Hiroshi Katsuki

3-Hydroxykynurenine, an endogenous oxidative stress generator, causes neuronal cell death with apoptotic features and region selectivity

Abstract: 3‐Hydroxykynurenine (3‐HK) is a potential endogenous neurotoxin whose increased levels have been described in several neurodegenerative disorders. Here, we characterized in vitro neurotoxicity of 3‐HK. Of the tested kynurenine pathway metabolites, only 3‐HK, and to a lesser extent 3‐hydroxyanthranilic acid, were toxic to primary cultured striatal neurons. 3‐HK toxicity was inhibited by various antioxidants, indicating that the generation of reactive oxygen species is essential to the toxicity. 3‐HK‐induced neuronal cell death showed several features of apoptosis, as determined by the blockade by macromolecule synthesis inhibitors, and by the observation of cell body shrinkage with nuclear chromatin condensation and fragmentation. In addition, 3‐HK toxicity was dependent on its cellular uptake via transporters for large neutral amino acids, because uptake inhibition blocked the toxicity. Cortical and striatal neurons were much more vulnerable to 3‐HK toxicity than cerebellar neurons, which may be attributable to the differences in transporter activities of these neurons. These results indicate that 3‐HK, depending on transporter‐mediated cellular uptake and on intracellular generation of oxidative stress, induces neuronal cell death with brain region selectivity and with apoptotic features, which may be relevant to pathology of neurodegenerative disorders.

Arachidonic acid as a neurotoxic and neurotrophic substance

In this article we summarize a wide variety of properties of arachidonic acid (AA) in the mammalian nervous system especially in the brain. AA serves as a biologically-active signaling molecule as well as an important component of membrane lipids. Esterified AA is liberated from the membrane by phospholipase activity which is stimulated by various signals such as neurotransmitter-mediated rise in intracellular Ca2+. AA exerts many biological actions which include modulation of the activities of protein kinases and ion channels, inhibition of neurotransmitter uptake, and enhancement of synaptic transmission. AA serves also as a precursor of a variety of eicosanoids, which are formed by oxidative metabolism of AA. AA cascade is activated under several pathological conditions in the brain such as ischemia and seizures, and may be involved in irreversible tissue damage. On the other hand, AA can show beneficial influences on brain tissues and cells in several situations. In a recent study using cultured brain neurons, we have found that AA shows quite distinct actions at a narrow concentration range, such as induction of cell death, promotion of cell survival and enhancement of neurite extension. The neurotoxic action is mediated by free radicals generated by AA metabolism, whereas the neurotrophic actions are exerted by AA itself. The observed in vitro actions of AA might be related to important roles of AA in brain pathogenesis and neural development.

Arachidonic acid: Toxic and trophic effects on cultured hippocampal neurons

Arachidonic acid (20:4) is a component of membrane lipids that has been implicated as a messenger both in physiological and pathophysiological processes, including ischemic injury and synaptic plasticity. In order to clarify direct trophic or toxic effects of arachidonic acid on central neurons, primary cultures of rat hippocampal neurons were exposed to arachidonic acid under chemically-defined conditions. Arachidonic acid present in the culture medium at concentrations over 5 x 10(-6) M showed profound toxicity, whereas at lower concentrations (10(-6) M) it significantly supported the survival of hippocampal neurons. These effects were not mimicked by oleic acid (18:1) or palmitic acid (16:0). The toxic action of 10(-5) M arachidonic acid was markedly and significantly prevented by a lipoxygenase inhibitor nordihydroguaiaretic acid (10(-6) M). AA861 and baicalein (each at 10(-6) M), a selective inhibitor for 5- and 12-lipoxygenase, respectively, also showed a significant protective effect, whereas cyclooxygenase inhibitor indomethacin (10(-5) M) had no effect. The toxic action was also prevented by an antioxidant alpha-tocopherol (10(-6) M), but not by superoxide dismutase (100 U/ml) or catalase (200 U/ml). The trophic effect of 10(-6) M arachidonic acid was not suppressed by the treatments listed above. At lower concentrations (10(-7)-10(-6) M), arachidonic acid promoted neurite elongation, which was not inhibited by nordihydroguaiaretic acid or indomethacin. Overall, arachidonic acid has both trophic and toxic actions on cultured hippocampal neurons, part of which involves its metabolism by lipoxygenases. The mechanisms and the physiological significance of these effects are discussed.

