Kandatege Wimalasena

Vesicular Monoamine Transporters: Structure-Function, Pharmacology, and Medicinal Chemistry

Vesicular monoamine transporters (VMAT) are responsible for the uptake of cytosolic monoamines into synaptic vesicles in monoaminergic neurons. Two closely related VMATs with distinct pharmacological properties and tissue distributions have been characterized. VMAT1 is preferentially expressed in neuroendocrine cells and VMAT2 is primarily expressed in the CNS. The neurotoxicity and addictive properties of various psychostimulants have been attributed, at least partly, to their interference with VMAT2 functions. The quantitative assessment of the VMAT2 density by PET scanning has been clinically useful for early diagnosis and monitoring of the progression of Parkinsons and Alzheimers diseases and drug addiction. The classical VMAT2 inhibitor, tetrabenazine, has long been used for the treatment of chorea associated with Huntingtons disease in the United Kingdom, Canada, and Australia, and recently approved in the United States. The VMAT2 imaging may also be useful for exploiting the onset of diabetes mellitus, as VMAT2 is also expressed in the β‐cells of the pancreas. VMAT1 gene SLC18A1 is a locus with strong evidence of linkage with schizophrenia and, thus, the polymorphic forms of the VMAT1 gene may confer susceptibility to schizophrenia. This review summarizes the current understanding of the structure–function relationships of VMAT2, and the role of VMAT2 on addiction and psychostimulant‐induced neurotoxicity, and the therapeutic and diagnostic applications of specific VMAT2 ligands. The evidence for the linkage of VMAT1 gene with schizophrenia and bipolar disorder I is also discussed.

Mechanisms of amphetamine action illuminated through optical monitoring of dopamine synaptic vesicles in Drosophila brain

Amphetamines elevate extracellular dopamine, but the underlying mechanisms remain uncertain. Here we show in rodents that acute pharmacological inhibition of the vesicular monoamine transporter (VMAT) blocks amphetamine-induced locomotion and self-administration without impacting cocaine-induced behaviours. To study VMATs role in mediating amphetamine action in dopamine neurons, we have used novel genetic, pharmacological and optical approaches in Drosophila melanogaster. In an ex vivo whole-brain preparation, fluorescent reporters of vesicular cargo and of vesicular pH reveal that amphetamine redistributes vesicle contents and diminishes the vesicle pH-gradient responsible for dopamine uptake and retention. This amphetamine-induced deacidification requires VMAT function and results from net H+ antiport by VMAT out of the vesicle lumen coupled to inward amphetamine transport. Amphetamine-induced vesicle deacidification also requires functional dopamine transporter (DAT) at the plasma membrane. Thus, we find that at pharmacologically relevant concentrations, amphetamines must be actively transported by DAT and VMAT in tandem to produce psychostimulant effects.

Autocatalytic Radical Ring Opening ofN-Cyclopropyl-N-phenylamines Under Aerobic Conditions − Exclusive Formation of the Unknown Oxygen Adducts,N-(1,2-Dioxolan-3-yl)-N-phenylamines

In contrast to the high stability of N-alkyl-N-cyclopropylamine derivatives, N-cyclopropyl-N-phenylamine (1a) has been found to slowly convert into the hitherto unknown product N-(1,2-dioxolan-3-yl)-N-phenylamine (1f) at room temperature under aerobic conditions. The rate of this conversion was found to be significantly increased by the presence of a catalytic amount of the single-electron oxidizing agent tris(1,10-phenanthroline)FeIII hexafluorophosphate or of the hydrogen-abstracting agents benzoyl peroxide or tert-butyl peroxide/UV light. Based on the regio- and stereochemical outcomes of aerobic ring-opening reactions of some specifically ring-methylated derivatives of 1a, namely N-(1-methylcyclopropyl)-N-phenylamine (2a), N-(trans-2-methylcyclopropyl)-N-phenylamine (3a), and N-(cis-2-methylcyclopropyl)-N-phenylamine (4a), as well as other experimental evidence, an autocatalytic mechanism analogous to that of the oxygenation of vinylcyclopropanes is proposed for the formation of the 1,2-dioxolane product. The oxidative radical ring opening and related chemistry of these novel derivatives could be valuable in mechanistic studies of heteroatom-oxidizing enzymes.

Copper ions disrupt dopamine metabolism via inhibition of V-H+-ATPase: a possible contributing factor to neurotoxicity.

The involvement of copper in the pathophysiology of neurodegeneration has been well documented but is not fully understood. Commonly, the effects are attributed to increased reactive oxygen species (ROS) production due to inherent redox properties of copper ions. Here we show copper can have physiological effects distinct from direct ROS production. First, we show that extragranular free copper inhibits the vesicular H+‐ATPase of resealed chromaffin granule ghosts. Extragranular ascorbate potentiates this inhibition. The inhibition is mixed type with Kis = 6.8 ± 2.8 µmol/L and Kii = 3.8 ± 0.6 µmol/L, with respect to ATP. Second, extracellular copper causes an inhibition of the generation of a pH‐gradient and rapid dissipation of pre‐generated pH and catecholamine gradients. Copper chelators and the ß‐amyloid peptide 1–42 were found to effectively prevent the inhibition. The inhibition is reversible and time‐independent suggesting the effects of extracellular copper on H+‐ATPase is direct, and not due to ROS. The physiological significance of these observations was shown by the demonstration that extracellular copper causes a dramatic perturbation of dopamine metabolism in SH‐SY5Y cells. Thus, we propose that the direct inhibition of the vesicular H+‐ATPase may also contribute to the neurotoxic effects of copper.

