Modulation of MPP<Superscript>+</Superscript> uptake by tea and some of its components in Caco-2 cells

The entry of most xeno/endobiotics into the organism is limited by their intestinal absorption. The interference of certain foods with the therapeutic efficacy of drugs or with chemical toxicity is becoming evident and growing attention is being given to these subjects. The aim of this work was to study the effect of green tea (GT) and black tea (BT), as well as some of their components, on the transport of organic cation molecules. For this purpose, ³H-MPP⁺ (radiolabeled 1-methyl-4-phenylpyridinium) was used as a model organic cation and Caco-2 cells were used as an intestinal epithelial model. Our results showed that both GT and BT significantly increased ³H-MPP⁺ absorption in these cells. Additionally, we studied the effect of epigallocatechin-3-gallate (EGCG), myricetin, caffeine, and theophylline. Whereas EGCG (2 mM) increased, myricetin (50 μM) and caffeine (1 mM) decreased, and theophylline (1 mM) had no effect on the uptake of ³H-MPP⁺ into Caco-2 cells. When GT was supplemented with caffeine or theophylline, we observed a partial loss of its effect. When BT was supplemented with EGCG, its ability to increase ³H-MPP⁺ uptake was much more pronounced than that observed with BT alone. In conclusion, this study showed that GT and BT might interfere with the absorption of the model organic cation MPP⁺ by the intestinal epithelium. Since important compounds are organic cations, the consequences of this interference may have an impact on human health. Although this constitutes only preliminary work and further studies are needed, tea should be included in the growing list of foodstuffs that have the potential to be involved in food–drug interactions.     

The extraneuronal transport mechanism for noradrenaline (uptake2) avidly transports 1-methyl-4-phenylpyridinium (MPP+)

Summary The corticosterone-sensitive extraneuronal transport mechanism for noradrenaline (uptake₂) removes the neurotransmitter from the extracellular space. Recently, an experimental model for uptake₂ has been introduced which is based on tissue culture techniques (human Caki-1 cells). The present study describes some properties of uptake₂ in Caki-1 cells and introduces a new substrate, i.e., 1-methyl-4-phenylpyridinium (MPP⁺).Experiments on Caki-1 cells disclosed disadvantages of tritiated noradrenaline as substrate for the investigation of uptake₂. The initial rate of ³H-noradrenaline transport [k_in = 0.58 l/(mg protein · min)] was low compared with other cellular transport systems and intracellular noradrenaline was subject to rapid metabolism (k_{O-methylation} = 0.54 min^–1). The neurotoxin MPP⁺ was found to be a good substrate of uptake₂. Initial rates of specific ³H-MPP⁺ transport into Caki-1 cells were saturable, the K_m being 24 mol/l and the V_max being 420 pmol/(mg protein · min). The rate constant of specific inward transport was 34 times higher [19.6 mol/l (mg protein · min)] than that of ³H-noradrenaline. The ratio specific over non-specific transport was considerably higher for ³H-MPP⁺ (12.6) than for ³H-noradrenaline (3.0). ³H-MPP⁺ transport into Caki-1 cells was inhibited by various inhibitors of uptake₂. The highly significant positive correlation (p < 0.001, r = 0.986, n = 7) between the IC₅₀'s for the inhibition of the transport of ³H-noradrenaline and ³H-MPP⁺, respectively, proves the hypothesis that MPP⁺ enters Caki-1 cells via uptake₂. ³H-MPP⁺ is taken up via uptake₂ not only by Caki-1 cells but also by the isolated perfused rat heart which is another established model of uptake₂.Tritiated MPP⁺ is a new and convenient tool for the investigation of uptake₂. The rate constant for inward transport, the factor of accumulation and the ratio specific over non-specific transport are considerably higher for ³H-MPP⁺ than for ³H-noradrenaline. In uptake studies with ³H-MPP⁺ inhibition of intracellular noradrenaline-metabolizing enzymes is not necessary. In tissues and tissue cultures which possess fewer uptake₂ carriers than Caki-1 cells or the rat heart, the identification and characterization of uptake₂ can be expected to be greatly facilitated by the use of ³H-MPP⁺.Supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to H. Russ at the above address     

