The Anticonvulant Effect of Cooling in Comparison to α-Lipoic Acid: A Neurochemical Study

Brain cooling has pronounced effects on seizures and epileptic activity. The aim of the present study is to evaluate the anticonvulsant effect of brain cooling on the oxidative stress and changes in Na⁺, K⁺-ATPase and acetylcholinesterase (AchE) activities during status epilepticus induced by pilocarpine in the hippocampus of adult male rat in comparison with α-lipoic acid. Rats were divided into four groups: control, rats treated with pilocarpine for induction of status epilepticus, rats treated for 3 consecutive days with α-lipoic acid before pilocarpine and rats subjected to whole body cooling for 30 min before pilocarpine. The present findings indicated that pilocarine-induced status epilepticus was accompanied by a state of oxidative stress as clear from the significant increase in lipid peroxidation (MDA) and superoxide dismutase (SOD) and significant decrease in reduced glutathione and nitric oxide (NO) levels and the activities of catalase, AchE and Na⁺, K⁺-ATPase. Pretreatment with α-lipoic acid ameliorated the state of oxidative stress and restored AchE to nearly control activity. However, Na⁺, K⁺-ATPase activity showed a significant decrease. Rats exposed to cooling for 30 min before the induction of status epilepticus revealed significant increases in MDA and NO levels and SOD activity. AchE returned to control value while the significant decrease in Na⁺, K⁺-ATPase persisted. The present data suggest that cooling may have an anticonvulsant effect which may be mediated by the elevated NO level. However, brain cooling may have drastic unwanted insults such as oxidative stress and the decrease in Na⁺, K⁺-ATPase activity.     

Neuroprotective Effects of Idebenone Against Pilocarpine-Induced Seizures: Modulation of Antioxidant Status, DNA Damage and Na+, K+-ATPase Activity in Rat Hippocampus

The current study investigated the neuroprotective activity of idebenone against pilocarpine-induced seizures and hippocampal injury in rats. Idebenone is a ubiquinone analog with antioxidant, and ATP replenishment effects. It is well tolerated and has low toxicity. Previous studies reported the protective effects of idebenone against neurodegenerative diseases such as Friedreich’s ataxia and Alzheimer’s disease. So far, the efficacy of idebenone in experimental models of seizures has not been tested. To achieve this aim, rats were randomly distributed into six groups. Two groups were treated with either normal saline (0.9 %, i.p., control group) or idebenone (200 mg/kg, i.p., Ideb200 group) for three successive days. Rats of the other four groups (P400, Ideb50 + P400, Ideb100 + P400, and Ideb200 + P400) received either saline or idebenone (50, 100, 200 mg/kg, i.p.) for 3 days, respectively followed by a single dose of pilocarpine (400 mg/kg, i.p.). All rats were observed for 6 h post pilocarpine injection. Latency to the first seizure, and percentages of seizures and survival were recorded. Surviving animals were sacrificed, and the hippocampal tissues were separated and used for the measurement of lipid peroxides, total nitrate/nitrite, glutathione and DNA fragmentation levels, in addition to catalase and Na⁺, K⁺-ATPase activities. Results revealed that in a dose-dependent manner, idebenone (100, 200 mg/kg) prolonged the latency to the first seizure, elevated the percentage of survival and diminished the percentage of pilocapine-induced seizures in rats. Significant increases in lipid peroxides, total nitrate/nitrite, DNA fragmentation levels and catalase activity, in addition to a significant reduction in glutathione level and Na⁺, K⁺-ATPase activity were observed in pilocarpine group. Pre-administration of idebenone (100, 200 mg/kg, i.p.) to pilocarpine-treated rats, significantly reduced lipid peroxides, total nitrate/nitrite, DNA fragmentation levels, and normalized catalase activity. Moreover, idebenone prevented pilocarpine-induced detrimental effects on brain hippocampal glutathione level, and Na⁺, K⁺-ATPase enzyme activity in rats. Data obtained from the current investigation emphasized the critical role of oxidative stress in induction of seizures by pilocarpine and elucidated the prominent neuroprotective and antioxidant activities of idebenone in this model.     

