A new assay technique for measuring receptor-mediated cyclic GMP formation by cultured mouse neutroblastoma cells was used to study the role of ions in, and the effects of local anesthetics on, the function of the muscarinic receptor. The technique involved radioactively labeling intracellular stores of GTP by incubating cells with [3H]guanine and isolating [3H]cyclic GMP with a cation exchange resin (Dowex-50+) column. High-pressure liquid chromatography of cell extracts and of eluates from the Dowex column showed that after 45 min the majority of the radioactivity in the cell extracts was [3H]GTP and that, for carbamylcholine-stimulated cells, greater than 90 per cent of the radioactivity in the eluates was [3H]cyclic GMP. In the absence of external Na+ ([Na+]e, with cesium chloride as osmotic filler) or external ([Ca2+]e), the carbamylcholine-stimulated formation of [3H]cyclic GMP was about 60 and 10 per cent of control, respectively, while removal of other ions had no significant effect. There was little difference in the responses at 10 mM vs 110 mM-[Na+]e, whereas the optimal [Ca2+], was around 5 mM. Ca2+ increased [3H]cyclic GMP formation in response to carbamylcholine without affecting the apparent affinity of this agonist for the receptor. Local anesthetics were apparently competitive inhibitors of carbamylcholine with equilibrium dissociation constants (KB) in the range of 6-250 μM. The rank order for the apparent affinity of local anesthetics for the muscarinic receptor was tetracaine = butacaine = procaine > dibucaine = lidocaine > ethyl aminobenzoate.
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