Trent J. Volz

Psychostimulant-induced alterations in vesicular monoamine transporter-2 function: Neurotoxic and therapeutic implications

The vesicular monoamine transporter-2 (VMAT-2) is an important regulator of intraneuronal monoamine concentrations and disposition as this protein sequesters critical cytoplasmic monoaminergic transmitters and contributes to their subsequent exocytotic release. This review primarily discusses the impact of psychoactive drugs (including those with abuse potential) on dopamine (DA)-related VMAT-2 and its function. In particular, the different responses by DA-related VMAT-2 and associated vesicles to plasmalemmal uptake blockers like methylphenidate and releasers like methamphetamine are presented. Recent preclinical findings suggest that vesicular transporter systems are highly regulatable, both by changes in localization as well as alterations in the kinetics of the VMAT-2 protein. The capacity for such shifts in VMAT-2 functions suggests the presence of physiological regulation that likely influences the activity of DA systems. In addition, these findings may contribute to our understanding of the pathogenesis of a variety of DA-related disorders such as substance abuse and Parkinsons disease and also suggest new therapeutic targets for treating such diseases.

The role of the plasmalemmal dopamine and vesicular monoamine transporters in methamphetamine-induced dopaminergic deficits.

Amphetamine (AMPH) and methamphetamine (METH) are members of a collection of phenethylamine psychostimulants that are commonly referred to collectively as “amphetamines.” Amphetamines exert their effects, in part, by affecting neuronal dopamine transport. This review thus focuses on the effects of AMPH and METH on the plasmalemmal dopamine transporter and the vesicular monoamine transporter‐2 in animal models with a particular emphasis on how these effects, which may vary for the different stereoisomers, contribute to persistent dopaminergic deficits.

Age-dependent differences in dopamine transporter and vesicular monoamine transporter-2 function and their implications for methamphetamine neurotoxicity.

The abuse of methamphetamine (METH) is a serious public health problem because METH can cause persistent dopaminergic deficits in the brains of both animal models and humans. Surprisingly, adolescent postnatal day (PND)40 rats are resistant to these METH‐induced deficits, whereas young adult PND90 rats are not. Studies described in this report used rotating disk electrode voltammetry and western blotting techniques to investigate whether there are age‐dependent differences in monoamine transporter function in PND38–42 and PND88–92 rats that could contribute to this phenomenon. The initial velocities of dopamine (DA) transport into, METH‐induced DA efflux from, and DA transporter (DAT) immunoreactivity in striatal suspensions are greater in PND38–42 rats than in PND88–92 rats. DA transport velocities into vesicles that cofractionate with synaptosomal membranes after osmotic lysis are also greater in PND38–42 rats. However, there is no difference in vesicular monoamine transporter‐2 (VMAT‐2) immunoreactivity between the two age groups in this fraction. This suggests that younger rats have a greater capacity to sequester cytoplasmic DA into membrane‐associated vesicles due to kinetically upregulated VMAT‐2 and also have increased levels of functionally active DAT. In the presence of METH, these may provide additional routes of cellular efflux for DA that is released from vesicles into the cytoplasm and thereby prevent cytoplasmic DA concentrations in younger rats from rising to neurotoxic levels after drug administration. These findings provide novel insight into the age‐dependent physiological regulation of neuronal DA sequestration and may advance the treatment of disorders involving abnormal DA disposition including substance abuse and Parkinsons disease. Synapse 63:147–151, 2009.

Measurement of kinetically resolved vesicular dopamine uptake and efflux using rotating disk electrode voltammetry

The vesicular monoamine transporter-2 (VMAT-2) sequesters cytoplasmic dopamine (DA) into vesicles for storage and subsequent release. VMAT-2 activity has traditionally been measured in small synaptic vesicles isolated from rat striatum by monitoring [3H] DA uptake and in cellular expression systems using fast scan cyclic voltammetry. This is the first report using rotating disk electrode (RDE) voltammetry to measure VMAT-2 DA uptake and efflux in small synaptic vesicles. DA uptake profiles followed mixed order kinetics with apparent zero order kinetics for the first 25 s and apparent first order kinetics thereafter. Vesicular DA uptake was temperature- and ATP-dependent and was blocked by the VMAT-2 inhibitor tetrabenazine. Initial velocities of DA uptake were kinetically resolved and displayed Michaelis-Menten kinetics with a Km and Vmax of 289 +/- 59 nM and 1.9 +/- 0.2 fmol/(s microg protein), respectively. Methamphetamine-induced DA efflux was blocked by tetrabenazine and kinetically resolved with an initial velocity of 0.54 +/- 0.08 fmol/(s microg protein). These results suggest that RDE voltammetry can be used to make kinetically resolved measurements of vesicular DA uptake and efflux and will allow the design of experiments that could reveal important information about the kinetics of VMAT-2 activity and its inhibition.

