Metabolic analysis of striatal tissues from Parkinson's disease-like rats by electrospray ionization ion mobility mass spectrometry.

Electrospray ionization ion mobility mass spectrometry (ESI-IMMS) was used to study the striatal metabolomes in a Parkinson's like disease (PD-like) rat model. Striatal tissue samples from Berlin Druckrey IV (BD-IV) with PD-like disease 20 dpn-affected and 15 dpn-affected rats (dpn: days postnatal) were investigated and compared with age-matched controls. An ion mobility mass spectrometer (IMMS) produced multidimensional spectra with mass to charge ratio (m/z), ion mobility drift time, and intensity information for each individual metabolite. Principle component analysis (PCA) was applied in this study for pattern recognition and significant metabolites selection (68% data was modeled in PCA). Both IMMS spectra and PCA results showed that there were clear global metabolic differences between PD-like samples and healthy controls. Nine metabolites were selected by PCA and identified as potential biomarkers using the Human Metabolome Database (HMDB). One targeted metabolite in this study was dopamine. Selected-mass mobility analysis indicated the absence of dopamine in PD-like striatal metabolomes. A major discovery of this work, however, was the existence of an isomer of dopamine. By using ion mobility spectrometry, the dopamine isomer, which has not previously been reported, was separated from dopamine.

Neuronal metabolomics by ion mobility mass spectrometry: cocaine effects on glucose and selected biogenic amine metabolites in the frontal cortex, striatum, and thalamus of the rat.

We report results of studies of global and targeted neuronal metabolomes by ambient pressure ion mobility mass spectrometry. The rat frontal cortex, striatum, and thalamus were sampled from control nontreated rats and those treated with acute cocaine or pargyline. Quantitative evaluations were made by standard additions or isotopic dilution. The mass detection limit was ~100 pmol varying with the analyte. Targeted metabolites of dopamine, serotonin, and glucose followed the rank order of distribution expected between the anatomical areas. Data was evaluated by principal component analysis on 764 common metabolites (identified by m/z and reduced mobility). Differences between anatomical areas and treatment groups were observed for 53 % of these metabolites using principal component analysis. Global and targeted metabolic differences were observed between the three anatomical areas with contralateral differences between some areas. Following drug treatments, global and targeted metabolomes were found to shift relative to controls and still maintained anatomical differences. Pargyline reduced 3,4-dihydroxyphenylacetic acid below detection limits, and 5-HIAA varied between anatomical regions. Notable findings were: (1) global metabolomes were different between anatomical areas and were altered by acute cocaine providing a broad but targeted window of discovery for metabolic changes produced by drugs of abuse; (2) quantitative analysis was demonstrated using isotope dilution and standard addition; (3) cocaine changed glucose and biogenic amine metabolism in the anatomical areas tested; and (4) the largest effect of cocaine was on the glycolysis metabolome in the thalamus confirming inferences from previous positron emission tomography studies using 2-deoxyglucose.

Potential role of α-synuclein in neurodegeneration: studies in a rat animal model.

Neuronal protein α-synuclein (α-syn) is an essential player in the development of neurodegenerative diseases called synucleinopathies. A spontaneous autosomal recessive rat model for neurodegeneration was developed in our laboratory. These rats demonstrate progressive increases in α-syn in the brain mesencephalon followed by loss of dopaminergic terminals in the basal ganglia (BG) and motor impairments. The severity of pathology is directly related to the overexpression of α-syn and parallel decrease in dopamine (DA) level in the striatum (ST) of affected rats. The neurodegeneration in this model is characterized by the presence of perikarya and neurites Lewis bodies (LB) and diffuse marked accumulation of perikaryal α-syn in the substantia nigra (SN), brain stem (BS), and striatum (ST) along with neuronal loss. Light and ultrastructural analyses revealed that the process of neuronal degeneration is a 'dying back' type. The disease process is accompanied by gliosis and release of inflammatory cytokines. This neurodegeneration is a multisystemic disease and implicate α-syn as a major factor in the pathogenesis of this inherited autosomal recessive animal model. Decrease dopamine (DA) and overexpression of α-syn in the brain mesencephalon may provide a naturally occurring animal model for Parkinson's disease (PD) and other synucleinopathies that reproduces significant pathological, neurochemical, and behavioral features of the human disease.

