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 ϩ -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.Methylphenidate (MPD) is a commonly prescribed psychostimulant used to treat attention-deficit hyperactivity disorder. It is well established that MPD binds with high affinity to the neuronal dopamine transporter (DAT) where it blocks the inward transport of dopamine (DA) (Wayment et al., 1999;. In addition, MPD indirectly affects DA transport by the vesicular monoamine transporter-2 (VMAT-2), a transporter protein that is responsible for the sequestration of cytoplasmic DA. Specifically, MPD administration increases [ 3 H]DA transport into nonmembrane-associated (referred to herein as cytoplasmic) vesicles purified from lysates of striatal synaptosomes prepared from treated rats (Sandoval et al., 2002(Sandoval et al., , 2003. MPD also increases DA content in the cytoplasmic vesicle subcellular fraction (Sandoval et al., 2002). These phenomena probably result from a redistribution of VMAT-2-containing vesicles within nerve terminals away from membranes and into the cytoplasm (Sandoval et al., 2002).Recent attention has focused on the regulation of cytop...

Objective Delays in HIV diagnosis and initiation of antiretroviral therapy (ART) are common even among symptomatic individuals in Africa. We hypothesized that ART delays might be more common if traditional healers were the first practitioners consulted. Design Cross-sectional study Methods We interviewed 530 newly diagnosed HIV-infected adults (≥18 years of age) who were clinically symptomatic at the time of HIV testing in two rural districts in Zambézia Province, Mozambique. We ascertained their prior health care seeking behavior, duration of their symptoms, CD4+ cell counts at the time of entry into care, and treatment provided by traditional healer(s). Results Of 517 patients (97.5%) with complete histories, 62% sought care from a healer before presenting to the local health facility. The median time to first health facility visit from first relevant symptom was 2 months (interquartile range [IQR]:1–4.5) for persons who had not visited a healer, 3 months [IQR:2–6] for persons visiting one healer, and 9 months [IQR: 5–12] for persons visiting >1 healer (p<0.001). Healers diagnosed 56% of patients with a social or ancestral curse and treated 66% with subcutaneous herbal remedies. A non-significant trend towards lower CD4+ cells for persons who had seen multiple healers was noted. Conclusion Seeking initial care from healers was associated with delays in HIV testing among symptomatic HIV-seropositive persons. We had no CD4 evidence that sicker patients bypass traditional healers, a potential inferential bias. Engaging traditional healers in a therapeutic alliance may facilitate the earlier diagnosis of HIV/AIDS.

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 Parkinson's disease. The abuse of methamphetamine (METH) is a serious public health problem with 7.3% of young adults (19-30 years of age) in the United States reporting lifetime illicit use of METH (Johnston et al., 2007b). By way of comparison, the percentages of adolescent 8th, 10th, and 12th graders in the United States reporting lifetime illicit use of METH are 2.7, 3.2, and 4.4%, respectively, (Johnston et al., 2007a). This prevalence of use is concerning because the behavioral consequences of METH differ as a function of age (Vorhees et al., 2005). METH and related stimulants can also cause long-term decreases in markers for monoaminergic neurons (Brown and Yamamoto, 2003). Specifically, multiple high-dose METH administrations cause long-term reductions in striatal dopamine (DA) content, tyrosine hydroxylase activity, and DA transporter (DAT) density in rodents (Hotchkiss et al.,

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 ϩ -stimulated DA release from striatal suspensions. Like MPD, the muscarinic receptor agonist, oxotremorine, increased K ϩ -stimulated DA release. Both eticlopride and the muscarinic receptor antagonist scopolamine blocked MPD-induced increases in K ϩ -stimulated DA release, whereas the N-methyl-D-aspartate receptor antagonist (Ϫ)-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 Parkinson's disease.The ritalinic acid psychostimulant methylphenidate (MPD) is frequently used to treat attention-deficit hyperactivity disorder. It is well established that MPD prevents the clearance of dopamine (DA) from the synaptic cleft by binding to the neuronal DA transporter (DAT) (Wayment et al., 1999;. In addition, MPD also indirectly affects the vesicular monoamine transporter (VMAT)-2, a protein that sequesters cytoplasmic DA inside the synaptic vesicles of nerve terminals. A single MPD treatment increases DA transport, VMAT-2 immunoreactivity, and binding of the VMAT-2 ligand [ 3 H]dihydrotetrabenazine in cytoplasmic vesicles purified from osmotic lysates of rat striatal synaptosomes (Sandoval et al., 2002(Sandoval et al., , 2003Volz et al., 2007a). These effects on cytoplasmic vesicles are D2 receptor-mediated, because the D2 receptor antagonist eticlopride (ETIC) attenuates or blocks these effects, and the D2 receptor agonist quinpirole (QUIN) mimics the effects of MPD (Sandoval et al., 2002;Truong et al., 2004).In co...

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 (due to 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, Parkinson's disease, and attention-deficit hyperactivity disorder.