Nature of nicotine binding to rat brain P 2 fraction

Abstract

(−)-Nicotine may bind to as many as 5 sites in the rat brain P 2 preparation: A very high affinity site (K D∼2.2×10 −11 M); a positive cooperativity site; a high affinity site (K D∼5.2×10 −9 M); a low affinity site (K D∼4.5×10 −5 M) and a very low affinity site. The curvilinear nature of both Scatchard plots and kinetic curves indicates the presence of multiple binding sites. Evidence for a positive cooperativity site includes: (1) The configuration of Scatchard plots (at low concentrations) of saturation as well as inhibition curves for (−)- and (+)-nicotine. (2) The Hill number of 1.37 for the binding of low concentrations of (±)-[ 3 H]nicotine . (3) Selectivity among cholinergic drugs for producing positive cooperativity. (4) Markedly different specificities of drugs for the positive cooperativity site. Thus while only (+)- and (−)-nicotine interacted with the very high affinity site, acetylcholine, atropine, mecamylamine, lobeline, carbachol, (+)-nicotine and (−)-nicotine enhanced the binding of (±)-[ 3 H]nicotine and cytisine, anabasine, cotinine and choline selectively inhibited binding at the high affinity site. Several lines of evidence indicate that there is stereospecificity. (+)-Nicotine was more potent than (−)-nicotine in inducing positive cooperativity whereas (−)-nicotine was 80 times more potent than (+)-nicotine in inhibiting binding at the high affinity site. Further, the specificity of the binding sites can be altered by changing the concentration of the buffer which gives additional evidence for the lability of the nicotine binding site. Although the pharmacologic significance of the different binding sites has not been determined, these data taken together indicate that (±)-[ 3 H]nicotine binds with specificity to multiple sites in the rat brain P 2 preparation with a complexity not addressed heretofore.