Effect of a purine derivative containing selenium to improve memory decline and anxiety through modulation of the cholinergic system and Na+/K+-ATPase in an Alzheimer’s disease model

Alzheimer’s disease (AD) is a worldwide problem, and there are currently no treatments that can stop this disease. To investigate the binding affinity of 6-((4-fluorophenyl) selanyl)-9H-purine (FSP) with acetylcholinesterase (AChE), to verify the effects of FSP in an AD model in mice and to evaluate the toxicological potential of this compound in mice. The binding affinity of FSP with AChE was investigated by molecular docking analyses. The AD model was induced by streptozotocin (STZ) in Swiss mice after FSP treatment (1 mg/kg, intragastrically (i.g.)), 1st-10th day of the experimental protocol. Anxiety was evaluated in an elevated plus maze test, and memory impairment was evaluated in the Y-maze, object recognition and step-down inhibitory avoidance tasks. The cholinergic system was investigated based on by looking at expression and activity of AChE and expression of choline acetyltransferase (ChAT). We evaluated expression and activity of Na⁺/K⁺-ATPase. For toxicological analysis, animals received FSP (300 mg/kg, i.g.) and aspartate aminotransferase, alanine aminotransferase activities were determined in plasma and δ-aminolevulinate dehydratase activity in brain and liver. FSP interacts with residues of the AChE active site. FSP mitigated the induction of anxiety and memory impairment caused by STZ. FSP protected cholinergic system dysfunction and reduction of activity and expression of Na⁺/K⁺-ATPase. FSP did not modify toxicological parameters evaluated and did not cause the death of mice. FSP protected against anxiety, learning and memory impairment with involvement of the cholinergic system and Na⁺/K⁺-ATPase in these actions.

     

Supplementation with vitamins E plus C or soy isoflavones in ovariectomized rats: effect on the activities of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase and cholinesterases

Since a previous study demonstrated that ovariectomized rats present an activation of Na⁺, K⁺-ATPase and acetylcholinesterase (AChE) activities, in the present study we investigated the influence of vitamins E plus C or soy isoflavones on the effects elicited by ovariectomy on the activities of these enzyme in hippocampus of ovariectomized rats. We also determined the effect of the same compounds on the reduction of serum butyrylcholinesterase (BuChE) activity caused by ovariectomy. Female adult Wistar rats were assigned to one of the following groups: sham (submitted to surgery without removal of the ovaries) and ovariectomized. Seven days after surgery, animals were treated for 30 days with a single daily intraperitoneous injection of vitamins E (40 mg/kg) plus C (100 mg/kg) or saline (control). In another set of experiments, the rats were fed for 30 days on a special diet with soy protein or a standard diet with casein (control). Rats were sacrificed after treatments and the hippocampus was dissected and serum was separated. Data demonstrate that vitamins E plus C reversed the activation of Na⁺, K⁺-ATPase and AChE in hippocampus of ovariectomized rats. Conversely, soy protein supplementation reversed the increase of AChE activity, but not of Na⁺, K⁺-ATPase activity, caused by ovariectomized group. Neither treatment was able to reverse the reduction of serum BuChE activity. Furthermore, treatments with vitamins E plus C or soy were unable to reverse the decrease in estradiol levels caused by ovariectomy. Our findings show that the treatment with vitamins E plus C significantly reversed the effect of ovariectomy on hippocampal Na⁺, K⁺-ATPase and AChE activities. However, a soy diet that was rich in isoflavones was able to reverse just the increase of AChE. Neither treatment altered the reduction in serum BuChE activity. Taken together, these vitamins and soy may have a protective role against the possible brain dysfunction observed in some menopause women. Vitamins E plus C and soy isoflavones may be a good alternative as a novel therapeutic strategy.     

Effects of adult-onset streptozotocin-induced diabetes on the rat brain antioxidant status and the activities of acetylcholinesterase, (Na+,K+)- and Mg2+-ATPase: modulation by L-cysteine

Uncontrolled diabetes is known to affect the nervous system. The aim of this study was to investigate the effect of the antioxidant L-cysteine (Cys) on the changes caused by adult-onset streptozotocin (STZ)-induced diabetes on the rat brain total antioxidant status (TAS) and the activities of acetylcholinesterase (AChE), (Na⁺,K⁺)-ATPase and Mg²⁺-ATPase. Thirty-eight male Wistar rats were divided into six groups: C_A (8-week-control), C_B (8-week-control + 1-week-saline-treated), C + Cys (8-week-control + 1-week-Cys-treated), D_A (8-week-diabetic), D_B (8-week-diabetic + 1-week-saline-treated) and D + Cys (8-week-diabetic + 1-week-Cys-treated). All diabetic rats were once treated with an intraperitoneal (i.p.) STZ injection (50 mg/kg body weight) at the beginning of the experiment, while all Cys-treated groups received i.p. injections of Cys 7 mg/kg body weight (daily, for 1-week, during the 9th-week). Whole rat brain parameters were measured spectrophotometrically. In vitro incubation with 0.83 mM of Cys or 10 mM of STZ for 3 h was performed on brain homogenate samples from groups C_B and D_B, in order to study the enzymes’ activities. Diabetic rats exhibited a statistically significant reduction in brain TAS (−28%, D_A vs C_A;−30%, D_B vs C_B) that was reversed after 1-week-Cys-administration into basal levels. Diabetes caused a significant increase in AChE activity (+27%, D_A vs C_A; +15%, D_B vs C_B), that was further enhanced by Cys-administration (+57%, D + Cys vs C_B). The C + Cys group exhibited no significant difference compared to the C_B group in TAS (+2%), but showed a significantly increased AChE activity (+66%, C + Cys vs C_B). Diabetic rats exhibited a significant reduction in the activity of Na⁺,K⁺-ATPase (−36%, D_A vs C_A;−48%, D_B vs C_B) that was not reversed after 1-week Cys administration. However, in vitro incubation with Cys partially reversed the diabetes-induced Na⁺,K⁺-ATPase inhibition. Mg²⁺-ATPase activity was not affected by STZ-induced diabetes, while Cys caused a significant inhibition of the enzyme, both in vivo (−14%, C + Cys vs C_B;−17%, D + Cys vs C_B) and in vitro (−16%, D_B + in vitro Cys vs C_B). In vitro incubation with STZ had no effect on the studied enzymes. The present data revealed a protective role for Cys towards the oxidative effect of diabetes on the adult rat brain. Moreover, an increase in whole brain AChE activity due to diabetes was recorded (not repeatedly established in the literature, since contradictory findings exist), that was further increased by Cys. The inhibition of Na⁺,K⁺-ATPase reflects a possible mechanism through which untreated diabetes could affect neuronal excitability, metabolic energy production and certain systems of neurotransmission. As concerns the use of Cys as a neuroprotective agent against diabetes, our in vitro findings could be indicative of a possible protective role of Cys under different in vivo experimental conditions.     

