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NEUROPHARMACOLOGY

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

Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah

Received June 7, 2007; accepted August 8, 2007.

	Abstract

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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 Parkinson's disease and substance abuse.

Recent attention has focused on the regulation of cytoplasmic VMAT-2-containing vesicles after in vivo pharmacological manipulation. 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 cytoplasmic vesicular DA transport, which is characterized by Michaelis-Menten kinetics, DA transport velocity versus substrate concentration curves in membrane-associated vesicles were of an unexpected sigmoidal shape, suggesting positive cooperativity with respect to DA transport (something uncommon in membrane transport proteins and not seen in any other vesicular neurotransmitter transporter). In addition, 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 significance, MPD increased DA transport into the membrane-associated vesicles despite rapidly decreasing VMAT-2 immunoreactivity (presumably by redistributing VMAT-2-associated vesicles into the cytoplasm); thus, it kinetically up-regulated the membrane-associated vesicles. MPD treatment also increased both the DA content of the membrane-associated vesicle fraction and K⁺-stimulated DA release from striatal suspensions. Taken together, these data suggest the existence of a previously uncharacterized pool of membrane-associated VMAT-2-containing vesicles that display heretofore unreported DA transport kinetics. Of functional relevance, DA transport in this pool, and thus its capacity to affect synaptic transmission, responds to in vivo pharmacological manipulation. These findings provide not only novel insight into the physiological regulation of vesicular DA sequestration and synaptic transmission but also into the mechanism of action of MPD. Accordingly, these data may advance the treatment of disorders involving abnormal DA disposition, including substance abuse and Parkinson's disease.

	Materials and Methods

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Animals. Male Sprague-Dawley rats (300–360 g) were purchased from Charles River Laboratories (Raleigh, NC), and they were housed in a light- and temperature-controlled room with free access to food and water. All animal procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals, and they were approved by the University of Utah Institutional Animal Care and Use Committee.

Rotating Disk Electrode Measurement of DA Transport Velocities. Rotating disk electrode (RDE) voltammetry (Schenk et al., 2005; Volz et al., 2006a,b) was used to measure the initial velocities of inwardly directed vesicular DA transport in vesicles purified (Erickson et al., 1990; Teng et al., 1997) from rat striata. Using this technique, DA transport in the clearance profiles is indicated by a downward sloping line as DA is transported into synaptosomes or vesicles where the RDE cannot detect it. Each sample consisted of both striata (60–70 mg of total wet weight) from a rat that were homogenized in ice-cold sucrose buffer and then centrifuged (800g for 12 min at 4°C) to remove nuclear debris. The resulting supernatant (S1) was centrifuged (22,000g for 15 min at 4°C) to obtain the synaptosomal pellet (P2). The P2 synaptosomal pellet was then resuspended and homogenized in ice-cold water to lyse the synaptosomal membranes. Ice-cold 25 mM HEPES, pH 7.5, and 100 mM potassium tartrate, pH 7.5, were then added to the synaptosomal pellet homogenate, and the resulting mixture was centrifuged (20,000g for 20 min at 4°C) to form the P3 pellet, which contained the membrane-associated vesicles, and a S3 supernatant. To isolate cytoplasmic vesicles, 1 mM ice-cold (pH 7.5) MgSO₄ was added to the S3 supernatant and the resulting mixture was centrifuged (100,000g for 45 min at 4°C) to obtain the cytoplasmic vesicle pellet (P4). To measure vesicular DA transport, the P3 (membrane-associated vesicles) and P4 (cytoplasmic vesicles) pellets were resuspended in 300 µl of VMAT-2 assay buffer. Some control experiments also monitored synaptosomal (i.e., DAT-mediated) DA transport by resuspending the P2 (synaptosomal) pellet in 300 µl of DAT assay buffer.

