Ethanol inhibits voltage-gated sodium channels in cultured superior cervical ganglion neurons.

To determine whether actions on sodium channels contribute to ethanol's depressant effects on the autonomic nervous system, the acute effects of ethanol on Na+ currents in primary cultured superior cervical ganglion were examined by whole-cell patch clamp recordings. Ethanol inhibited Na+ currents concentration dependently, and decreased action potential firing. Ethanol (100 mM) did not affect activation curve, but resulted in a left shift of the inactivation curve and prolonged the recovery from inactivation. This finding indicates that the channels in the inactivated state are more susceptible to ethanol than those in the resting state. For the first time, this study demonstrates acute inhibitory effects of ethanol on sodium channel gating in sympathetic neurons.

[Enhancement of NMDA receptor sensitivity by thrombin and its relationship with tissue transglutaminase].

OBJECTIVE: To explore the possible relationship between the thrombin induced potentiation of rat cerebellum granular cell N-methyl-D-aspartate (NMDA) receptor function and the change of intracellular tissue transglutaminase (tTG). METHODS: Primary cultured rat cerebellum granular cells were pretreated by using thrombin (0.01 U/ml) with or without co-pretreatment of 0.5 x 10(-4) mol/L MDC (monodansylcadaverine, a tTG inhibitor) 30 minutes. Induce cells mentioned above to undergo apoptosis with different concentration of NMDA (0.1 x 10(-4) mol/L, 0.25 x 10(-4) mol/L and 0.5 x 10(-4) mol/L). Flowcytometer was applied 3 hours later to detect the changed function of NMDA receptor by counting percentage of apoptotic cells. RT-PCR was used to compare the tTG mRNA level in the cerebellum granular cells treated with NMDA of same dosage with/without thrombin pretreatment. RESULTS: Thrombin pretreatment increased the percentage of apoptosis cells induced by NMDA [(8.3 +/- 0.5)% to (15.1 +/- 0.8)% at 0.1 x 10(-4) mol/L, (14.1 +/- 1.1)% to (23.8 +/- 0.7)% at 0.25 x 10(-4) mol/L, (26.8 +/- 1.9)% to (33.7 +/- 2.1)% at 0.5 x 10(-4) mol/L)]; and this effect was inhibited by co-pretreatment MDC with thrombin, cell apoptosis decreased to (5.8 +/- 1.2)%, (11.5 +/- 1.5)% and (19.0 +/- 1.7)%; Thrombin increased intracellular tTG mRNA levels comparing with those without thrombin pretreatment. CONCLUSION: Thrombin potentiates rat cerebellum granular cell NMDA receptor function by increasing intracellular tissue transglutaminase.

Effects of extracellular Ca(2+) influx and intracellular Ca(2+) release on ethanol-induced cytoplasmic Ca(2+) overload in cultured superior cervical ganglion neurons.

The present research was designed to investigate the interference of Ca(2+) homeostasis by ethanol on the primary cultured superior cervical ganglion (SCG) neurons. (1) Using the whole cell patch clamp recording, the amplitudes of voltage-dependent Ca(2+) channel (VDCC) currents could be reduced by ethanol in a concentration-dependent manner. Ethanol (100mM) inhibited about 25% of Ca(2+) channel current. However, the activation of Ca(2+) channel was not affected by ethanol at those concentrations. (2) The similar extent inhibitions of 100mM ethanol on the increments of intracellular Ca(2+) concentration ([Ca(2+)](i)) induced by 40 mM KCl and 1 microM A23187 were also observed in the fluo-3-AM loaded superior cervical ganglia (SCG) via detecting the change of [Ca(2+)](i) with a laser scanning confocal microscopy. In contrast, the basal [Ca(2+)](i) was significantly increased by ethanol alone in a concentration-dependent manner. These phenomena were also observed even under Ca(2+) free bath solution or the solution added 300 microM cadmium chloride conditions. Together with above results, our data suggest that ethanol increases basal [Ca(2+)](i), but it also inhibits the extracellular Ca(2+) influx through VDCC and ionophore channel. And the augment of basal [Ca(2+)](i) induced by ethanol might attribute to the Ca(2+) releasing from intracellular Ca(2+) pools.