The Reduction of Membrane-bound Dopamine β-Monooxygenase in Resealed Chromaffin Granule Ghosts IS INTRAGRANULAR ASCORBIC ACID A MEDIATOR FOR EXTRAGRANULAR REDUCING EQUIVALENTS?

The role of internal and external reductants in the dopamine β-monooxygenase (DβM)-catalyzed conversion of dopamine to norepinephrine has been investigated in resealed chromaffin granule ghosts. The rate of norepinephrine production was not affected by the exclusion of internal ascorbate. The omission of ascorbate from the external medium drastically reduced the norepinephrine production without affecting the net rate of dopamine uptake. In the presence of the external reductant, the internal ascorbate levels were constant throughout the incubation period. The rate of norepinephrine production was not affected when ghosts were resealed to contain the DβM reduction site inhibitor, imino-D-glucoascorbate. Ghosts incubated with external imino-D-glucoascorbate reduced the norepinephrine production. The weak DβM reductant, 6-amino-L-ascorbic acid, was found to be a good external reductant for granule ghosts. The outcome of the above experiments was not altered when dopamine was replaced with the reductively inactive DβM substrate, tyramine. These results and the known topology of membrane-bound DβM disfavor the direct reduction of the enzyme by the external reductant. Our observations are consistent with the hypothesis that external ascorbate is the sole source of reducing equivalents for DβM monooxygenation and that internal soluble ascorbate (or dopamine) may not directly reduce or mediate the reduction of membrane-bound DβM in resealed granule ghosts.

N,N,N′,N′-Tetramethyl-1,4-phenylenediamine: a facile electron donor and chromophoric substrate for dopamine β-monooxygenase

Abstract Dopamine β-monooxygenase is shown to catalyze the oxidation of N,N,N′,N′-tetramethyl-1,4-phenylenediamine (TMPD) to its cation radical in the presence of a regular substrate and molecular oxygen. The enzyme-mediated oxidation of TMPD is stoichiometrically coupled with the hydoxylation of the substrate to the corresponding enzymatic product. TMPD is kinetically well behaved as an alternate electron donor for the enzyme with a potency comparable to that of the most efficient electron donor, ascorbate. Dopamine β-monooxygenase mediated oxidation of TMPD has been employed to design a convenient and sensitive spectrophotometric assay for the enzyme. The finding that TMPD is a well behaved facile alternate electron donor for dopamine β-monooxygenase raises some interesting novel questions regarding the specificity and chemistry of the reduction site, which may have important implications on the reduction of active site coppers of the enzyme.

Reduction of Dopamine β-Monooxygenase A UNIFIED MODEL FOR APPARENT NEGATIVE COOPERATIVITY AND FUMARATE ACTIVATION

The interactions of reductants with dopamine β-monooxygenase (DβM) were examined using two novel classes of reductants. The steady-state kinetics of the previously characterized DβM reductant, N,N-dimethyl-1,4-p-phenylenediamine (DMPD), were parallel to the ascorbic acid-supported reaction with respect to pH dependence and fumarate activation. DMPD also displayed pH and fumarate-dependent apparent negative cooperativity demonstrating that the previously reported cooperative behavior of DβM toward the reductant is not unique to ascorbic acid. The 6-OH phenyl and alkylphenyl-substituted ascorbic acid derivatives were more efficient reductants for the enzyme than ascorbic acid. Kinetic studies suggested that these derivatives behave as pseudo bisubstrates with respect to ascorbic acid and the amine substrate. The lack of apparent cooperative behavior with these derivatives suggests that this behavior of DβM is not common for all the reductants. Based on these findings and additional kinetic evidence, the proposal that the apparent negative cooperativity in the interaction of ascorbic acid with DβM was due to the presence of a distinct allosteric regulatory site has been ruled out. In contrast to previous models, where fumarate was proposed to interact with a distinct anion binding site, the effect of fumarate on the steady-state kinetics of these novel reductants suggests that fumarate and the reductant may interact with the same site of the enzyme. In accordance with these observations and mathematical analysis of the experimental data, a unified model for the apparent negative cooperativity and fumarate activation of DβM in which both fumarate and the reductant interact with the same site of all forms of the enzyme with varying affinities under steady-state turnover conditions has been proposed.