Preconcentration of f-elements from aqueous solution utilizing a modified carbon paste electrode.

An evaluation using paraffin oil based, Acheson 38 carbon paste electrodes modified with α-hydroxyisobutyric acid (HIBA) to preconcentrate f-elements cathodically is described. The modified paste was made by directly mixing solid HIBA into the carbon paste. A chemically reversible cyclic voltammogram for HIBA was observed on this modified carbon paste, which was found to be a non-Nerstian, single electron transfer process. Lanthanides (less promethium) were found to accumulate onto the electrode surface during a 30 s electrodeposition step at -0.4 V vs Ag/AgCl from 0.1 M LiCl. The elements were then stripped off into a 2% HNO(3) solution by an oxidative step at +0.8 V vs Ag/AgCl; quantitative removal from the electrode was confirmed by ICPMS. Ultratrace solutions with initial concentrations down to 5 parts per quadrillion (ppq) were preconcentrated in 5 min above our instrumental limit of detection (LOD) of around 1 ppt for lanthanides.

Cathodic preconcentration of f-elements on a mercury film carbon fiber disk microelectrode.

Field detection and quantification of f-elements is an important problem in radioanalytical chemistry requiring small, portable devices. Here, characterization of a 10 µm Hg film carbon fiber disk microelectrode to accumulate f-elements is described. Accumulation was performed by cathodic deposition and evaluated by anodic stripping and subsequent ICPMS analyses. La(3+) was used as the model element, and subsequent studies were conducted on a 17 element mixture (Sc, Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, and Th). In the model studies, La(3+) undergoes a sorption phenomenon, and as in other studies and confirmed by ICPMS, a monolayer of atoms on the electrode surface is formed. Dissolved O(2) was found to have no effect on the cathodic accumulation of La(3+). Consideration of electrode reaction conditions is made, and reactions are hypothesized. The limit of detection (LOD) was found to be 10(-7) M with mass detection of 10(9) atoms, approximately 5 orders of magnitude less than at conventionally sized electrodes. To solve a dilution problem in follow-on analyses, a suggestion to use microelectrode chip-based sensors was made.

The kinetic mechanism for cytochrome P450 metabolism of type II binding compounds: evidence supporting direct reduction.

The metabolic stability of a drug is an important property that should be optimized during drug design and development. Nitrogen incorporation is hypothesized to increase the stability by coordination of nitrogen to the heme iron of cytochrome P450, a binding mode that is referred to as type II binding. However, we noticed that the type II binding compound 1 has less metabolic stability at sub-saturating conditions than a closely related type I binding compound 3. Three kinetic models will be presented for type II binder metabolism; (1) Dead-end type II binding, (2) a rapid equilibrium between type I and II binding modes before reduction, and (3) a direct reduction of the type II coordinated heme. Data will be presented on reduction rates of iron, the off rates of substrate (using surface plasmon resonance) and the catalytic rate constants. These data argue against the dead-end, and rapid equilibrium models, leaving the direct reduction kinetic mechanism for metabolism of the type II binding compound 1.

Mass transport at rotating disk electrodes: effects of synthetic particles and nerve endings.

An unstirred layer (USL) exists at the interface of solids with solutions. Thus, the particles in brain tissue preparations possess a USL as well as at the surface of a rotating disk electrode (RDE) used to measure chemical fluxes. Time constraints for observing biological kinetics based on estimated thicknesses of USLs at the membrane surface in real samples of nerve endings were estimated. Liposomes, silica, and Sephadex were used separately to model the tissue preparation particles. Within a solution stirred by the RDE, both diffusion and hydrodynamic boundary layers are formed. It was observed that the number and size of particles decreased the following: the apparent diffusion coefficient excluding Sephadex, boundary layer thicknesses excluding silica, sensitivity excluding diluted liposomes (in agreement with results from other laboratories), limiting current potentially due to an increase in the path distance, and mixing time. They have no effect on the detection limit (6 ± 2 nM). The RDE kinetically resolves transmembrane transport with a timing of approximately 30 ms.