Inward transport of 3H-MPP+ in freshly isolated rat hepatocytes: evidence for interaction with catecholamines

1-Methyl-4-phenylpyridinium (MPP⁺), the neurotoxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), is efficiently taken up and accumulated by rat hepatocytes. However, the nature of the mechanism(s) involved in the hepatic uptake of MPP⁺ remains partially unknown. The aim of the present study was to further characterize the hepatic uptake of ³H-MPP⁺, namely by investigating the interactions of catecholamines (which are also efficiently taken up by rat hepatocytes) with MPP¹ transport.The accumulation of ³H-MPP⁺ in isolated rat hepatocytes occurred through saturable and non-saturable mechanisms. The kinetics of the saturable component of ³H-MPP⁺ uptake was as follows: V_max = 181.3 ± 11.1 pmol mg protein^–1 min^–1 and K_m = 47.1 M (27.9, 66.3) (n = 5). The diffusion constant (in ml mg protein^–1 min^–1) for the non-saturable uptake of ³H-MPP⁺ was 0.00068 (0.00052, 0.00083) (n = 5). From the analysis of the time course of ³H-MPP⁺ accumulation at a substrate concentration of 100 nM ³H-MPP⁺, it was found that the rate constant of inward transport of ³H-MPP⁺ into hepatocytes (k_in) was 15.7 ± 3.8 l mg protein^–1 min^–1, the rate constant of outward transport of ³H-MPP⁺ from hepatocytes (k_out) was 0.077 ± 0.023 min^–1 and the equilibrium accumulation (A_max) of ³H-MPP⁺ was 20.2 ± 2.0 pmol mg protein^–1 (n = 36). Decynium22 (1,1-diethyl-2,2-cyanide; 1 M) significantly reduced k_in to 6.1 ± 1.8 l mg protein^–1 min^–1 (P < 0.05) and the equilibrium accumulation (A_max) of ³H-MPP⁺ to 9.6 ± 1.3 pmol mg protein^–1 (P < 0.005) (n = 36). ³H-MPP⁺ accumulation (in cells incubated with 200 nM ³H-MPP⁺) was sensitive to (–)-adrenaline, (–)-isoprenaline, (–)-dopamine, (±)-adrenaline and (–)-noradrenaline. The most potent catecholamine in inhibiting ³H-MPP⁺ uptake was (–)-adrenaline, with an IC₅₀ of 99 (22, 449) M (n = 6). (–)-Adrenaline competitively inhibited ³H-MPP⁺ uptake, as it significantly increased the K_m value of ³H-MPP⁺ uptake (to 125.4 M (63.6; 187.1); P < 0.02; n = 3) but did not change the V_max value. The cyanide-derivatives decynium22 and cyanine863 (1-ethyl-2-([1,4-dimethyl-2-phenyl-6-pyrimidinylidene]methyl)quinolinium), which inhibit uptake₂ as well as the apical type of the renal transporter for organic cations, potently inhibited ³H-MPP⁺ uptake with IC₅₀'s of 1.4 (0.4–5.3) (n = 6) and 6.5 (2.6–16) (n = 4) M, respectively. Under conditions of monoamine oxidase (MAO) and catechol-O-methyl transferase (COMT) inhibition with either pargyline (500 M + Ro01-2812) (3,5-dinitropyrocatechol; 2 M) or pargyline (500 M) + U-0521(3,4-dihidroxy-2-methyl-propiophenone; l2 M)), (–)-adrenaline (up to 1 mM) had no inhibitory effect on the uptake of ³H-MPP⁺. Moreover, the uptake of ³H-MPP⁺ in the presence of pargyline + Ro 01-2812 was significantly lower (66.9 ± 30.4%; P < 0.05; n = 4) than in the absence of these compounds. Therefore, the effect of these MAO and COMT inhibitors on ³H-MPP⁺ uptake was examined. Interestingly enough, pargyline, Ro 01-2812 and U-0521 were found to inhibit the uptake of ³H-MPP⁺ (in cells incubated with 200 nM ³H-MPP⁺): 500 M pargyline, 2 M Ro 012812 and 100 M U-0521 decreased the accumulation of ³H-MPP⁺ to 38.1 ± 6.8 (n = 5), 60.5 ± 10.1(n = 7) and 71.3 ± 14.5 (n = 7) % of control, respectively.It is concluded that ³H-MPP⁺ is efficiently taken up by rat hepatocytes by a carrier-mediated mechanism sensitive to catecholamines, decynium22 and cy anine863, and to the enzyme inhibitors pargyline, Ro 01-2812 and U-0521.     

