1-methyl-4-phenylpyridinium-induced alterations of glutathione status in immortalized rat dopaminergic neurons

Decreased glutathione levels associated with increased oxidative stress are a hallmark of numerous neurodegenerative diseases, including Parkinson's disease. GSH is an important molecule that serves as an anti-oxidant and is also a major determinant of cellular redox environment. Previous studies have demonstrated that neurotoxins can cause changes in reduced and oxidized GSH levels; however, information regarding steady state levels remains unexplored. The goal of this study was to characterize changes in cellular GSH levels and its regulatory enzymes in a dopaminergic cell line (N27) following treatment with the Parkinsonian toxin, 1-methyl-4-phenylpyridinium (MPP(+)). Cellular GSH levels were initially significantly decreased 12 h after treatment, but subsequently recovered to values greater than controls by 24 h. However, oxidized glutathione (GSSG) levels were increased 24 h following treatment, concomitant with a decrease in GSH/GSSG ratio prior to cell death. In accordance with these changes, ROS levels were also increased, confirming the presence of oxidative stress. Decreased enzymatic activities of glutathione reductase and glutamate-cysteine ligase by 20-25% were observed at early time points and partly account for changes in GSH levels after MPP(+) exposure. Additionally, glutathione peroxidase activity was increased 24 h following treatment. MPP(+) treatment was not associated with increased efflux of glutathione to the medium. These data further elucidate the mechanisms underlying GSH depletion in response to the Parkinsonian toxin, MPP(+).     

Comparison of cytotoxicity of a quaternary pyridinium metabolite of haloperidol (HP+) with neurotoxinN-methyl-4-phenylpyridinium (MMP+) towards cultured dopaminergic neuroblastoma cells

Haloperidol has recently been found to be metabolized to its pyridinium ion (HP⁺). This conversion of haloperidol to HP⁺ appears to be similar to the activation of the dopaminergic neurotoxinN-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toN-methyl-4-phenyl pyridinium ion (MPP⁺). MPP⁺ is responsible for the damage of striatal dopaminergic neurons induced by MPTP in humans and animals. It seemed sensible to investigate whether or not HP⁺ might be toxic towards dopaminergic neurons and perhaps associated with some of the residual motofunction side effects of haloperidol. We therefore investigated the neurotoxicity of HP⁺ toward cultured human dopamine neuroblastoma cells (SH-SY5Y) and compared it with that of MPP⁺. HP⁺ reduced the viability as measured by MTT and [³H]thymidine incorporation methods in SH-SY5Y cells. Cell membrane integrity is reduced by the treatment of HP⁺ as measured by intracellular LDH levels. The toxicity was concentration and time dependent. Interestingly, HP⁺ appeared to be more toxic than MPP⁺ towards the SH-SY5Y cells in early phase in cultures. The toxicity of MPP⁺ appear to be progressive and subsequently become more than HP⁺ with prolonged cultivation. In contrary to MPP⁺, the toxic effect of HP⁺ towards a dopamine transporter transfected SK-N-MC cell line is not different from its wild type. This indicates that dopamine uptake system is probably not involved in the cytotoxicity caused by HP⁺.     

Aquaporin-4 knockout enhances astrocyte toxicity induced by 1-methyl-4-phenylpyridinium ion and lipopolysaccharide via increasing the expression of cytochrome P4502E1

The role of aquaporin-4 (AQP4) in the regulation of astrocytes function has been widely investigated. However, there is little information about its contribution to the drug metabolism enzymes such as Cytochrome P4502E1. In the present study, we investigated whether AQP4 is involved in the process of the cell damage caused by MPP⁺ and LPS through regulating the expression of CYP2E1 in astrocytes. Compared to the wild-type, in primary astrocytes, AQP4 knockout increased the cell damage and the reactive oxygen species (ROS) production which were induced by MPP⁺, LPS and ethanol. Notably, AQP4 knockout enhanced the up-regulation of the expression of CYP2E1 in astrocytes exposed to MPP⁺, LPS and ethanol. Furthermore, Diallylsulphide (DAS), a CYP2E1 inhibitor, partially or almost abolished the cell injury and the ROS production of the astrocytes induced by MPP⁺ and LPS. These findings indicate AQP4 protects astrocytes from the damage caused by MPP⁺ and LPS through reducing the ROS production correlation to the diminished expression of CYP2E1.     