The contribution of low-affinity transport mechanisms to serotonin clearance in synaptosomes.

Although many studies assert that the serotonin (5-HT) transporter (SERT) is the predominant mechanism controlling extracellular 5-HT concentrations, accumulating evidence suggests that low affinity, high capacity transport mechanisms may contribute more to 5-HT clearance than previously thought. The goal of this study was to quantify the contributions of SERT relative to other mechanisms in clearing extracellular 5-HT concentrations ranging from 50 nM to 1 µM in synaptosomes prepared from wild-type and SERT knockout mice using rotating disk electrode voltammetry. SERT inhibitors combined with decynium-22 (D-22), a blocker of several low-affinity transporters, blocked all uptake of 5-HT into synaptosomes. We found that SERT is responsible for the majority of synaptosomal uptake only at relatively low 5-HT concentrations, but comprises a diminishing proportion of 5-HT clearance when extracellular 5-HT increases above 100 nM. The effect of D-22 was similar in wild-type and SERT knockout synaptosomes. Thus, there was no evidence of upregulation of low-affinity mechanisms in knockout mice across the concentrations of 5-HT tested. These are surprising results, in light of the prevailing view that SERT is the primary uptake mechanism for extracellular 5-HT at physiological concentrations. We conclude that non-SERT mediated 5-HT uptake is substantial even at modest 5-HT concentrations. These findings, in conjunction with other studies, have important implications for understanding serotonergic disorders and may explain the variable efficacy and stability of patients' responses to antidepressants, such as the selective serotonin reuptake inhibitors.

Preconcentration of trivalent lanthanide elements on a mercury film from aqueous solution using rotating disk electrode voltammetry.

An approach to concentrate trivalent lanthanide elements into or onto mercury film electrodes supported on rotating disk glassy carbon electrodes in small volumes (

Neuronal dopamine transporter activity, density and methamphetamine inhibition are differentially altered in the nucleus accumbens and striatum with no changes in glycosylation in rats behaviorally sensitized to methamphetamine.

Animals sensitized to methamphetamine (METH) have altered dopaminergic systems, including dopamine transporter (DAT) activity. We investigated the effects induced by a sensitizing dose (5 mg/kg, i.p. per day for 5 days) of METH on rat behavior, DA transport by the DAT, DAT density, and inhibition of DA transport by METH in both the nucleus accumbens and striatum. We further investigated possible changes to glycosylation of the DAT after METH sensitization. The dosing paradigm caused an increased stereotyped response in rats treated with METH compared with saline controls. In animals treated with METH, DA transport velocities were increased by 6.4% in the nucleus accumbens and decreased by 21% in the striatum. Western blots demonstrated that DAT density was unchanged in the nucleus accumbens of METH-treated animals, but striatal DAT density was decreased by 20%. Further studies investigating METH inhibition of DA transport found that in the nucleus accumbens of METH-treated animals, the IC(50) was shifted to a larger value (from 0.81 to 1.45 microM). In the striatum, the IC(50) was decreased by 19% (from 1.00 to 0.81 microM) in METH-treated animals. Studies using glycosidase treatments and Western blots revealed that glycosylation was effectively removed by N-glycanase and neuraminidase, but not O-glycosidase or alpha-mannosidase. These studies also suggest that glycosylation was not altered in METH-treated animals. This study demonstrates that in animals sensitized to METH, the DAT is differentially regulated in different areas of the brain important for drug abuse, and that DA transport changes induced by METH are not due to DAT density, but to changes in the kinetics of the DAT. Additionally, this study suggests that glycosylation may not play a role in DAT activity changes after METH exposure.

Time-of-day differences in dopamine clearance in the rat medial prefrontal cortex and nucleus accumbens.

