Trapping of Nicotinic Acetylcholine Receptor Ligands Assayed by In Vitro Cellular Studies and In Vivo PET Imaging

A question relevant to nicotine addiction is how nicotine and other nicotinic receptor membrane-permeant ligands, such as the anti-smoking drug varenicline (Chantix), distribute in brain. Ligands, like varenicline, with high pKa and high affinity for cc4P2-type nico-tinic receptors (cc4P2Rs) are trapped in intracellular acidic vesicles containing cc4P2Rs in vitro. Nicotine, with lower pKa and cc4P2R affinity, is not trapped. Here, we extend our results by imaging nicotinic PET ligands in vivo in male and female mouse brain and identifying the trapping brain organelle in vitro as Golgi satellites (GSats). Two PET 18F-labeled imaging ligands were chosen: [18F]2-FA85380 (2-FA) with varenicline-like pKa and affinity and [18F]Nifene with nicotine-like pKa and affinity. [18F]2-FA PET-imaging kinetics were very slow consistent with 2-FA trapping in cc4P2R-containing GSats. In contrast, [18F]Nifene kinetics were rapid, consist-ent with its binding to cc4P2Rs but no trapping. Specific [18F]2-FA and [18F]Nifene signals were eliminated in P2 subunit knock-out (KO) mice or by acute nicotine (AN) injections demonstrating binding to sites on P2-containing receptors. Chloroquine (CQ), which dissipates GSat pH gradients, reduced [18F]2-FA distributions while having little effect on [18F]Nifene distributions in vivo consistent with only [18F]2-FA trapping in GSats. These results are further supported by in vitro findings where dissipation of GSat pH gradients blocks 2-FA trapping in GSats without affecting Nifene. By combining in vitro and in vivo imaging, we mapped both the brain-wide and subcellular distributions of weak-base nicotinic receptor ligands. We conclude that ligands, such as varenicline, are trapped in neurons in cc4P2R-containing GSats, which results in very slow release long after nicotine is gone after smoking.

Automated summary

The distribution of nicotine and other nicotinic receptor ligands in the brain is investigated. Ligands with high pKa and affinity for cc4P2Rs, like varenicline, are trapped in intracellular acidic vesicles in vitro, while nicotine, with lower pKa and affinity, is not trapped. In vivo imaging of PET ligands in mouse brain reveals that the trapping organelle is Golgi satellites (GSats) and provides insights into the subcellular distribution of these ligands.