Validation of a probe for assessing deconjugation of glucuronide and sulfate phase II metabolites assayed through LC-MS/MS in biological matrices.

LC-MS/MS has been proposed in various areas such as Therapeutic Drug Monitoring (TDM), Human Biomonitoring (HBM), disease diagnosis, clinical toxicology and doping control to identify and quantify chemical parents and their metabolites in biological matrices. To determine the total content of a xenobiotic (unconjugated+conjugated forms), an enzymatic hydrolysis step is required. Most studies in the literature have not controlled the effectiveness of the deconjugation process because no method has been described for that purpose. Therefore the aim of this study was to develop and validate a deconjugation probe using a LC-MS/MS method. In order to estimate deconjugation using ß-glucuronidase and/or sulfatase, 4-methyl-umbelliferone (MU) and its conjugates were used as markers. Glucuronidase/sulfatase was added to plasma or urine spiked with 4-methylumbelliferyl-ß-d-glucuronide (MUG) and 4-methylumbelliferyl sulfate (MUS) and umbelliferone, which was used as the internal standard. After incubation at 37°C during 90min, MU appears as a result of the deconjugation of MUG and MUS. The concentrations of the 3 markers were determined using LC-MS/MS. Trueness and precision of the LC-MS/MS method were determined by quality control analysis at three different levels of concentration covering the whole range of calibration. In both matrices, the analytical method allows quantification of the different compounds, with good linearity, trueness and precision and negligible matrix effects. The method was applied with success to deconjugation assay using active glucuronidase/sulfatase in plasma and urine. The probe developed in this study allows to ensure that enzymatic preparation is working properly in the frame of a quality system.

Ganglioside and related-sphingolipid profiles are altered in a cellular model of Alzheimer's disease.

Sphingolipid-related issues are increasingly discussed to contribute to the neuropathological process of Alzheimer's disease (AD). In this study, gangliosides and related-sphingolipids (ceramides, neutral glycosphingolipids and sphingomyelins) were analyzed in neuroglioma (H4) cells expressing the Swedish mutation of the human amyloid precursor protein (H4APPsw) and compared with those of wild-type control H4 cells. These cells were chosen since H4APPsw cells were previously reported to reproduce well some essential features of AD. We found that H4APPsw cells exhibited a striking elevation of the simplest ganglioside GM3, an abnormality that was consistently reported in AD patients and animal models of AD. Concomitantly, the levels of both lactosylceramide (the immediate metabolic precursor of GM3) and ganglioside GD1a increased, suggesting a deregulation in the biosynthesis of gangliosides in the H4APPsw cells. Moreover, while the total ceramide level remained unaltered in H4APPsw cells, a shift in ceramide composition from long chain - to very long chain fatty acid-ceramide species was recorded. Because sphingolipid alterations occurring in H4APPsw cells were similar to those observed in transgenic mice and in human brains, this cellular model might be useful to further explore the complex role of sphingolipids in AD pathogenesis.

Anti-amyloidogenic effects of glycosphingolipid synthesis inhibitors occur independently of ganglioside alterations.

Evidence has suggested that ganglioside abnormalities may be linked to the proteolytic processing of amyloid precursor protein (APP) in Alzheimer's disease (AD) and that pharmacological inhibition of ganglioside synthesis may reduce amyloid ß-peptide (Aß) production. In this study, we assessed the usefulness of two well-established glycosphingolipid (GSL) synthesis inhibitors, the synthetic ceramide analog D-PDMP (1-phenyl 2-decanoylamino-3-morpholino-1-propanol) and the iminosugar N-butyldeoxynojirimycin (NB-DNJ or miglustat), as anti-amyloidogenic drugs in a human cellular model of AD. We found that both GSL inhibitors were able to markedly inhibit Aß production, although affecting differently the APP cleavage. Surprisingly, the L-enantiomer of PDMP, which promotes ganglioside accumulation, acted similarly to D-PDMP to inhibit Aß production. Concurrently, both D- and L-PDMP strongly and equally reduced the levels of long-chain ceramides. Altogether, our data suggested that the anti-amyloidogenic effects of PDMP agents are independent of the altered cellular ganglioside composition, but may result, at least in part, from their ability to reduce ceramide levels. Moreover, our current study established for the first time that NB-DNJ, a drug already used as a therapeutic for Gaucher disease (a lysosomal storage disorder), was also able to reduce Aß production in our cellular model. Therefore, our study provides novel information regarding the possibilities to target amyloidogenic processing of APP through modulation of sphingolipid metabolism and emphasizes the potential of the iminosugar NB-DNJ as a disease modifying therapy for AD.