EFFECT OF BILE DUCT OBSTRUCTION ON HEPATIC UPTAKE OF 1-METHYL-4-PHENYLPYRIDINIUM IN THE RAT

Previous studies have demonstrated that the organic cation 1-methyl-4-phenylpyridinium (MPP⁺) is avidly taken up by rat freshly isolated hepatocytes through at least two distinct transport mechanisms: the type I hepatic transporter of organic cations and P-glycoprotein. In this study, the effects of extrahepatic cholestasis induced by bile duct ligation for 4 days on the uptake of [³H]MPP⁺by rat freshly isolated hepatocytes and liver slices were determined. Bile duct ligation produced no significant alterations in the characteristics of [³H]MPP⁺uptake by freshly isolated hepatocytes. The strong correlation found between the effect of various drugs on [³H]MPP⁺uptake by hepatocytes from control and treated rats (r=0.958;P<0.0001;n=15) suggests that neither the type I hepatic transporter of organic cations nor P-glycoprotein were affected by bile duct ligation. On the contrary, uptake of [³H]MPP⁺by liver slices was markedly changed after bile duct ligation: (1) there was a significant increase (≅40%) in the amount of [³H]MPP⁺taken up by liver slices from bile duct-ligated rats; (2) there was no correlation between the effect of various drugs on [³H]MPP⁺uptake by liver slices from control and treated rats (r=0.772;P=0.072;n=6). On the basis of (1) the lack of effect of bile duct ligation on [³H]MPP⁺uptake by isolated hepatocytes; and (2) the profound morphological alterations of liver tissue observed 4 days after bile duct ligation (increase in volume density of bile ductules, ductular cells and infiltration of inflammatory cells), we suggest that non-parenchymal liver cells have an important participation in the hepatic uptake of [³H]MPP⁺after bile duct ligation in the rat.     

Effects of methylmercury on dopamine release in MN9D neuronal cells

Abstract

Epidemiological evidence has shown associations between prevalence of Parkinson’s disease (PD) and exposure to environmental pollutants, but the mechanisms of pathogensis are still unclear. The objective of this study is to investigate effects of methylmercury (MeHg) on a dopaminergic neuronal cell line, MN9D and compare that to 1-methyl-4-phenylpyridinium (MPP⁺), a well-established agent associated with pathogenesis of PD. MN9D cells were exposed to MeHg (1–10?µM) and MPP⁺ (10–400?µM) for 24 or 48?h. Our results showed that MeHg induced cell death dose-dependently. MeHg also decreased the release of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) similar to the effects of MPP⁺. There was an increase in DOPAC?+?HVA/DA ratio. At the same time, both MeHg and MPP⁺ decreased the synthesis of tyrosine hydroxylase and dopamine transporter at the mRNA and protein levels. Expression of the α-Synuclein (α-Syn), a hallmark neuropathological indicator of PD, was also up-regulated at the mRNA level but not at the protein level after both MeHg and MPP⁺ dosing. Monoamine oxidase-B activity was suppressed in all MeHg treatments and MPP⁺ (1?µM)-treated cells. These findings suggest that MeHg can disrupt the synthesis, the uptake of DA and the metabolism as well as alter the biology of α-Syn similar to MPP⁺. Exposure to MeHg may potentially be a risk factor for the development of PD.     

Cocaine inhibits the release of MPP+ but not dopamine through the rat dopamine transporter

Transporter-mediated release of dopamine and the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺) was examined in COS cells, a cell line derived from monkey kidney, expressing the rat dopamine transporter. The release of preloaded [³H]MPP⁺ but not [³H]dopamine was dose-dependently inhibited by cocaine and other compounds known as dopamine uptake inhibitor, 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine (GBR 12935) and nomifensine, although the uptake of both [³H]dopamine and [³H]MPP⁺ was sensitive to these compounds. The different sensitivity of the outward transport of dopamine and MPP⁺ to the uptake inhibitors might provide a clue to the identity of the specific site(s) for cocaine on the transporter.     

