Ko-ichi Ohtaki

Carrier-mediated processes in blood-brain barrier penetration and neural uptake of paraquat

Due to the structural similarity to N-methyl-4-phenyl pyridinium (MPP(+)), paraquat might induce dopaminergic toxicity in the brain. However, its blood--brain barrier (BBB) penetration has not been well documented. We studied the manner of BBB penetration and neural cell uptake of paraquat using a brain microdialysis technique with HPLC/UV detection in rats. After subcutaneous administration, paraquat appeared dose-dependently in the dialysate. In contrast, MPP(+) could not penetrate the BBB in either control or paraquat pre-treated rats. These data indicated that the penetration of paraquat into the brain would be mediated by a specific carrier process, not resulting from the destruction of BBB function by paraquat itself or a paraquat radical. To examine whether paraquat was carried across the BBB by a certain amino acid transporter, L-valine or L-lysine was pre-administered as a co-substrate. The pre-treatment of L-valine, which is a high affinity substrate for the neutral amino acid transporter, markedly reduced the BBB penetration of paraquat. When paraquat was administered to the striatum through a microdialysis probe, a significant amount of paraquat was detected in the striatal cells after a sequential 180-min washout with Ringers solution. This uptake was significantly inhibited by a low Na(+) condition, but not by treatment with putrescine, a potent uptake inhibitor of paraquat into lung tissue. These findings indicated that paraquat is possibly taken up into the brain by the neutral amino acid transport system, then transported into striatal, possibly neuronal, cells in a Na(+)-dependent manner.

Paraquat induces long-lasting dopamine overflow through the excitotoxic pathway in the striatum of freely moving rats

The herbicide paraquat is an environmental factor that could be involved in the etiology of Parkinsons disease. We have previously shown that paraquat penetrates through the blood-brain barrier and is taken up by neural cells. In this study, we examined the in vivo toxic mechanism of paraquat to dopamine neurons. GBR-12909, a selective dopamine transporter inhibitor, reduced paraquat uptake into the striatal tissue including dopaminergic terminals. The subchronic treatment with systemic paraquat significantly decreased brain dopamine content in the striatum and slightly in the midbrain and cortex, and was accompanied by the diminished level of its acidic metabolites in rats. When paraquat was administered through a microdialysis probe, a transitory increase in the extracellular levels of glutamate, followed by long-lasting elevations of the extracellular levels of NO(x)(-) (NO(2)(-) plus NO(3)(-)) and dopamine were detected in the striatum of freely moving rats. This dopamine overflow lasted for more than 24 h after the paraquat treatment. Dopamine overflow was inhibited by N(G)-nitro-L-arginine methyl ester, dizocilpine, 6,7-dinitroquinoxaline-2,3-dione and L-deprenyl. The toxic mechanism of paraquat involves glutamate induced activation of non-NMDA receptors, resulting in activation of NMDA receptor-channels. The influx of Ca(2+) into cells stimulates nitric oxide synthase. Released NO would diffuse to dopaminergic terminals and further induce mitochondrial dysfunction by the formation of peroxynitrite, resulting in continuous and long-lasting dopamine overflow. The constant exposure to low levels of paraquat may lead to the vulnerability of dopaminergic terminals in humans, and might potentiate neurodegeneration caused by the exposure of other substances, such as endogenous dopaminergic toxins.

Endogenously Occurring β‐Carboline Induces Parkinsonism in Nonprimate Animals: A Possible Causative Protoxin in Idiopathic Parkinson's Disease

