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 Parkinson's and Alzheimer's diseases and drug addiction. The classical VMAT2 inhibitor tetrabenazine has long been used for the treatment of chorea associated with Huntington's disease in UK, Canada and Australia and recently approved in the US. The VMAT2 imaging may also be useful for exploiting the onset of diabetes mellitus, since 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 structurefunction 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 are also discussed.
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 VMAT's 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.
Specific uptake through dopamine transporter (DAT) followed by the inhibition of the mitochondrial complex-I have been accepted as the cause of the specific dopaminergic toxicity of 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 ROS 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 towards mitochondrial toxins that lead to the excessive production of ROS. Therefore the specific dopaminergic toxicity of MPP+ must also at least be 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.
In an initial communication [May, S. W., Mueller, P. W., Padgette, S. R., Herman, H. H., & Phillips, R. S. (1983) Biochem. Biophys. Res. Commun. 110, 161-168], we reported that 1-phenyl-1-(aminomethyl)ethene hydrochloride (PAME) is an olefinic substrate for dopamine beta-monooxygenase (DBM; EC 1.14.17.1) which inactivates the enzyme in an apparent mechanism-based manner. The present study further characterizes this reaction. The inactivation reaction yields kinact = 0.23 min-1 at pH 5.0 and 37 degrees C and is strictly dependent on reductant (ascorbate) and oxygen. The DBM/PAME substrate reaction (apparent kcat = 14 s-1), shown to be stimulated by fumarate, gives the corresponding epoxide as product, identified by derivatization with 4-(p-nitrobenzyl)pyridine. However, the lack of DBM inhibition by alpha-methylstyrene oxide, and the observation of identical PAME/DBM inactivation rates in the absence and presence of preformed enzymatic PAME epoxide, indicates that free epoxide is not the inactivating species. A structure-activity study revealed that 4-hydroxylation of PAME (to give 4-HOPAME) increases both kinact (0.81 min-1) and apparent kcat (56 s-1) values, while 3-hydroxylation (to give 3-HOPAME) greatly diminishes inactivation activity while retaining substrate activity (apparent kcat = 47 s-1). 4-Hydroxy-alpha-methylstyrene was found to be a DBM inhibitor (kinact = 0.53 min-1) with weak substrate activity (apparent kcat = 0.71 s-1), while 3-hydroxy-alpha-methylstyrene and alpha-(cyanomethyl) styrene were found not to exhibit detectable DBM substrate activity and only weak inhibitory activity. 3-Phenylpropargylamine hydrochloride showed no detectable DBM substrate activity but rapidly inactivated the enzyme. A new substrate activity for DBM was discovered, N-dealkylation of N-phenylethylenediamine and N-methyl-N-phenylethylenediamine, and the lack of O-dealkylation activity with phenyl 2-aminoethyl ether and 4-hydroxyphenyl 2-aminoethyl ether indicates that DBM N-dealkylation proceeds via initial one-electron abstraction from the benzylic nitrogen heteroatom. With this new substrate and inhibitor reactivity information in hand, along with the other known substrate reactions, a DBM oxygenation mechanism analogous to that for cytochrome P-450 is proposed.
The early proposal that P450-catalyzed N-dealkylation of N,N-dialkylamines proceeds through a single-electron-transfer (SET) mechanism was later challenged in favor of the C(alpha)-H abstraction mechanism. In the present study, a series of N-alkyl-N-cyclopropyl-p-chloroaniline probes have been used to examine whether the P450-catalyzed N-dealkylations proceed through a C(alpha)-H abstraction and/or a SET mechanism, using phenobarbital-induced rat liver microsomal P450 enzymes as a model system. While the findings are highly consistent with a C(alpha)-H abstraction mechanism, further experimental evidence may be necessary to completely rule out the SET mechanism.