Transport Characteristics of Ceftibuten,a New Cephalosporin Antibiotic,via the Apical H+/Dipeptide Cotransport System in Human Intestinal Cell Line Caco-2: Regulation by Cell Growth

Purpose. The intestinal epithelial cell line Caco-2 possesses the H⁺/ dipeptide cotransport system responsible for uptake of oral cephalosporins. In this study, the transport characteristics of ceftibuten were examined from the viewpoint of cell growth in the Caco-2 cells. Methods. The uptake of cephalosporins by Caco-2 cell monolayers grown on plastic dishes was measured and analyzed kinetically. Results. The uptake of ceftibuten was increased by lowering pH of the incubation medium and was inhibited by excess dipeptide. The transport activity of ceftibuten was dependent on the duration of culture, being maximal on the 14th day after inoculation. Kinetic analysis revealed that the development of ceftibuten uptake was due to not only a decrease in Km but also to an increase in Vmax value. Conclusions. The uptake of ceftibuten is mediated by the apical H⁺/ dipeptide cotransport system which is regulated by cell growth and/or differentiation in the Caco-2 cells.     

Double-transfected MDCK cells expressing human OCT1/MATE1 or OCT2/MATE1: determinants of uptake and transcellular translocation of organic cations

BACKGROUND AND PURPOSE

The organic cation transporters 1 (OCT1) and 2 (OCT2) mediate drug uptake into hepatocytes and renal proximal tubular cells, respectively. Multidrug and toxin extrusion protein 1 (MATE1) is a major component of subsequent export into bile and urine. However, the functional interaction of OCTs and MATE1 for uptake and transcellular transport of the oral antidiabetic drug metformin or of the cation 1-methyl-4-phenylpyridinium (MPP⁺) has not fully been characterized.

EXPERIMENTAL APPROACH

Single-transfected Madin-Darby canine kidney (MDCK) cells as well as double-transfected MDCK-OCT1-MATE1 and -OCT2-MATE1 cells were used to study metformin and MPP⁺ uptake into and transcellular transport across cell monolayers, along with their concentration and pH dependence.

KEY RESULTS

Cellular accumulation of MPP⁺ and metformin was significantly reduced by 31% and 46% in MDCK-MATE1 single-transfected cells compared with MDCK control cells (10 µM; P < 0.01). Over a wide concentration range (10–2500 µM) metformin transcellular transport from the basal into the apical compartment was significantly higher in the double-transfected cells compared with the MDCK control and MDCK-MATE1 monolayers. This process was not saturated up to metformin concentrations of 2500 µM. In MDCK-OCT2-MATE1 cells basal to apical MPP⁺ and metformin transcellular translocation decreased with increasing pH from 6.0 to 7.5.

CONCLUSIONS AND IMPLICATIONS

Our data demonstrate functional interplay between OCT1/OCT2-mediated uptake and efflux by MATE1. Moreover, MATE1 function in human kidney might be modified by changes in luminal pH values.     

A novel synthetic compound PHID (8-Phenyl-6a, 7, 8, 9, 9a, 10-hexahydro-6H-isoindolo [5, 6-g] quinoxaline-7, 9-dione) protects SH-SY5Y cells against MPP(+)-induced cytotoxicity through inhibition of reactive oxygen species generation and JNK signaling