Methylphenidate Administration Alters Vesicular Monoamine Transporter-2 Function in Cytoplasmic and Membrane-Associated Vesicles

In vivo methylphenidate (MPD) administration increases vesicular monoamine transporter-2 (VMAT-2) immunoreactivity, VMAT-2-mediated dopamine (DA) transport, and DA content in a nonmembrane-associated (referred to herein as cytoplasmic) vesicular subcellular fraction purified from rat striatum: a phenomenon attributed to a redistribution of VMAT-2-associated vesicles within nerve terminals. In contrast, the present study elucidated the nature of, and the impact of MPD on, VMAT-2-associated vesicles that cofractionate with synaptosomal membranes after osmotic lysis (referred to herein as membrane-associated vesicles). Results revealed that, in striking contrast to the cytoplasmic vesicles, DA transport velocity versus substrate concentration curves in the membrane-associated vesicles were sigmoidal, suggesting positive cooperativity with respect to DA transport. Additionally, DA transport into membrane-associated vesicles was greater in total capacity in the presence of high DA concentrations than transport into cytoplasmic vesicles. Of potential therapeutic relevance, MPD increased DA transport into the membrane-associated vesicles despite rapidly decreasing (presumably by redistributing) VMAT-2 immunoreactivity in this fraction. Functional relevance was suggested by findings that MPD treatment increased both the DA content of the membrane-associated vesicle fraction and K+-stimulated DA release from striatal suspensions. In summary, the present data demonstrate the existence of a previously uncharacterized pool of membrane-associated VMAT-2-containing vesicles that displays novel transport kinetics, has a large sequestration capacity, and responds to in vivo pharmacological manipulation. These findings provide insight into both the regulation of vesicular DA sequestration and the mechanism of action of MPD, and they may have implications regarding treatment of disorders involving abnormal DA disposition, including Parkinsons disease and substance abuse.

METHYLPHENIDATE-INDUCED INCREASES IN VESICULAR DOPAMINE SEQUESTRATION AND DOPAMINE RELEASE IN THE STRIATUM: THE ROLE OF MUSCARINIC AND DOPAMINE D2 RECEPTORS

Methylphenidate (MPD) administration alters the subcellular distribution of vesicular monoamine transporter-2 (VMAT-2)-containing vesicles in rat striatum. This report reveals previously undescribed pharmacological features of MPD by elucidating its receptor-mediated effects on VMAT-2-containing vesicles that cofractionate with synaptosomal membranes after osmotic lysis (referred to herein as membrane-associated vesicles) and on striatal dopamine (DA) release. MPD administration increased DA transport into, and decreased the VMAT-2 immunoreactivity of, the membrane-associated vesicle subcellular fraction. These effects were mimicked by the D2 receptor agonist quinpirole and blocked by the D2 receptor antagonist eticlopride. Both MPD and quinpirole increased vesicular DA content. However, MPD increased, whereas quinpirole decreased, K+-stimulated DA release from striatal suspensions. Like MPD, the muscarinic receptor agonist, oxotremorine, increased K+-stimulated DA release. Both eticlopride and the muscarinic receptor antagonist scopolamine blocked MPD-induced increases in K+-stimulated DA release, whereas the N-methyl-d-aspartate receptor antagonist (-)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) was without effect. This suggests that D2 receptors mediate both the MPD-induced redistribution of vesicles away from synaptosomal membranes and the MPD-induced up-regulation of vesicles remaining at the membrane. This results in a redistribution of DA within the striatum from the cytoplasm into vesicles, leading to increased DA release. However, D2 receptor activation alone is not sufficient to mediate the MPD-induced increases in striatal DA release because muscarinic receptor activation is also required. These novel findings provide insight into the mechanism of action of MPD, regulation of DA sequestration/release, and treatment of disorders affecting DA disposition, including attention-deficit hyperactivity disorder, substance abuse, and Parkinsons disease.

Methylphenidate-induced alterations in synaptic vesicle trafficking and activity.

The psychostimulant, methylphenidate (MPD), is commonly prescribed to treat attention‐deficit hyperactivity disorder. MPD binds to the neuronal dopamine (DA) transporter, where it blocks the inward transport of DA. The present study expands upon these findings by examining the effects of in vivo MPD administration on the vesicular monoamine transporter‐2 (VMAT‐2) in membrane‐associated vesicle and cytoplasmic vesicle subcellular fractions (i.e., those vesicles that do and do not co‐fractionate with synaptosomal membranes after osmotic lysis, respectively) isolated from lysates of rat striatal synaptosomes. The results indicate that a single MPD administration redistributes VMAT‐2 and associated vesicles within nerve terminals away from the synaptosomal membranes and into the cytoplasm, as assessed 1 hour after treatment. DA transport is also increased by MPD in both vesicle fractions (on account of vesicle trafficking in the cytoplasmic vesicles and to kinetic upregulation of the VMAT‐2 in the membrane‐associated vesicles). This, in turn, leads to an increase in the DA content of both vesicle fractions as well as an increase in the velocity and magnitude of K+‐stimulated DA release from striatal suspensions. Taken together, these data show that the trafficking, DA sequestration function, DA content, and exocytotic DA release function of synaptic vesicles can all be pharmacologically manipulated by in vivo MPD treatment. These findings may provide important insights useful for understanding and treating disorders involving abnormal DA transmission including drug abuse, Parkinsons disease, and attention‐deficit hyperactivity disorder.

