Changes in the Levels of Dolichol and Dolichyl Phosphate in a Murine Model of Niemann-Pick''s Type C Disease

Abstract: The distributions of mevalonate pathway lipids in various organs of a mouse strain used as a model for Niemann-Pick's type C disease were analyzed. Extensive accumulation of cholesterol was observed in all tissues with the exception of the brain, where the content of this lipid was decreased. The changes in total dolichol contents of most organs varied from a 50% decrease in the lung to a twofold increase in kidney and heart. There was relative enrichment of longer-chain dolichols, but no increase in the relative amount of α-unsaturated polyprenols was observed. The levels of dolichyl phosphate in all organs were increased, and most of this lipid was associated with bound oligosaccharides or proteins. Ubiquinone levels were largely unchanged. Subfractionation studies revealed that heavy and light lysosomes exhibited a 10-fold increase in cholesterol level, the amount of dolichol was decreased in lysosomes and increased in microsomes, and there was an increase in the dolichyl phosphate levels of all three of these subfractions. These results indicate that in diseased mice cholesterol accumulation in various organs is paralleled by an increase in the dolichyl phosphate concentration, whereas dolichol transport from the endoplasmic reticulum to lysosomes is inhibited.     

Acute and Repeated Systemic Amphetamine Administration: Effects on Extracellular Glutamate, Aspartate, and Serine Levels in Rat Ventral Tegmental Area and Nucleus Accumbens

Abstract: Recent work indicates an important role for excitatory amino acids in behavioral sensitization to amphetamine. We therefore examined, using in vivo microdialysis in awake rats, the effects of amphetamine on efflux of glutamate, aspartate, and serine in the ventral tegmental area and nucleus accumbens, brain regions important for the initiation and expression of amphetamine sensitization, respectively. Water-pretreated and amphetamine-pretreated rats were compared to determine if sensitization altered such effects. In both brain regions, Ca²⁺-dependent efflux of glutamate accounted for ∼20% of basal glutamate efflux. A challenge injection of water or 2.5 mg/kg of amphetamine did not significantly alter glutamate, aspartate, or serine efflux in the ventral tegmental area or nucleus accumbens of water- or amphetamine-pretreated rats. However, 5 mg/kg of amphetamine produced a gradual increase in glutamate efflux in both regions that did not reverse, was observed in both water- and amphetamine-pretreated rats, and was prevented by haloperidol. Although increased glutamate efflux occurred with too great a delay to mediate acute behavioral responses to amphetamine, it is possible that repeated augmentation of glutamate efflux during repeated amphetamine administration results in compensatory changes in levels of excitatory amino acid receptors in the ventral tegmental area and nucleus accumbens that contribute to development or expression of amphetamine sensitization.     

Differential Effects of δ- and μ-Opioid Receptor Antagonists on the Amphetamine-Induced Increase in Extracellular Dopamine in Striatum and Nucleus Accumbens

Abstract: The specific opioid receptor antagonist naloxone attenuates the behavioral and neurochemical effects of amphetamine. Furthermore, the amphetamine-induced increase in locomotor activity is attenuated by intracisternally administered naltrindole, a selective δ-opioid receptor antagonist, but not by the irreversible μ-opioid receptor antagonist β-funaltrexamine. Therefore, this research was designed to determine if naltrindole would attenuate the neurochemical response to amphetamine as it did the behavioral response. In vivo microdialysis was used to monitor the change in extracellular concentrations of dopamine in awake rats. Naltrindole (3.0, 10, or 30 µg) or vehicle was given 15 min before and β-funaltrexamine (10 µg) or vehicle 24 h before the start of cumulative dosing, intracisternally in a 10-µl volume, while the rats were lightly anesthetized with methoxyflurane. Cumulative doses of subcutaneous d-amphetamine (0.0, 0.1, 0.4, 1.6, and 6.4 mg/kg) followed pretreatment injections at 30-min intervals. Dialysate samples were collected every 10 min from either the striatum or nucleus accumbens and analyzed for dopamine content by HPLC. Amphetamine dose-dependently increased dopamine content in both the striatum and nucleus accumbens, as reported previously. Naltrindole (3.0, 10, and 30 µg) significantly reduced the dopamine response to amphetamine in the striatum. In contrast, 30 µg of naltrindole did not modify the dopamine response to amphetamine in the nucleus accumbens. On the other hand, β-funaltrexamine (10 µg) had no effect in the striatum but significantly attenuated the amphetamine-induced increase in extracellular dopamine content in the nucleus accumbens. These data suggest that δ-opioid receptors play a relatively larger role than μ-opioid receptors in mediating the amphetamine-induced increase in extracellular dopamine content in the striatum, whereas μ-opioid receptors play a larger role in mediating these effects in the nucleus accumbens.     