The resuspended pellet was placed in a cylindrical glass chamber (10-mm internal diameter with a height of 20 mm) maintained at 37°C by a VWR (West Chester, PA) model 1104 Heating Recirculator, and RDE voltammetry was used to measure DA transport as described previously (Volz et al., 2006a,b). A Pine Instruments, Inc. (Grove City, PA) AFMD03GC glassy carbon electrode (5 mm in total diameter with a 3-mm-diameter glassy carbon electrode shrouded in Teflon) attached to a Pine Instruments, Inc. MSRX high-precision rotator was lowered into the glass chamber and rotated at 2000 rpm. A Bioanalytical Systems (West Lafayette, IN) LC3D (Petite Ampere) potentiostat was used to apply a potential of +450 mV relative to a Ag/AgCl reference electrode, and a detection current baseline was obtained in approximately 5 min. Then, an aqueous DA solution was injected using a Hamilton (Reno, NV) CR-700-20 constant rate syringe, and the resulting current outputs were recorded onto a Tektronix (Beaverton, OR) TDS 1002 digital storage oscilloscope. The initial velocities of DA transport were calculated from the linear slope of the initial apparent zero order portion of a plot of [DA] versus time as described previously (Earles et al., 1998; Volz et al., 2006b).

The resulting vesicular DA transport velocities were fit to either the Michaelis-Menten equation

(1)

or the Hill equation

(2)

with nonlinear regression using GraphPad Prism version 4.0 (GraphPad Software Inc., San Diego, CA) as described previously (Segel, 1993; Motulsky and Christopoulos, 2003). In these equations, v is the transport velocity, V_max is the maximal transport velocity, K_m is the Michaelis-Menten constant, [DA] is the initial extravesicular concentration of exogenously added DA, K_0.5 is formally defined as the substrate concentration at half-maximal transport velocity in sigmoidal response curves, and n_H is the Hill coefficient. The density of kinetically active VMAT-2, the catalytic rate constant, and the rate constant for DA binding to the VMAT-2 were calculated as described previously (Volz et al., 2006a). Protein concentrations were measured using a Bio-Rad (Hercules, CA) Bradford protein assay. Indicators of precision are standard errors of the mean, and statistical comparisons of the results were made using a t test.

The total amount of DA transported via the cytoplasmic and membrane-associated vesicles was determined by using RDE voltammetry to measure the amount of DA transported in the cytoplasmic and membrane-associated vesicle fractions per unit time (i.e., picomoles of DA per second). These values were then normalized to the original wet weight of the tissues used to prepare the vesicle fractions [i.e., picomoles of DA/(second x gram of wet weight)]. The cytoplasmic and membrane-associated vesicle fractions contained all of the tissue recoverable from two rat striata, and thus the amounts of DA that are ultimately partitioned into each vesicle fraction were readily compared despite large differences in the amount of protein in each fraction. Indicators of precision are standard errors of the mean.

Immunoreactivity. After RDE measurement of DA transport velocities, SDS-polyacrylamide gel electrophoresis and Western blot analysis were performed on the synaptosome, membrane-associated vesicle, and cytoplasmic vesicle fractions as described previously (Riddle et al., 2002; Sandoval et al., 2002). To compare DAT and VMAT-2 levels in the synaptosome, membrane-associated vesicle, and cytoplasmic vesicle fractions, the pellets were resuspended at 50 mg of original striatal wet weight per milliliter, and 80 µl was loaded per well. For analysis of actin and VMAT-2 trafficking in the vesicle fractions, 40 µg of protein from each membrane-associated vesicle sample and 5 µg of protein from each cytoplasmic vesicle sample were used. For analysis of Na⁺/K⁺-ATPase and piccolo in the vesicle fractions, 32 µg of protein from each membrane-associated vesicle sample and 7 µg of protein from each cytoplasmic vesicle sample were used. Bound antibody was visualized with horseradish peroxidase-conjugated secondary antibody [rabbit secondary from BioSource International (Camarillo, CA) or mouse secondary from Chemicon International (Temecula, CA)], and bands on blots were quantified by densitometry using a FluorChem SP Imaging System from Alpha Inotech (San Leandro, CA). Indicators of precision are standard errors of the mean, and statistical comparisons of the results were made using a t test.

