Phenoxybenzamine and Benextramine, but Not 4-Diphenylacetoxy-N-[2-chloroethyl]piperidine Hydrochloride, Display Irreversible Noncompetitive Antagonism at G Protein- Coupled Receptors Johannes Bodenstein, 1 Daniel P. Venter, and Christiaan B. Brink Division of Pharmacology, The Northwest University (PUK), Potchefstroom, South Africa Received January 12, 2005; accepted April 21, 2005 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 mecha- nisms of antagonism have been demonstrated but not thor- oughly investigated. We studied noncompetitive antagonism by phenoxybenzamine and benextramine at  2A -adrenoceptors in stably transfected Chinese hamster ovary cells, benextramine and 4-diphenylacetoxy-N-[2-chloroethyl]piperidine hydrochlo- ride (4-DAMP mustard) at endogenous muscarinic acetylcho- line (mACh) receptors in human neuroblastoma SH-SY5Y cells, and benextramine at serotonin 5-HT 2A receptors in stably transfected SH-SY5Y cells. Primary binding sites were pro- tected by reversible competitive antagonists during pretreat- ment 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-[ 35 S]thio)triphosphate ([ 35 S]GTPS)-G o binding assays to determine remaining G protein activity. Phenoxybenzamine (100 M; 20 min) and benextramine (10 or 100 M; 20 min) at  2A -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 noncompeti- tively inhibits agonist-induced responses at three different GPCR types ( 2A , mACh, and 5-HT 2A receptors) that signal via three families of G proteins (G i/o ,G s , and G q/11 ). We conclude that, where irreversible antagonists are utilized to study drug- receptor interaction mechanisms, the presence of significant irreversible noncompetitive antagonism may influence the in- terpretation of results under the experimental conditions used. Irreversible competitive antagonists are generally ac- cepted to bind pharmacological receptors irreversibly, pre- sumably by forming strong covalent bonds with the receptor. Thereby, they prevent other ligands from binding to the primary (orthosteric) binding site(s) on these receptors [the reader may consult Neubig et al. (2003) regarding specific terminology]. The classical irreversible -adrenoceptor- blocking drug phenoxybenzamine (Dibenzyline; Wellspring Pharmaceutical Corporation, Neptune, NJ) has been used since the 1960s in the clinical setting to treat pheochromo- cytoma (Crago et al., 1967). Most irreversible competitive antagonists, however, found application in experimental pharmacology to investigate and eliminate spare receptors, implementing the Furchgott analysis (Furchgott, 1966) to estimate the relative intrinsic efficacy of agonists and the apparent equilibrium dissociation constants of agonist-recep- This project was funded by a grant from the Medical Research Council (MRC) of South Africa. 1 Current address: Department of Pharmacology, University of Michigan Medical School, Ann Arbor, MI. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.083568. ABBREVIATIONS: mACh, muscarinic acetylcholine; 5-HT, 5-hydroxytryptamine; 4-DAMP mustard, 4-diphenylacetoxy-N-[2-chloroethyl]piperi- dine hydrochloride;  2A -H,  2A -adrenoceptors expressed at relative high numbers;  2A -L,  2A -adrenoceptors expressed at relative low numbers; SQ 30,741, [1S-[1,2(5Z),3,4]]-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; IP x , 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; [ 35 S]GTPS, guanosine 5-O-(3-[ 35 S]thio)triphosphate; DTT, dithiothreitol; U-73122, 1-[6-[((17)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole- 2,5-dione. 0022-3565/05/3142-891–905$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 314, No. 2 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 83568/3040349 JPET 314:891–905, 2005 Printed in U.S.A. 891 at ASPET Journals on July 20, 2018 jpet.aspetjournals.org Downloaded from