Characterization of human recombinant alpha(2A)-adrenoceptors expressed in Chinese hamster lung cells using extracellular acidification rate changes

Abstract

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.

Figure 1

Acidification rate changes evoked by agonists at human α_2A-adrenoceptors expressed in CHL cells. In each experiment different chambers of cells were challenged with noradrenaline or other agonists. Data are the mean±s.e.mean of three to six separate experiments.

Figure 2

Acidification rate changes evoked by noradrenaline, A-54741, UK-14304, dexmedetomidine and B-HT 920 were abolished by pertussis toxin. Concentration-effect curves to agonists were constructed in control CHL cells expressing the human α_2A-adrenoceptor (closed symbols) or in cells exposed to pertussis toxin (open symbols; 500 ng ml⁻¹ for 24 h). Data are the mean±s.e.mean of 4–6 separate experiments.

Figure 3

Antagonism by rauwolscine of (A) noradrenaline and (B) dexmedetomidine induced acidification rate increases mediated by human α_2A-adrenoceptors expressed in CHL cells. E/[A] curves to agonists were constructed by non-cumulative exposure of the agonist in the absence or presence of rauwolscine which had been added 75 min earlier. (A) and (B) show representative data from one experiment only while the Schild plots (C, vsand the different symbols represent data from each experiment.

Figure 4

Effect of prazosin (A) and doxazosin (B) on noradrenaline-induced acidification rate increases mediated by human α_2A-adrenoceptors expressed in CHL cells. E/[A] curves to agonists were constructed by non-cumulative exposure of the agonist in the absence or presence of antagonist which had been added 75 min earlier (n=3).