Vesicular DA Content. Vesicular DA content was measured by high-performance liquid chromatography as described previously (Sandoval et al., 2003; Truong et al., 2005). The P3 (membrane-associated vesicles) and P4 (cytoplasmic vesicles) pellets were prepared as described above, and they were resuspended in ice-cold tissue buffer at 50 and 100 mg of the original striatal wet weight per milliliter of tissue buffer, respectively. The resuspended vesicle preparations were then sonicated for 10 s and centrifuged (22,000g for 15 min at 4°C). A 100-µl aliquot of the resulting supernatant was injected onto a high-performance liquid chromatograph system [4.6 x 250-mm Whatman (Maidstone, England) Partisphere C18 column] that was coupled to an electrochemical detector (+730 mV relative to a Ag/AgCl reference electrode). The mobile phase, pH 2.86, consisted of 50 mM sodium phosphate, 30 mM citric acid, 0.16 mM EDTA, 1.5 mM sodium octyl sulfate, and 10% (v/v) methanol (Chapin et al., 1986). Indicators of precision are standard error of the mean, and statistical comparisons of the results were made using one-way analysis of variance with a Tukey post test.

RDE Measurement of K⁺-Stimulated DA Release. RDE voltammetry was used to measure K⁺-stimulated DA release in striatal suspensions (Volz and Schenk, 2004; Volz et al., 2004) prepared from treated rats as described previously (McElvain and Schenk, 1992b). Each sample consisted of one striata (28–38 mg of wet weight) that was placed on an ice-cold watch glass and chopped by hand with an ice-cold razor blade for 30 s. The chopped striatum was then placed in 500 µl of DAT assay buffer inside the RDE glass chamber, and it was disrupted by repetitive pipetting for approximately 1 min. The resulting striatal suspension was allowed to stand for 12 min, and then it was washed by the addition and subsequent removal of 250 µl of fresh DAT assay buffer six times. A detection current baseline was obtained as described above in approximately 18 min, and then a small quantity of DAT assay buffer containing an elevated KCl concentration (resulting in 40 mM K⁺ inside the RDE glass chamber) was added to the striatal suspension to stimulate DA release (McElvain and Schenk, 1992b). The initial velocity of K⁺-stimulated DA release (obtained from the first 3 s of release) and the magnitude of K⁺-stimulated DA release (taken as the maximum amount of DA released) were calculated as described previously (McElvain and Schenk, 1992b). Indicators of precision are standard errors of the mean, and statistical comparisons of the results were made using a t test.

Drugs and Chemicals. Solutions were made using university-supplied deionized water that was further purified to 18 M with a DIamond Water Purification System from Barnstead (Dubuque, IA). Tetrabenazine was a generous gift from Drs. Jeffrey Erickson and Helene Varoqui (Louisiana State University Health Sciences Center, New Orleans, LA), and it was first dissolved in absolute ethanol before being diluted to final concentration in assay buffer as reported previously (Volz et al., 2006b). The ethanol did not affect DA transport in either the synaptosomal or membrane-associated vesicle fractions (data not shown). (±)-MPD hydrochloride and (–)-cocaine hydrochloride were supplied by the National Institute on Drug Abuse (Bethesda, MD). MPD doses were calculated as the free base, and they were dissolved in 0.9% (w/v) saline before being administered at 1 ml/kg as indicated in the figure legends. The sucrose buffer, pH 7.4, contained 320 mM sucrose, 3.8 mM NaH₂PO₄, and 12.7 mM Na₂HPO₄ (Sandoval et al., 2001). The VMAT-2 assay buffer, pH 7.5 (Erickson et al., 1990; Teng et al., 1997), consisted of 25 mM HEPES, 100 mM potassium tartrate, 0.05 mM EGTA, 0.1 mM EDTA, and 2 mM Mg²⁺-ATP (with the exception of experiments designed to investigate the ATP dependence of DA transport, where the assay buffer contained no Mg²⁺-ATP; Volz et al., 2006b). The DAT assay buffer, pH 7.4, consisted of 126 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl₂, 16 mM sodium phosphate, 1.4 mM MgSO₄, and 11 mM dextrose (Sandoval et al., 2001). The tissue buffer, pH 2.5, consisted of 50 mM sodium phosphate, 30 mM citric acid, and 10% (v/v) methanol (Sandoval et al., 2003; Truong et al., 2005). VMAT-2 antibody (AB1767) was purchased from Chemicon International, Na⁺/K⁺-ATPase antibody was from BD Biosciences (San Jose, CA), piccolo antibody was from Abcam Inc. (Cambridge, MA), actin antibody was from ICN Biotechnologies (Costa Mesa, CA), and DAT antibody was generously provided by Dr. Roxanne Vaughan (University of North Dakota, Grand Forks, ND).