The novel squamosamide derivative (compound FLZ) attenuated 1-methyl, 4-phenyl-pyridinium ion (MPP+)-induced apoptosis and alternations of related signal transduction in SH-SY5Y cells

Compound FLZ (cFLZ) is a synthetic novel derivative of natural squamosamide. Previous pharmacological study found that cFLZ improved the abnormal behavior and the decrease of dopamine content in striatum in 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) model mice. 1-Methyl 4-phenylpyridinium (MPP+) is the active metabolite of MPTP to cause Parkinsonism in experimental animals. The purpose of this paper was to further study the protective action of cFLZ against MPP+-induced apoptosis and alternations of related signaling transduction. The results indicated that cFLZ at concentrations of 0.1 microM and 1 microM prevented 100 microM MPP+-induced apoptosis of SH-SY5Y cells, and inhibited the release of cytochrome C and apoptosis-inducing factor (AIF), and the activation of caspase 3 and NF-kappaB as well as alpha-synuclein gene and protein expressions. The results suggest that cFLZ possesses potent neuroprotective activity and may be a potential anti-Parkinson's disease drug worthy for further study.     

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.     

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     

Allopurinol suppresses 2-bromoethylamine and 1-methyl-4-phenylpyridinium ion (MPP(+))-induced hydroxyl radical generation in rat striatum

The present study was examined whether or not 2-bromoethyamine, a semicarbazide-sensitive amine oxidase (SSAO, EC; 1.4.3.6) inhibitor, would increase an active dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium ion (MPP(+))-induced hydroxyl radical ((*)OH) generation in the rat striatum. Rats were anesthetized, and sodium salicylate (0.5 mM or 0.5 nmol/microl/min) was infused through a microdialysis probe to detect the generation of (*)OH as reflected by the non-enzymatic formation of 2,3-dihydroxybenzoic acid (DHBA) in the striatum. Infusion of 2-bromoethylamine (100 microM or 100 pmol/microl/min) into the striatum drastically increased the formation of (*)OH products, trapped as DHBA by the action of MPP(+). Further, I studied the effect of allopurinol, a xanthine oxidase inhibitor, an 2-bromoethylamine and MPP(+)-induced (*)OH generation. Allopurinol (10 microM or 10 pmol/microl/min) significantly suppressed 2-bromoethyamine and MPP(+)-induced (*)OH. These results suggest that a definite mechanism is not clear at the moment, after inhibition of tissue-bound and/or blood plasma SSAO activity, with consequent increases in bioactive amine levels, enhances the formation of (*)OH products of efflux/oxidation due to MPP(+).     

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.     

Protective effect of verbascoside on 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells

The neuroprotective effects of verbascoside, one of phenylpropanoid glucoside isolated from the Chinese herbal medicine Buddleja officinalis Maxim, on 1-methyl-4-phenylpyridinium ion (MPP(+)) induced apoptosis and oxidative stress in PC12 neuronal cells were investigated. Treatment of PC12 cells with MPP(+) for 48 h induced apoptotic death as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, the activation of caspase-3 measured by the caspase-3 activity assay kit, the reduction in mitochondrial membrane potential with laser scanning confocal microscopy and the increase in the extracellular hydrogen peroxide level. Simultaneous treatment with verbascoside markedly attenuated MPP(+)-induced apoptotic death, increased extracellular hydrogen peroxide level, the activation of caspase-3 and the collapse of mitochondrial membrane potential. These results strongly indicate that verbascoside may provide a useful therapeutic strategy for the treatment of oxidative stress-induced neurodegenerative disease such as Parkinson's disease.     

