Pharmacological characterization and cross talk of alpha1a- and alpha1b-adrenoceptors coexpressed in human embryonic kidney 293 cells.
We established three human embryonic kidney (HEK) 293 cell lines stably expressing alpha(1)-adrenoceptor (AR) subtypes, one (alpha(1A), (1B)-AR) coexpressing both receptors and the other two (alpha(1A)-AR and alpha(1B)-AR) expressing each receptor in isolation. In the alpha(1A), (1B)-AR cells, both receptors were clearly distinguished by the alpha(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 alpha(1A,1B)-AR cells, compared with the responses of cells expressing either receptor individually (alpha(1A)-AR or alpha(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 alpha(1A)-AR cells (prazosin, 9.3; KMD-3213, 10.5) and alpha(1B)-AR cells (prazosin, 11.0; KMD-3213, 8.1). The inhibitor pK(b) values in alpha(1A), (1B)-AR also differed from their ligand binding affinities measured in alpha(1A)-AR and alpha(1B)-AR cells. In contrast, the alpha(1A)-selective agonist methoxamine, which did not activate alpha(1B)-AR cells, stimulated either alpha(1A,) (1B)-AR or alpha(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.
Israilova
Malika
M
Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, Matsuoka, Fukui 910-1193, Japan.
Tanaka
Takashi
T
Suzuki
Fumiko
F
Morishima
Shigeru
S
Muramatsu
Ikunobu
I
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D006801:Humans; D007668:Kidney; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D020239:Receptor Cross-Talk; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects / Q000378:metabolism*
C547713:ADRA1A protein, human; C547716:ADRA1B protein, human; D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D018340:Receptors, Adrenergic, alpha-1; D020928:Mitogen-Activated Protein Kinases