Nrf2 Mediates the Anti-apoptotic and Anti-inflammatory Effects Induced by Gastrodin in Hydrogen Peroxide-Treated SH-SY5Y Cells.

Redox impairment, inflammation, and increased rates of cell death are central players during neurodegeneration. In that context, activation of the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) has been viewed as an interesting strategy in order to reduce the impact of redox dysfunction and neuroinflammation on cell fate. There is evidence indicating that the benefits caused by natural products in the brain may be due to the ability of these agents in upregulating Nrf2. Gastrodin (GAS) induces anti-oxidant, anti-inflammatory, and anti-apoptotic actions in brain cells. Nonetheless, the mechanisms underlying such effects are not clear yet. Therefore, we investigated here whether GAS would affect apoptosis and inflammation in the human neuroblastoma cell line (SH-SY5Y) exposed to hydrogen peroxide (H2O2). GAS at 1-25 µM was administrated to the cells during 30 min before a challenge with H2O2 at 300 µM for additional 24 h. GAS prevented the activation of the intrinsic apoptotic pathway by modulating the levels of Bcl-2 and Bax, causing a decrease in the release of cytochrome c to the cytosol. GAS also prevented the activation of the pro-apoptotic enzymes caspase-9 and caspase-3. Consequently, GAS abrogated poly (ADP-ribose) polymerase (PARP) cleavage and DNA fragmentation in the H2O2-treated SH-SY5Y cells. Moreover, GAS reduced the levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) and the activity of nuclear factor-κB in H2O2-treated cells. Silencing of Nrf2 by small interfering RNA (siRNA) suppressed the GAS-induced cytoprotection. Thus, GAS elicited anti-apoptotic and anti-inflammatory effects by a mechanism involving Nrf2 in SH-SY5Y cells.

Inhibition of the Nrf2/HO-1 Axis Suppresses the Mitochondria-Related Protection Promoted by Gastrodin in Human Neuroblastoma Cells Exposed to Paraquat.

Mitochondria are double-membrane organelles involved in the transduction of energy from different metabolic substrates into adenosine triphosphate (ATP) in mammalian cells. The oxidative phosphorylation system is comprised by the activity of the respiratory chain and the complex V (ATP synthase/ATPase). This system is dependent on oxygen gas (O2) in order to maintain a flux of electrons in the respiratory chain, since O2 is the final acceptor of these electrons. Electron leakage from this complex system leads to the continuous generation of reactive species in the cells. The mammalian cells exhibit certain mechanisms to attenuate the consequences originated from the constant exposure to these reactive species. In this context, the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and one of the enzymes whose expression is modulated by Nrf2, heme oxygenase-1 (HO-1), take a central role in inducing cytoprotection in humans. Mitochondrial abnormalities are observed during intoxication and in certain diseases, including neurodegeneration. Mitochondrial protection promoted by natural compounds has attracted the attention of researchers due to the promising effects these agents induce experimentally. In this regard, we examined here whether and how gastrodin (GAS), a phenolic glucoside, would prevent the paraquat (PQ)-induced mitochondrial impairment in the SH-SY5Y cells. The cells were exposed to GAS (25 µM) for 4 h prior to the challenge with PQ at 100 µM for additional 24 h. The silencing of Nrf2 by siRNA or the inhibition of HO-1 by ZnPP IX suppressed the GAS-elicited cytoprotection. Therefore, GAS promoted mitochondrial protection by an Nrf2/HO-1-dependent manner.

Gastrodin induced HO-1 and Nrf2 up-regulation to alleviate H2O2-induced oxidative stress in mouse liver sinusoidal endothelial cells through p38 MAPK phosphorylation.