	Results

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Results presented in Fig. 1A confirmed previous findings that a single injection of MPD (40 mg/kg s.c., a dose used previously to investigate MPD-induced vesicular trafficking; Sandoval et al., 2002) redistributes VMAT-2 within nerve terminals, as assessed 1 h after treatment. MPD decreased VMAT-2 immunoreactivity in the membrane-associated vesicle fraction and increased VMAT-2 immunoreactivity in the cytoplasmic vesicle fraction. Actin immunoreactivity, used to demonstrate equal protein loading onto the gels, was not altered by MPD (Fig. 1B).

View larger version (30K): [in this window] [in a new window]   Fig. 1. MPD redistributes VMAT-2 immunoreactivity. Rats received a single administration of MPD (40 mg/kg s.c.) or saline vehicle (1 ml/kg s.c.), and they were killed 1 h later. A, MPD decreases VMAT-2 immunoreactivity in membrane-associated vesicles and increases VMAT-2 immunoreactivity in cytoplasmic vesicles. Each column represents the average of four independent densitometric determinations, and asterisks indicate a statistical difference, p < 0.05, between immunoreactivity in saline- and MPD-treated animals. Molecular mass, in kilodaltons, is shown to the side of the representative VMAT-2 blots. B, the representative actin blots demonstrate that MPD does not change actin immunoreactivity in either the membrane-associated or cytoplasmic vesicles (densitometric data not shown).

Western blot analysis was conducted to estimate the relative amounts of DAT and VMAT-2 in both vesicle fractions. Virtually all of the DAT isolated from the synaptosomal fraction was present in the membrane-associated vesicle fraction (Fig. 2A), whereas VMAT-2 isolated from the synaptosome was distributed between the cytoplasmic and membrane-associated vesicle fractions (Fig. 2B), with the membrane-associated vesicle fraction containing the majority of the VMAT-2 isolated from the synaptosome. The membrane-associated vesicle fraction also contained the plasmalemmal membrane marker Na⁺/K⁺-ATPase and the readily releasable/active zone marker piccolo (data not shown). Neither of these markers were detected under the assay conditions used (see Materials and Methods) in the cytoplasmic vesicle fraction.

View larger version (27K): [in this window] [in a new window]   Fig. 2. Virtually all of the DAT isolated from the synaptosome is present in the membrane-associated vesicle fraction, whereas VMAT-2 isolated from the synaptosome is distributed between the cytoplasmic and membrane-associated vesicle fractions. A and B, DAT and VMAT-2 immunoreactivity, respectively, in the synaptosomal (synap), membrane-associated vesicle (memb), and cytoplasmic vesicle (cyto) subcellular fractions. Each column represents the average of four independent densitometric determinations, and molecular mass, in kilodaltons, is shown to the side of the representative blots. ND, DAT levels that were not detectable.

Before characterizing the kinetics of DA transport in the two vesicle populations, the specificity of transport was established. This had been accomplished previously for cytoplasmic vesicles, where vesicular DA transport was demonstrated to be ATP-dependent and blocked by the VMAT-2 inhibitor tetrabenazine (Volz et al., 2006b). In the present study, 60 nM tetrabenazine, a concentration that was without effect on synaptosomal (i.e., DAT-mediated) DA transport (Fig. 3A), completely blocked DA transport in the membrane-associated vesicles (Fig. 3B). The absence of ATP likewise inhibited DA transport in the membrane-associated vesicles (Fig. 3C). Additionally, 100 µM cocaine completely blocked DAT-mediated synaptosomal DA transport (Fig. 4A), but it was without effect on DA transport into the membrane-associated vesicles (Fig. 4B). These results demonstrate that membrane-associated vesicular DA transport is selectively mediated by the VMAT-2 and that the DAT does not contribute to the measured DA transport in this preparation.