Inhibition by 5-HT7 receptor stimulation of GABAA receptor-activated current in cultured rat suprachiasmatic neurones.

1. Whole‐cell voltage‐clamp recordings were made from postnatal rat suprachiasmatic (SCN) neurones to investigate possible modulation by 5‐hydroxytryptamine (5‐HT) of gamma‐aminobutyric acid (GABA)‐activated current (IGABA). 2. 5‐HT reversibly inhibited IGABA in a concentration‐dependent manner (10(‐10) to 10(‐6) M). (+/‐)‐8‐Hydroxy‐2‐N,N‐dipropylaminotetralin (8‐OH‐DPAT, 10(‐10) to 10(‐5) M) and 5‐carboxamidotryptamine (10(‐6) M) also inhibited IGABA, whereas 1‐(2,5‐dimethyl‐4‐iodophenyl)‐2‐aminopropane (DOI, 10(‐6) M) had no significant effect. 3. The effect of 8‐OH‐DPAT (10(‐7) M) was blocked by ritanserin (10(‐7) M), but not by pindolol (10(‐7) M). The effect of 5‐HT was also suppressed by ritanserin, but not by pindolol, ketanserin (10(‐7) M) or ICS 205‐930 (10(‐6) M). 4. 8‐Bromo‐cAMP (10(‐3) M) or forskolin (5 x 10(‐5) M) suppressed IGABA. The effects of forskolin and 5‐HT were not additive. Furthermore, the effect of 5‐HT (10(‐7) M) was significantly reduced by N‐[2‐(methylamino)ethyl]‐5‐isoquinoline sulphonamide (H‐8, 10(‐6) M). 5. It is concluded that 5‐HT inhibits IGABA in the SCN neurones, which involves the activation of 5‐HT7 receptors and cAMP‐coupled systems.

GABAA receptor stimulation promotes survival of embryonic rat striatal neurons in culture

In order to clarify the functional role of gamma-aminobutyric acid (GABA) in developing brain, we investigated the effect of GABA on the survival of embryonic rat striatal neurons in dissociated cell culture. Chronic exposure of striatal cultures to GABA resulted in a significant increase in the number of surviving neurons. The effect of GABA was concentration-dependent (1-1000 microM) and was blocked by a GABAA receptor antagonist, bicuculline (100 microM), or a GABAA chloride channel blocker, picrotoxin (100 microM), but not by a GABAB receptor antagonist, 2-hydroxysaclofen (100 microM). In addition, the GABAA receptor agonist muscimol mimicked the effect of GABA, promoting cell survival in a concentration-dependent manner (0.01-100 microM), while the GABAB receptor agonist baclofen (up to 100 microM) had no significant effect. The GABA-induced enhancement of neuronal survival was suppressed by the L-type voltage-dependent Ca2+ channel blockers nifedipine (1-3 microM) and nicardipine (1-5 microM). Protein kinase inhibitors, H-7 (10-30 microM) or genistein (3 microM), also suppressed GABA-induced enhancement of neuronal survival. These results suggest that stimulation of GABAA receptors enhances survival of embryonic striatal neurons, and that the effect is mediated by Ca2+ influx through L-type voltage-dependent Ca2+ channels, initiating intracellular signaling cascades that involve activation of H-7- and genistein-sensitive protein kinases.

Neurotrophic Activity of Organosulfur Compounds Having a Thioallyl Group on Cultured Rat Hippocampal Neurons

Several organosulfur compounds found in garlic extract promoted the survival of rat hippocampal neurons in vitro. From the analysis of structure-activity relationship, thioallyl group in these compounds is essential for the manifestation of neurotrophic activity. S-Allyl-L-cysteine (SAC), one of the organosulfur compounds having thioallyl group in garlic extract, also promoted the axonal branching of cultured neurons. These results suggest that thioallyl compounds make a unique group of neurotrophic factors.

