Influence of allosteric modulators of muscarinic receptors on the dissociation characteristics of radioligands of A1-adenosine-, α1 - and β-adrenergic receptors.
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文摘
In order to demonstrate the specificity of allosteric modulators of the M2-muscarinic receptor, the dissociation characteristics of ligands of adenosine A1-, α1- and β-adrenergic receptors have been studied. For this purpose the effects of three representative allosteric modulators, alcuronium (ALC), W84 (N,N,N′,N′-tetramethyl-N,N′-bis-(3-phthalimido-propyl)-N,N′-hexane-1,6,-diyl-bis-ammonium dibromide), and gallamine (GAL) on the equilibrium binding and the dissociation of the adenosine A1-receptor ligand [3H]-cyclopentyl-dipropylxanthin (CPX), the α1-adrenoceptor ligand [3H]-prazosin (PRAZ) and the β-adrenoceptor ligand (−)-[125I]-iodocyanopindolol (ICYP) have been investigated. The membrane fraction of homogenized rat cardiac left ventricle (ICYP, PRAZ) or rat whole forebrain (CPX) was incubated for 45 min with PRAZ or for one hour with ICYP or CPX. At the end of the incubation period the equilibrium binding or, after the addition of an excess concentration of unlabelled cyclopentyl-dimethylxanthin (A1, 10 μM), phentolamine (α, 10 μM) or (±)-CGP 12,177 (β, 10μM) the dissociation was determined in the presence or the absence of the allosteric modulators. All values are given as mean ± SEM of 4-6 observations. CPX showed a monophasic dissociation pattern with a half-life of 6.0 ± 0.3 min in the absence and of 6.0 ± 0.2, 6.5 ± 0.1 and 6.0 ± 0.1 min. in the presence of 30 μM ALC, 30 μM W84 and 1000 μM GAL, respectively. No influence on the CPX-equilibrium binding was detectable. The same dissociation pattern was observed for PRAZ with the respective half-life of 16 ± 1 min in the absence and of 17 ± 1, 8 ± 1 and 13 ± 1 min. In the presence of the allosteric modulators, the PRAZ equilibrium binding amounted to 36 ± 2 % (ALC), 93 ± 2 % (W84) and 42 ± 2 % (GAL) of control. Apparently, ALC and GAL interfere with the association of PRAZ. Since the dissociation of ICYP was slow and after 3 hours more than 40 % of the radioligand were still bound, no reliable half-life could be calculated. However, at no time point the binding of ICPY in the absence and the presence of 30 μM W84 showed differences. In the presence of GAL and ALC, the dissociation curve of ICYP revealed an initial offset from the starting level followed by the major phase of dissociation being parallel to the control curve. The equilibrium binding of ICYP amounted to 92 ± 6 % (ALC), 83 ± 8 % (W84) and 91 ± 5 % (GAL) of control. In conclusion, the allosteric alteration of radioligand dissociation by ALC, W84, and GAL does not appear to be a common feature of G-protein coupled receptors.