View larger version (13K): [in this window] [in a new window]   Fig. 3. DA transport in the membrane-associated vesicles is ATP-dependent, and it is blocked by tetrabenazine. Each panel depicts representative DA concentration versus time clearance profiles of exogenously applied DA. DA transport in the clearance profiles is indicated by a downward sloping line as DA is transported into synaptosomes or vesicles where the RDE cannot detect it. A, 60 nM tetrabenazine does not block DAT-mediated DA transport in striatal synaptosomes when added 30 s before DA. B, 60 nM tetrabenazine completely blocks DA transport in membrane-associated vesicles when added 30 s before DA. C, DA transport in membrane-associated vesicles is ATP-dependent.

View larger version (15K): [in this window] [in a new window]   Fig. 4. DA transport in the membrane-associated vesicles is not blocked by cocaine. As in Fig. 3, each panel depicts representative DA concentration versus time clearance profiles of exogenously applied DA. A, 100 µM cocaine completely blocks DAT-mediated DA transport in striatal synaptosomes when added 30 s before DA. B, 100 µM cocaine does not block DA transport in membrane-associated vesicles when added 30 s before DA.

View larger version (24K): [in this window] [in a new window]   Fig. 5. DA transport displays Michaelis-Menten kinetics in cytoplasmic vesicles and non-Michaelis-Menten cooperativity in membrane-associated vesicles. Rats received a single administration of MPD (40 mg/kg s.c.) or saline vehicle (1 ml/kg s.c.), and they were killed 1 h later. Each datum point represents the average of four independent determinations, and asterisks indicate a statistical difference, p < 0.05, between DA transport velocities in saline- and MPD-treated animals. A, MPD increases DA transport velocities in cytoplasmic vesicles. The solid lines represent the best fits of the Michaelis-Menten equation (eq. 1) to the observed data with r2 values of 0.9985 (saline) and 0.9971 (MPD). B, MPD also increases DA transport velocities in membrane-associated vesicles. The solid lines represent the best fits of the Hill equation (eq. 2) to the observed data with r2 values of 0.9736 (saline) and 0.9876 (MPD). DA transport velocities at the lowest concentrations of DA are magnified in the inset.

Results presented in Fig. 6 demonstrate that MPD administration increased DA content, as assessed ex vivo, in both the cytoplasmic and membrane-associated vesicle fractions. MPD administration increased both the magnitude and velocity of K⁺-stimulated DA release in striatal suspensions as well (Fig. 7). However, MPD administration did not affect the duration of K⁺-stimulated DA release (6 ± 1 versus 7.0 ± 0.2 s for saline versus MPD, respectively) (n = 4).

View larger version (14K): [in this window] [in a new window]   Fig. 6. MPD increases DA content in both the cytoplasmic and membrane-associated vesicle fractions. Rats received a single administration of MPD (40 mg/kg s.c.) or saline vehicle (1 ml/kg s.c.), and they were killed 1 h later. Each column represents the average of six independent determinations, and asterisks indicate a statistical difference, p < 0.05, between DA content in saline- and MPD-treated animals.

View larger version (21K): [in this window] [in a new window]   Fig. 7. MPD increases both the magnitude and velocity of K+-stimulated DA release. Rats received a single administration of MPD (40 mg/kg s.c.) or saline vehicle (1 ml/kg s.c.), and they were killed 1 h later. Each column represents the average of three independent determinations, and asterisks indicate a statistical difference, p < 0.05, between the magnitude and velocity of K+-stimulated DA release in saline- and MPD-treated animals.

	Discussion

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In the course of investigating the impact of MPD on DA transport into cytoplasmic vesicles, an additional unique population of vesicles was identified that exhibited prominently distinct properties as discussed below. DA transport in these membrane-associated vesicles was completely inhibited in the presence of tetrabenazine and in the absence of ATP (Fig. 3), indicating that the DA transport is mediated exclusively by the VMAT-2. The tetrabenazine-induced inhibition occurred at a concentration that was without effect on synaptosomal (i.e., DAT-mediated) DA transport (Fig. 3A) demonstrating that the DAT in the membrane-associated vesicle fraction (Fig. 2A) does not contribute to the observed DA transport. Further supporting this conclusion, DA transport in the membrane-associated vesicles was unaffected by cocaine at a concentration that completely blocked synaptosomal DA transport (Fig. 4). Additionally, DA transport by the DAT is Na⁺- and Cl^–-dependent (McElvain and Schenk, 1992a), and there was virtually no Na⁺ or Cl^– in the VMAT-2 assay buffer. Although the possibility that there may be some small amount of DA binding to the DAT present in the membrane-associated vesicle fraction (Fig. 2A) cannot be excluded, this negligible amount is not detectable by RDE voltammetry when VMAT-2-mediated DA transport is inhibited (Fig. 3, B and C) and therefore does not influence the analysis of VMAT-2 kinetics in the membrane-associated vesicle fraction.