Methamphetamine Administration Reduces Hippocampal Vesicular Monoamine Transporter-2 Uptake

Repeated high-dose injections of methamphetamine (METH) rapidly decrease dopamine uptake by the vesicular monoamine transporter-2 (VMAT-2) associated with dopaminergic nerve terminals, as assessed in nonmembrane-associated vesicles purified from striata of treated rats. The purpose of this study was to determine whether METH similarly affects vesicular uptake in the hippocampus; a region innervated by both serotonergic and noradrenergic neurons and profoundly affected by METH treatment. Results revealed that repeated high-dose METH administrations rapidly (within 1 h) reduced hippocampal vesicular dopamine uptake, as assessed in vesicles purified from treated rats. This reduction was likely associated with serotonergic nerve terminals because METH did not further reduce vesicular monoamine uptake in para-chloroamphetamine-lesioned animals. Pretreatment with the serotonin transporter inhibitor fluoxetine blocked both this acute effect on VMAT-2 and the decrease in serotonin content observed 7 days after METH treatment. In contrast, there was no conclusive evidence that METH affected vesicular dopamine uptake in noradrenergic neurons or caused persistent noradrenergic deficits. These findings suggest a link between METH-induced alterations in serotonergic hippocampal vesicular uptake and the persistent hippocampal serotonergic deficits induced by the stimulant.

Cocaine Alters Vesicular Dopamine Sequestration and Potassium-Stimulated Dopamine Release: The Role of D2 Receptor Activation

Cocaine is a psychostimulant that inhibits the inward transport of dopamine (DA) via the neuronal DA transporter, thereby increasing DA concentrations in the synaptic cleft. Cocaine administration also causes a redistribution of striatal vesicular monoamine transporter (VMAT)-2-containing vesicles that co-fractionate with synaptosomal membranes after osmotic lysis (referred to herein as membrane-associated vesicles) to a nonmembrane-associated, cytoplasmic subcellular fraction. Although previous studies from our laboratory have focused on the impact of cocaine on cytoplasmic vesicles, the present report describes the pharmacological effects of cocaine on the membrane-associated vesicle population. Results revealed that the redistribution of VMAT-2 and associated vesicles away from synaptosomal membranes is associated with a decrease in total DA transport and DA content in the membrane-associated VMAT-2-containing subcellular fraction. Cocaine also decreases the velocity and magnitude of K+-stimulated exocytotic DA release from whole striatal suspensions. The cocaine-induced VMAT-2 redistribution, decrease in DA release, and decrease in total DA transport are mediated by D2 receptors as these events were prevented by pretreatment with the D2 receptor antagonist, eticlopride [S-(-)-3-chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2-methoxybenzamide hydrochloride]. These data suggest that after cocaine administration, D2 receptors are activated because of increased synaptic DA, resulting in a redistribution of DA-containing vesicles away from synaptosomal membranes, thus leading to less DA released after a depolarizing stimulus. These findings provide insight into not only the mechanism of action of cocaine but also mechanisms underlying the regulation of dopaminergic neurons.

Measurement of plasmalemmal dopamine transport, vesicular dopamine transport, and K+-stimulated dopamine release in frozen rat brain tissue

This report describes experiments designed to (1) establish the specificity of dopamine (DA) transporter (DAT)-mediated plasmalemmal DA transport, vesicular monoamine transporter-2 (VMAT-2)-mediated vesicular DA transport, and K+-stimulated DA release in samples prepared from frozen rat striata, and (2) characterize the time-course of the effects of freezing on these processes. The procedure described herein uses a simple method of freezing brain tissue (first cooling in ice-cold buffer and then freezing at -80 degrees C) that allows for the storage of rat striata followed by the assay of DA transport and K+-stimulated DA release using rotating disk electrode voltammetry. Plasmalemmal DA transport into samples prepared from frozen striata was blocked by the DAT inhibitor, cocaine, and vesicular DA transport was blocked by the VMAT-2 inhibitor, dihydrotetrabenazine. Additionally, K+-stimulated DA release was Ca+2-dependent. Freezing decreases DAT-mediated DA transport, VMAT-2-mediated DA transport, and K+-stimulated DA release. However activity is still measurable even after 3 weeks of storage. These results suggest that rat striata retain some DA transport and DA release activity even when stored frozen for a few weeks. Frozen storage of rat striata may thus be valuable for experiments requiring lengthy assays, the accumulation of material, or the transport of samples from one laboratory to another for analysis. These results may also be applicable to the study of frozen human brain tissue.