Brain inflammation enhances 1-methyl-4-phenylpyridinium-evoked neurotoxicity in rats

Experimental Parkinson's disease and Parkinson's disease in humans include a CNS inflammatory component that may contribute to the pathogenesis of the disease. CNS inflammation produces a loss in cytochrome P450 metabolism and may impair the brain's protection against neurotoxins. We have examined if preexisting inflammation in the brain could increase the toxicity of the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP(+)). Lipopolysaccharide (LPS, 25 microg) or saline (control) was injected into the left lateral cerebral ventricle. A single injection of MPP(+) into the median forebrain bundle followed 48 h later and produced a reduction in striatal dopamine content that was dose- and time-dependent. Two-days after 5 microg of MPP(+) was administered, a 90% decrease in striatal dopamine content was observed in saline- and LPS-pretreated rats. However, 4 and 7 days after 5 microg MPP(+) treatment, striatal dopamine recovered up to 70-80% of control values in saline-pretreated rats but remained depressed (80-90%) in rats treated with LPS. These results suggested that CNS inflammation might create an increased risk factor for drug-induced CNS toxicity or chemically mediated Parkinson's disease. The prolonged toxicity of MPP(+) may be due to a decrease in brain cytochrome P450 metabolism that occurs during inflammation. As a second objective for the study, we examined if the CNS lesion produced by MPP(+) altered cytochrome P450 metabolic activity in the liver, kidney, and lung. We have demonstrated a novel mechanism whereby the brain pathology produced by MPP(+) treatment contributes to a reduction in cytochrome P450 metabolism in the kidney but not the liver or lung. Therefore, a chemically evoked CNS disorder with a chronic inflammatory component might have major effects on the renal metabolism of drugs or endogenous substrates.     

Different effects of 1-methyl-4-phenyl-pyridinium (MPP+) on monoamine oxidase of dopaminergic terminals in caudate nucleus slices from pigmented and from albino rabbits

Summary Slices of caudate nucleus from pigmented and from albino rabbits were preincubated in vitro for 24 h with different concentrations of the neurotoxic compound MPP⁺. Subsequently, endogenous dopamine (DA) in the slices was determined by HPLC. MPP⁺ (1 and 3.2 mol/1) was more effective in diminishing DA levels in caudate nucleus slices from albino than in slices from pigmented rabbits. Following 24 h pretreatment with MPP⁺, the accumulation of [³H]-DA in caudate nucleus slices from pigmented rabbits was either enhanced (at 0.32 mol/1, 1 mol/l and 3.2 mol/l MPP⁺) or reduced (at 32 mol/l MPP⁺). In contrast, MPP⁺ did not enhance the accumulation of [³H]-DA in caudate nucleus tissue from albino rabbits and was more potent in reducing the [³H]-DA content in slices from albino than in slices from pigmented rabbits. When the selective type A monoamine oxidase (MAO) inhibitor clorgyline was present during pre-incubation, but not when the selective type B MAO inhibitor deprenyl was, the concentration-response curve for MPP⁺ with caudate nucleus slices from pigmented rabbits was similar to that obtained with slices from albino rabbits. Clorgyline and deprenyl did not change the effects of MPP⁺ in caudate nucleus slices from albino rabbits. These findings are compatible with the hypothesis that the MAO within dopaminergic terminals in the caudate nucleus of pigmented, but not of albino, rabbits is of type A since MAO-A is preferentially inhibited by MPP⁺. In line with this hypothesis, the accumulation of the preferential MAOA substrate [³H]-5-HT in caudate nucleus slices from pigmented rabbits was about 39% lower than that in slices from albino rabbits. Clorgyline abolished this difference.These findings show that MPP+, by inhibiting MAO-A, may mask its own depletory effect on DA terminals in the caudate nucleus of pigmented rabbits. Since the MAO in the striatum of albino rabbits is not blocked by MPP⁺, in contrast to the enzyme in the striatum of pigmented rabbits, it may be mainly of the B type and may, or may not, be present in the dopaminergic nerve terminals. Whether or not differences in the activity of MAO within dopaminergic neurons of the nigro-striatal system of humans, possibly leading to subsequent metabolic steps which result in a different profile of cytotoxic DA metabolites, could account for the different incidence of Parkinsons's disease in darker-skinned compared with lighter-skinned people needs further investigation.Correspondence to T.J. Feuerstein at the above address     