Use of Calcium Antagonism for the Characterization of Drug-Evoked Dopamine Release from the Brain of Conscious Rats Determined by Microdialysis

Dopamine was determined by microdialysis of the striatum of conscious rats. We investigated whether the release of dopamine, induced by nine different pharmacological treatments, was sensitive to calcium antagonism. Calcium antagonism was determined by Mg2+ or Cd2+ infusion. The following conditions were investigated: haloperidol, haloperidol plus GBR 12909, nomifensine, (+)-amphetamine (all administered intraperitoneally), KCl, 1-methyl-4-phenyl-pyridinium ion (MPP+), glutamate, ouabain, and 120 mmol/L magnesium (all applied by infusion through the dialysis membrane). The results on calcium antagonism were combined with data on tetrodotoxin (TTX) sensitivity. With the combined data, three different types of dopamine release were characterized. First, action potential-dependent dopamine release was observed in animals treated with saline, haloperidol, haloperidol plus GBR 12909, nomifensine, and ouabain. Second, action potential-independent release was established in the case of (+)-amphetamine, glutamate, MPP+, and 120 mmol/L Mg2+. Finally, K+-induced dopamine release was classified as TTX independent and calcium dependent. It is concluded that brain dialysis is a powerful method for differentiating between different types of neurotransmitter release.     

Relationships Between the Catechol Substrate Binding Site and Amphetamine, Cocaine, and Mazindol Binding Sites in a Kinetic Model of the Striatal Transporter of Dopamine In Vitro

Abstract: Experiments were conducted to determine how (−)-cocaine and S (+)-amphetamine binding sites relate to each other and to the catechol substrate site on the striatal dopamine transporter (sDAT). In controls, m -tyramine and S (+)-amphetamine caused release of dopamine from intracellular stores at concentrations ≥12-fold those observed to inhibit inwardly directed sDAT activity for dopamine. In preparations from animals pretreated with reserpine, m -tyramine and S (+)-amphetamine caused release of preloaded dopamine at concentrations similar to those that inhibit inwardly directed sDAT activity. S (+)-Amphetamine and m -tyramine inhibited sDAT activity for dopamine by competing for a common binding site with dopamine and each other, suggesting that phenethylamines are substrate analogues at the plasmalemmal sDAT. (−)-Cocaine inhibited sDAT at a site separate from that for substrate analogues. This site is mutually interactive with the substrate site ( K _int = 583 n M ). Mazindol competitively inhibited sDAT at the substrate analogue binding site. The results with (−)-cocaine suggest that the (−)-cocaine binding site on sDAT is distinct from that of hydroxyphenethylamine substrates, reinforcing the notion that an antagonist for (−)-cocaine binding may be developed to block (−)-cocaine binding with minimal effects on dopamine transporter activity. However, a strategy of how to antagonize drugs of abuse acting as substrate analogues is still elusive.     

