Novel dopamine releasing response of an anti-convulsant agent with possible anti-Parkinson's activity.

We used cerebral microdialysis to assess the ability of the anticonvulsant drug Zonisamide (ZNS) to release striatal dopamine in 6-hydroxydopamine nigrotomized rats. Following exogeneously administered ZNS we measured dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels in striatal dialysates obtained from the ipsilateral side of the nigrotomy. ZNS administration alone had no effect on levels of DA and its metabolites or rotational behavior. Administration of carbidopa-levodopa alone led to small but insignificant increases in rotational behavior contralateral to the side of the nigrotomy but no corresponding increases in indices of striatal catecholamine release. In contrast, if animals were preloaded with carbidopa and ZNS was co-administered with levodopa 30 minutes later significant increases in contralateral rotational behavior occurred within 20 minutes of ZNS-levodopa injection that lasted for at least 90 minutes. In contrast to the uniform rotational behavioral responses observed in all our nigrotomized animals, less than half demonstrated neurochemical evidence of DA release. In these "responder" animals DOPAC levels increased 300% following carbidopa-levodopa-ZNS administration. We conclude that these results support previously reported findings and provide additional evidence that the anticonvulsant ZNS appears to possess anti-Parkinson's properties. ZNS could therefore be a novel agent for the treatment of PD that could delay the use of or reduce the amount of levodopa needed to treat patients with PD.

Parallel increases in lipid and protein oxidative markers in several mouse brain regions after methamphetamine treatment.

The neurotoxic actions of methamphetamine (METH) may be mediated in part by reactive oxygen species (ROS). Methamphetamine administration leads to increases in ROS formation and lipid peroxidation in rodent brain; however, the extent to which proteins may be modified or whether affected brain regions exhibit similar elevations of lipid and protein oxidative markers have not been investigated. In this study we measured concentrations of TBARs, protein carbonyls and monoamines in various mouse brain regions at 4 h and 24 h after the last of four injections of METH (10 mg/kg/injection q 2 h). Substantial increases in TBARs and protein carbonyls were observed in the striatum and hippocampus but not the frontal cortex nor the cerebellum of METH-treated mice. Furthermore, lipid and protein oxidative markers were highly correlated within each brain region. In the hippocampus and striatum elevations in oxidative markers were significantly greater at 24 h than at 4 h. Monoamine levels were maximally reduced within 4 h (striatal dopamine [DA] by 95% and serotonin [5-HT] in striatum, cortex and hippocampus by 60-90%). These decrements persisted for 7 days after METH, indicating effects reflective of nerve terminal damage. Interestingly, NE was only transiently depleted in the brain regions investigated (hippocampus and cortex), suggesting a pharmacological and non-toxic action of METH on the noradrenergic nerve terminals. This study provides the first evidence for concurrent formation of lipid and protein markers of oxidative stress in several brain regions of mice that are severely affected by large neurotoxic doses of METH. Moreover, the differential time course for monoamine depletion and the elevations in oxidative markers indicate that the source of oxidative stress is not derived directly from DA or 5HT oxidation.

CNS oxidative stress associated with the kainic acid rodent model of experimental epilepsy.