Nuclear factor erythroid-related factor 2 (Nrf2) has been implicated in several detoxifying and antioxidant defense processes. Nrf2-mediated heme oxygenase-1 (HO-1) expression was demonstrated to play a key role against oxidative stress. Gastrodin (GSTD) is a well-known active compound isolated from the roots of Rhizoma gastrodiae, a plant used in ancient Chinese traditional medicine. The aim of this work was to investigate whether GSTD could alleviate H2O2-induced oxidative stress in mouse liver sinusoidal endothelial cells (LSECs). In LSECs exposed to 1 mM H2O2, treatment with GSTD (1, 10, or 50 µM) resulted in higher cell viability than the untreated control. Treated cells maintained a higher Bcl2/Bax ratio and suppressed caspase-9 expression compared with untreated cells, reducing cell apoptosis. GSTD was protective for H2O2-induced oxidative injury by reducing the generation of intracellular reactive oxygen species and malondialdehyde. HO-1 and Nrf2 expressions were synergistically upregulated by GSTD. Inhibition of HO-1 by 10 µM zinc protoporphyrin resulted in less protective effects on cell viability and malondialdehyde reduction by GSTD treatment in H2O2-exposed LSECs. Additionally, phosphorylated p38 in LSECs exposed to H2O2 was elevated by GSTD. Inhibition of p38 phosphorylation by SB203580 did not induce Nrf2 and HO-1 expression after 1 or 10 µM GSTD treatment and the protective effect on cell viability and malondialdehyde reduction in H2O2-exposed LSECs was reduced. The data conclusively demonstrated that GSTD-induced HO-1 and Nrf2 expression is involved in protection of LSECs from H2O2-induced oxidative injury, which may be regulated by p38 phosphorylation.

Evaluation of the Mitochondria-Related Redox and Bioenergetics Effects of Gastrodin in SH-SY5Y Cells Exposed to Hydrogen Peroxide.

Mitochondrion is the main site of ATP production in animal cells and also orchestrates signaling pathways associated with cell survival and death. Mitochondrial dysfunction has been linked to bioenergetics and redox impairment in human diseases, such as neurodegeneration and cardiovascular disease. Protective agents able to attenuate mitochondrial impairment are of pharmacological interest. Gastrodin (GAS; 4-hydroxybenzyl alcohol 4-O-beta-D-glucoside) is a phenolic glucoside obtained from the Chinese herbal medicine Gastrodia elata Blume and exhibits antioxidant, anti-inflammatory, and antiapoptotic effects in several cell types. GAS is able to cross the blood-brain barrier, reducing the impact of different stressors on the cognition of experimental animals. In the present work, we investigated whether GAS would protect mitochondria of human SH-SY5Y neuroblastoma cells against an exposure to a pro-oxidant agent. The cells were treated with GAS at 25 µM for 30 min before the administration of hydrogen peroxide (H2O2) at 300 µM for an additional 3 or 24 h, depending on the assay. We evaluated both mitochondrial redox state and function parameters and analyzed the mechanism by which GAS protected mitochondria in this experimental model. Silencing of the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor suppressed the GAS-induced mitochondrial protection seen here. Moreover, Nrf2 knockdown abrogated the effects of GAS on cell viability, indicating a potential role for Nrf2 in both mitochondrial and cellular protection promoted by GAS. Further research would be necessary to investigate whether GAS would be able to induce similar effects in in vivo experimental models.

Gastrodin induced HO-1 and Nrf2 up-regulation to alleviate H2O2-induced oxidative stress in mouse liver sinusoidal endothelial cells through p38 MAPK phosphorylation

Nuclear factor erythroid-related factor 2 (Nrf2) has been implicated in several detoxifying and antioxidant defense processes. Nrf2-mediated heme oxygenase-1 (HO-1) expression was demonstrated to play a key role against oxidative stress. Gastrodin (GSTD) is a well-known active compound isolated from the roots of Rhizoma gastrodiae, a plant used in ancient Chinese traditional medicine. The aim of this work was to investigate whether GSTD could alleviate H2O2-induced oxidative stress in mouse liver sinusoidal endothelial cells (LSECs). In LSECs exposed to 1 mM H2O2, treatment with GSTD (1, 10, or 50 µM) resulted in higher cell viability than the untreated control. Treated cells maintained a higher Bcl2/Bax ratio and suppressed caspase-9 expression compared with untreated cells, reducing cell apoptosis. GSTD was protective for H2O2-induced oxidative injury by reducing the generation of intracellular reactive oxygen species and malondialdehyde. HO-1 and Nrf2 expressions were synergistically upregulated by GSTD. Inhibition of HO-1 by 10 µM zinc protoporphyrin resulted in less protective effects on cell viability and malondialdehyde reduction by GSTD treatment in H2O2-exposed LSECs. Additionally, phosphorylated p38 in LSECs exposed to H2O2 was elevated by GSTD. Inhibition of p38 phosphorylation by SB203580 did not induce Nrf2 and HO-1 expression after 1 or 10 µM GSTD treatment and the protective effect on cell viability and malondialdehyde reduction in H2O2-exposed LSECs was reduced. The data conclusively demonstrated that GSTD-induced HO-1 and Nrf2 expression is involved in protection of LSECs from H2O2-induced oxidative injury, which may be regulated by p38 phosphorylation.