Iron-induced oxidative damage and apoptosis in cerebellar granule cells: attenuation by tetramethylpyrazine and ferulic acid.

Tetramethylpyrazine and ferulic acid are two active ingredients of a Chinese herbal medicine Ligusticum wallichi Franchat. In the present investigation, iron-induced oxidative neuronal damage and the protective effects of tetramethylpyrazine and ferulic acid against this induction were studied in primary cultures of rat cerebellar granule cells. When neurons were treated with 200 microM of FeSO(4) for 1 h, lipid peroxidation in neurons increased time dependently, as measured with the thiobarbituric acid assay. Thirty-six hours after iron treatment, the cell viability decreased to 43.6% and the percentage of apoptotic cells increased to 50.6%. Transmission electron microscopic examination showed a disrupted nuclear envelope and condensed chromatin in iron-treated neurons. Analysis of DNA extracted from iron-treated cells by agarose gel electrophoresis showed the typical "ladder pattern", which indicated the formation of mono- and oligonucleosomes. After iron treatment, caspase 3 activity increased significantly, as measured in a fluoregenic assay. The results above suggested that iron treatment triggered oxidative stress and apoptosis in neurons. Western blot revealed that iron treatment up-regulated the apoptosis-related gene p53 as well as its effector gene p21(waf1/cip1). Pretreatment of the cells with 100 microM of tetramethylpyrazine or ferulic acid effectively decreased the activation of caspase 3 as well as the expression of p53 and p21(waf1/cip1), and attenuated iron-induced oxidative damage and apoptosis. The results suggest that tetramethylpyrazine and ferulic acid might be used as preventive agents against neuronal diseases associated with oxidative stress.

Tetramethylpyrazine scavenges superoxide anion and decreases nitric oxide production in human polymorphonuclear leukocytes.

Tetramethylpyrazine is one of the active ingredients of the Chinese herb Ligusticum wallichii Franchat. By electron spin resonance spin trapping methods, effects of tetramethylpyrazine on superoxide anion and nitric oxide generated by human polymorphonuclear leukocytes were studied. During the respiratory burst of polymorphonuclear leukocytes induced by N-formylmethionyl-leucyl-phenylalanine, tetramethylpyrazine scavenges superoxide anion dose-dependently, and decreases the production of nitric oxide significantly, but shows no influence on oxygen consumption. These results suggest that the effective protection of tetramethylpyrazine against ischemic brain injury might be due to its scavenging of reactive oxygen species and regulation on nitric oxide production, and consequent prevention of peroxynitrite formation.

[Content of nitric oxide in cerebrospinal fluid from patients with neurocysticercosis].

OBJECTIVE: To evaluate the relationship between the concentration of nitric oxide (NO) and cytological change in cerebrospinal fluid (CSF) and the onset of neurocysticercosis in patients. METHODS: NO Level in CSF was detected in 30 cases of neurocysticercosis, 20 cases of tuberculous meningitis (TBM) and 20 healthy people as control by using Griess method. The cytological components in CSF were also examined. RESULTS AND CONCLUSION: Griess method was proved to be a rapid and accurate technique for the detection of NO content in CSF. The NO concentration in cases of neurocysticercosis and TBM was significantly higher than that of control(P < 0.01). The neurocysticercosis cases showed the highest CST NO level which was considerably higher than that of TBM cases(P < 0.05). Among the cytological changes, the neurocysticercosis cases also showed a higher count of eosinophils than that of TBM cases and control (P < 0.01). The NO level and eosinophils count in CSF increased significantly in patients with neurocysticercosis.