Regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide

The regulation of muscarinic receptor binding by guanine nucleotides and N-ethylmaleimide (NEM) was investigated using the agonist ligand, [³H] cis methyldioxolane ([³H]CD). Characterization studies on rat forebrain homogenates showed that [³H]CD binding was linear with tissue concentration and was unaffected by a change in pH form 5.5 to 8.0. The regional variation in [³H]CD binding in the rat brain correlated generally with [³H](-)3-quinuclidinyl benzilate ([³H](-)QNB) binding, although the absolute variation in binding was somewhat less. At a concentration of 100 μM, the GTP analogue, guanyl-5'-yl imidodiphosphate [Gpp(NH)p], caused a 43-77% inhibition of [³H]CD binding in the corpus striatum, ileum, and heart. The results of binding studies using several Gpp(NH)p concentrations demonstrated that the potency of this guanine nucleotide for inhibition of [³H]CD binding was greater in the heart than in the ileum. In contrast to its effects on [³H]CD binding, Gpp(NH)p caused an increase in [³H](-)QNB binding in the heart and ileum and no change in [³H](-)QNB binding in the corpus striatum. When measured by competitive inhibition of [³H](-)QNB binding to the longitudinal muscle of the ileum, Gpp(NH)p (100μM) caused an increase in the IC₅₀ values of a series of agonists in a manner that was correlated with the efficacy of these compounds. The results of binding studies in NEM treated forebrain homogenates revealed an enhancement of [³H]CD binding by NEM.