The Tyr216 phosphorylated form of GSK3ß contributes to tau phosphorylation at PHF-1 epitope in response to Aß in the nucleus of SH-SY5Y cells.

AIMS: GSK3ß activation in Aß conditions leading to tau phosphorylation at pathological sites is a well-known phenomenon. However, the serine/tyrosine phosphorylation processes implied in Aß-induced GSK3ß activation and responsible for tau phosphorylation, especially at the GSK3ß specific Ser396/Ser404 (PHF-1) site, are still debated. MAIN METHODS: Experiments were performed on SH-SY5Y cells exposed to 20µM Aß1-42 in a time ranging from 5min to 8h. The phophorylated forms (Ser9 and Tyr216) of GSK3ß and pTau at PHF-1 epitope were measured by immunoblotting in nuclear extracts. KEY FINDINGS: We showed a superimposable time-dependent increase of nuclear pGSK3ßTyr216 and nuclear pTau at PHF-1 site, both reaching their maximal level after 8h of Aß1-42 exposure. In addition, nuclear accumulation of pTau is accompanied by its cytoplasmic decrease suggesting that pTau is translocated in response to Aß treatment. Besides, our experiments showed that specific pGSK3ßTyr216 inhibition is required to drop nuclear pTau, ensuring the involvement of Tyr216 phosphorylation in Aß-mediated tau phosphorylation at PHF-1 epitope. SIGNIFICANCE: These data suggested that in response to Aß exposure in SH-SY5Y cells, GSK3ß activation is performed through Tyr216 phosphorylation and resulted in tau phosphorylation at PHF-1 epitope and in its translocation.

Inhibition of GSK3ß by pharmacological modulation of sphingolipid metabolism occurs independently of ganglioside disturbance in a cellular model of Alzheimer's disease.

Accumulating evidence implicates ganglioside and/or related-sphingolipid disturbance in the pathogenesis of Alzheimer's disease (AD). However, it is not known whether these lipidic alterations are connected with other important features of AD, such as deregulated insulin/Akt/GSK3 signaling. In this study, we have treated neuroglioma cells expressing the double Swedish mutation of human amyloid precursor protein (H4APPsw) with several glycosphingolipid (GSL)-modulating agents, and we have analyzed the impact of the aberrant ganglioside composition on the GSK3 activation state. We found that both ceramide analogs D- and L-PDMP (1-phenyl 2-decanoylamino-3-morpholino-1-propanol), which have opposite effects on ganglioside synthesis, selectively inhibited GSK3ß via Ser9 phosphorylation independently of the upstream insulin/Akt pathway. Conversely, the iminosugar N-butyldeoxynojirimycin (NB-DNJ) which displayed similar reduction of gangliosides as D-PDMP, did not affect the phosphorylation state of GSK3ß. Concurrently, while NB-DNJ did not modify the cellular ceramide content, both PDMP enantiomers strongly and equally reduced the levels of long-chain ceramide species. Altogether, our findings led us to hypothesize that the PDMP-induced altered ganglioside composition is not the principal mechanism involved in the inhibition of GSK3ß, but seems to implicate, at least in part, their ability to reduce ceramide levels. Nevertheless, this study provides new information regarding the possibilities to target GSK3ß through modulation of sphingolipid metabolism.

