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CELLULAR AND MOLECULAR

Phenoxybenzamine and Benextramine, but Not 4-Diphenylacetoxy-N-[2-chloroethyl]piperidine Hydrochloride, Display Irreversible Noncompetitive Antagonism at G Protein-Coupled Receptors

Division of Pharmacology, The Northwest University (PUK), Potchefstroom, South Africa

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 _2A-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-HT_2A 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]GTPS)-G_o binding assays to determine remaining G protein activity. Phenoxybenzamine (100 µM; 20 min) and benextramine (10 or 100 µM; 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 noncompetitively 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 drugreceptor interaction mechanisms, the presence of significant irreversible noncompetitive antagonism may influence the interpretation of results under the experimental conditions used.

Address correspondence to: Christiaan B. Brink, Division of Pharmacology, Northwest University (PUK), Potchefstroom, 2520 South Africa. E-mail: fklcbb{at}puknet.puk.ac.za