Increasing evidence suggests that the dopamine transporter is situated almost exclusively on dopamine neurons. Accordingly, it is an valuable marker for Parkinson's disease and other pathological states of dopamine neurons. We previously demonstrated that the potent dopamine transport inhibitor [125I]altropane (IACFT:E-N-iodoallyl-2beta-carbomethoxy-3beta-(4-fluor ophenyl)tropane) is a high affinity selective probe for the dopamine transporter in monkey brain and an effective SPECT imaging agent in nonhuman primate brain. We now report the binding properties of [125I]altropane in postmortem tissue of normal human brain and compare the findings to Parkinson's diseased brain. In homogenates of human brain putamen, [125I]altropane bound with high affinity (KD: 4.96 +/- 0.38 nM, n = 4) and site density (BMAX: 212 +/- 41.1 pmol/g original wet tissue weight) well within the density range reported previously for the dopamine transporter in this brain region. Drugs inhibited [125I]altropane binding with a rank order of potency that corresponded closely to their rank order for blocking dopamine transport (r 0.98, P < 0.001). In postmortem Parkinson's diseased brain, bound [125I]altropane (1 nM) was markedly reduced (89%, 99% in putamen, depending on measures of nonspecific binding) compared with normal aged-matched controls (normal putamen: 49.2 +/- 8.1 pmol/g; Parkinson's diseased putamen: 0.48 +/- 0.33 pmol/g; n = 4). In vitro autoradiography, conducted in tissue sections at a single plane of the basal ganglia, revealed high levels of [125I]altropane binding the caudate nucleus and putamen, but lower levels (73% of the caudate-putamen) in the nucleus accumbens (n = 7). In Parkinson's diseased brains (n = 4), [125I]altropane binding was 13% of the levels detected in normal putamen, 17% of normal values in the caudate nucleus, and 25% of normal levels in nucleus accumbens. The association of [125I]altropane to the dopamine transporter in human postmortem tissue, the marked reduction of [125I]altropane binding in Parkinson's diseased brains, its rapid entry into brain and highly localized distribution in dopamine-rich brain regions, support its use as a probe for monitoring the dopamine transporter in vitro and in vivo by SPECT imaging.

Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP1 synaptic vesicles banding at 1.055 g/mL actively transported [3H]acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more [3H]AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP1 vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP2 synaptic vesicle originally described in intact Torpedo marmorata electric organ [Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 695-714; Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 715-730]. The properties of the receptor for vesamicol were studied by measuring binding of [3H]vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP1 vesicles the VP2 vesicles had a ratio of [3H]AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. Based on an antibody standardization, in a typical preparation the VP1 vesicles contained 237 +/- 15 pmol of receptor/mg of protein whereas VP2 vesicles contained 102 +/- 3 pmol of receptor/mg of protein, and VP2 vesicles transported AcCh 2-3-fold more actively than VP1 vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

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The dopamine transporter in brain, localized almost exclusively on dopamine neurons, is an effective window on dopamine neurons. SPECT or PET imaging of the transporter in brain requires selective imaging agents that display appropriate pharmacokinetic properties. We previously reported that [125I]altropane ([125I]IACFT,2beta-carbomethoxy-3beta-(4-fluorophenyl)-n-(1- iodoprop-1-en-3-yl)nortropane) bound with high affinity (Kd: 5.33 nM) to a single site on the dopamine transporter and was selective for dopamine over the serotonin transporter in homogenates of monkey striatum. To determine whether the selective binding of [125I]altropane is reflected in its brain distribution, the in vitro and ex vivo distribution of [125I]altropane in squirrel monkey (Saimiri sciureus) brain was determined by quantitative autoradiography of coronal brain sections. In vitro, [125I]altropane (2 nM) distribution was discrete and was detectable primarily in the dopamine-rich putamen, caudate nucleus, and nucleus accumbens. The resulting putamen:cerebellum ratio exceeded 120:1 (n = 3). The selective in vitro binding of [125I]altropane to the dopamine transporter, at concentrations approaching its Kd value (Kd: 5.33 nM, a single high affinity site), highlight its suitability for investigating the density of the dopamine transporter in various brain regions in vitro. Ex vivo autoradiography was conducted in monkeys to determine whether the brain distribution of [125I]altropane in vitro was predictive of its brain distribution pattern after intravenous administration. Thirty minutes after intravenous injection, highest levels of [125I]altropane (0.3 nmol/kg) were detected in the caudate-putamen and nucleus accumbens and lowest levels in the cerebellum and cortex. The putamen or caudate:cerebellum ratio was 7. SPECT imaging of the brain within 30 min of i.v. injection confirmed the rapid and selective accumulation of [123I]altropane to the striatum. The selective binding of altropane to the dopamine-rich striatum within 30 min of i.v. administration indicates that it is uniquely suited for SPECT or PET imaging of the dopamine transporter and associated dopamine neurons.