1-Methyl-4-phenylpyridinium ion (MPP⁺), a neurotoxin selective to dopaminergic neurons and an inhibitor of mitochondrial complex I, has been widely used as an etiologic model of Parkinson's disease. In this study, we investigated the protective effects of a novel synthetic compound, 8-Phenyl-6a,7,8,9,9a,10-hexahydro-6H-isoindolo[5,6-g]quinoxaline-7,9-dione (PHID), on MPP⁺-induced cytotoxicity in SH-SY5Y cells. MPP⁺ induced apoptosis characterized by generation of reactive oxygen species, caspase-3 activation, poly ADP ribose polymerase proteolysis and increase in Bax/Bcl-2 ratio were blocked by PHID in a dose-dependent fashion. Furthermore, MPP⁺-mediated activation of stress-activated protein kinase/c-Jun N-terminal kinase (JNK) was also inhibited by PHID in a dose-dependent manner. The results indicate that PHID protects against MPP⁺-induced apoptosis by blocking reactive oxygen species stimulation and JNK signaling pathways in SH-SY5Y cells, implicating the novel compound in the prevention of progressive neurodegenerative diseases such as Parkinson's disease.     

Rg1 protects the MPP-treated MES23.5 cells via attenuating DMT1 up-regulation and cellular iron uptake

Ginsenoside-Rg1 is one of the pharmacologically active component isolated from ginseng. Our previous study observed the protective effect of Rg1 on iron accumulation in the substantia nigra (SN) in 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-treated Parkinson's disease (PD) mice. However, the mechanisms of this neuroprotective effect of Rg1 are unknown. In this study, we elucidated possible mechanisms for this effect using 1-methyl-4-phenylpyridinium (MPP⁺)-treated MES23.5 cells. Previous study showed MPP⁺ treatment induced up-regulation of divalent metal transporter 1 without iron responsive element (DMT1-IRE) in MES23.5 cells. In the present study, we observed that pretreatment with Rg1 could inhibit MPP⁺-induced up-regulation of DMT1-IRE in MES23.5 cells. Up-regulation of DMT1-IRE by MPP⁺ treatment was associated with ROS production and translocation of nuclear factor-kappaB (NF-κB) to nuclei, both of which were significantly inhibited by Rg1 pretreatment. The role of ROS and NF-κB in the up-regulation of DMT1-IRE was supported by application of an antioxidant NAC and BAY 11-7082, an inhibitor of IκBα phosphorylation. Furthermore, we also showed Rg1 could decrease DMT1-mediated ferrous iron uptake and iron-induced cell damage by inhibiting the up-regulation of DMT1-IRE. These results indicate that Rg1 protected the MPP⁺-treated MES23.5 cells via attenuating DMT1-IRE up-regulation likely through inhibition of ROS-NF-κB pathway; Attenuation of DMT1-IRE expression decreased the iron influx and iron-induced oxidative stress.     

Requirement of a dopaminergic neuronal phenotype for toxicity of low concentrations of 1-methyl-4-phenylpyridinium to human cells

LUHMES cells are conditionally-immortalized non-transformed human fetal cells that can be differentiated to acquire a dopaminergic neuron-like phenotype under appropriate growth conditions. After differentiation by GDNF and cyclic adenosine monophosphate, LUHMES were sensitive to 1-methyl-4-phenylpyridinium (MPP⁺) toxicity at ≤ 5 μM, but resistant to the parental compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The high homogeneity and purity of the cultures allowed the detection of metabolic changes during the degeneration. Cellular ATP dropped in two phases after 24 and 48 h; cellular glutathione (GSH) decreased continuously, paralleled by an increase in lipid peroxidation. These events were accompanied by a time-dependent degeneration of neurites. Block of the dopamine transporter by GBR 12909 or mazindol completely abrogated MPP⁺ toxicity. Inhibition of de novo dopamine synthesis by α-methyl-l-tyrosine or 3-iodo-l-tyrosine attenuated toxicity, but did not reduce the initial drop in ATP. Inhibition of mixed lineage kinases by CEP1347 completely prevented the MPP⁺-induced loss of viability and intracellular GSH, but failed to attenuate the initial drop of ATP. For the quantitative assessment of neurite degeneration, an automated imaging-based high content screening approach was applied and confirmed the findings made by pharmacological interventions in this study. Our data indicate that inhibition of mitochondrial ATP synthesis is not sufficient to trigger cell death in MPP⁺-treated LUHMES.     