Acetyl-L-carnitine cytoprotection against 1-methyl-4-phenylpyridinium toxicity in neuroblastoma cells

Acetyl-L-carnitine (ALCAR) plays an integral role in the transport of long chain fatty acids across the inner mitochondrial membrane for oxidative phosphorylation. In non-human primates, administration of ALCAR was reported to prevent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurological injury to the substantia nigra. The present study investigates the effects of ALCAR against the toxicity of 1-methyl-4-phenylpyridinium (MPP(+)), the neurotoxic metabolite of MPTP, in murine brain neuroblastoma cells. MPP(+), a potent mitochondrial toxin, induced a dose-dependent reduction in mitochondrial oxygen consumption and cell viability, corresponding to an accelerated rate of cellular glucose utilization. Treatment with ALCAR, but not L-carnitine, prevented MPP(+) toxicity and partially restored intracellular ATP concentrations, but did not reverse the MPP(+)-induced loss of mitochondrial oxygen consumption. These data indicate that protective effects are independent of oxidative phosphorylation. ALCAR had a substantial glucose sparing effect in both controls and MPP(+)-treated groups, demonstrating a potential role in enhancing glucose utilization through glycolysis. Antagonizing the entry of fatty acids into the mitochondria, with either insulin or malonyl CoA, did not interfere with ALCAR protection against MPP(+). On the contrary, insulin potentiated the protective effects of ALCAR. In conclusion, these data indicate that ALCAR protects against MPP(+) toxicity, independent of mitochondrial oxidative capacity or beta-oxidation of fatty acids. In contrast, the protective effects of ALCAR appear to involve potentiation of energy derived from glucose through anaerobic glycolysis.     

Obligatory role for complex I inhibition in the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice and nonhuman primates causes a parkinsonian disorder characterized by a loss of dopamine-producing neurons in the substantia nigra and corresponding motor deficits. MPTP has been proposed to exert its neurotoxic effects through a variety of mechanisms, including inhibition of complex I of the mitochondrial respiratory chain, displacement of dopamine from vesicular stores, and formation of reactive oxygen species from mitochondrial or cytosolic sources. However, the mechanism of MPTP-induced neurotoxicity is still a matter of debate. Recently, we reported that the yeast single-subunit nicotinamide adenine dinucleotide (reduced) dehydrogenase (NDI1) is resistant to rotenone, a complex I inhibitor that produces a parkinsonian syndrome in rats, and that overexpression of NDI1 in SK-N-MC cells prevents the toxicity of rotenone. In this study, we used viral-mediated overexpression of NDI1 in SK-N-MC cells and animals to determine the relative contribution of complex I inhibition in the toxicity of MPTP. In cell culture, NDI1 overexpression abolished the toxicity of 1-methyl-4-phenylpyridinium, the active metabolite of MPTP. Overexpression of NDI1 through stereotactic administration of a viral vector harboring the NDI1 gene into the substantia nigra protected mice from both the neurochemical and behavioral deficits elicited by MPTP. These data identify inhibition of complex I as a requirement for dopaminergic neurodegeneration and subsequent behavioral deficits produced by MPTP. Furthermore, combined with reports of a complex I defect in Parkinson's disease (PD) patients, the present study affirms the utility of MPTP in understanding the molecular mechanisms underlying dopaminergic neurodegeneration in PD.     

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.     

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.     