Inhibition of Na(+),K(+)-ATPase activity in hippocampus of rats subjected to acute administration of homocysteine is prevented by vitamins E and C treatment

In the present study we evaluated the effect of acute homocysteine (Hcy) administration on Na⁺,K⁺-ATPase activity, as well as on some parameters of oxidative stress such as total radical-trapping antioxidant potential (TRAP) and on activities of antioxidant enzymes catalase (CAT), superoxide dismutase and glutathione peroxidase in rat hippocampus. Results showed that Hcy significantly decreased TRAP, Na⁺,K⁺-ATPase and CAT activities, without affecting the activities of superoxide dismutase and glutathione peroxidase. We also verified the effect of chronic pretreatment with vitamins E and C on the reduction of TRAP, Na⁺,K⁺-ATPase and CAT activities caused by Hcy. Vitamins E and C per se did not alter these parameters, but prevented the reduction of TRAP, Na⁺,K⁺-ATPase and CAT activities caused by Hcy. Our results indicate that oxidative stress is probably involved in the pathogenesis of homocystinuria and that reduction of Na⁺,K⁺-ATPase activity may be related to the neuronal dysfunction found in homocystinuric patients.     

The Decrease on Na+, K+-ATPase Activity in the Cortex, but not in Hippocampus, is Reverted by Antioxidants in an Animal Model of Sepsis

In the present study, we investigated whether sepsis induced by cecal ligation and puncture (CLP) modifies Na⁺, K⁺-ATPase activity, mRNA expression, and cerebral edema in hippocampus and cerebral cortex of rats and if antioxidant (ATX) treatment prevented the alterations induced by sepsis. Rats were subjected to CLP and were divided into three groups: sham; CLP??rats were subjected to CLP without any further treatment; and ATX?CCLP plus administration of N-acetylcysteine plus deferoxamine. Several times (6, 12, and 24) after CLP or sham operation, the rats were killed and hippocampus and cerebral cortex were isolated. Na⁺, K⁺-ATPase activity was inhibited in the hippocampus 24?h after sepsis, and ATX treatment was not able to prevent this inhibition. The Na⁺, K⁺-ATPase activity also was inhibited in cerebral cortex 6, 12, and 24?h after sepsis. No differences on Na⁺, K⁺-ATPase catalytic subunit mRNA levels were found in the hippocampus and cerebral cortex after sepsis. ATX treatment prevents Na⁺, K⁺-ATPase inhibition only in the cerebral cortex. Na⁺, K⁺-ATPase inhibition was not associated to increase brain water content. In conclusion, the present study demonstrated that sepsis induced by CLP inhibits Na⁺, K⁺-ATPase activity in a mechanism dependent on oxidative stress, but this is not associated to increase brain water content.     

The activity of erythrocyte and brain Na+/K+ and Mg2+-ATPases in rats subjected to acute homocysteine and homocysteine thiolactone administration

Hyperhomocysteinemia is associated with various pathologies including cardiovascular disease, stroke, and cognitive dysfunctions. Systemic administration of homocysteine can trigger seizures in animals, and patients with homocystinuria suffer from epileptic seizures. Available data suggest that homocysteine can be harmful to human cells because of its metabolic conversion to homocysteine thiolactone, a reactive thioester. A number of reports have demonstrated a reduction of Na⁺/K⁺-ATPase activity in cerebral ischemia, epilepsy and neurodegeneration possibly associated with excitotoxic mechanisms. The aim of this study was to examine the in vivo effects of d,l-homocysteine and d,l-homocysteine thiolactone on Na⁺/K⁺- and Mg²⁺-ATPase activities in erythrocyte (RBC), brain cortex, hippocampus, and brain stem of adult male rats. Our results demonstrate a moderate inhibition of rat hippocampal Na⁺/K⁺-ATPase activity by d,l-homocysteine, which however expressed no effect on the activity of this enzyme in the cortex and brain stem. In contrast,d,l-homocysteine thiolactone strongly inhibited Na⁺/K⁺-ATPase activity in cortex, hippocampus and brain stem of rats. RBC Na⁺/K⁺-ATPase and Mg²⁺-ATPase activities were not affected by d,l-homocysteine, while d,l-homocysteine thiolactone inhibited only Na⁺/K⁺-ATPase activity. This study results show that homocysteine thiolactone significantly inhibits Na⁺/K⁺-ATPase activity in the cortex, hippocampus, and brain stem, which may contribute at least in part to the understanding of excitotoxic and convulsive properties of this substance.     

