In vitro binding of³H-acetylcholine to nicotinic receptors in rodent and human brain

The binding of³H-acetylcholine (³H-ACh) to nicotinic receptors in rodent and human brain was measured in the presence of atropine to prevent binding to muscarinic binding sites.³H-ACh binds specifically and saturably to rodent brain. From saturation binding K_d was 30 nM in rat cerebral cortex, which is close to that calculated from kinetic experiments. The binding was temperature-dependent, being highest at low temperatures and decreasing at higher temperatures. The regional distribution of binding in mouse brain was not uniform. The binding was highest in the midbrain, intermediate in the cerebral cortex and striatum, and lowest in the cerebellum, hippocampus, hypothalamus and medulla oblongata. No significant correlation was found between the regional³H-ACh binding and the regional binding of³H-alpha-bungarotoxin (³H-BTX),³H-nicotine (³H-NIC),³H-tubocurarine and the endogenous acetylcholine content, although the correlation value for³H-ACh/³H-NIC was at the limit for significance.³H-ACh also bound specifically to human cerebral cortical tissue and this binding was approximately three times lower than in rodent brain, when a low³H-ACh concentration was used. In contrast to rat brain there appears to exist multiple binding sites for³H-ACh in human cerebral cortex as suggested by the curvelinear nature of the Scatchard plot. It was calculated that³H-ACh bound with K_d 4 nM and B_max 8 pmol/g protein and K_d 112 nM and B_max 67 pmol/g protein. The Hill number of 1.5 for the binding of low concentration and 2.5 for high concentration of³H-ACh also suggest that the³H-ACh-binding sites interaction exhibit positive cooperativity.