The role of oxidative stress in seizure-induced brain injury was investigated in a kainic acid model of experimental epilepsy. Kainic acid (12.5 mg/kg) or saline was injected intraperitoneally into 12-week-old male Fischer 344 rats and sacrificed by decapitation at 4 and 24 h after injection. Markers of oxidative stress including protein carbonyls, thiobarbituric acid reactive material (TBARs), glutathione (GSH) and glutathione disulfide (GSSG) were measured in hippocampus, cortex, cerebellum and basal ganglia. Four hours after treatment, protein carbonyls were elevated by 103, 55, 52 and 32% in cortex, hippocampus, basal ganglia and cerebellum, respectively. TBARs were increased by 30-45% in all areas. After 24 h, elevated protein and lipid oxidative markers persisted in the hippocampus and cerebellum; by contrast, in the cortex, TBARs almost normalized to control values and protein carbonyls trended downward by one-half compared with measurements at 4 h, although this reduction relative to the 4 h timepoint did not reach statistical significance. In the basal ganglia, protein carbonyls approached control values at 24 h. GSSG levels were only increased statistically in the cortex after 4 h, GSH levels in all the regions were unchanged after treatment with kainic acid. However, in cortex, GSH levels correlated negatively with increases in protein and lipid oxidation (r = -0.69, P < 0.002). In contrast, significant correlations between GSH, protein carbonyls and TBARs measured in the hippocampus or cerebellum were not observed. Our data suggests that kainic acid induced similar oxidative stress in all of the brain regions that were examined, and that GSH plays a major antioxidant role in the cerebral cortex but not the hippocampus.

Unaltered cytochrome oxidase, glutamate dehydrogenase and glutaminase activities in platelets from patients with sporadic amyotrophic lateral sclerosis--a study of potential pathogenetic mechanisms in neurodegenerative diseases.

Sporadic Amyotrophic Lateral Sclerosis (SALS) is a fatal neurologic disease characterized by degeneration of motor neurons in the spinal cord, brainstem and cortex. While familial cases of ALS exist, the sporadic form accounts for the majority of adult-onset cases. It has been hypothesized that the neurodegenerative mechanisms underlying SALS might arise from glutamate-mediated excitotoxicity and mitochondrial dysfunction. Studies on autopsied SALS spinal cord and brain have reported decreased cytochrome oxidase activity, decreased astrocytic glutamate-transporter protein, and alterations of glutamate levels and glutamate metabolizing enzyme activities. We conjectured that if alterations in glutamate metabolism and cytochrome oxidase activity occur in the SALS central nervous system these alterations may also be manifested in peripheral tissues such as platelets in living SALS patients. In this study we compared the activities of cytochrome oxidase, citrate synthase, glutamate dehydrogenase and glutaminase in platelets from SALS and control subjects. We found that there were no differences in any of the enzyme activities measured between the two groups. Our data argue against generalized ubiquitous biochemical alterations of these enzymes in SALS patients.

Studies on the characterization of the inhibitory mechanism of 4'-alkylated 1-methyl-4-phenylpyridinium and phenylpyridine analogues in mitochondria and electron transport particles.

1-Methyl-4-phenylpyridinium (MPP+), the toxic agent in MPTP-induced dopaminergic neurotoxicity, is thought to act by inhibiting mitochondrial electron transport at complex I. This study examined this latter action further with a series of 4'-alkylated analogues of MPP+. These derivatives had IC50 values that ranged from 0.5 to 110 microM and from 1.6 to 3,300 microM in mitochondria and electron transport particles (ETPs), respectively. The IC50 values of corresponding 4'-alkylated phenylpyridine derivatives to inhibit NADH-linked oxidation ranged from 10 to 205 microM in mitochondria and from 1.7 to 142 microM in ETPs. The potencies of both classes of inhibitors directly correlated with their ability to partition between 1-octanol and water. In mitochondria, increased hydrophobicity resulted in greater inhibition of NADH dehydrogenase but a smaller dependence on the transmembrane electrochemical gradient for accumulation of the pyridiniums as evidenced by an approximately 600-fold, versus only a 36-fold, increase in the IC50 of MPP+ versus 4'-pentyl-MPP+, respectively, in the presence of uncoupler. In ETPs, the analogous increase in potencies of the more hydrophobic analogues was also consistent with an inhibitory mechanism that relied on differential partitioning into the lipid environment surrounding NADH dehydrogenase. However, the pyridinium charge must play a major role in explaining the inhibitory mechanism of the pyridiniums because their potencies are much greater than would be predicted based solely on hydrophobicity. For example, in ETPs, 4'-decyl-MPP+ was nearly 80-fold more potent than phenylpyridine although the latter compound partitions twice as much into 1-octanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the inhibitory mechanism of 1-methyl-4-phenylpyridinium and 4-phenylpyridine analogs in inner membrane preparations.