Lithium chloride and staurosporine potentiate the accumulation of phosphorylated glycogen synthase kinase 3ß/Tyr216, resulting in glycogen synthase kinase 3ß activation in SH-SY5Y human neuroblastoma cell lines.

Glycogen synthase kinase 3ß (GSK3ß) activity is regulated by phosphorylation processes and regulates in turn through phosphorylation several proteins, including eukaryotic initiation factor 2B (eIF2B). Serine 9 phosphorylation of GSK3ß (pGSK3ßSer9), usually promoted by activation of the PI3K/Akt survival pathway, triggers GSK3ß inhibition. By contrast, tyrosine 216 phosphorylation of GSK3ß (pGSK3ßTyr216) increases under apoptotic conditions, leading to GSK3ß activation. Lithium chloride (LiCl) is usually described to increase pGSK3ßSer9 through the PI3K/Akt pathway, resulting in GSK3ß inhibition. The purpose of this study is to demonstrate that in some cases LiCl is also able to increase pGSK3ßTyr216, resulting in GSK3ß activation. For this, we used SH-SY5Y cells and primary neuronal cultures and investigated the effects of LiCl on the two phosphorylated forms of GSK3ß under staurosporine (STS)-intoxicated conditions. The ratios between the phosphorylated and total forms of GSK3ß and eIF2B were determined by Western blotting. Our results revealed that, besides its ability to increase pGSK3ßSer9, LiCl is also able to increase pGSK3ßTyr216 greatly in STS-intoxicated SH-SY5Y cells but not in STS-intoxicated primary neuronal cultures. This accumulation of both Ser9 and Tyr216 phosphorylation results in GSK3ß activation in STS-intoxicated SH-SY5Y cells in spite of the presence of LiCl. These findings indicate that LiCl treatment is not necessarily correlated with GSK3ß inhibition even though it generates Ser9 phosphorylation. Consequently, the ratio pGSK3ßSer9/pGSK3ßTyr216, which takes into account the balance between the two inactive (Ser9) and active (Tyr216) forms of GSK3ß, could be more useful for predicting GSK3ß inhibition.

Ceramide and Related-Sphingolipid Levels Are Not Altered in Disease-Associated Brain Regions of APP and APP/PS1 Mouse Models of Alzheimer's Disease: Relationship with the Lack of Neurodegeneration?

There is evidence linking sphingolipid abnormalities, APP processing, and neuronal death in Alzheimer's disease (AD). We previously reported a strong elevation of ceramide levels in the brain of the APP(SL)/PS1Ki mouse model of AD, preceding the neuronal death. To extend these findings, we analyzed ceramide and related-sphingolipid contents in brain from two other mouse models (i.e., APP(SL) and APP(SL)/PS1(M146L)) in which the time-course of pathology is closer to that seen in most currently available models. Conversely to our previous work, ceramides did not accumulate in disease-associated brain regions (cortex and hippocampus) from both models. However, the APP(SL)/PS1Ki model is unique for its drastic neuronal loss coinciding with strong accumulation of neurotoxic Aß isoforms, not observed in other animal models of AD. Since there are neither neuronal loss nor toxic Aß species accumulation in APP(SL) mice, we hypothesized that it might explain the lack of ceramide accumulation, at least in this model.

Gender-dependent accumulation of ceramides in the cerebral cortex of the APP(SL)/PS1Ki mouse model of Alzheimer's disease.

Altered sphingolipid metabolism plays an emergent role in the etiology of Alzheimer's disease (AD). In this study, we determined the levels of ceramides and other related-sphingolipids (sphingomyelins, sulfatides and galactosylceramides) in the cerebral cortex of an APP(SL)/PS1Ki mouse model of AD. The results demonstrate that ceramides accumulated in the cortex of APP(SL)/PS1Ki mice, but not in PS1Ki mice, whereas all others major sphingolipids (except galactosylceramides) were not altered in comparison with those from age-matched wild-type mice. Furthermore, as early as 3 months of age, female mice but not males, exhibit a strong increase in 2-hydroxy fatty acid-containing ceramides, whereas males display a significant elevation of non-hydroxy fatty acid ceramide species. Therefore, the gender differences in ceramide accumulation in the brain of mice expressing APP(SL) suggest that additional factors like modified ceramide metabolism may contribute to the increased propensity of females to develop AD.

