Hack Seang Kim

Changes of GABAA receptor binding and subunit mRNA level in rat brain by infusion of subtoxic dose of MK-801

In the present study, we have investigated the effects of prolonged inhibition of NMDA receptor by infusion of subtoxic dose of MK-801 to examine the modulation of GABA(A) receptor binding and GABA(A) receptor subunit mRNA level in rat brain. It has been reported that NMDA-selective glutamate receptor stimulation alters GABA(A) receptor pharmacology in cerebellar granule neurons in vitro by altering the levels of selective subunit. However, we have investigated the effect of NMDA antagonist, MK-801, on GABA(A) receptor binding characteristics in discrete brain regions by using autoradiographic and in situ hybridization techniques. The GABA(A) receptor bindings were analyzed by quantitative autoradiography using [3H]muscimol, [3H]flunitrazepam, and [35S]TBPS in rat brain slices. Rats were infused with MK-801 (1 pmol/10 microl per h, i.c.v.) for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2 ML). The levels of [3H]muscimol binding were highly elevated in almost all of brain regions including cortex, caudate putamen, thalamus, hippocampus, and cerebellum. However, the [3H]flunitrazepam binding and [35S]TBPS binding were increased only in specific regions; the former level was increased in parts of the cortex, thalamus, and hippocampus, while the latter binding sites were only slightly elevated in parts of thalamus. The levels of beta2-subunit were elevated in the frontal cortex, thalamus, hippocampus, brainstem, and cerebellar granule layers while the levels of beta3-subunit were significantly decreased in the cortex, hippocampus, and cerebellar granule layers in MK-801-infused rats. The levels of alpha6- and delta-subunits, which are highly localized in the cerebellum, were increased in the cerebellar granule layer after MK-801 treatment. These results show that the prolonged suppression of NMDA receptor function by MK-801-infusion strongly elevates [3H]muscimol binding throughout the brain, increases regional [3H]flunitrazepam and [35S]TBPS binding, and alters GABA(A) receptor subunit mRNA levels in different directions. The chronic MK-801 treatment has differential effect on various GABA(A) receptor subunits, which suggests involvement of differential regulatory mechanisms in interaction of NMDA receptor with the GABA receptors.

Modulation of the Levels of NMDA Receptor Subunit mRNA and the Bindings of [3H]MK-801 in Rat Brain by Chronic Infusion of Subtoxic Dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 μl/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [3H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [3H]MK-801 binding are involved in the MK-801-infused adult rats.

Cytotoxic activities of various fractions extracted from some pharmaceutical insect relatives

This research was performed to screen the cytotoxic activities of some pharmaceutical insect relatives. Cytotoxic activities of total extract and fractions of hexane, ethyl acetate, methanol, water and boiling water were extracted from four pharmaceutical insect relatives: the Chinese gall, the cicada slough, the hornet nest and the batryticated silkworm. These extracts were investigated against the cancer cell lines of L1210, P388 and SNU-1in vitro tests. Results showed that, ED50 against the cancer cell lines of L1210, P388 and SNU-1 were 0.55, 0.50, and 0.83 μg/ml in the ethyl acetate fraction from the Chinese gall; 1.07, 2.19, and 2.24 μg/ml in the ethyl acetate fraction, 1.51, 1.26, and 1.45 μg/ml in the water fraction and 1.48, 2.29, and 1.29 μg/ml in the boiling water fraction from the cicada slough; 3.31, 2.00, and 6.61 μg/ml in the water fraction from the hornet nest and 13.80, 19.95, and 31.62 μg/ml in the hexane fraction and 33.88, 21.88, and 25.12 μg/ml in the ethyl acetate fraction from the batryticated silkworm, respectively. All of the fractions metioned above showed high cytotoxic activities and could be suggested for further studiesin vivo tests.

Inhibition of Tyrosine Hydroxylase by Palmatine

Palmatine, an protoberberine isoquinoline alkaloid, has been found to inhibit dopamine biosynthesis by reducing tyrosine hydroxylase (TH) activity in PC12 cells (Lee and Kim, 1996). We have therefore investigated the effects of palmatine on bovine adrenal TH. Palmatine showed a mild inhibition on bovine adrenal TH (36.4% inhibition at concentration of 200 μM). Bovine adrenal TH was inhibited competitively by palmatine with a substrate L-tyrosine. The Ki value was found to be 0.67 mM. This result suggests that the inhibition of TH activity by palmatine may be partially involved in the reduction of dopamine biosynthesis in PC12 cells.

Inhibitory effect of fangchinoline on excitatory amino acids-induced neurotoxicity in cultured rat cerebellar granule cells.

Glutamate receptors-mediated excitotoxicity is believed to play a role in the pathophysiology of neurodegenerative diseases. The present study was performed to evaluate the inhibitory effect of fangchinoline, abis-benzylisoquinoline alkaloid, which has a characteristic as a Ca2+ channel blocker, on excitatory amino acids (EAAs)-induced neurotoxicity in cultured rat cerebellar granule neuron. Fangchinoline (1 and 5 μM) inhibited glutamate (1 mM), N-methyl-D-aspartate (NMDA; 1 mM) and kainate (100 μM)-induced neuronal cell death which was measured by trypan blue exclusion test. Fangchinoline (1 and 5 μM) inhibited glutamate release into medium induced by NMDA (1 mM) and kainate (100 μM), which was measured by HPLC. And fangchinoline (5 μM) inhibited glutamate (1 mM)-induced elevation of intracellular calcium concentration. These results suggest that inhibition of Ca2+ influx by fangchinoline may contribute to the beneficial effects on neurodegenerative effect of glutamate in pathophysiological conditions.

