Abstract
Many irreversible antagonists have been shown to inactivate G protein-coupled receptors (GPCRs) and used to study agonists and spare receptors. Presumably, they bind to primary (agonist) binding sites on the GPCR, although noncompetitive mechanisms of antagonism have been demonstrated but not thoroughly investigated. We studied noncompetitive antagonism by phenoxybenzamine and benextramine at α2A-adrenoceptors in stably transfected Chinese hamster ovary cells, benextramine and 4-diphenylacetoxy-N-[2-chloroethyl]piperidine hydrochloride (4-DAMP mustard) at endogenous muscarinic acetylcholine (mACh) receptors in human neuroblastoma SH-SY5Y cells, and benextramine at serotonin 5-HT2A receptors in stably transfected SH-SY5Y cells. Primary binding sites were protected by reversible competitive antagonists during pretreatment with irreversible antagonists. We conducted appropriate radioligand binding assays by measuring remaining primary binding sites and agonist affinity, functional assays to evaluate agonist-induced responses, and constitutive guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS)-Gαo binding assays to determine remaining G protein activity. Phenoxybenzamine (100 μM; 20 min) and benextramine (10 or 100 μM; 20 min) at α2A-adrenoceptors, but not 4-DAMP mustard (100 nM; 120 min) at mACh receptors, displayed irreversible noncompetitive antagonism in addition to their known irreversible competitive antagonism. Although agonist binding affinity is not influenced, signal transduction is modulated in a G protein-dependent manner via allotopic interactions. Benextramine noncompetitively inhibits agonist-induced responses at three different GPCR types (α2A, mACh, and 5-HT2A receptors) that signal via three families of G proteins (Gi/o, Gs, and Gq/11). We conclude that, where irreversible antagonists are utilized to study drugreceptor interaction mechanisms, the presence of significant irreversible noncompetitive antagonism may influence the interpretation of results under the experimental conditions used.
Footnotes
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This project was funded by a grant from the Medical Research Council (MRC) of South Africa.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.083568.
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ABBREVIATIONS: mACh, muscarinic acetylcholine; 5-HT, 5-hydroxytryptamine; 4-DAMP mustard, 4-diphenylacetoxy-N-[2-chloroethyl]piperidine hydrochloride; α2A-H, α2A-adrenoceptors expressed at relative high numbers; α2A-L, α2A-adrenoceptors expressed at relative low numbers; SQ 30,741, [1S-[1α,2α(5Z),3α,4α]]-7-[[[[[(oxaheptyl)amino]acetyl]amino]-methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; GPCR, G protein-coupled receptor; CHO, Chinese hamster ovary; PLC, phospholipase C; IPx, total inositol phosphate; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PTX, pertussis toxin; IBMX, 3-isobutyl-1-methylxanthine; UK 14,304, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (brimonidine); TCA, trichloroacetic acid; UltraMEM, reduced serum minimum essential medium; [35S]GTPγS, guanosine 5′-O-(3-[35S]thio)triphosphate; DTT, dithiothreitol; U-73122, 1-[6-[((17β)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione.
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↵1 Current address: Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI.
- Received January 12, 2005.
- Accepted April 21, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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