A series of 3-amino-2-phenylpropene (APP) derivatives have been synthesized and characterized as novel competitive inhibitors, with K(i) values in the microM range, for the bovine chromaffin granule membrane monoamine transporter(s) (bVMAT). Although, these inhibitors are structurally similar to the bVMAT substrate tyramine, none of them were measurably transported into the granule. Structure-activity studies have revealed that, while the 3'- or 4'-OH groups on the aromatic ring enhance the inhibition potency, Me or OMe groups in these positions reduce the inhibition potency. Halogen substitution on the 4'-position of the aromatic ring causes gradual increase of the inhibition potency parallel to the electron donor ability of the halogen. Substituents on the NH(2) as well as on the 3-position of the alkyl chain reduce the inhibition potency. Comparative structure-activity analyses of APP derivatives with tyramine and the neurotoxin 1-methyl-4-phenylpyridinium suggest that the flexibility of the side chain and the relative orientation of the NH(2) group may be critical for the efficient transport of the substrate through the bVMAT. Comparable bVMAT affinities of these inhibitors to that of DA and other pharmacologically active amines suggest that they are suitable for the structure-activity and mechanistic studies of monoamine transporters and may also be useful in modeling the mechanism of action of amphetamine-related derivatives.
In previous work we have established that the "w-hydroxylation" system of P. oleouorans readily converts terminal olefins to the corresponding 1,2-oxides and does so stereoselectively. We also demonstrated loss of olefin configuration during enzymatic epoxidation, a result inconsistent with a concerted epoxidation mechanism ( J . Am. Chem. SOC. 1977,99, 2017-2024. Since loss of olefin configuration is unprecedented for monooxygenase-catalyzed epoxidations, these studies have been confirmed with isolated enzymes and further extended in order to probe the mechanism of non-heme iron monooxygenase catalysis. Enzymatic epoxidation of both cis-and trans-1-deuterio-1-octene proceeds with about 70% inversion of the olefinic configuration, with corresponding results being obtained for the two olefins. As we reported in a preliminary communication (Bio/ Technology 1983, I , 677-686), the w-hydroxylation system also produces aldehydes from olefins. Aldehyde formation exhibits the reaction characteristics expected for the usual oxygenase pathway. Deuterium migration from C-1 to C-2 occurs in formation of aldehyde from olefin, although loss of deuterium also occurs. The w-hydroxylation system was found to efficiently catalyze 0-demethylation of heptyl methyl ether, the first demonstration of such activity for a non-heme iron monooxygenase of this type. Taken together, the results provide support for a two-step mechanism involving enzyme-generated species with cationic and/or radical character, which accounts for the stereoselectivity, configurational loss, substrate specificity, formation of aldehydes with deuterium migration, and demethylation activity exhibited by this enzyme system.The mechanism by which oxygenases catalyze the insertion of molecular oxygen into organic molecules has been the subject of intense scrutiny in recent years, due to the importance of these enzymes in detoxification, oncogenesis, biosynthesis, and metabolism in general. However, while the P-450 and the flavin-containing monooxygenases have been intensively studied at the molecular level, the state of our understanding of the molecular basis of non-heme iron monooxygenase catalysis is, by comparison, poor indeed. Non-heme iron monooxygenases comprise a large class of enzymes which, in addition to the P. oleouorans system discussed here, includes the phenylalanine, tyrosine, and tryptophan monoxygenases as well as squalene epoxidase.1,2The non-heme iron monooxygenase system from P. oleouorans which catalyzes terminal methyl group hydroxylation of alkanes and fatty acids was first shown by Coon and co-workers to consist of three protein components: rubredoxin, a flavoprotein reductase, and non-heme iron m o n~x y g e n a s e .~-~~ In previous work from (1) Comprehensive reviews on oxygenases can be found in: (a) "The Enzymes", 3rd ed.; Boyer, P. D., Ed.; Academic Press: New York, 1975; vol. 12. (b) 'Molecular Mechanisms of Oxygen Activation"; Hayaishi, O., Ed.; Academic Press: New York, 1974.
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