A cell-permeable peptide inhibitor TAT-JBD reduces the MPP+-induced caspase-9 activation but does not prevent the dopaminergic degeneration in substantia nigra of rats.

Many studies showed that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) which was widely used to produce Parkinson's disease (PD)-like models in animals can elicit apoptosis with increase of caspase activity via its neurotoxic metabolite 1-methyl-4-phenylpyridinium ion (MPP(+)). Another pathway shown in MPTP-mediated nigrostriatal dopaminergic cell death involved the c-Jun-N-terminal kinases (JNKs) which are stress-activated protein kinases (SAPKs). Activation of the JNKs leads to the activation of transcription factors such as c-Jun that regulates its own expression. However, it is not known whether the activation of c-Jun is crucial in the stimulation of caspases leading to apoptosis observed in PD-like models. The aim of this study was to investigate the cellular expression and phosphorylation of c-Jun and the caspase-9 activity in rat injured with an intranigral injection of MPP(+). Furthermore, we determined the effects of a cell-permeable peptide TAT-JBD, inhibiting selectively JNKs, on apoptosis markers and on the expression of tyrosine hydroxylase (TH). Our results showed that MPP(+) induced not only an activation of c-Jun but also an early and robust stimulation of caspase-9 in midbrain of rats. Furthermore, a preliminary intravenous injection of TAT-JBD reduced the caspase-9 activation specifically induced by MPP(+) suggesting a control of the JNKs pathway on the intrinsic way of apoptosis in MPP(+)-toxicity. However, the inhibition of the JNK pathway did not prevent TH inhibition, DNA fragmentation and Bad expression in MPP(+)-lesioned substantia nigra of rats. Therefore, the possibility of intervention on the JNK pathway as a therapeutic strategy in Parkinson's disease is questionable.

The oxindole/imidazole derivative C16 reduces in vivo brain PKR activation.

Inhibition of double-stranded RNA-dependent protein kinase (PKR) represents an interesting strategy for neuroprotection. However, inhibiting this kinase which triggers the apoptotic process could favour in counterpart cell proliferation and tumorigenesis. Here, we use an in vivo model of 7-day-old rat displaying a high activation of brain PKR to investigate the effects of a new PKR inhibitor identified as an oxindole/imidazole derivative (C16). We show for the first time that acute systemic injection of C16 specifically inhibits the apoptotic PKR/eIF2alpha signaling pathway without stimulating the proliferative mTOR/p70S6K signaling mechanism.

Regulation of initiation factors controlling protein synthesis on cultured astrocytes in lactic acid-induced stress.

The goals of this work were first to assess whether the lactic acidosis observed in vivo in ischemia may by itself explain the inhibition of protein synthesis described in the literature and second to study the factors controlling the initiation of protein synthesis under lactic acid stress. Primary rat astrocyte cultures exposed to pH 5.25 underwent cell death and a strong inhibition of protein synthesis assessed by [3H]methionine incorporation, which was solely due to acidity of the extracellular medium and was not related to lactate concentrations. This result was associated with a weak phosphorylation of eukaryotic initiation factor (eIF)4E and a rapid phosphorylation of eIF2alpha via the kinases PKR and PKR-like endoplasmic reticulum kinase. The inhibition of PKR by PRI led first to a significant but not complete dephosphorylation of eIF2alpha that probably contributed to maintain the inhibition of the protein synthesis and second to surprising phosphorylations of extracellular signal-regulated protein kinase, p70S6K and eIF4E, suggesting a possible cross-link between the two pathways. Conversely, cell death was weak at pH 5.5. Protein synthesis was decreased to a lesser extent, the phosphorylation of eIF2alpha was limited, extracellular signal-regulated protein kinase 1/2 was activated and its downstream targets, p70S6K and eIF4E, were phosphorylated. However, the strong phosphorylation of eIF4E was not associated with an activation of the eIF4F complex. This last result may explain why protein synthesis was not stimulated at pH 5.5. However, when astrocytes were exposed at pH 6.2, corresponding to the lower pH observed in hyperglycemic ischemia, no modification in protein synthesis was observed. Consequently, lactic acidosis cannot, by itself, provide an explanation for the decrease in protein synthesis previously reported in vivo in ischemia.