CHEMICAL CHARACTERIZATION OF A HYPOGLYCEMIC EXTRACT FROM CUCURBITA FICIFOLIA BOUCHE THAT INDUCES LIVER GLYCOGEN ACCUMULATION IN DIABETIC MICE.

BACKGROUND: The aqueous extract of Cucurbita ficifolia (C. ficifolia) fruit has demonstrated hypoglycemic effect, which may be attributed to some components in the extract. However, the major secondary metabolites in this fruit have not yet been identified and little is known about its extra-pancreatic action, in particular, on liver carbohydrate metabolism. Therefore, in addition to the isolation and structural elucidation of the principal components in the aqueous extract of C. ficifolia, the aim of this study was to determine whether or not the hypoglycemic effect of the aqueous extract of Cucurbita ficifolia (C. ficifolia) fruit is due to accumulation of liver glycogen in diabetic mice. MATERIALS AND METHODS: The aqueous extract from fruit of C. ficifolia was fractionated and its main secondary metabolites were purified and chemically characterized (NMR and GC-MS). Alloxan-induced diabetic mice received daily by gavage the aqueous extract (30 days). The liver glycogen content was quantified by spectroscopic method and by PAS stain; ALT and AST by spectrometric method; glycogen synthase, glycogen phosphorylase and GLUT2 by Western blot; the mRNA expression of GLUT2 and glucagon-receptor by RT-PCR; while serum insulin was quantified by ELISA method. A liver histological analysis was also performed by H&E stain. RESULTS: Chemical fingerprint showed five majoritarian compounds in the aqueous extract of C. ficifolia: p-coumaric acid, p-hydroxybenzoic acid, salicin, stigmast-7,2,2-dien-3-ol and stigmast-7-en-3-ol. The histological analysis showed accumulation of liver glycogen. Also, increased glycogen synthase and decreased glycogen phosphorylase were observed. Interestingly, the histological architecture evidenced a liver-protective effect due the extract. CONCLUSION: Five compounds were identified in C. ficifolia aqueous extract. The hypoglycemic effect of this extract may be partially explained by liver glycogen accumulation. The bioactive compound responsible for the hypoglycemic effect of this extract will be elucidated in subsequent studies.

Trypanosoma cruzi cell death induced by the Morita-Baylis-Hillman adduct 3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile).

Chagas disease, caused by the protozoan Trypanosoma cruzi, remains a serious health concern due to the lack of effective vaccines or satisfactory treatment. In the search for new compounds against this neglected disease, we have previously demonstrated that the compound 3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile) (MBHA3), derived from the Morita-Baylis-Hillman reaction, effectively caused a loss of viability in both the epimastigote and trypomastigote forms. However, the mechanisms of parasite death elicited by MBHA3 remain unknown. The aim of this study was to better understand the morphophysiological changes and the mechanism of cell death induced by MBHA3 treatment on T. cruzi. To perform this analysis, we used confocal microscopy and flow cytometry to monitor the fluorescent probes such as annexin-V/propidium iodide (AV/PI), calcein-AM/ethidium homodimer (CA/EH), acridine orange (AO) and rhodamine 123 (Rho 123). Lower concentrations of MBHA3 led to alterations in the mitochondrial membrane potential and AO labeling, but did not decrease the viability of the epimastiogote forms, as determined by the CA/EH and AV/PI assays. Conversely, treatment with higher concentrations of MBHA3 led to extensive plasma membrane damage, loss of mitochondrion membrane potential, DNA fragmentation and acidification of the cytoplasm. Our findings suggest that at higher concentrations, MBHA3 induces T. cruzi epimastigote death by necrosis in a mitochondrion-dependent manner.

