CHARACTERIZATION OF [H-3] CYTISINE BINDING TO HUMAN BRAIN MEMBRANE PREPARATIONS

The binding characteristics of [H-3]cytisine, a putative CNS nicotinic receptor ligand, were examined in 4 regions of the human brain. [H-3]Cytisine was found to bind non-cooperatively with high affinity to a single site in tissue homogenates and to exhibit low non-specific association. The binding characteristics of this ligand were evaluated in thalamus at 4-degrees-C and 24-degrees-C. The association constants were found to be 0.234 and 0.308 min-1 nM-1, while the dissociation constants were 0.007 and 0.098 min-1, respectively. Saturation analysis of thalamus revealed the equilibrium K(d) to be 147 pM (4-degrees-C) and 245 pM (24-degrees-C), values in good agreement with those determined kinetically. The Hill coefficient varied slightly between brain regions; however, the mean values in all regions examined were close to 1.0 at 0.95 +/- 0.03 (4-degrees-C) and 0.91 +/- 0.04 (24-degrees-C). [H-3]Cytisine binding could be displaced using both nicotinic agonists and antagonists. Cytisine was the most potent displacer of [H-3]cytisine binding with an K(i) of 250 pM. Nicotine and acetylcholine were also potent displacers with K(i) values of 1.8 and 8.1 nM, respectively. The nicotinic antagonists alpha-bungarotoxin and mecamylamine were ineffective competitors for the [H-3]cytisine binding site while dihydro-beta-erythroidine had an K(i) value of 109 nM. Thalamus showed the highest density of cytisine binding sites of all the regions examined (48 fmol/mg protein) while the hippocampus, cingulate gyrus and the cortex showed B(max) values of 18.9, 19.3 and 8.8 fmol/mg protein, respectively. The results of this characterization of [H-3]cytisine binding are in excellent agreement with previously published reports on the nicotinic cholinergic receptor population in the human brain. We conclude that [H-3]cytisine exhibits both high affinity for the receptor and a very low non-specific binding component which makes this a useful ligand for the quantitation of nicotinic receptors in tissues with low receptor density.