Abstract
We established three human embryonic kidney (HEK) 293 cell lines stably expressing α1-adrenoceptor (AR) subtypes, one (α1A, 1B-AR) coexpressing both receptors and the other two (α1A-AR and α1B-AR) expressing each receptor in isolation. In the α1A, 1B-AR cells, both receptors were clearly distinguished by the α1A-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 α1A,1B-AR cells, compared with the responses of cells expressing either receptor individually (α1A-AR or α1B-AR). Furthermore, the antagonistic affinities of prazosin (pKb of 10.1) and KMD-3213 (9.4) for inhibiting the phenylephrine response were intermediate between the values for inhibition in α1A-AR cells (prazosin, 9.3; KMD-3213, 10.5) and α1B-AR cells (prazosin, 11.0; KMD-3213, 8.1). The inhibitor pKb values in α1A, 1B-AR also differed from their ligand binding affinities measured in α1A-AR and α1B-AR cells. In contrast, the α1A-selective agonist methoxamine, which did not activate α1B-AR cells, stimulated either α1A,1B-AR or α1A-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.
Footnotes
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This work was supported by grants-in-aid for scientific research and 21st Century Centers of Excellence Program (Medical Science) from the Ministry of Education, Culture, Sports, Science and Technology and by a grant from the Smoking Research Foundation.
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DOI: 10.1124/jpet.103.061796.
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ABBREVIATIONS: AR, adrenoceptor; MAPK, mitogen-activated protein kinase; KMD-3213, (-)-1(3-hydroxypropyl)-5-((2R)-2-{[2-(2,2,2-trifluoroethyl]oxy]phenyl}oxy)ethyl]amino}propyl)-2,3-dihydro-1H-indole-7-carboxamide; HEK, human embryonic kidney; IP, inositol phosphate; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor.
- Received October 17, 2003.
- Accepted December 8, 2003.
- The American Society for Pharmacology and Experimental Therapeutics
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