Genotype-related changes of ganglioside composition in brain regions of transgenic mouse models of Alzheimer's disease.

In this study, brain gangliosides of different transgenic mouse models of Alzheimer's disease (AD) were analyzed and compared with age-matched wild-type mice. Gangliosides were analyzed in cerebral cortex, a region with extensive A beta plaques, and cerebellum, a non-vulnerable region with no A beta containing plaques. There was a marked increase in simple gangliosides GM2 and GM3 only within the cortex of all mice expressing APP(SL). Additionally, loss of complex "a" gangliosides (GT1a, GD1a and GM1) was recorded in APP/PS1Ki model, whereas in APP(SL) and APP/PS1 mice, the complex "b" gangliosides (GQ1b, GT1b and GD1b) moderately decreased. Surprisingly, expression of either mutant PS1(M146L) or PS1 mutant FAD (Ki model) alone tended to lower the levels of both GM2 and GM3 within the cortex. Conversely, only slight changes of the ganglioside pattern were found in the cerebellum. Because ganglioside alterations occurring in APP transgenic mice were similar to those observed in human AD brain, these transgenic models would represent valuable tools to further investigate the role of altered ganglioside metabolism in the pathogenesis of AD.

Fluoro-Jade B staining as useful tool to identify activated microglia and astrocytes in a mouse transgenic model of Alzheimer's disease.

Fluoro-Jade B is known as a high affinity fluorescent marker for the localization of neuronal degeneration during acute neuronal distress. However, one study suggested that fluoro-Jade B stains reactive astroglia in the primate cerebral cortex. In this study, we analyzed the staining of fluoro-Jade B alone or combined with specific markers for detection of glial fibrillary acidic protein (GFAP) or activated CD68 microglia in the double APP(SL)/PS1 KI transgenic mice of Alzheimer's disease (AD), which display a massive neuronal loss in the CA1 region of the hippocampus. Our results showed that fluoro-Jade B did not stain normal and degenerating neurons in this double mouse transgenic model. Fluoro-Jade B was co-localized with Abeta in the core of amyloid deposits and in glia-like cells expressing Abeta. Furthermore, fluoro-Jade B was co-localized with CD68/macrosialin, a specific marker of activated microglia, and with GFAP for astrocytes in APP(SL)/PS1 KI transgenic mice of AD. Taken together, these findings showed that fluoro-Jade B can be used to label activated microglia and astrocytes which are abundant in the brain of these AD transgenic mice. It could stain degenerating neurons as a result of acute insult while it could label activated microglia and astrocytes during a chronic neuronal degenerative process such as AD for example.

Group I metabotropic glutamate receptors activate the p70S6 kinase via both mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK 1/2) signaling pathways in rat striatal and hippocampal synaptoneurosomes.