Gastrodia elata Blume and its pure compounds protect BV-2 microglial-derived cell lines against ß-amyloid: the involvement of GRP78 and CHOP.

OBJECTIVES: Gastrodia elata (GE) Blume (Orchidaceae) has been previously known for its therapeutic benefits against neurodegenerative diseases. Microglial activation and death have been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease. In this study, GE and its pure components, gastrodin and 4-hydroxybenzyl alcohol (4HBA), were applied to ß-amyloid-induced BV2 mouse microglial cells. MATERIALS AND METHODS: Cell viability was assessed by the MTT assay and Western blotting was also performed. RESULTS: ß-amyloid-induced cell death was shown to be induced time- and dose-dependently. To examine the cell death mechanism, we confirmed the involvement of ER stress signaling. C/EBP homologous protein (CHOP), a pro-apoptotic ER stress protein, was expressed at high levels but glucose-regulated protein 78 (GRP78), an anti-apoptotic ER stress protein with chaperone activity, was only slightly affected by treatment with ß-amyloid. However, pretreatment with GE and its components inhibited the expression of CHOP but increased that of GRP78 in ß-amyloid-treated cells. This study also showed that a single treatment with GE extracts, gastrodin, or 4HBA induced the expression of GRP78, a marker for enhanced protein folding machinery, suggesting a protective mechanism for GE against ß-amyloid. CONCLUSIONS: This study reveals the protective effects of GE against ß-amyloid-induced cell death, possibly through the enhancement of protein folding machinery of a representative protein, GRP78, and the regulation of CHOP in BV2 mouse microglial cells.

Gastrodia elata Blume and its pure compounds protect BV-2 microglial-derived cell lines against β-amyloid: The involvement of GRP78 and CHOP

Objectives: Gastrodia elata (GE) Blume (Orchidaceae) has been previously known for its therapeutic benefits against neurodegenerative diseases. Microglial activation and death have been implicated in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease. In this study, GE and its pure components, gastrodin and 4-hydroxybenzyl alcohol (4HBA), were applied to β-amyloid-induced BV2 mouse microglial cells. Materials and Methods Cell viability was assessed by the MTT assay and Western blotting was also performed. Results: β-amyloid-induced cell death was shown to be induced time- and dose-dependently. To examine the cell death mechanism, we confirmed the involvement of ER stress signaling. C/EBP homologous protein (CHOP), a pro-apoptotic ER stress protein, was expressed at high levels but glucose-regulated protein 78 (GRP78), an anti-apoptotic ER stress protein with chaperone activity, was only slightly affected by treatment with β-amyloid. However, pretreatment with GE and its components inhibited the expression of CHOP but increased that of GRP78 in β-amyloid-treated cells. This study also showed that a single treatment with GE extracts, gastrodin, or 4HBA induced the expression of GRP78, a marker for enhanced protein folding machinery, suggesting a protective mechanism for GE against β-amyloid. Conclusions: This study reveals the protective effects of GE against β-amyloid-induced cell death, possibly through the enhancement of protein folding machinery of a representative protein, GRP78, and the regulation of CHOP in BV2 mouse microglial cells.

3-Hydroxy-2-methylene-3-(4-nitrophenylpropanenitrile): A new highly active compound against epimastigote and trypomastigote form of Trypanosoma cruzi.

We have synthesized the Morita-Baylis-Hillman adduct (MBHA) 3-hydroxy-2-methylene-3-(4-nitrophenyl)-propanenitrile (3) in quantitative yield and evaluated on Trypanosoma cruzi epimastigote and bloodstream trypomastigote forms. Compound 3 strongly inhibited epimastigote growth, with IC(50)/72hof 28.5 microM and also caused intense trypomastigotes lysis, with an IC(50)/24h of 25.5 microM. Ultrastructural analysis showed significant morphological changes on both parasite forms treated with 3, including increase of cell volume and rounding of cell body as well as intense intracellular disorganization. Morphological changes indicative of apoptosis, autophagy or necrosis were observed in most affected cells. Docking calculations of 1, 2 and 3 pointed out the possibility of T. cruzi Farnesyl Pyrophosphate Synthase (TcFPPS) enzyme inhibition in 3 mechanism of action.