Circadian rhythms influence cocaine-seeking behavior in rats, and this behavior may be mediated by variability in the rate of extracellular dopamine clearance across the day:night cycle. We used rotating disk electrode voltammetry to examine dopamine clearance and inhibition of clearance by cocaine in the rat medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). Rats were housed under light:dark conditions (LD, 12 h:12 h) or in constant darkness (DD), the latter given just prior to the day of sacrifice. Tissue was collected at 4-h intervals under LD and DD conditions. Under LD, dopamine clearance in both brain regions was greatest at 4h after lights on. Under DD, there was a blunted but still rhythmic pattern of dopamine clearance across the 24-h cycle. Cocaine-induced inhibition of dopamine clearance in the mPFC was not different across the day:night cycle in rats under LD. Paradoxically, under DD, dopamine clearance in the mPFC was enhanced by cocaine at ZT16, 4 h into the subjective night, and only minimally inhibited at other times. In the NAc, cocaine inhibition of dopamine clearance was lowest at ZT4 under LD, and did not vary under DD. We conclude that dopamine clearance varies both in a diurnal and possibly in a circadian manner in the mPFC, and in a diurnal manner in the NAc. These results indicate that light itself may be used to manipulate molecules implicated in drug addiction.

Differential effects of Zn2+ on the kinetics and cocaine inhibition of dopamine transport by the human and rat dopamine transporters.

Zn2+ may play a major role in the modulation of neurotransmission because it modulates membrane receptors and channels. Recent literature has shown Zn2+ inhibits dopamine transport by the dopamine transporter (DAT), the main target of cocaine and some other drugs of abuse. Cocaine inhibits DAT and modulation of the DAT by Zn2+ may alter effects of cocaine on dopamine neurotransmission. This study investigates how Zn2+ changes DAT kinetics and its inhibition by cocaine. Steady-state and pre-steady-state kinetics of DAT activity were investigated using rotating disk electrode voltammetry. Values of KM and Vmax in hDAT and effects of cocaine match those in the literature. Zn2+ allosterically inhibited transport in the human DAT (hDAT) with a KI=7.9+/-0.42 microM. Removal of endogenous Zn2+ with penicillamine in hDAT increased transport values. In contrast, Zn2+ did not alter transport by rat DAT (rDAT), with KM and Vmax values of 1.2+/-0.49 microM and 15.7+/-2.57 pmol/(sx10(6) cells), respectively, and removal of Zn2+ did not increase dopamine transport values. Zn2+ allosterically reduced the inhibition by cocaine in hDAT. Results of pre-steady-state studies demonstrated that Zn2+ increases the second order binding rate constant for dopamine to hDAT (3.5 fold to 19.2x10(6) M-1 s-1 for hDAT). In rat striatal homogenates Zn2+ increased initial dopamine transport velocity and decreased cocaine inhibition providing evidence for differences in sensitivity to Zn2+ between the three different preparations. Modulation of the DAT by Zn2+ needs to be assessed further in development of cocaine antagonists.

Unraveling neuronal dopamine transporter mechanisms with rotating disk electrode voltammetry.

Herein we describe how the rotating disk electrode voltammetric technique can be used to examine the mechanism(s) of the inward transport of dopamine by the neuronal transporter for dopamine (DAT). The usefulness of making measurements kinetically resolving dopamine transport, interpretations of changes in Km and Vmax, approaches to defining pre-steady-state binding of dopamine to DAT, interactions between competing inhibitors, chemical modification of functional groups within DAT, and a presentation of a hypothetical multi-state model of dopamine transport are presented and discussed.

What can be learned from studies of multisubstrate mechanisms of neuronal dopamine transport?

The dopamine transporter (DAT) is a Na+- and Cl--dependent transporter and, with respect to its three apparent substrates, both partially random sequential as well as ordered mechanisms have been reported. Here we describe some of the features of DAT, such as the coupling of energy to concentrate dopamine and the properties of slippage and leakage. Further, in considering the regulation of transport velocities by DAT few have considered issues related to substrate regulation of DAT activity. Specifically, what effect do changes in the constants (K) for the participation of Na+ and Cl- have on dopamine transport velocity? It is shown that DAT may possess properties of slippage, an argument is made that leakage may be important in neuronal systems containing DAT, and the influence of changing values of K for the participation of Na+ and Cl- in transport is shown to produce large effects on DAT activity depending on the multisubstrate kinetic mechanism.