Group I metabotropic glutamate receptors (mGluRs) have been demonstrated to play a role in synaptic plasticity via a rapamycin-sensitive mRNA translation signaling pathway. Various growth factors can stimulate this pathway, leading to the phosphorylation and activation of mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that modulates the activity of several translation regulatory factors, such as p70S6 kinase. However, little is known about the cellular and molecular mechanisms that bring the plastic changes of synaptic transmission after stimulation of group I mGluRs. Here, we investigated the role of the mTOR-p70S6K and the ERK1/2-p70S6K pathways in rat striatal and hippocampal synaptoneurosomes after group I mGluR stimulation. Our findings show that (S)-3,5-dihydroxyphenylglycine (DHPG) increases significantly the activation of mTOR and p70S6K (Thr389, controlled by mTOR) in both brain areas. The mTOR activation is dose-dependent and requires the stimulation of mGluR1 subtype receptors as for the p70S6K activation observed in striatum and hippocampus. In addition, the p70S6K (Thr421/Ser424) activation via the ERK1/2 activation is increased and involved also mGluR1 receptors. These results demonstrate that group I mGluRs are coupled to mTOR-p70S6K and ERK1/2-p70S6K pathways in striatal and hippocampal synaptoneurosomes. The translational factor p70S6K could be involved in the group I mGluRs-modulated synaptic efficacy.

Lactic acidosis stimulates ganglioside and ceramide generation without sphingomyelin hydrolysis in rat cortical astrocytes.

Acidosis is a ubiquitous feature of cerebral ischemia, and triggers a cascade of biochemical events that results in neuronal injury. The purpose of this study was to evaluate the effects of lactic acidosis on the ganglioside composition, the ceramide and sphingomyelin (SM) levels in rat cortical astrocytes. Primary astrocyte cultures were exposed to lactic acid (pH 5.5) for 2, 5 and 17 h, and cell death was evaluated at each time point. Gangliosides, ceramides and SM were analyzed by high-performance thin layer chromatography. Lactic acidosis caused a progressive increase of both GM3 and GD3 gangliosides up to 5 h of treatment. However, at 17 h of acidosis, GM3 tented to return to the normal level whereas GD3 accumulated. Additionally, ceramides were gradually generated, whereas no significant decrease of SM occured for 17 h of acidosis. These results suggest that ceramides were not produced by the breakdown of SM and may be served as metabolic precursor for the biosynthesis of GM3 and GD3. Since these lipids are important messengers of the adaptative responses to stress, accumulation of sphingolipids triggered by lactic acid exposure of astrocytes might play an important role in determining the outcomes of injurious processes.

The up-regulation of the striatal dopamine transporter's activity by cAMP is PKA-, CaMK II- and phosphatase-dependent.

The neuronal dopamine transporter (DAT) is a presynaptic plasma membrane protein mediating the re-uptake of dopamine released from synaptic cleft into the nerve terminals. While the regulation of its activity by protein kinase C signalling is well-characterized, there is controversial debate about its regulation by protein kinase A (PKA) signalling. In rat striatal synaptosomes, we showed that a cell-permeable cyclic adenosine 3',5'-monophosphate analogue up-regulated the DAT capacity without modification of its efficiency. This acute effect was PKA-, calcium calmodulin dependent kinase II- and phosphatase-dependent. Together, these results suggest that the activity of DAT may depend on a state of the transporter with both specific phosphorylated and dephosphorylated sites.

Effect of ischemia on TBARS and lactate production in several cerebral regions of anaesthetised and awake rats.

The premise of neuroprotective therapy for acute ischemic stroke is based upon the possibility to interfere with the cellular ischemic cascade, so the understanding of the mechanisms and consequences of cerebral ischemia is necessary. The relationship between lipid peroxidation and acidosis was investigated in several regions of rat brain following ischemia without reperfusion. Male Wistar rats (280-300 g) were anaesthetised (Ketalar 33 mg/kg and Rompun 6.66 mg/kg) or not and underwent a four-vessel occlusion for 5 minutes. Then, thiobarbituric acid-reactive substances (TBARS) and lactate levels were measured in different brain regions (cerebellum, bulb, striatum, hippocampus, cortex). Induction of ischemia by ligation of two common carotid arteries and two vertebral arteries resulted in a production of TBARS (40-120%, p < 0.05) and lactate (20-60%, p < 0.05) in all cerebral regions of awake rats, especially in striatum, suggesting a potential link between lipid peroxidation and acidosis. When ischemia was realised on anaesthetised animals, an increase of lactate levels (30-50%, p < 0.05) was shown in all brain regions but TBARS were produced only in striatum (82%, p < 0.05). These data showed the particular vulnerability of striatum to ischemia and the possible opposite effects of an anaesthesia.