Amphetamine and D1 Dopamine Receptor Agonists Produce Biphasic Effects on Glutamate Efflux in Rat Ventral Tegmental Area: Modification by Repeated Amphetamine Administration

Abstract: Amphetamine or selective D1 and D2 dopamine receptor agonists and antagonists were administered to the ventral tegmental area (VTA) through a microdialysis probe to determine their effects on glutamate and aspartate efflux in rats pretreated for 5 days with vehicle or 5 mg/kg (+)-amphetamine sulfate. In vehicle rats, glutamate efflux declined during 2 h of perfusion with the D1 receptor agonist SKF-82958 (10 and 100 µ M ). After SKF-82958 perfusion ended, glutamate efflux rebounded to basal levels and continued to increase gradually over the next 2 h. A similar biphasic pattern was observed with intra-VTA amphetamine (10 and 100 µ M ) and with another D1 agonist (100 µ M SKF-38393). The biphasic effects of SKF-82958 were prevented by coperfusion with a D1 antagonist (SCH-23390; 30 µ M ). Glutamate efflux was unaffected by a D2 agonist (100 µ M quinpirole) and by D1 or D2 antagonists administered alone (SCH 23390 and eticlopride; 30 µ M ). In amphetamine-pretreated rats tested 2 days after the last injection, both the decrease during SKF-82958 perfusion and the delayed increase in glutamate efflux were attenuated. In rats tested 12–14 days after the last amphetamine injection, only the decrease during SKF-82958 perfusion was attenuated. None of these drug treatments produced consistent effects on aspartate efflux. We showed previously that systemic amphetamine (5 mg/kg, i.p.) has no immediate effect on VTA glutamate efflux but produces a delayed increase in glutamate efflux that reaches statistical significance 2–3 h after injection. Because behavioral sensitization can be elicited either by repeated systemic or repeated intra-VTA administration, neurochemical effects common to both routes (such as the delayed increase in glutamate efflux) are most likely to contribute to its induction.     

Studies on the metabolism and toxicological detection of the amphetamine-like anorectic mefenorex in human urine by gas chromatography-mass spectrometry and fluorescence polarization immunoassay

Studies on the metabolism and on the toxicological analysis of mefenorex [R,S-N-(3-chloropropyl)-α-methylphenethylamine, MF] using gas chromatography-mass spectrometry (GC-MS) and fluorescence polarization immunoassay (FPIA) are described. The metabolites were identified in urine samples of volunteers by GC-MS. Besides MF, thirteen metabolites including amphetamine (AM) could be identified and three partially overlapping metabolic pathways could be postulated. For GC-MS detection, the systematic toxicological analysis procedure including acid hydrolysis, extraction at pH 8-9 and acetylation was suitable (detection limits 50 ng/ml for MF and 100 ng/ml for AM). Excretion studies showed, that only AM but neither MF nor its specific metabolites were detectable between 32 and 68 h after ingestion of 80 mg of MF. Therefore, misinterpretation can occur. The Abbott TDx FPIA amphetamine/methamphetamine II gave positive results up to 68 h. All the positive immunoassay results could be confirmed by the described GC-MS procedure.     

Analysis of benzphetamine and its metabolites in rat urine by liquid chromatography–electrospray ionization mass spectrometry

An analytical method to identify and determine benzphetamine (BMA) and its five metabolites in urine was developed by liquid chromatography–electrospray ionization mass spectrometry (LC–ESI–MS) using the solid-phase extraction column Bond Elut SCX. Deuterium-labeled compounds, used as internal standards, were separated chromatographically from each corresponding unlabeled compound in the alkaline mobile phase with an alkaline-resistant ODS column. This method was applied to the identification and determination of BMA and its metabolites in rat urine collected after oral administration of BMA. Under the selected ion monitoring mode, the limit of quantitation (signal-to-noise ratio 10) for BMA, N-benzylamphetamine (BAM), p-hydroxybenzphetamine (p-HBMA), p-hydroxy-N-benzylamphetamine (p-HBAM), methamphetamine (MA) and amphetamine (AM) was 700 pg, 300 pg, 500 pg, 1.4 ng, 6 ng and 10 ng in 1 ml of urine, respectively. This analytical method for p-HBMA, structurally closer to the unchanged drug of all the metabolites, was very sensitive, making this a viable metabolite for discriminating the ingestion of BMA longer than the parent drug or other metabolites in rat.