In vitro characterization of [

Vesicular acetylcholine transporter plays a crucial role in the cholinergic system, and its alterations is implicated in several neurodegenerative disorders. We recently developed a PET imaging tracer [(18)F]VAT to target VAChT in vivo with high affinity and selectivity. Here we report in vitro characterization of [(3)H]VAT, a tritiated counterpart of [(18)F]VAT. Using human VAChT-rich cell membrane extracts, a saturated binding curve was obtained for [(3)H]VAT with K(d) = 6.5 nM and B(max) = 22.89 pmol/mg protein. In the [(3)H]VAT competition-binding assay with a panel of CNS ligands, binding inhibition of [(3)H]VAT was observed using VAChT ligands, the K(i) values ranged from 5.41 to 33.3 nM. No inhibition was detected using a panel of other CNS ligands. In vitro [(3)H]VAT autoradiography of rat brain sections showed strong signals in the striatum, moderate to high signals in vermis, thalamus, cortex, and hippocampus, and weak signals in cerebellum. Strong [(3)H]VAT ARG signals were also observed from striatal sections of normal nonhuman primates and human brains. Competitive ARG study with human striatal sections demonstrated strong ARG signals of [(3)H]VAT in caudate and putamen were blocked significantly by either VAChT ligand TZ659 or (−)-vesamicol, but not by the σ(1) receptor ligand Yun-122. ARG study also indicated that signal in the striatal sections from PSP human brains was lower than normal human brains. These data provide solid evidence supporting [(18)F]VAT as a suitable PET radiotracer for quantitative assessment of VAChT levels in vivo.