Probing the selectivity of allosteric modulators of muscarinic receptors at other G-protein-coupled receptors

1 The aim of the present investigation was to analyse whether three prototy pe allosteric modulators of ligand binding to muscarinic receptors, i.e. al curonium, gallamine, and the alkane-bis-ammonium compound W84 (hexane-1,6-b is[dimethyl-3'-phthalimidopropylammonium bromide]), may have allosteric eff ects on radioligand-binding characteristics at other G-protein-coupled rece ptors, such as cerebral A, adenosine receptors (G(i)-coupled), cardiac left ventricular alpha (1)-adrenoceptors (G(q)), and beta -adrenoceptors (G(s)) . 2 The modulators were applied at concentrations known to be high with regar d to the allosteric delay of the dissociation of the antagonist [H-3]-N-met hylscopolamine (NMS) from muscarinic M-2-receptors: 30 mu mol l(-1) W84, 30 mu mol l(-1) alcuronium, 1000 mu mol l(-1) gallamine. As radioligands, we used the adenosine A(1)-receptor ligand [H-3]-cyclopentyl-dipropylxanthine (CPX), the alpha (1)-adrenoceptor ligand [H-3]-prazosin (PRAZ), and the bet a -adrenoceptor ligand (-)-[I-125]-iodocyanopindolol (ICYP). Allosteric act ions on ligand dissociation and the equilibrium binding were measured in th e membrane fractions of rat whole forebrain (CPX) and of rat cardiac left v entricle (PRAZ, ICYP, NMS), respectively. 3 CPX and PRAZ showed a monophasic dissociation with half-lives of 5.88 +/- 0.15 and 12.27 +/- 0.46 min, respectively. In the case of CPX, neither the binding at equilibrium nor the dissociation characteristics were influence d by the allosteric agents. With PRAZ, the binding at equilibrium remained almost unaltered in the presence of W84, whereas it was reduced to 36 +/- 2 % of the control value with alcuronium and to 42 +/- 2% with gallamine. The dissociation of PRAZ was not affected by W84, whereas it was moderately ac celerated by alcuronium and gallamine. In the case of ICYP, the binding at equilibrium was not affected by the allosteric modulators. The dissociation of ICYP was slow, and after 3 h, more than 50% of the radioligand was stil l bound, so that a reliable half-life could not be calculated. ICYP dissoci ation was not affected by W84. In the presence of alcuronium and gallamine, the dissociation curve of ICYP revealed an initial drop from the starting level, followed by the major phase of dissociation being parallel to the co ntrol curve. 4 In summary, the allosteric action of the applied agents is not a common f eature of G-protein-coupled receptors and appears to be specific for muscar inic receptors.