Cross-talk between guanidinoacetate neurotoxicity,memory and possible neuroprotective role of creatine

Guanidinoacetate Methyltransferase deficiency is an inborn error of metabolism that results in decreased creatine and increased guanidinoacetate (GAA) levels. Patients present neurological symptoms whose mechanisms are unclear. We investigated the effects of an intrastriatal administration of 10 μM of GAA (0.02 nmol/striatum) on energy metabolism, redox state, inflammation, glutamate homeostasis, and activities/immunocontents of acetylcholinesterase and Na⁺,K⁺-ATPase, as well as on memory acquisition. The neuroprotective role of creatine was also investigated. Male Wistar rats were pretreated with creatine (50 mg/kg) or saline for 7 days underwenting stereotactic surgery. Forty-eight hours after surgery, the animals (then sixty-days-old) were divided into groups: Control, GAA, GAA + Creatine, and Creatine. Experiments were performed 30 min after intrastriatal infusion. GAA decreased SDH, complexes II and IV activities, and ATP levels, but had no effect on mitochondrial mass/membrane potential. Creatine totally prevented SDH and complex II, and partially prevented COX and ATP alterations. GAA increased dichlorofluorescein levels and decreased superoxide dismutase and catalase activities. Creatine only prevented catalase and dichlorofluorescein alterations. GAA increased cytokines, nitrites levels and acetylcholinesterase activity, but not its immunocontent. Creatine prevented such effects, except nitrite levels. GAA decreased glutamate uptake, but had no effect on the immunocontent of its transporters. GAA decreased Na⁺,K⁺-ATPase activity and increased the immunocontent of its α3 subunit. The performance on the novel object recognition task was also impaired. Creatine partially prevented the changes in glutamate uptake and Na⁺,K⁺-ATPase activity, and completely prevented the memory impairment. This study helps to elucidate the protective effects of creatine against the damage caused by GAA.     

Clomipramine Treatment and Repeated Restraint Stress Alter Parameters of Oxidative Stress in Brain Regions of Male Rats

This study aimed to compare the effects of repeated restraint stress alone and the combination with clomipramine treatment on parameters of oxidative stress in cerebral cortex, striatum and hippocampus of male rats. Animals were divided into control and repeated restraint stress, and subdivided into treated or not with clomipramine. After 40 days of stress and 27 days of clomipramine treatment with 30 mg/kg, the repeated restraint stress alone reduced levels of Na⁺, K⁺-ATPase in all tissues studied. The combination of repeated restraint stress and clomipramine increased the lipid peroxidation, free radicals and CAT activity as well as decreased levels of NP-SH in the tissues studied. However, Na⁺, K⁺-ATPase level decreased in striatum and cerebral cortex and the SOD activity increased in hippocampus and striatum. Results indicated that clomipramine may have deleterious effects on the central nervous system especially when associated with repeated restraint stress and chronically administered in non therapeutic levels.     

Changes in Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Activity and Alpha 3 Subunit Expression in CNS After Administration of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Inhibitors

The expression of Na⁺, K⁺-ATPase α3 subunit and synaptosomal membrane Na⁺, K⁺-ATPase activity were analyzed after administration of ouabain and endobain E, respectively commercial and endogenous Na⁺, K⁺-ATPase inhibitors. Wistar rats received intracerebroventricularly ouabain or endobain E dissolved in saline solution or Tris–HCl, respectively or the vehicles (controls). Two days later, animals were decapitated, cerebral cortex and hippocampus removed and crude and synaptosomal membrane fractions were isolated. Western blot analysis showed that Na⁺, K⁺-ATPase α3 subunit expression increased roughly 40% after administration of 10 or 100 nmoles ouabain in cerebral cortex but remained unaltered in hippocampus. After administration of 10 μl endobain E (1 μl = 28 mg tissue) Na⁺, K⁺-ATPase α3 subunit enhanced 130% in cerebral cortex and 103% in hippocampus. The activity of Na⁺, K⁺-ATPase in cortical synaptosomal membranes diminished or increased after administration of ouabain or endobain E, respectively. It is concluded that Na⁺, K⁺-ATPase inhibitors modify differentially the expression of Na⁺, K⁺-ATPase α3 subunit and enzyme activity, most likely involving compensatory mechanisms.     