Biochemical basis for the low sensitivity of the rat heart to digitalis

Summary The concentration of cardiac glycosides to produce positive inotropic effects in the rat heart is markedly higher than that in other species. Such a low digitalis sensitivity of the rat heat is attributed to the low affinity of cardiac Na⁺, K⁺-ATPase for digitalis in this species. In the present study the biochemical cause which is responsible for the formation of the unstable complex between the glycosides and Na⁺, K⁺-ATPase or positive inotropic, receptor in the rat heart was examined using Na⁺, K⁺-ATPase preparations obtained from rat hearts, guinea-pig hearts and rat brains as well as isolated, electrically stimulated atrial preparations obtained from these animals. Monensin, which alters transmembrane Na⁺ movements without interacting with the cardiotonic sites on Na⁺, K⁺-ATPase, had equivalent potencies in guinea-pig and rat hearts. Cassaine, which lacks a lactone ring but interacts with cardiotonic sites on Na⁺, K⁺-ATPase, increased the force of contraction in guinea-pig hearts at low, but in rat hearts only at high, concentrations. AY-22,241 (Actodigin) and prednisolone-3,20-bisguanylhydrazone (PBGH) bind to cardiotonic sites on Na⁺, K⁺-ATPase and had a similar spectrum as cassaine in these two species. Actodigin has an altered lactone ring resulting in a marked reduction of the inotropic potency, and PBGH is devoid of this structure. With the latter agent, the rabbit was as insensitive as the rat, although both rabbit and guinea-pig are equally sensitive to digitalis. K⁺ delayed the development of the positive inotropic action of ouabain with a minimal effect on the plateau response in guinea-pig hearts. In rat hearts, however, K⁺ markedly lowered the plateau response without affecting the time course of the response. These results indicate that the low sensitivity of the rat heart to digitalis is due to a difference in the glycoside binding sites on Na⁺, K⁺-ATPase; but the difference cannot be explained by the lack of a lactone ring complementary binding sites. The difference seems to result from the absence of lipid barrier which regulates the rate of release of cardiac glycosides from their binding sites on Na⁺, K⁺-ATPase.This work was supported by U.S. Public Health Service grant, HL-16052 and by the Michigan Heart Association     

Reduction of the equilibrium binding of cardiac glycosides and related compounds to Na+, K+-ATPase as a possible mechanism for the potassium-induced reversal of their toxicity

Summary The influence of potassium ions on the equilibrium state of the binding of cardiac glycosides and their derivatives to partially purified dog heart and rat brain enzyme preparations was studied in vitro. The addition of potassium to the incubation mixture containing enzyme preparation, ³H-ouabain, Na⁺, Mg²⁺ and ATP, at the time when the binding reaction is close to equilibrium, caused an immediate reduction of the bound drug concentration; the concentration apparently shifting toward a lower equilibrium state. The degree of the potassium-induced reduction in bound drug concentration was dependent on the potassium concentration and on the chemical structure of the compound. The binding of aglycones, pentacetyl-gitoxin and cassaine was affected to a greater extent than that of the glycosides. These data suggest that one of the mechanisms by which potassium antagonizes the toxic actions of digitalis on the heart is to reduce the drug binding to cardiac Na⁺, K⁺-ATPase.This work was supported by a U.S. Public Health Service Grant, HL-16052     

The toxicity of H2O2 on the ionic homeostasis of airway epithelial cells in vitro

Oxygen species may be formed in the air spaces of the respiratory tract in response to environmental pollution such as particulate matter. The mechanisms and target molecules of these oxidants are still mainly unknown but may involve modifications of the ionic homeostasis in epithelial cells. Cytosolic concentrations of Ca²⁺ (Fura2) and Na⁺ (SBFI) and short-circuit current (Isc) were followed in primary cultures of human nasal epithelial cells and in the cell line 16HBE14o⁻ after exposure to H₂O₂ or ·OH (H₂O₂+Fe²⁺). Cells were grown on glass coverslips for ionic imaging or on permeable snapwell inserts for Isc studies. Exposure of the apical as well as the basal side of the cultures to H₂O₂ or ·OH induced a concentration-dependent transient increase in Isc which is due to a transient secretion of Cl⁻. Ca_i also increased transiently with approximately the same kinetics. The response was dependent on the release of calcium from intracellular stores. Na_i on the contrary increased steadily over more than an hour. When the apical membrane was permeabilized with gramicidin, ·OH inhibited the Na⁺ current (a measure of Na⁺-K⁺-ATPase activity in the baso-lateral membrane). The arrest of the pump was significant after 30 min exposure to oxidant. On the other hand no increase in the apical or baso-lateral sodium conductances could be detected. The progressive arrest of the Na⁺/K⁺-pump may contribute to the sustained elevation of Na_i. This strong modification in the cellular ionic homeostasis may participate in the stress response of the respiratory epithelium through alterations in signal transduction pathways.     