Further study of the membrane-associated vesicles revealed several novel findings. First, and in striking contrast to cytoplasmic vesicular DA transport (Fig. 5A), initial velocity versus DA concentration curves for membrane-associated vesicles from both saline- and MPD-treated animals were of an unexpected sigmoidal shape, and they were modeled by the Hill equation with a Hill coefficient of approximately 5 (Fig. 5B). This is inconsistent with Michaelis-Menten kinetics (Segel, 1993; Fersht, 1998), and it suggests that, in contrast to cytoplasmic vesicles, the membrane-associated vesicles display a unique positive cooperativity with respect to DA transport. Substrate cooperativity, especially of this large magnitude (Hill coefficient of 5), is uncommon among membrane transport proteins, and this is the first report of cooperativity involving a vesicular neurotransmitter transporter.

One important consequence of the cooperativity exhibited by vesicles in the membrane-associated preparation under study is that at intermediate substrate concentrations, the sigmoidal response would provide a much more sensitive control of substrate transport rates than the rectangular hyperbolic response of cytoplasmic vesicles. Cytoplasmic DA concentrations have been estimated (in pheochromocytoma-12 cells) to be in the range of 0.5 to 1 µM (Perlman and Sheard, 1982). This is near the middle of the rectangular hyperbolic curve of cytoplasmic vesicles (Fig. 5A) and in the lower portion of the sigmoidal curve of membrane-associated vesicles (Fig. 5B). At these concentrations, total DA transport (see Materials and Methods for description of calculation) via the cytoplasmic and membrane-associated vesicles is comparable [i.e., 6 ± 3 versus 5 ± 2 pmol/(s x g wet weight), respectively]. If the concentration of intraneuronal DA were to rise (i.e., after drug treatment or as a consequence of a particular microenvironment), then DA transport in the cytoplasmic vesicles would be saturated, whereas DA transport in the membrane-associated vesicles would increase dramatically. This allows for the possibility that at least a subpopulation of the membrane-associated vesicles may function as a reserve sequestration capacity or "DA sink" to prevent cytoplasmic DA from rising to aberrant levels.

A second related novel finding of this study is that membrane-associated vesicles are capable, at high DA concentrations, of sequestering in total far more DA than the cytoplasmic vesicles. The maximal amount of DA transported in the entire cytoplasmic vesicle fraction was 9 ± 2 pmol/(s x g wet weight). In contrast, DA transport values for the membrane-associated vesicles ranged from 47 ± 4 (at a DA concentration observed at the plateau value for cytoplasmic vesicles) to 81 ± 11 pmol/(s x g wet weight) (at a DA concentration observed at the plateau value for membrane-associated vesicles; see Results). Thus, total DA transport via the cytoplasmic and membrane-associated vesicles was comparable at "typical" cytoplasmic DA concentrations of 0.5 to 1 µM (see above), but membrane-associated vesicles were capable of sequestering, in total, 5- to 9-fold more DA than cytoplasmic vesicles at higher DA concentrations. This large DA transport capacity of membrane-associated vesicles further underscores their potential reserve sequestration capacity as discussed above.