2, 2′- and 4, 4′-Cyanines are transporter-independent in vitro dopaminergic toxins with the specificity and mechanism of toxicity similar to MPP+

Specific uptake through dopamine transporter followed by the inhibition of the mitochondrial complex‐I have been accepted as the cause of the specific dopaminergic toxicity of 1‐methyl‐4‐phenylpyridinium (MPP+). However, MPP+ is taken up into many cell types through other transporters, suggesting that, in addition to the efficient uptake, intrinsic vulnerability of dopaminergic cells may also contribute to their high sensitivity to MPP+ and similar toxins. To test this possibility, two simple cyanines were employed in a comparative study based on their unique characteristics and structural similarity to MPP+. Here, we show that they freely accumulate in dopaminergic (MN9D and SH‐SY5Y) as well as in liver (HepG2) cells, but are specifically and highly toxic to dopaminergic cells with IC50s in the range of 50–100 nM, demonstrating that they are about 1000‐fold more toxic than MPP+ under similar experimental conditions. They cause mitochondrial depolarization non‐specifically, but increase the reactive oxygen species specifically in dopaminergic cells leading to the apoptotic cell death parallel to MPP+. These and other findings suggest that the specific dopaminergic toxicity of these cyanines is due to the inherent vulnerability of dopaminergic cells toward mitochondrial toxins that lead to the excessive production of reactive oxygen species. Therefore, the specific dopaminergic toxicity of MPP+ must also be, at least partly, due to the specific vulnerability of dopaminergic neurons. Thus, these cyanines could be stronger in vivo dopaminergic toxins than MPP+ and their in vivo toxicities must be evaluated.

Perturbation of Dopamine Metabolism by 3-Amino-2-(4′-halophenyl)propenes Leads to Increased Oxidative Stress and Apoptotic SH-SY5Y Cell Death

We have recently characterized a series of 3-amino-2-phenyl-propene (APP) derivatives as reversible inhibitors for the bovine adrenal chromaffin granule vesicular monoamine transporter (VMAT) that have been previously characterized as potent irreversible dopamine-β-monooxygenase (DβM) and monoamine oxidase (MAO) inhibitors. Halogen substitution on the 4′-position of the aromatic ring gradually increases VMAT inhibition potency from 4′-F to 4′-I, parallel to the hydrophobicity of the halogen. We show that these derivatives are taken up into both neuronal and non-neuronal cells, and into resealed chromaffin granule ghosts efficiently through passive diffusion. Uptake rates increased according to the hydrophobicity of the 4′-substituent. More importantly, these derivatives are highly toxic to human neuroblastoma SH-SY5Y but not toxic to M-1, Hep G2, or human embryonic kidney 293 non-neuronal cells at similar concentrations. They drastically perturb dopamine (DA) uptake and metabolism in SH-SY5Y cells under sublethal conditions and are able to deplete both vesicular and cytosolic catecholamines in a manner similar to that of amphetamines. In addition, 4′-IAPP treatment significantly increases intracellular reactive oxygen species (ROS) and decreases glutathione (GSH) levels in SH-SY5Y cells, and cell death is significantly attenuated by the common antioxidants α-tocopherol, N-acetyl-l-cysteine and GSH, but not by the nonspecific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. DNA fragmentation analysis further supports that cell death is probably due to a caspase-independent ROS-mediated apoptotic pathway. Based on these and other findings, we propose that drastic perturbation of DA metabolism in SH-SY5Y cells by 4′-halo APP derivatives causes increased oxidative stress, leading to apoptotic cell death.

The inherent high vulnerability of dopaminergic neurons toward mitochondrial toxins may contribute to the etiology of Parkinson's disease.

Although the exact mechanism(s) of the degeneration of dopaminergic neurons in Parkinsons disease (PD) is not well understood, mitochondrial dysfunction is proposed to play a central role. This proposal is strongly strengthened by the findings that compromised mitochondrial functions and/or exposure to mitochondrial toxins such as rotenone, paraquat, or MPTP causes degeneration of the midbrain dopaminergic system and manifest symptoms similar to Parkinsons disease in primates and rodents (Goldman, 2014). In fact, the specific dopaminergic toxin MPTP is one of the most commonly used models in the mechanistic studies of environmental factors associated with the etiology of PD, particularly due to the availability of direct and unequivocal clinical and biochemical evidence from human and primate subjects. Several decades of intense studies in many laboratories have led to the proposition of a general mechanism for the specific dopaminergic toxicity of MPTP (Przedborski and Jackson-Lewis, 1998). The salient features of this mechanism are (a) lipophilic pro-toxin, MPTP, freely crosses the blood-brain barrier and enters the brain; (b) in glial cells monoamine oxidase-B converts it to the toxic metabolite MPP+; (c) the polar MPP+ is extruded into the extracellular space through organic cation transporter-3; (d) presynaptic dopamine transporter (DAT) takes it up specifically into dopaminergic neurons; (e) in dopaminergic neurons, MPP+ accumulates in the synaptic vesicles and/or mitochondria; (f) mitochondrial MPP+ inhibits the mitochondrial complex-I of the electron transport chain, leading to cellular ATP depletion and excessive reactive oxygen species (ROS) production causing apoptotic cell death (Lotharius and O’Malley, 2000; Storch et al., 2004). Although this mechanism is generally well accepted, numerous recent studies seriously challenge the central dogma of this proposal that the specific dopaminergic toxicity of MPP+ is primarily due to the specific uptake into dopaminergic neurons through presynaptic DAT.