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 from 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 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 (100 μM) 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 on NEM treated forebrain homogenates revealed an enhancement of ³H] CD binding by NEM.