Pharmacological Characterization and Cross Talk of α_1A- and α_1B-Adrenoceptors Coexpressed in Human Embryonic Kidney 293 Cells

Abstract

We established three human embryonic kidney (HEK) 293 cell lines stably expressing α₁-adrenoceptor (AR) subtypes, one (α_1A, _1B-AR) coexpressing both receptors and the other two (α_1A-AR and α_1B-AR) expressing each receptor in isolation. In the α_1A, _1B-AR cells, both receptors were clearly distinguished by the α_1A-selective ligands (-)-1(3-hydroxypropyl)-5-((2R)-2-{[2-(2,2,2-trifluoroethyl]oxy]phenyl}oxy)ethyl]amino}propyl)-2,3-dihydro-1H-indole-7-carboxamide (KMD-3213) and methoxamine, but not by the subtype-nonselective ligands prazosin and phenylephrine. In all three cell lines, phenylephrine caused a concentration-dependent increase in inositol phosphates and an increase in extracellular signal-regulated kinase 1/2 (ERK1/2) activation. However, there was a 2-fold or greater maximal response to phenylephrine and a somewhat higher agonist potency in ERK1/2 activation in the α_1A,1B-AR cells, compared with the responses of cells expressing either receptor individually (α_1A-AR or α_1B-AR). Furthermore, the antagonistic affinities of prazosin (pK_b of 10.1) and KMD-3213 (9.4) for inhibiting the phenylephrine response were intermediate between the values for inhibition in α_1A-AR cells (prazosin, 9.3; KMD-3213, 10.5) and α_1B-AR cells (prazosin, 11.0; KMD-3213, 8.1). The inhibitor pK_b values in α_1A, _1B-AR also differed from their ligand binding affinities measured in α_1A-AR and α_1B-AR cells. In contrast, the α_1A-selective agonist methoxamine, which did not activate α_1B-AR cells, stimulated either α_1A,1B-AR or α_1A-AR cells with a comparable potency and maximum effectiveness. Our data indicate that when coexpressed in the same cell, the activation of common pathways by individual AR receptor subtypes by a nonselective agonist can exhibit enhanced responsiveness and a distinct antagonist affinity compared with the parameters for the same receptors, when expressed alone in the same cell background.