.
MacLennan
S J
SJ
Department of Molecular Pharmacology, Center for Biological Research, Neurobiology Unit, Roche Bioscience, 3401 Hillview Avenue, Palo Alto, California, CA 94304, USA. stephen.maclennan@roche.com
Reynen
P H
PH
Martin
R S
RS
Eglen
R M
RM
Martin
G R
GR
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D000068438:Brimonidine Tartrate; D002460:Cell Line; D006224:Cricetinae; D020927:Dexmedetomidine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D017292:Doxazosin / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D009638:Norepinephrine / Q000494:pharmacology; D011224:Prazosin / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D018341:Receptors, Adrenergic, alpha-2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D013764:Tetrahydronaphthalenes / Q000494:pharmacology; D015016:Yohimbine / Q000494:pharmacology
C547768:ADRA2A protein, human; D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D001381:Azepines; D007093:Imidazoles; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D013764:Tetrahydronaphthalenes; C056298:5,6-dihydroxy-1-(2-imidazolinyl)tetralin; D015016:Yohimbine; D000068438:Brimonidine Tartrate; D020927:Dexmedetomidine; C024763:talipexole; D017292:Doxazosin; D009638:Norepinephrine; D011224:Prazosin