Influence of nutrients on enhancing laccase production by Botryosphaeria rhodina MAMB-05.

The physiological requirements needed to enhance the production of laccases by the ascomycete Botryosphaeria rhodina MAMB-05 in submerged cultivation were examined under non-induced and induced (veratryl alcohol, VA) conditions. Under non-induced conditions (-VA), the initial pH, C:N ratio, and inorganic N source did not influence laccase production, in contrast to Tween 80, soybean oil, and copper, which significantly increased laccase production, and proline and urea, which suppressed laccase formation. In addition, Tween 60 could serve as the sole carbon source for the production of these enzymes. Under VA-induced conditions of fungal growth, factors such as inoculum type, time-point of addition of inducer, initial pH, C:N ratio, and type of N source, influenced the production of laccases; however, unlike the non-induced conditions, proline and urea did not act as suppressors. Each of these physiological conditions exerted different effects on biomass production. The nutritional conditions examined for B. rhodina MAMB-05 are discussed in relation to their influence on fungal growth and laccase production.

The effect of carbohydrate carbon sources on the production of constitutive and inducible laccases by Botryosphaeria sp.

The influence of carbohydrates: glucose, fructose, galactose, galacturonic acid, xylose, lactose, sucrose, pectin and inulin, were evaluated as sole carbon source for the production of laccases by the ascomycete, Botryosphaeria sp. Veratryl alcohol, a laccase inducer, was added to culture media to study inducible laccase production on the same carbon sources. Inulinase and pectinase were also produced when Botryosphaeria sp. was grown on inulin, and galacturonic acid and pectin, respectively, and their levels were less in the presence of veratryl alcohol. Botryosphaeria sp. produced constitutive laccases on all carbon sources examined, and veratryl alcohol increased the laccase production on most of carbon sources studied except for inulin and galacturonic acid. Evidence is presented that Botryosphaeria sp. is also pectinolytic.

Kinetic studies on veratryl alcohol transformation by horseradish peroxidase.

A number of peroxidases, such as lignin peroxidase and manganese peroxidase have proved to be useful for industrial applications. Some studies on the effects of temperature and pH stability have been carried out. It is known that veratryl alcohol increases their stability in the range 28-50 degrees C and is oxidized, leading to veratryl aldehyde formation. Similar results with horseradish peroxidase (HRP) in the presence of cofactors were found, but the oxidation of veratryl alcohol in the absence of cofactors was extremely labile at acid pH and inactivated in a few minutes. Considering the growing industrial application of HRP, knowledge of its stability and denaturation kinetics is required. In this study, horseradish peroxidase pool (HRP-VI) and its isoenzymes HRP-VIII (acid) and HRP-IX (basic) have been shown to catalyze the oxidation of veratryl alcohol to veratryl aldehyde in the presence of hydrogen peroxide at pH 5.8 in the 35-45 degrees C range and in the absence of any cofactors. Heat and pH denaturation experiments in the presence and absence of veratryl alcohol incubation were conducted with HRP-VI and HRP-IX isoenzymes. HRP-IX was the most active isoenzyme acting on veratryl alcohol but HRP-VI was the most stable for the temperature range tested. At 35 degrees C the HRP pool presented decay constant (Kd) values of 5.5 x 10(-2) h(-1) and 1.4 10(-2) h(-1) in the absence and presence of veratryl alcohol, respectively, with an effective ratio of 3.9. These results present a new feature of peroxidases that opens one more interesting application of HRP to industrial processes.

Veratryl alcohol stimulates fruiting body formation in the oyster mushroom, Pleurotus ostreatus.

The oyster mushroom, Pleurotus ostreatus, cultivated in solid state on sugarcane bagasse-wheat bran (5:1) medium in the presence of veratryl alcohol resulted in an increased production of the fruiting body at earlier times compared to when the fungus was grown in the absence of veratryl alcohol. The results indicate a new physiological role for veratryl alcohol in stimulating fruiting body formation. Veratryl alcohol also stimulated laccase production during the mycelial growth stage. Evidence is also presented that laccases were involved in the physiological development of the fruiting body.

Mechanism of lysis of Trypanosoma brucei gambiense by human serum.