Alterations on Na+,K+-ATPase and Acetylcholinesterase Activities Induced by Amyloid-β Peptide in Rat Brain and GM1 Ganglioside Neuroprotective Action

Alzheimer’s disease (AD) is a neurodegenerative disorder whose pathogenesis involves production and aggregation of amyloid-β peptide (Aβ). Aβ-induced toxicity is believed to involve alterations on as Na⁺,K⁺-ATPase and acetylcholinesterase (AChE) activities, prior to neuronal death. Drugs able to prevent or to reverse these biochemical changes promote neuroprotection. GM1 is a ganglioside proposed to have neuroprotective roles in AD models, through mechanisms not yet fully understood. Therefore, this study aimed to investigate the effect of Aβ1-42 infusion and GM1 treatment on recognition memory and on Na⁺,K⁺-ATPase and AChE activities, as well as, on antioxidant defense in the brain cortex and the hippocampus. For these purposes, Wistar rats received i.c.v. infusion of fibrilar Aβ1-42 (2 nmol) and/or GM1 (0.30 mg/kg). Behavioral and biochemical analyses were conducted 1 month after the infusion procedures. Our results showed that GM1 treatment prevented Aβ-induced cognitive deficit, corroborating its neuroprotective function. Aβ impaired Na⁺,K⁺-ATPase and increase AChE activities in hippocampus and cortex, respectively. GM1, in turn, has partially prevented Aβ-induced alteration on Na⁺,K⁺-ATPase, though with no impact on AChE activity. Aβ caused a decrease in antioxidant defense, specifically in hippocampus, an effect that was prevented by GM1 treatment. GM1, both in cortex and hippocampus, was able to increase antioxidant scavenge capacity. Our results suggest that Aβ-triggered cognitive deficit involves region-specific alterations on Na⁺,K⁺-ATPase and AChE activities, and that GM1 neuroprotection involves modulation of Na⁺,K⁺-ATPase, maybe by its antioxidant properties. Although extrapolation from animal findings is difficult, it is conceivable that GM1 could play an important role in AD treatment.     

Inhibition of Na+,K+-ATPase Activity from Rat Hippocampus by Proline

Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the synaptic plasma membranes from hippocampus of rats subjected to chronic and acute proline administration. Na⁺,K⁺-ATPase activity was significantly reduced in chronic and acute treatment by 33% and 40%, respectively. Mg²⁺-ATPase activity was not altered by any treatment. In another set of experiments, synaptic plasma membranes were prepared from hippocampus and incubated with proline or glutamate at final concentrations ranging from 0.2 to 2.0 mM. Na⁺,K⁺-ATPase, but not Mg²⁺-ATPase was inhibited (30%) by the two amino acids. In addition, competition between proline and glutamate for the enzyme activity was observed, suggesting a common binding site for these amino acids. Considering that Na⁺,K⁺-ATPase activity is critical for normal brain function, the results of the present study showing a marked inhibition of this enzyme by proline may be associated with the neurological dysfunction found in patients affected by type II hyperprolinemia.     

Inhibition of Rat Brain Na+, K+-ATPase Activity Induced by Homocysteine Is Probably Mediated by Oxidative Stress

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na⁺, K⁺-ATPase activity, whereas methionine had no effect. Mg²⁺-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na⁺, K⁺-ATPase activity. Tested compounds did not alter Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na⁺, K⁺-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.     

A missense mutation converts the Na+,K+-ATPase into an ion channel and causes therapy-resistant epilepsy

The ion pump Na⁺,K⁺-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na⁺,K⁺-ATPase catalytic α1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg²⁺. We found that membrane expression of the mutant α1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na⁺,K⁺-ATPase transport demonstrated massive water inflow into mutant α1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant α1 detected a nonspecific cation current. Finally, neurons expressing mutant α1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na⁺,K⁺-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.     

The chlorpromazine inhibition of transport ATPase and acetylcholinesterase activities in the microsomal membranes of rat in vitro and in vivo

Chlorpromazine, an antipsychotic drug, is found to inhibit Na⁺,K⁺-ATPase activity in rat brain microsomal membranes in vitro in concentration and time dependent manner but some inconsistency is observed when the effect was studied with respect to different temperatures. Various ligands and/or substrate affect the inhibition by chlorpromazine in different ways. The in vivo study with this drug shows that the activities of Na⁺,K⁺-ATPase, Ca^–2-ATPase and acetylcholinesterase in the microsomal membranes of different organs are inhibit with increases in concentration or lengths of time of treatment and then levels off.     