A third novel finding of this study is that although MPD treatment traffics VMAT-2 and associated vesicles away from membranes (Fig. 1A), it also kinetically up-regulates the decreased number of VMAT-2 remaining in the membrane-associated fraction such that a larger quantity of DA is transported (Fig. 5B). Consistent with this finding, DA content in the membrane-associated vesicle fraction is also increased (Fig. 6). One functional consequence of these data relates to findings that MPD post-treatment protects against the persistent dopaminergic deficits caused by treatment with the psychostimulant methamphetamine. In particular, it has been suggested that methamphetamine promotes aberrant cytoplasmic DA accumulation and the subsequent formation of DA-associated reactive oxygen species, thus leading to long-term damage (Cubells et al., 1994; Cadet and Brannock, 1998; Fumagalli et al., 1999; Hanson et al., 2004; Volz et al., 2007a,b). Previous studies indicated that MPD prevented this damage by increasing DA sequestration in cytoplasmic vesicles (Sandoval et al., 2003). The present study expands on these findings by suggesting that the DA sequestration-promoting capacity of MPD is not limited to cytoplasmic vesicles and that MPD-affected membrane-associated vesicles may serve as an additional, and perhaps higher capacity, DA sink with enhanced ability to afford neuroprotection. Because abnormal DA disposition probably contributes to the development of Parkinson's disease (Cubells et al., 1994; Jenner, 1998), an MPD-induced kinetic up-regulation of DA transport in membrane-associated vesicles may afford protection in this disease state as well.

In addition to increasing DA transport velocities and DA content in the membrane-associated vesicle fraction, MPD increased the magnitude and initial velocity of K⁺-stimulated DA release in striatal suspensions (Fig. 7). Because both the degree of vesicle loading and the speed of neurotransmitter release can influence receptor activation (Liu, 2003), these findings suggest the functional consequence that in the striatum MPD treatment influences quantal synaptic transmission by increasing the rate at which DA receptors are exposed to DA, and perhaps the duration of this effect. The MPD-induced increase in DA release was not due to an inhibition of the DAT by residual MPD introduced by the original subcutaneous injection, because 1) MPD administration did not change the duration of K⁺-stimulated DA release (see Results) and 2) the striatal suspensions were washed six times before the measurement of DA release to remove any MPD that was introduced by the original subcutaneous injection. Instead, the increase was probably due to the MPD-induced kinetic up-regulation of, and enhanced DA sequestration afforded by, VMAT-2.

The membrane-associated vesicles cofractionate with synaptosomal membranes after osmotic lysis: this is supported by the observations that plasmalemmal membrane marker Na⁺/K⁺-ATPase, the DAT, and the readily releasable/active zone marker piccolo are among the proteins found in this fraction. Taken together with previous findings that K⁺-stimulated DA release in rat striatal suspensions is both temperature- and Ca²⁺-dependent (McElvain and Schenk, 1992b), this suggests that the membrane-associated vesicle fraction may contain the readily releasable population of vesicles. Whether this population contains additional pools of vesicle remains to be determined. Future studies will further address this possibility.

In summary, this is the first report of membrane-associated VMAT-2-containing synaptic vesicles that 1) have large positive substrate cooperativity, 2) are capable of sequestering greater amounts of DA than cytoplasmic vesicles under conditions of elevated concentrations of DA, and 3) are both kinetically and functionally up-regulated by MPD. Vesicles of the membrane-associated fraction are probably of importance as regulators of both intraneuronal DA and of vesicular DA-release capacity. Because these vesicles can be regulated pharmacologically, they may provide an intriguing target for understanding and treating disorders involving abnormal DA transmission, including drug abuse, Parkinson's disease, and attention-deficit hyperactivity disorder.

	Acknowledgements

 
We thank Drs. Jeffery Erickson and Helene Varoqui for the gift of tetrabenazine and Dr. Roxanne Vaughan for the gift of DAT antibody. We are also grateful to Dr. James O. Schenk (Department of Chemistry, Washington State University) for helpful discussions on transporter kinetics and for enthusiastic support of our use of RDE voltammetry.

	Footnotes

 
This work was supported by Grants DA 00869, DA 04222, DA 13367, DA 11389, DA 019447, and DA 00378 from the National Institute on Drug Abuse as well as a Focused Funding Gift from Johnson & Johnson.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.126888.

ABBREVIATIONS: MPD, methylphenidate; DAT, dopamine transporter; DA, dopamine; VMAT, vesicular monoamine transporter; RDE, rotating disk electrode.

Address correspondence to: Dr. Annette E. Fleckenstein, Department of Pharmacology and Toxicology, University of Utah, 30 South 2000 East, Room 201, Salt Lake City, UT 84112. E-mail: fleckenstein{at}hsc.utah.edu

	References

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