title: Functional assessment of recombinant human alpha(2)-adrenoceptor subtypes with cytosensor microphysiometry.
abstract: We applied the Cytosensor Microphysiometry system to study the three human alpha(2)-adrenoceptor subtypes, alpha(2A), alpha(2B) and alpha(2C), expressed in Chinese hamster ovary (CHO) cells, and assessed its potential in the quantitative monitoring of agonist activity. The natural full agonist, (-)-noradrenaline, was used to define agonist efficacy. The imidazole derivative dexmedetomidine was a potent full agonist of all three receptor subtypes. The imidazolines clonidine and UK 14,304 (5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) appeared to be partial agonists at alpha(2B)-adrenoceptors (E(max) approximately 60% of (-)-noradrenaline) but full agonists at alpha(2A)- and alpha(2C)-adrenoceptors. The responses mediated by all three alpha(2)-adrenoceptor subtypes were partly inhibited by the sodium-hydrogen (Na(+)/H(+)) exchange inhibitor, MIA (5-(N-methyl-N-isobutyl)-amiloride). The agonist responses were totally abolished by pretreatment with pertussis toxin in cells with alpha(2A)- and alpha(2C)-adrenoceptors, and partly abolished in cells with alpha(2B)-adrenoceptors. The residual signal in alpha(2B)-cells was sensitive to the intracellular Ca(2+)chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester). Cholera toxin (which acts on G(s)-proteins) had no effect on the agonist responses. The results suggest that the extracellular acidification responses mediated by all three human alpha(2)-adrenoceptor subtypes are dependent on Na(+)/H(+)exchange and G(i/o) pathways, and that alpha(2B)-adrenoceptors are capable of coupling to another, G(i/o)-independent and Ca(2+)-dependent signaling pathway.
authors.affiliation: Department of Pharmacology and Clinical Pharmacology, University of Turku, MediCity, Tykistökatu 6 A, FIN-20520, Turku, Finland. mpihla@utu.fi
authors.forename: M
authors.initials: M
authors.lastname: Pihlavisto
authors.forename: M
authors.initials: M
authors.lastname: Scheinin
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000378:metabolism / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D015374:Biosensing Techniques; D000068438:Brimonidine Tartrate; D016466:CHO Cells; D002772:Cholera Toxin / Q000494:pharmacology; D003000:Clonidine / Q000378:metabolism / Q000494:pharmacology; D006224:Cricetinae; D020927:Dexmedetomidine / Q000378:metabolism / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009638:Norepinephrine / Q000378:metabolism / Q000494:pharmacology; D037342:Pertussis Toxin; D011810:Quinoxalines / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: C547768:ADRA2A protein, human; C547771:ADRA2B protein, human; C547774:ADRA2C protein, human; D000316:Adrenergic alpha-Agonists; D011810:Quinoxalines; D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; D000068438:Brimonidine Tartrate; D004533:Egtazic Acid; D020927:Dexmedetomidine; D000584:Amiloride; D002772:Cholera Toxin; C056369:5-(N-methyl-N-isobutyl)amiloride; D037342:Pertussis Toxin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D003000:Clonidine; D009638:Norepinephrine