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-2associated 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.