Physiological and biochemical response of Aedes aegypti tolerance to Bacillus thuringiensis

Bacillus thuringiensis subsp. israelensis (Bti) represents the only eco-friendly bio-degradable insecticide for mosquito-borne disease control. Our research aims to identify if mosquito detoxification enzymes play an important role in Bti tolerance mechanisms in the dengue vector, Aedes aegypti. Several enzymes, such as amylase, cytochromes P450, Na⁺/K⁺-ATPase, acetylcholinesterase, protease and glutathione S-transferase (GST) were analysed and level of activity determined in Ae. aegypti larvae after Bti treatment. Bti exposure significantly increased the level of amylase (183.2%) as well as the activity of cytochromes P450 (177.5%), and Na⁺/K⁺-ATPase (142.9%). On the other hand, there was a decrease of 8.6% and 11.4% in acetylcholinesterase and GST activity, and no significant effect in the total level of protease activity. We suggest that the variation in amylase, cytochromes P450, Na⁺/K⁺-ATPase, acetylcholinesterase, protease and GST activity may be associated with the Bti insecticidal mechanism. This study provides the basis of detoxifying enzymes in Bti tolerance.     

Reduction of hippocampal Na+, K+-ATPase activity in rats subjected to an experimental model of depression

The effect of a model of depression using female rats on Na⁺, K⁺-ATPase activity in hippocampal synaptic plasma membranes was studied. In addition, the effect of further chronic treatment with fluoxetine on this enzyme activity was verified. Sweet food consumption was measured to evaluate the efficacy of this model in inducing a state of reduced response to rewarding stimili. After 40 days of mild stress, a reduction in sweet food ingestion was observed. Reduction of hippocampal Na⁺, K⁺-ATPase activity was also observed. Treatment with fluoxetine increased this enzyme activity and reversed the effect of stress. Chronic fluoxetine decreased the ingestion of sweet food in both groups. This result is in agreement with suggestions that reduction of Na⁺, K⁺-ATPase activity is a caracteristic of depressive disorders. Fluoxetine reversed this effect. Therefore it is possible that altered Na⁺, K⁺-ATPase activity may be involved in the pathophysiology of depression in patients.     

Long lasting effects of intrauterine growth retardation on basal and 5-HT-stimulated Na+/K+-ATPase in the brain of developing rats

Intrauterine growth retardation induced by ligation of the uterine vessels in pregnant rats on the 5th day before delivery was associated with brain and body weights of hypotrophic offspring significantly lower than those of pair-aged control rats, even after 6 weeks of postnatal rearing under normal conditions. In vitro measurements in homogenates indicated that Na⁺/K⁺-ATPase in the forebrain, cerebellum and hippocampus was less active in hypotrophic rats than in pair-aged controls for at least the first month after birth. However, 5-HT and related agonists (RU-24969, bufotenine, and to a lower extent, tryptamine) stimulated Na⁺/K⁺-ATPase activity more efficiently in tissues from hypotrophic rats than in those from control animals. Opposite changes were noted in the brain stem: basal Na⁺/K⁺-ATPase activity was higher in hypotrophic rats during the second half of the first postnatal month but the stimulatory effect of 5-HT was lower than in pair-aged control animals. Since potent 5-HT antagonists such as cinanserin, methiothepin and methysergide, prevented the 5-HT induced-activation of Na⁺/K⁺-ATPase in brain homogenates, these results are discussed in relation with the possible existence of a specific 5-HT receptor controlling Na⁺/K⁺-ATPase activity in the rat brain.     

Reduction of Na(+),K(+)-ATPase activity in hippocampus of rats subjected to chemically induced hyperhomocysteinemia

Hyperhomocysteinemia occurs in homocystinuria, an inherited metabolic disease clinically characterized by thromboembolic episodes and a variable degree of neurological dysfunction whose pathophysiology is poorly known. In this study, we induced elevated levels of homocysteine (Hcy) in blood (500 M), comparable to those of human homocystinuria, and in brain (60 nmol/g wet tissue) of young rats by injecting subcutaneously homocysteine (0.3-0.6 mol/g of body weight) twice a day at 8-hr intervals from the 6th to the 28th postpartum day. Controls received saline in the same volumes. Na⁺,K⁺-ATPase and Mg²⁺-ATPase activities were determined in the hippocampus of treated Hcy- and saline-treated rats. Chronic administration of Hcy significantly decreased (40%) Na⁺,K⁺-ATPase activity but did not alter Mg²⁺-ATPase activity. Considering that Na⁺,K⁺-ATPase plays a crucial role in the central nervous system, our results suggest that the brain dysfunction found in homocystinuria may be related to the reduction of brain Na⁺,K⁺-ATPase activity.     