Lactic acidosis progressively impairs dopamine uptake in rat striatal synaptosomes by a mechanism partially independent of the Na+/K+-ATPase dysfunction.

Previous experiments reported that incubation of rat striatal synaptosomes with lactic acid (pH 5.5) resulted in an inhibition of dopamine (DA) uptake partially mediated by free radical damage. Since the DA uptake process is highly dependent on the functionality of Na+/K+-ATPase, the present study investigated whether this inhibition of DA uptake could be related to an alteration of the Na+/K+-ATPase activity. Striatal lactic acidosis was performed by direct addition of lactic acid in the incubation medium to obtain a pH as close as possible to that observed in ischemia. Acidosis (pH 5.5) induced a progressive decline in the specific DA uptake and a decrease of Na+/K+-ATPase activity in striatal synaptosomes. However, whereas loss of Na+/K+-ATPase activity was totally prevented by Trolox, a powerful antioxidant, DA uptake remained partially inhibited. Taken together, these data suggest that acidosis, in a degree encountered during ischemia, alters the high-affinity DA uptake in part by a mechanism that does not involve a Na+/K+ pump deficiency.

Evidence that acidosis alters the high-affinity dopamine uptake in rat striatal slices and synaptosomes by different mechanisms partially related to oxidative damage.

Several experimental studies have shown that acidosis impairs neurotransmitter uptake processes. The purpose of this study was to determine the mechanism underlying acidosis-induced alterations of the high-affinity dopamine (DA) uptake in rat striatal synaptosomes and slices. Acidosis (pH 5.5) performed either by lactic acid or phosphoric acid induced a decrease in the high-affinity DA uptake in the two striatal models, slices being lesser affected than synaptosomes. Addition of the acid prior to uptake measurement led to a strong reduction of the DA uptake velocity. This early inhibitory effect was completely reversed when acid was removed from the medium by washings. Conversely, when slices and synaptosomes were pre-incubated for different times with each acid, DA uptake remained inhibited in spite of washings. This later inhibition was accompanied by the production of thiobarbituric acid reactive substances, a marker of lipid peroxidation, and was partially prevented by the antioxidant Trolox. Taken together, these results suggest that acidosis, in a degree encountered during ischemia, alters the high-affinity DA uptake by at least two ways: an early and direct effect of H(+) ions on the DA transporters, and subsequently an inhibition partially mediated by free radical damage.

Sulfatide and GM1 ganglioside modulate the high-affinity dopamine uptake in rat striatal synaptosomes: evidence for the involvement of their ionic charges.

The present study was undertaken to examine the effects of the anionic glycolipids GM1 ganglioside and sulfatide on the high-affinity dopamine (DA) uptake in rat striatal synaptosomes. After 1h of incubation, GM1 stably bound to synaptosomes and modified the activity of the neuronal dopamine transporter (DAT). With 1.2 and 12 microM GM1, V(max) decreased by 13 and 23%, respectively, reflecting a slight reduction of the number of functional uptake sites and K(m) was lowered by 21 and 33%, thus showing an increase of the affinity. Treatment of synaptosomes with 1.2 microM of sulfatide, which possesses an anionic sulfated group, led to a similar decrease of V(max) (19%) than GM1, but to a significantly higher reduction of K(m) (35%). In fact, sulfatide associated to synaptosomes in a 3.5-fold higher extent than GM1. Conversely, when GM1 and sulfatide were replaced by GM1 alcohol and galactosylceramide, respectively, no modification of the DA uptake occurred, although these neutral glycolipids incorporated into the synaptosomes to the same extent as the related anionic compounds.Altogether, these results demonstrate the key role of negative charges linked to the oligosaccharide chains of glycolipids in the modulation of DA transport across the synaptosomal membrane.