Allixin, a phytoalexin produced by garlic, and its analogues as novel exogenous substances with neurotrophic activity

Effects of allixin, a phytoalexin of garlic, and its analogues were studied on the survival and morphology of primary cultured neurons from fetal rat brain. Addition of allixin (1-100 ng/ml) to medium significantly promoted the survival of neurons derived from various regions of brain and increased the number of branching points per axon in hippocampal neurons. Allixin, however, was cytotoxic at higher concentrations (>1 microg/ml). Among the analogues of allixin, 2,6-dimethyl-3-hydroxy-4H-pyran-4-one (DHP) possessed potent neurotrophic activity at concentrations over 10 ng/ml without any obvious cytotoxicity up to 10 microg/ml. DHP also retained the activity to promote axonal branching. These results indicate that DHP is a novel exogenous low molecular weight neurotrophic substance without apparent cytotoxicity. This compound may be a useful prototype leading chemical for developing therapeutic and/or prophylactic drugs for neurodegenerative disorders.

Pharmacological characteristics of GABAA responses in postnatal suprachiasmatic neurons in culture

The suprachiasmatic nucleus (SCN) is considered to be an endogenous circadian pacemaker. Previous studies have suggested functional roles of gamma-aminobutyric acid (GABA) in the control of circadian rhythms. In this study, the responses to applied GABA in cultured SCN neurons dissociated from postnatal rat hypothalamus were investigated using whole-cell voltage-clamp techniques. GABA and muscimol induced a large current response (EC50 values 5.3 and 1.6 microM, respectively), which was blocked by the GABAA antagonist bicuculline. This current response was also blocked by Zn2+ (0.5-50 microM) in a concentration-dependent manner, but was not potentiated by diazepam (10 microM) or ethanol (21 mM). These characteristics seem to correspond to those of GABAA receptors that lack gamma-type subunits.

Biphasic effect of hydrogen peroxide on field potentials in rat hippocampal slices

In the CA1 region of rat hippocampal slices, H2O2 (0.294-2.94 mM) caused initial augmentation, and subsequent long-lasting depression, of population spikes and excitatory postsynaptic potentials. The effect of H2O2 may not be mediated by its degradation product, hydroxyl radicals, because an iron chelator deferoxamine did not block the effect. A catalase inhibitor 3-amino-1,2,4-triazole only modestly attenuated the initial augmentation, suggesting that the effect of H2O2 is not attributable to catalase-dependent O2 generation, either. An N-methyl-D-aspartate receptor antagonist DL-2-amino-5-phosphonovaleric acid had no influence on the effect of H2O2, whereas a gamma-aminobutyric acid type A receptor channel blocker picrotoxin attenuated long-lasting depression, indicating that gamma-aminobutyric acid-mediated inhibition is altered during the depression phase. The initial augmentation but not subsequent depression was attenuated by a phospholipase A2/C inhibitor 4-bromophenacyl bromide, suggesting the involvement of lipid signaling molecule(s) in the enhancement of excitatory synaptic transmission. These results suggest that H2O2 regulates hippocampal synaptic transmission via multiple mechanisms.

The involvement of muscarinic, β-adrenergic and metabotropic glutamate receptors in long-term potentiation in the fimbria-CA3 pathway of the hippocampus

Possible modulatory actions of endogenous neurotransmitters on long-term potentiation (LTP) were investigated in the fimbria-CA3 pathway of rat hippocampal slices. Bath application of atropine (10 microM), but neither timolol (10 microM) nor D,L-2-amino-3-phosphonopropionate (AP3, 100 microM), significantly attenuated LTP induced by 20 pulses of 50 Hz stimulation. When stronger stimulation (3 trains of 100 Hz, 100 pulses) was used for the induction of LTP, timolol significantly attenuated LTP, but atropine and AP3 did not. These results suggest that, under specified conditions, endogenous acetylcholine through muscarinic receptors, and noradrenaline through beta-adrenergic receptors may modulate the generation of LTP in the fimbria-CA3 pathway. Metabotropic glutamate receptors may be involved not in the generation of LTP but in low-frequency synaptic transmission, since 300-1,000 microM AP3 greatly reduced, or abolished synaptic transmission in this pathway.