Salidroside protects PC12 cells from MPP-induced apoptosis via activation of the PI3K/Akt pathway

Oxidative stress plays an important role in the pathogenesis of Parkinson’s disease (PD). Salidroside (SAL), a phenylpropanoid glycoside isolated from Rhodiola rosea L., can exert potent antioxidant properties. In this study, we investigated the protective effects, and the possible mechanism of action, of SAL against 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell damage in rat adrenal pheochromocytoma PC12 cells. Pretreatment of PC12 cells with SAL significantly reduced the ability of MPP⁺ to induce apoptosis in a dose and time-dependent manner. SAL significantly and dose-dependently inhibited MPP⁺-induced chromatin condensation and MPP⁺-induced release of lactate dehydrogenase by PC12 cells. SAL enhanced Akt phosphorylation in PC12 cells, and the protective effects of SAL against MPP⁺-induced apoptosis were abolished by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K) phosphorylation. These findings suggest that SAL prevents MPP⁺-induced apoptosis in PC12 cells, at least in part through activation of the PI3K/Akt pathway.     

p53-mediated ferroptosis is required for 1-methyl-4-phenylpyridinium-induced senescence of PC12 cells

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and striatum. Aging is the most important risk factor of PD. Ferroptosis is an iron-dependent form of cell death associated with PD. However, it is not clear whether ferroptosis accelerates PD by promoting cellular senescence. This study investigated the mechanism of 1-methyl-4-phenylpyridinium (MPP⁺) -induced PC12 cells injury. We found that MPP⁺ induced cell senescence with increased β-galactosidase activity and the expression of p53, p21 and p16 activation in cells. In addition, MPP⁺ treatment showed smaller mitochondria and increased membrane density, downregulation of ferritin heavy chain 1 expression and upregulation of acyl-CoA synthetase long chain family member 4 expression, and enhanced levels of oxidative stress, which were important characteristics of ferroptosis. Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, was tested to eliminate MPP⁺-induced cell senescence. Fer-1 downregulated the expression of p53 and upregulated the expression of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase-4 (GPX4) in MPP⁺-induced ferroptosis. Inhibition of p53 eliminated cell senescence by upregulation the expression of of SLC7A11 and GPX4. Thus, these results suggest that MPP⁺ induces senescence in PC12 cells via the p53/ SLC7A11/ GPX4 signaling pathway in the ferroptosis regulation mechanism.     

Transport of small organic cations in the rat liver

The kidneys and the liver are the principal organs for the inactivation of circulating organic cations. Recently, an organic cation transporter (OCT1) has been cloned from rat kidney. In order to answer the question whether OCT1 is involved also in hepatic uptake of organic cations, the pharmacological characteristics of organic cation transport in hepatocytes were compared to the characteristics of transiently expressed OCT1.Primary cultures of rat hepatocytes avidly accumulated the small organic cation ³H-1-methyl-4-phenylpyridinium (³H-MPP⁺). At equilibrium, the hepatocytes accumulated ³H-MPP⁺ 56-fold. Initial rates of specific ³H-MPP⁺ transport in hepatocytes were saturable. The half-saturating concentration was 13 mol/l. ³H-MPP⁺ transport was sensitive to quinine (K_i = 0.79 mol/l) and cyanine863 (K_i = 0.097 µmol/l). Quinine and cyanine863 are known inhibitors of type I hepatic transport of cationic drugs and of renal excretion of organic cations, respectively. To compare the functional characteristics of ³H-MPP⁺ transport in hepatocytes with those of OCT1, OCT1 has been heterologously expressed and characterized in a mammalian cell line (293 cells). Initial rates of ³H-MPP⁺ transport were saturable, the K_m being 13 mol/l. The rank order of inhibitory potencies of various inhibitors was almost identical in hepatocytes and 293 cells transiently transfected with OCT1. There was a positive correlation between the K_i's for the inhibition of ³H-MPP⁺ transport in isolated hepatocytes and transfected 293 cells (r = 0.85; P<0.01; n = 8).The results indicate that OCT1 is functionally expressed not only in the kidney but also in hepatocytes where it is responsible for the transport of small organic cations which, in the past, have been classified as type I substrates.     