Effects of enhancing mitochondrial oxidative phosphorylation with reducing equivalents and ubiquinone on 1-methyl-4-phenylpyridinium toxicity and complex I-IV damage in neuroblastoma cells

The effects of increasing mitochondrial oxidative phosphorylation (OXPHOS), by enhancing electron transport chain components, were evaluated on 1-methyl-4-phenylpyridinium (MPP+) toxicity in brain neuroblastoma cells. Although glucose is a direct energy source, ultimately nicotinamide and flavin reducing equivalents fuel ATP produced through OXPHOS. The findings indicate that cell respiration/mitochondrial O(2) consumption (MOC) (in cells not treated with MPP+) is not controlled by the supply of glucose, coenzyme Q(10) (Co-Q(10)), NADH+, NAD or nicotinic acid. In contrast, MOC in whole cells is highly regulated by the supply of flavins: riboflavin, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), where cell respiration reached up to 410% of controls. In isolated mitochondria, FAD and FMN drastically increased complex I rate of reaction (1300%) and (450%), respectively, having no effects on complex II or III. MPP+ reduced MOC in whole cells in a dose-dependent manner. In isolated mitochondria, MPP+ exerted mild inhibition at complex I, negligible effects on complexes II-III, and extensive inhibition of complex IV. Kinetic analysis of complex I revealed that MPP+ was competitive with NADH, and partially reversible by FAD and FMN. Co-Q(10) potentiated complex II ( approximately 200%), but not complex I or III. Despite positive influence of flavins and Co-Q(10) on complexes I-II function, neither protected against MPP+ toxicity, indicating inhibition of complex IV as the predominant target. The nicotinamides and glucose prevented MPP+ toxicity by fueling anaerobic glycolysis, evident by accumulation of lactate in the absence of MOC. The data also define a clear anomaly of neuroblastoma, indicating a preference for anaerobic conditions, and an adverse response to aerobic. An increase in CO(2), CO(2)/O(2) ratio, mitochondrial inhibition or O(2) deprivation was not directly toxic, but activated metabolism through glycolysis prompting depletion of glucose and starvation. In conclusion, the results of this study indicate that the mechanism of action for MPP+, involves the inhibition of complex I and and more specifically complex IV, leading to impaired OXPHOS and MOC. Moreover, flavin dervatives control the rate of complex I/cellular respiration and Co-Q10 augments complex II [corrected].     

Oxidative stress-dependent toxicity of silver nanoparticles in human hepatoma cells

Cytotoxicity induced by silver nanoparticles (AgNPs) and the role that oxidative stress plays in this process were demonstrated in human hepatoma cells. Toxicity induced by silver (Ag⁺) ions was studied in parallel using AgNO₃ as the Ag⁺ ion source. Using cation exchange treatment, we confirmed that the AgNP solution contained a negligible amount of free Ag⁺ ions. Metal-responsive metallothionein 1b (MT1b) mRNA expression was not induced in AgNP-treated cells, while it was induced in AgNO₃-treated cells. These results indicate that AgNP-treated cells have limited exposure to Ag⁺ ions, despite the potential release of Ag⁺ ions from AgNPs in cell culture. AgNPs agglomerated in the cytoplasm and nuclei of treated cells, and induced intracellular oxidative stress. AgNPs exhibited cytotoxicity with a potency comparable to that of Ag⁺ ions in in vitro cytotoxicity assays. However, the toxicity of AgNPs was prevented by use of the antioxidant N-acetylcysteine, and AgNP-induced DNA damage was also prevented by N-acetylcysteine. AgNO₃ treatment induced oxidative stress-related glutathione peroxidase 1 (GPx1) and catalase expression to a greater extent than AgNP exposure, but treatment with AgNO₃ and AgNPs induced comparable superoxide dismutase 1 (SOD1) expression levels. Our findings suggest that AgNP cytotoxicity is primarily the result of oxidative stress and is independent of the toxicity of Ag⁺ ions.