A simple and rapid radio-receptor assay for the estimation of acetylcholine

The high potency with which acetylcholine (ACh) inhibits the binding of the specific muscarinic agonist, ³H]$\text{cis}$ methyldioxolane (³H]CD), has provided the basis for the development of a radio-receptor assay for estimation of ACh. A synaptosomal preparation of the rat cerebral cortex was used as a source of muscarinic receptors. When binding assays were run at 0°C, the IC₅₀ value of ACh was approximately 5 × 10^?9 M, which corresponds to 2.5 – 10 pmoles of ACh, depending upon the assay volume. The ACh content of the rat cerebral cortex and corpus striatum was measured following fast microwave irradiation. By measuring the displacement of ³H]CD binding caused by aliquots of the supernatant from tissue homogenates and comparing the displacement values with an ACh standard curve, the ACh content of the cerebral cortex and corpus striatum was calculated to be 19 and 55 nmoles/g wet tissue weight, respectively.