Effects of MPP+ on the molecular pathways involved in cell cycle control in B65 neuroblastoma cells

The toxicity caused by cell exposure to 1-methyl-4-phenylpyridinium ion (MPP⁺) is a useful model in the study of Parkinson's disease (PD). However, the exact molecular mechanisms triggered by MPP⁺ in cell death are currently unclear. In the present research, we show that exposure to MPP⁺ induce the cell death of neuroblastoma-derived dopaminergic B65 cells, which is not reversed by the widely known caspase inhibitor Z-VADfmk or by calpain inhibition. Likewise, when B65 cells were treated with MPP⁺, the DNA damage pathway that involves p53 was activated, and cells were arrested in the G₂/M phase of the cell cycle. Interestingly, MPP⁺ has two effects on the expression of cell cycle-related proteins. It increases the content of cyclins A, E, cdk2 and the phosphorylated form of pRb (serine 780). However, MPP⁺ 5 mM decreased the expression of cyclin D1, B1 and cdk4. The decrease in the expression of cyclin B1 may be related to the arrest of cells observed in the G₂/M phase of cell cycle. The increase in S phase cell cycle proteins and retinoblastoma protein phosphorylation was an unexpected result. As the antioxidant trolox attenuated the process of cell loss and changes in the cell cycle, as measured by flow cytometry, we concluded that oxidative stress was involved in the effects of MPP⁺ in this cell line. In summary, the present work characterizes the molecular changes involved in damage caused by MPP⁺ in B65 cells, and highlights the effects of MPP⁺ on molecules involved in the control of cell cycle progression.     

Saturable Absorptive Transport of the Hydrophilic Organic Cation Ranitidine in Caco-2 Cells: Role of pH-Dependent Organic Cation Uptake System and P-Glycoprotein

Purpose The purpose of this work was to investigate the involvement of carrier-mediated apical (AP) uptake and efflux mechanisms in the absorptive intestinal transport of the hydrophilic cationic drug ranitidine in Caco-2 cells. Methods Absorptive transport and AP uptake of ranitidine were determined in Caco-2 cells as a function of concentration. Permeability of ranitidine in the absorptive and secretory directions was assessed in the absence or presence of the P-glycoprotein (P-gp) inhibitor, GW918. Characterization of the uptake mechanism was performed with respect to inhibitor specificity, pH, energy, membrane potential, and Na⁺ dependence. Efflux from preloaded monolayers was evaluated over a range of concentrations and in the absence or presence of high extracellular ranitidine concentrations. Results Saturable absorptive transport and AP uptake of ranitidine were observed with K_m values of 0.27 and 0.45 mM, respectively. The ranitidine absorptive permeability increased and secretory permeability decreased upon inhibition of P-gp. AP ranitidine uptake was inhibited in a concentration-dependent fashion by a diverse set of organic cations including tetraethylammonium, 1-methyl-4-phenylpyridinium, famotidine, and quinidine. AP ranitidine uptake was pH and membrane potential dependent and reduced under conditions that deplete metabolic energy. Efflux of [³H]ranitidine across the basolateral membrane was neither saturable as a function of concentration nor trans stimulated by unlabeled ranitidine. Conclusions Saturable absorptive transport of ranitidine in Caco-2 cells is partially mediated via a pH-dependent uptake transporter for organic cations and is subject to attenuation by P-gp. Inhibition and driving force studies suggest the uptake carrier exhibits similar properties to cloned human organic cation transporters. The results also imply ranitidine transport is not solely restricted to the paracellular space.     

Glycyrrhizin Attenuates MPTP Neurotoxicity in Mouse and MPP+-Induced Cell Death in PC12 Cells