Inhibitory effects of ginseng total saponins on hypoxia-induced dysfunction and injuries of cultured astrocytes

The effects of ginseng total saponins (GTS) on hypoxic damage of primary cultures of astrocytes were studied. Hypoxia was created by placing cultures in an air tight chamber that was flushed with 95% N2/5% CO2 for 15 min before being sealed. Cultures showed evidence of significant cell injury after 24 h of hypoxia (increased lactate dehydrogenase (LDH) content in the culture medium, cell swelling and decreased glutamate uptake and protein content). Addition of GTS (0.1, 0.3 mg/ml) to the cultures during the exposure to hypoxic conditions produced dose-dependent inhibition of the LDH efflux. GTS (0.1, 0.3 mg/ml) also produced significant inhibition of the increased cell volume of astrocytes measured by [3H]O-methyl-D-glucose uptake under the hypoxic conditions. Decreased glutamate uptake and protein content was inhibited by GTS. These data suggest that GTS prevents astrocytic cell injury induced by severe hypoxiain vitro.

Effects of ginsenosides on the glutamate release and intracellular calcium levels in cultured rat cerebellar neuronal cells

These studies were designed to examine the effects of ginsenosides on glutamate neurotransmission. In primary cultures of rat cerebellar granule cells, ginsenosides (Rb1, Rc and Rg1, 500 μg/ml) increased glutamate release which was measured by HPLC, but Re did not show an elevation of glutamate release. However, all of these ginsenosides down-regulated N-methyl-D-aspartate (NMDA)-induced glutamate release. Rc strongly increased glutamate release and elevated intracellular calcium concentrations (Ca2+i) which was measured by ratio fluorometry with FURA-2 AM. These, results indicate that ginsenosides have a homeostatic effect on glutamate neurotransmission, and there is a structure-function relationship among the ginsenosides tested.

The time course of NMDA-and kainate-induced cGMP elevation and glutamate release in cultured neuron

The levels of extracellular glutamate, intracellular Ca2+ ([Ca2+]i) and cGMP were determined for 1 h with the excitatory amino acids, N-methyl-D-aspartate (NMDA) or kainate in cultured cerebellar granule cells. Both NMDA and kainate produced a time-dependent release of glutamate, and kainate was more potent than NMDA in glutamate elevation. The elevation of extracellular glutamate was not purely governed by intracellular Ca2+ concentration. However, in opposite to the time-dependent elevation of glutamate, the elevation of cGMP by NMDA and kainate were at maximum level in short-time (1 min) incubation then remarkably decreased with longer incubation times. Post-applications (30 min after agonist) of EAA antagonst did not block EAAs-induced glutamate elevation. However, NMDA antagonist, phencyclidine (PCP), blocked NMDA-induced cGMP elevation at pre- or post-application, but kainate antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), paradoxically augmented kainate-induced cGMP elevation for 1 h incubation. These results show that NMDA or kainate induces time-dependent elevations of extracellular glutamate, while the elevations of cGMP by these EAAs are remarkably decreased with longer incubation times. However, NMDA- and kainate-induced glutamate release was blocked by pre-application of each receptor antagonist but not by post-application while EAA-induced [Ca2+]i was blocked by post-application of antagonist. These observations suggest that EAA-induced elevation of [Ca2+]i is not parallel with elevation of glutamate release or cGMP.

Inhibitory effect of the root ofCoptis japonica on catecholamine biosynthesis in PC12 cells

The effect of the root ofCoptis japonica (COPT), both the dichloromethane soluble (CH2Cl2) and insoluble (H2O) fractions, on catecholamine contents and tyrosine hydroxylase (TH) activity in PC12 cells was investigated. CH2Cl2 and H2O fractions showed 21 and 53% inhibitions on dopamine content, respectively, at a concentration of 40 μg/ml in medium: the H2O fraction provided a greater inhibitory effect. The TH activity was reduced by the treatment of COPT (H2O fraction). These results suggest that COPT has an inhibitory effect on the catecholamine biosynthesis by the reduction of TH activity in PC12 cells.

Effects of L-trans-pyrrolidine-2,4-dicarboxylate, a glutamate uptake inhibitor, on NMDA receptor-mediated calcium influx and extracellular glutamate accumulation in cultured cerebellar granule neurons.

Glutamate uptake inhibitor, L-trans-pyrrolidine-2,4-dicarboxylate (PDC, 20 μM) elevated basal and N-methyl-D-aspartate (NMDA, 100 μM)-induced extracellular glutamate accumulation, while it did not augment kainate (100 μM)-induced glutamate accumulation in cultured cerebellar granule neurons. However, pretreatment with PDC for 1 h significantly reduced NMDA-induced glutamate accumulation, but did not affect kainate-induced response. Pretreatment with glutamate (5 μM) for 1 h also reduced NMDA-induced glutamate accumulation, but did not kainate-induced response. Upon a brief application (3–10 min), PDC did neither induce elevation of intracellular calcium concentration ([Ca2+]i) nor modulate NMDA-induced [Ca2+]i elevation. Pretreatment with PDC for 1 h reduced NMDA-induced [Ca2+]i elevation, but it did not reduce kainate-induced [Ca2+]i elevation. These results suggest that glutamate concentration in synaptic clefts of neuronal cells is increased by prolonged exposure (1 h) of the cells to PDC, and the accumulated glutamate subsequently induces selective desensitization of NMDA receptor.