Na+, K+ ATPase Activity Is Reduced in Amygdala of Rats with Chronic Stress-Induced Anxiety-Like Behavior

In this study, we examined the effects of two chronic stress regimens upon anxiety-like behavior, Na⁺, K⁺-ATPase activity and immunocontent, and oxidative stress parameters (antioxidant enzymes and reactive oxygen species production) in the amygdala. Male rats were subjected to chronic unpredictable and to chronic restraint stress for 40 days. Subsequently, anxiety-like behavior was examined. Both stressed groups presented increased anxiety-like behavior. Reduced amygdalal Na⁺, K⁺-ATPase activity in the synaptic plasma membranes was also observed, without alterations in the amygdala immunocontent. In addition, when analyzing oxidative stress parameters, only superoxide dismutase activity was decreased in the amygdala of animals subjected to unpredictable stress. We conclude that both models of chronic stress lead to anxiety-like behavior and decreased amygdalal Na⁺, K⁺-ATPase activity, which appears not to be related to oxidative imbalance. The relationship between this decreased activity and anxiety-like behavior remains to be studied.     

Physicochemical approaches to the alcohol-membrane interaction in brain

The effects of ethanol on physicochemical and enzymatic perturbations of neuronal membranes were examined. Using synaptic plasma membrane (SPM) isolated from cerebral cortex of Sprague-Dawley rats, a biphasic mode of action for ethanol was observed with (Na⁺+K⁺)-ATPase, but not with Ca²⁺-ATPase or acetylcholinesterase. (Na⁺+K⁺)-ATPase was found to be more sensitive to low concentration of sodium deoxycholate treatment than Ca²⁺-ATPase. A sharp transition break of (Na⁺+K⁺)-ATPase activity in response to temperature changes was found with SPM preparation. Arrhenius plots of the response also indicated that (Na⁺+K⁺)-ATPase is more sensitive to temperature changes than Ca²⁺-ATPase. The fluorescence polarization of TNS-membrane complex decreases as ethanol concentration increases, indicating an increase in membrane fluidity. However, ethanol, at low concentration (<0.3%) appears to elevate TNS fluorescence, but a hhigher concentration (3%) ethanol tends to lower the intensity of maximal emission. The results of this study indicate that ethanol may interact with the synaptic plasma membranes and elicit specific biochemical responses depending on the concentration of the alcohol used.     

Disruption of Ion-Trafficking System in the Cochlear Spiral Ligament Prior to Permanent Hearing Loss Induced by Exposure to Intense Noise: Possible Involvement of 4-Hydroxy-2-Nonenal as a Mediator of Oxidative Stress

Noise-induced hearing loss is at least in part due to disruption of endocochlear potential, which is maintained by various K⁺ transport apparatuses including Na⁺, K⁺-ATPase and gap junction-mediated intercellular communication in the lateral wall structures. In this study, we examined the changes in the ion-trafficking-related proteins in the spiral ligament fibrocytes (SLFs) following in vivo acoustic overstimulation or in vitro exposure of cultured SLFs to 4-hydroxy-2-nonenal, which is a mediator of oxidative stress. Connexin (Cx)26 and Cx30 were ubiquitously expressed throughout the spiral ligament, whereas Na⁺, K⁺-ATPase α1 was predominantly detected in the stria vascularis and spiral prominence (type 2 SLFs). One-hour exposure of mice to 8 kHz octave band noise at a 110 dB sound pressure level produced an immediate and prolonged decrease in the Cx26 expression level and in Na⁺, K⁺-ATPase activity, as well as a delayed decrease in Cx30 expression in the SLFs. The noise-induced hearing loss and decrease in the Cx26 protein level and Na⁺, K⁺-ATPase activity were abolished by a systemic treatment with a free radical-scavenging agent, 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl, or with a nitric oxide synthase inhibitor, N^ω-nitro-L-arginine methyl ester hydrochloride. In vitro exposure of SLFs in primary culture to 4-hydroxy-2-nonenal produced a decrease in the protein levels of Cx26 and Na⁺, K⁺-ATPase α1, as well as Na⁺, K⁺-ATPase activity, and also resulted in dysfunction of the intercellular communication between the SLFs. Taken together, our data suggest that disruption of the ion-trafficking system in the cochlear SLFs is caused by the decrease in Cxs level and Na⁺, K⁺-ATPase activity, and at least in part involved in permanent hearing loss induced by intense noise. Oxidative stress-mediated products might contribute to the decrease in Cxs content and Na⁺, K⁺-ATPase activity in the cochlear lateral wall structures.