The present study examined the inhibitory effect of licorice compounds glycyrrhizin and a metabolite 18β-glycyrrhetinic acid on the neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the mouse and on the 1-methyl-4-phenylpyridinium (MPP⁺)-induced cell death in differentiated PC12 cells. MPTP treatment increased the activities of total superoxide dismutase, catalase and glutathione peroxidase and the levels of malondialdehyde and carbonyls in the brain compared to control mouse brain. Co-administration of glycyrrhizin (16.8 mg/kg) attenuated the MPTP effect on the enzyme activities and formation of tissue peroxidation products. In vitro assay, licorice compounds attenuated the MPP⁺-induced cell death and caspase-3 activation in PC12 cells. Glycyrrhizin up to 100µM significantly attenuated the toxicity of MPP⁺. Meanwhile, 18β-glycyrrhetinic acid showed a maximum inhibitory effect at 10µM; beyond this concentration the inhibitory effect declined. Glycyrrhizin and 18β-glycyrrhetinic acid attenuated the hydrogen peroxide- or nitrogen species-induced cell death. Results from this study indicate that glycyrrhizin may attenuate brain tissue damage in mice treated with MPTP through inhibitory effect on oxidative tissue damage. Glycyrrhizin and 18β-glycyrrhetinic acid may reduce the MPP⁺ toxicity in PC12 cells by suppressing caspase-3 activation. The effect seems to be ascribed to the antioxidant effect.     

Similar potency of catechin and its enantiomers in alleviating 1-methyl-4-phenylpyridinium ion cytotoxicity in SH-SY5Y cells

Objectives Previously, the flavonoid (±)‐catechin was shown to exert potent neuroprotective action in the mouse 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐induced Parkinson's disease model. The purpose of this study was to investigate whether the different enantiomers of catechin ((+)‐catechin, (?)‐catechin and (±)‐catechin, a 50 : 50 mixture of (+)‐catechin and (?)‐catechin) could protect SH‐SY5Y cells against 1‐methyl‐4‐phenylpyridinium ion (MPP⁺) toxicity by decreasing the generation of oxygen free radicals. The inhibitive effect of (±)‐catechin on JNK/c‐Jun activation was investigated. Methods The effects of (+)‐catechin, (?)‐catechin or (±)‐catechin in protecting against MPP⁺ toxicity were evaluated and compared in SH‐SY5Y cells by testing the release of lactate dehydrogenase. The generation of reactive oxygen species (ROS) was measured by immunochemistry and the phosphorylation level of JNK/c‐Jun was determined by Western blotting. Key findings In SH‐SY5Y cells, (+)‐catechin, (?)‐catechin or (±)‐catechin reduced apoptosis induced by MPP⁺ and decreased ROS generation caused by MPP⁺. Different enantiomers of catechin showed protective effects at similar potency. Moreover (±)‐catechin decreased JNK/c‐Jun phosphorylation which was increased by MPP⁺. Conclusions Catechin and its two enantiomers could protect SH‐SY5Y cells against MPP⁺ cytotoxicity at a similar potency. Antioxidative stress and inhibition of the JNK/c‐Jun signalling pathway might have been involved in the neuroprotective mechanisms of catechin against MPP⁺ cytotoxicity in SH‐SY5Y cells.     

Expression of the extraneuronal monoamine transporter (uptake2) in human glioma cells

Tritiated methylphenylpyridinium ([³H]MPP⁺), a substrate of the neuronal and extraneuronal noradrenaline transporter (uptake₁ and uptake₂, respectively) and of the organic cation transporter (OCT1), was used to characterize the amine transport system of the established human glioma cell line SK-MG-1.Uptake of [³H]MPP⁺ (25 nM) into SK-MG-1 cells increased linearly with time for up to 15 min. Selective uptake₁ inhibitors (e.g. (+)oxaprotiline) or omission of Na⁺ or Cl^– ions did not affect [³H]MPP⁺ uptake, whereas uptake₂ inhibitors such as O-methyl-isoprenaline (OMI) or corticosterone as well as depolarizing concentrations of K⁺ or Ba²⁺ strongly reduced [³H]MPP⁺ uptake. Initial rates of OMI(100 M)-sensitive [³H]MPP⁺ uptake were saturable, with a K_m of about 17 M and a maximal rate of about 50 pmol/ (min × mg protein). IC₅₀ (or K_i) values for inhibition of [³H]MPP⁺ uptake by substrates and inhibitors of uptake₂ or OCTI were highly significantly correlated with published IC₅₀ values for inhibition of uptake₂ but not with corresponding values for inhibition of OCT1.The results presented here clearly demonstrate that human glioma cells express an uptake₂ transporter. Thus, glial cells in the human central nervous system endowed with this transporter are likely to contribute to the inactivation of neuronally released noradrenaline.