Functional assessment of recombinant human alpha(2)-adrenoceptor subtypes with Cytosensor microphysiometry

We applied the Cytosensor Microphysiometry system to study the three human alpha(2)-adrenoceptor subtypes, alpha(2A), alpha(2B) and alpha(2C), express ed 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 derivativ e 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-imidaz ol-2-yl)-6-quinoxalinamine) appeared to be partial agonists at alpha(2B)-ad renoceptors (E-max approximate to 60% of (-)-noradrenaline) but full agonis ts at alpha(2A)- and alpha(2C)-adrenoceptors. The responses mediated by all three alpha(2)-adrenoceptor subtypes were partly inhibited by the sodium-h ydrogen (Na+/H+) exchange inhibitor, MIA (5-(N-methyl-N-isobutyl)-amiloride ). The agonist responses were totally abolished by pretreatment with pertus sis toxin in cells with alpha(2A)- and alpha(2C)-adrenoceptors, and partly abolished in cells with alpha(2B)-adrenoceptors. The residual signal in alp ha(2B)-cells was sensitive to the intracellular Ca2+ chelator, BAPTA (1,2-b is(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 re sponses. The results suggest that the extracellular acidification responses mediated by all three human alpha(2)-adrenoceptor subtypes are dependent o n Na+/H(+)exchange and G(i/o) pathways, and that alpha(2B)-adrenoceptors ar e capable of coupling to another, G(i/o)-independent and Ca2+-dependent sig naling pathway. (C) 1999 Elsevier Science B.V. All rights reserved.