The functioning neuronal transporter for dopamine: kinetic mechanisms and effects of amphetamines, cocaine and methylphenidate.

The dopamine transporter (DAT) is a transmembrane spanning protein that catalyzes the transport of dopamine across the neuronal membrane to concentrate the neurotransmitter inside the cell. Although the uptake of dopamine has been studied since the 1960s, more recent advances in knowledge of the protein itself and in making kinetically resolved measurements of its action have led to more insights into its mechanism and pharmacology. The literature of the kinetics of transporters and kinetic measurements of DAT activity is reviewed to provide an overview of the multisubstrate mechanism of DAT activity, its pharmacology with regard to amphetamine, cocaine and methylphenidate, and correlations of DAT activity with some behavioral outputs.

Potential role of cardiac calsequestrin in the lethal arrhythmic effects of cocaine.

BACKGROUND: Cocaine-related deaths are continuously rising and its overdose is often associated with lethal cardiotoxic effects. METHODS AND RESULTS: Our approach, employing isothermal titration calorimetry (ITC) and light scattering in parallel, has confirmed the significant affinity of human cardiac calsequestrin (CASQ2) for cocaine. Calsequestrin (CASQ) is a major Ca(2+)-storage protein within the sarcoplasmic reticulum (SR) of both cardiac and skeletal muscles. CASQ acts as a Ca(2+) buffer and Ca(2+)-channel regulator through its unique Ca(2+)-dependent oligomerization. Equilibrium dialysis and atomic absorption spectroscopy experiments illustrated the perturbational effect of cocaine on CASQ2 polymerization, resulting in substantial reduction of its Ca(2+)-binding capacity. We also confirmed the accumulation of cocaine in rat heart tissue and the substantial effects cocaine has on cultured C2C12 cells. The same experiments were performed with methamphetamine as a control, which displayed neither affinity for CASQ2 nor any significant effects on its function. Since cocaine did not have any direct effect on the Ca(2+)-release channel judging from our single channel recordings, these studies provide new insights into how cocaine may interfere with the normal E-C coupling mechanism with lethal arrhythmogenic consequences. CONCLUSION: We propose that cocaine accumulates in SR through its affinity for CASQ2 and affects both SR Ca(2+) storage and release by altering the normal CASQ2 Ca(2+)-dependent polymerization. By this mechanism, cocaine use could produce serious cardiac problems, especially in people who have genetically-impaired CASQ2, defects in other E-C coupling components, or compromised cocaine metabolism and clearance.

Mechanism of action of methamphetamine within the catecholamine and serotonin areas of the central nervous system.

Addiction to methamphetamine (METH) is thought to be mediated by dopaminergic effects in the reward pathway in the brain via the A10 dopaminergic pathway. Herein we describe an overview of the results of the basic preclinical science undertaken to provide mechanistic insights into the action of amphetamines in general and METH in particular. A brief history of amphetamine and METH use and abuse is given, and an overview of the relevant chemical aspects of amphetamine as they relate to neurotransmitters in general is made. A review of the methods used to study the biochemical effects of METH is outlined. Finally, a focused analysis of the kinetic mechanisms of action of the amphetamines in general, and METH in particular, at the transmembrane transporters and at the intracellular vesicular storage sites is made. A description of how catecholaminergic and serotonergic nerve signaling may be altered by METH is proposed. Overall, the emphasis here is on differences in effects observed between the striatal (the A9 substantia nigral dopamine pathway) and nucleus accumbens (the A10, ventral tegmental pathway) areas of the brain following acute as well as repeated dosing and withdrawal.

Rotating disk electrode voltammetric measurements of serotonin transporter kinetics in synaptosomes.