We have investigated the mechanism of the inhibition of membrane-bound NADH dehydrogenase by 1-methyl-4-phenylpyridinium (MPP+) and a series of its 4'-alkyl-substituted analogs of increasing hydrophobicity, as well as their neutral, desmethyl congeners. Comparison of hydrophobicity, as measured by partition coefficients, with the IC50 for the inhibition of NADH oxidase activity in mitochondrial inner membrane preparations shows a negative correlation, but the cationic inhibitors are more effective than the neutral analogs with similar hydrophobicity. The presence of 10 microM tetraphenylboron (TPB-) potentiates the inhibitory power of positively charged analogs up to 4'-pentyl-MPP+, while the neutral inhibitors are unaffected by TPB-. Without TPB-, the more hydrophilic analogs give incomplete inhibition, but the inclusion of TPB- permits the attainment of complete inhibition, accompanied by the appearance of sigmoidal titration curves. These data support the hypothesis that MPP+ analogs, like rotenone, are bound at two sites on the enzyme and occupancy of both is required for complete inhibition. TPB-, by forming ion pairs with the cationic analogs, facilitates their equilibration to both sites in membrane preparations. When present in molar excess over the MPP+ analog, TPB- partially reverses the inhibition by decreasing its concentration in the more hydrophilic binding site. The effect of temperature and of pH on the IC50 values for inhibition support the concept of dual binding sites, and the pH dependence of the inhibition reveals the participation of two ionized protein groups in the binding, one of which may be a thiol group.

Interaction of 1-methyl-4-phenylpyridinium ion (MPP+) and its analogs with the rotenone/piericidin binding site of NADH dehydrogenase.

Nigrostriatal cell death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease results from the inhibition of mitochondrial respiration by 1-methyl-4-phenylpyridinium (MPP+). MPP+ blocks electron flow from NADH dehydrogenase to coenzyme Q at or near the same site as do rotenone and piericidin and protects against binding of and loss of activity due to these inhibitors. The 4'-analogs of MPP+ showed increasing affinity for the site with increasing length of alkyl chain, with the lowest Ki, for 4'-heptyl-MPP+, being 6 microM. The 4'-analogs compete with rotenone for the binding site in a concentration-dependent manner. They protect the activity of the enzyme from inhibition by piericidin in parallel to preventing its binding, indicating that the analogs and piericidin bind at the same inhibitory site(s). The optimum protection, however, was afforded by 4'-propyl-MPP+. The lesser protection by the more lipophilic MPP+ analogs with longer alkyl chains may involve a different orientation in the hydrophobic cleft, allowing rotenone and piericidin to still bind even when the pyridinium cation is in a position to interrupt electron flow from NADH to coenzyme Q.

4'-alkylated analogs of 1-methyl-4-phenylpyridinium ion are potent inhibitors of mitochondrial respiration.

1-Methyl-4-phenylpyridinium ion, a major brain metabolite of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, is an inhibitor of Complex I of the mitochondrial respiratory chain. We have synthesized several analogs of 1-methyl-4-phenylpyridinium ion containing various alkyl groups in the 4' position of the phenyl ring and have tested them for their abilities to inhibit the oxidation of NADH-linked substrates by intact mouse liver mitochondria. These compounds are considerably more potent inhibitors than MPP+ itself, with potency increasing as the length of the alkyl chain increases. The most potent inhibitor, 1-methyl-4-(4'heptylphenyl)pyridinium ion, was about 200 times as effective as MPP+. These analogs should prove to be useful tools for studying the nature of the process whereby MPP+ and its pyridinium analogs interact with Complex I to inhibit mitochondrial respiration.