Abstract: To examine whether simple β‐carbolines induce parkinsonian‐like symptoms in vivo via N‐methylation, the simple β‐carbolines norharman (NH), 2‐mono‐N‐methylated norharmanium cation (2‐MeNH+), and 9‐mono‐N′‐methylnorharman (9‐MeNH) were systematically administered to C57BL/6 mice for 7 days. These substances induced bradykinesia with reduction of locomotion activity. NH or 2‐MeNH+ decreased dopamine (DA) contents to 50–70% of values in controls in the striatum and midbrain. 9‐MeNH potently decreased not only DA but also serotonin content in various regions. Immunohistochemical examination revealed that the numbers of tyrosine hydroxylase (TH)‐positive cells in the substantia nigra pars compacta of NH‐ and 9‐MeNH‐treated mice were diminished to 76 and 66% of values in control mice, respectively. The formation of a toxic metabolite, 2,9‐di‐N,N′‐methylated norharmanium cation (2,9‐Me2NH+), was 14 and eight times higher in the brain of mice receiving 9‐MeNH than that in NH‐ and 2‐MeNH+‐treated mice, respectively. In cultured mesencephalic cells from rat embryo, 2,9‐Me2NH+ selectively killed TH‐positive neurons only at a lower dose but was toxic to all neurons at higher doses. Thus, the excess formation of 2,9‐Me2NH+ would induce nonspecific neurotoxicity. These results indicated that 9‐indole nitrogen methylation should be the limiting step in the development of the toxicity. NH, a selective dopaminergic toxin precursor, is sequentially methylated to form 2,9‐Me2NH+, which could be an underlying factor in idiopathic Parkinsons disease.

Structural significance of azaheterocyclic amines related to Parkinson's disease for dopamine transporter

We have evaluated the neuronal uptake of 12 neutral and quaternary azaheterocyclic amines that are possible candidates for idiopathic Parkinsons disease via dopamine transporter of striatal synaptosomes. The double-reciprocal plots for dopamine transporter obtained from Wistar rat and C57BL/6 mouse synaptosomes with N-methyl-4-phenylpyridinium cation (MPP+) as a substrate were identical to each other. Neutral beta-carbolines and tetrahydroisoquinolines were unfavorable substrates for dopamine transporter. The quarternization of these compounds strikingly increased the affinity for dopamine transporter with 2-10 times greater Km and 10 times smaller Vmax values than MPP+. Although catechol tetrahydroisoquinolines were weak substrates, their quarternization reduced their original properties as substrates for dopamine transporter. These results provide both topographic and electrogenic information of azaheterocyclic amines for the dopamine transporter-mediated influx. The intramolecular distance between the N-atom and the centroid of the benzene ring could be an important factor for the recognition of binding site of dopamine transporter, and an adequate net charge similar to dopamine would be further required for translocation into the cells.

Cefoselis, a β-lactam antibiotic, easily penetrates the blood-brain barrier and causes seizure independently by glutamate release

Summary.Cefoselis is a widely used β-lactam antibiotic, but occasionally induces seizures and convulsion in elder and renal failure patients. However, β-lactams are known not to pass through the blood-brain barrier (BBB). In this study, we examined the BBB penetration of cefoselis in normal and renal failure rats by means of brain microdialysis. Cefoselis was dose-dependently appeared in brain extracellular fluid in proportion to its blood level. The elimination constant from brain extracellular fluid (apparent) was slightly lower than that from blood. These results indicated that cefoselis might penetrate the BBB or be discharged by a certain transport system. In contrast to the result of cefoselis, cefazolin, a leading drug of cephalosporins, could not be detected in the brain extracellular fluid after an intravenous injection. In renal dysfunction rats, the elimination half-lives of cefoselis from both blood and brain were extensively prolonged. This would be one of responsible factors inducing seizures seen in patients. However, the additional factor, such as decrease in brain function related to aging, would be involved in seizures in patient received cefoselis, because an extremely high dose was required to induce seizures even in renal failure rats. A local administration of cefoselis into the hippocampus through the microdialysis probe caused a striking elevation of extracellular glutamate, with a minimum increase in γ-aminobutyric acid (GABA). However, a systematic cefoselis administration via the tail vein did not elevate extracellular glutamate and GABA concentrations in the hippocampus of renal failure rats that exhibited marked seizures. These results suggested that not the stimulation of glutamate release, but the blockade of GABA receptors might be responsible for the seizure induced by cefoselis.