Altered serotonin (5-HT) signaling is implicated in several neuropsychiatric disorders, including depression, anxiety, obsessive-compulsive disorder, and autism. The 5-HT transporter (SERT) modulates 5-HT neurotransmission strength and duration. This is the first study using rotating disk electrode voltammetry (RDEV) to measure 5-HT clearance. SERT kinetics were measured in whole brain synaptosomes. Uptake kinetics of exogenous 5-HT were measured using glassy carbon electrodes rotated in 500 µL glass chambers containing synaptosomes from SERT-knockout (-/-), heterozygous (+/-), or wild-type (+/+) mice. RDEV detected 5-HT concentrations of 5nM and higher. Initial velocities were kinetically resolved with K(m) and V(max) values of 99±35 standard error of regression (SER) nM and 181±11 SER fmol/(s×mg protein), respectively in wild-type synaptosomes. The method enables control over drug and chemical concentrations, facilitating interpretation of results. Results are compared in detail to other techniques used to measure SERT kinetics, including tritium labeled assays, chronoamperometry, and fast scan cyclic voltammetry. RDEV exhibits decreased 5-HT detection limits, decreased vulnerability to 5-HT oxidation products that reduce electrode sensitivity, and also overcomes diffusion limitations via forced convection by providing a continuous, kinetically resolved signal. Finally, RDEV distinguishes functional differences between genotypes, notably, between wild-type and heterozygous mice, an experimental problem with other experimental approaches.

Repeated cocaine effects on learning, memory and extinction in the pond snail Lymnaea stagnalis.

The persistence of drug addiction suggests that drugs of abuse enhance learning and/or impair extinction of the drug memory. We studied the effects of repeated cocaine on learning, memory and reinstatement in the pond snail, Lymnaea stagnalis. Respiratory behavior can be operantly conditioned and extinguished in Lymnaea, and this behavior is dependent on a critical dopamine neuron. We tested the hypothesis that repeated cocaine exposure promotes learning and memory or attenuates the ability to extinguish the memory of respiratory behavior that relies on this dopaminergic neuron. Rotating disk electrode voltammetry revealed a K(m) and V(max) of dopamine uptake in snail brain of 0.9 micromol l(-1) and 558 pmol s(-1) g(-1) respectively, and the IC(50) of cocaine for dopamine was approximately 0.03 micromol l(-1). For operant conditioning, snails were given 5 days of 1 h day(-1) immersion in water (control) or 0.1 micromol l(-1) cocaine, which was the lowest dose that maximally inhibited dopamine uptake, and snails were trained 3 days later. No changes were found between the two groups for learning or memory of the operant behavior. However, snails treated with 0.1 micromol l(-1) cocaine demonstrated impairment of extinction memory during reinstatement of the behavior compared with controls. Our findings suggest that repeated exposure to cocaine modifies the interaction between the original memory trace and active inhibition of this trace through extinction training. An understanding of these basic processes in a simple model system may have important implications for treatment strategies in cocaine addiction.

A comprehensive atlas of the topography of functional groups of the dopamine transporter.

The neuronal dopamine transporter (DAT) is a transmembrane transporter that clears DA from the synaptic cleft. Knowledge of DAT functional group topography is a prerequisite for understanding the molecular basis of transporter function, the actions of psychostimulant drugs, and mechanisms of dopaminergic neurodegeneration. Information concerning the molecular interactions of drugs of abuse (such as cocaine, amphetamine, and methamphetamine) with the DAT at the functional group level may also aid in the development of compounds useful as therapeutic agents for the treatment of drug abuse. This review will provide a cumulative and comprehensive focus on the amino acid functional group topography of the rat and human DATs, as revealed by protein chemical modification and the techniques of site-directed mutagenesis. The results from these studies, represented mostly by site-directed mutagenesis, can be classified into several main categories: modifications without substantial affects on substrate transport, DAT membrane expression, or cocaine analog binding; those modifications which alter both substrate transport and cocaine analog binding; and those that affect DAT membrane expression. Finally, some modifications can selectively affect either substrate transport or cocaine analog binding. Taken together, these literature results show that domains for substrates and cocaine analogs are formed by interactions with multiple and sometimes distinct DAT functional groups.