Resistance to lysis by human serum (HS) is an important parameter used to distinguish Trypanosoma brucei brucei from both Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Neither the exact nature of the trypanolytic factor (TLF) nor the mechanism of action by which HS lyses susceptible trypanosomes is well understood. This report tries to elucidate the role played by the variable surface glycoprotein (VSG) coat and trypanosome surface-related processes in the mechanism of HS lysis of HS-sensitive (HSS) and HS-resistant (HSR) trypanosomes. Procyclic forms of T. brucei gambiense transformed from either HSS or HSR bloodstream stages were found to be HSR. These procyclic forms were shown to have lost their VSG coat. However, the addition of excess soluble VSG from HSS trypanosomes did not block lysis of HSS trypanosomes. Human serum lysis was significantly inhibited if the trypanosomes were incubated with membrane stabilizers, i.e., including cytochalasins (B, D, and E specifically), zinc acetate, vinblastine, and benzyl alcohol, or with the lysosomotropic agents ammonium chloride and chloroquine. The inhibition exerted by these compounds was always reversible. The results in this report, taken together, strengthen the hypothesis that the lytic factor interacts with and moves along the trypanosome surface to be internalized eventually.

Acetylcholine receptor channel properties are modified by benzyl alcohol.

The effect of the general anaesthetic, benzyl alcohol, on the nicotinic cholinergic receptor (AChR) was evaluated at the single channel level using the patch-clamp technique. Benzyl alcohol decreases both the conductance (about 2-fold with 40 mM benzyl alcohol) and the mean open time (about 2.5-fold) of the AChR channels. When modified channels are activated by high ACh concentrations, groups of brief channel openings are observed. Each group is in turn composed of a higher number of openings than in non-treated receptors. Similar modifications are observed when benzyl alcohol is applied from the cytoplasmic side of the membrane, suggesting that the general anaesthetic interacts with a nonspecific site, possibly the lipid-protein interface.

Influences of estrogen and/or progesterone on some dopamine related behavior in rats.

1. The effects of ovariectomy and/or of female hormonal treatments on open-field behavior, apomorphine-induced stereotypies and haloperidol-induced catalepsy were studied in rats. 2. Rat's locomotion frequency was significantly decreased by 17 beta-estradiol and estradiol plus progesterone treatments; this open-field parameter was not affected by progesterone administration per se. 3. 17 beta-estradiol and progesterone treatments, alone or in combination decreased apomorphine-stereotyped behavior. 4. Haloperidol effects were higher in both 17 beta-estradiol and 17 beta-estradiol plus progesterone treated rats. 5. Progesterone treatment alone, decreased the duration of catalepsy induced by the minor (1.0 mg/kg) neuroleptic dose. 6. These results were discussed in the light of a possible interference of estrogen and/or progesterone on dopaminergic transmission at the level of the nigroestratial pathway.

Fluidization of brain membranes by A2C does not produce anesthesia and does not augment muscimol-stimulated 36Cl- influx.

Intravenous administration of 2-[2-methoxyethoxy]-ethyl 8-[cis-2-n-octylcyclopropyl]-octanoate (A2C) was found to disorder brain membranes but did not produce intoxication or anesthesia in mice. The abilities of A2C and an anesthetic (benzyl alcohol) to inhibit [35S]t-butylbicyclophosphorothionate (TBPS) binding, and modify gamma-aminobutyric acid (GABA) receptor-mediated 36Cl- influx into brain vesicles were then compared. Both of the perturbants inhibited [35S]TBPS binding at the same concentrations at which they reduced membrane order; however, the anesthetic was nearly 4 times more effective in reducing [35S]TBPS binding than was A2C. Muscimol-stimulated 36Cl- uptake was enhanced by benzyl alcohol at a concentration which produced little or no change in membrane order. Concentrations of both A2C and benzyl alcohol which reduced membrane order inhibited muscimol-stimulated 36Cl- influx. Similarly, membrane order and muscimol-activated 36Cl- uptake were reduced in brain vesicles prepared from mice which had received A2C in vivo. The effects of anesthetics on the GABAA receptor-chloride channel complex were analyzed by a two site model of action in which a 'perturbant' site is responsible for decreased 36Cl- uptake; but a distinct 'anesthetic' site is responsible for augmentation of chloride flux and anesthesia.