Experimentally-induced Wernicke’s encephalopathy modifies crucial rat brain parameters: the importance of Na+,K+-ATPase and a potentially neuroprotective role for antioxidant supplementation

Wernicke’s encephalopathy (WE) is a serious neuropsychiatric syndrome caused by chronic alcoholism and thiamine (T) deficiency. Our aim was to shed more light on the pathophysiology of WE, by introducing a modified in vivo experimental model of WE and by focusing on changes provoked in the total antioxidant status (TAS) and three crucial brain enzyme activities in adult rats. Rats were placed on ethanol (EtOH) consumption (20 %?v/v) for a total of 5 weeks. By the end of the third week, rats were fed a T-deficient diet (TDD) and were treated with pyrithiamine (PT; 0.25 mg/kg) for the remaining 2 weeks. Following the induction of WE symptomatology, rats were treated with three consecutive (every 8 h) injections of saline or T (100 mg/kg) and were sacrificed. Brain homogenates were generated and used for spectrophotometrical evaluation of TAS and enzymatic activities. Additionally, in vitro experiments were conducted on brain homogenates or pure enzymes incubated with T or neuromodulatory antioxidants. Pre-exposure to EtOH provided a successful protocol modification that did not affect the expected time of WE symptomatology onset. Administration of T ameliorated this symptomatology. WE provoked oxidative stress that was partially limited by T administration, while T itself also caused oxidative stress to a smaller extent. Brain acetylcholinesterase (AChE) was found inhibited by WE and was further inhibited by T administration. In vitro experiments demonstrated a potential neuroprotective role for L-carnitine (Carn). Brain sodium-potassium adenosine triphosphatase (Na⁺,K⁺-ATPase) activity was found increased in WE and was reduced to control levels by in vivo T administration; this increase was also evident in groups exposed to PT or to TDD, but not to EtOH. In vitro experiments demonstrated a potential neuroprotective role for this Na⁺,K⁺-ATPase stimulation through T or L-cysteine (Cys) administration. Brain magnesium adenosine triphosphatase (Mg²⁺-ATPase) activity was found decreased by prolonged exposure to EtOH, but was not affected by the experimental induction of WE. Our data suggest that T administration inhibits AChE, which is also found inhibited in WE. Moreover, increased brain Na⁺,K⁺-ATPase activity could be a marker of T deficiency in WE, while combined T and antioxidant co-supplementation of Cys and/or Carn could be neuroprotective in terms of restoring the examined crucial brain enzyme activities to control levels.     

Combined thirty-day exposure to thioacetamide and choline-deprivation alters serum antioxidant status and crucial brain enzyme activities in adult rats

Choline (Ch) is an essential nutrient that seems to be involved in a wide variety of metabolic reactions and functions that affect the nervous system, while thioacetamide (TAA) is a well-known hepatotoxic agent. The induction of prolonged Ch-deprivation (CD) in rats receiving TAA (through the drinking water) provides an experimental model of mild progressive hepatotoxicity that could simulate commonly-presented cases in clinical practice. In this respect, the aim of this study was to investigate the effects of a 30-day dietary CD and/or TAA administration (300 mg/L of drinking water) on the serum total antioxidant status (TAS) and the activities of brain acetylcholinesterase (AChE), Na⁺,K⁺-ATPase and Mg²⁺-ATPase of adult rats. Twenty male Wistar rats were divided into four groups: A (control), B (CD), C (TAA), D (CD+TAA). Dietary CD was provoked through the administration of Ch-deficient diet. Rats were sacrificed by decapitation at the end of the 30-day experimental period and whole brain enzymes were determined spectrophotometrically. Serum TAS was found significantly lowered by CD (−11% vs Control, p < 0.01) and CD+TAA administration (−19% vs Control, p < 0.001), but was not significantly altered due to TAA administration. The rat brain AChE activity was found significantly increased by TAA administration (+11% vs Control, p < 0.01), as well as by CD+TAA administration (+14% vs Control, p < 0.01). However, AChE was not found to be significantly altered by the 30-day dietary CD. On the other hand, CD caused a significant increase in brain Na⁺,K⁺-ATPase activity (+16% vs Control, p < 0.05) and had no significant effect on Mg²⁺-ATPase. Exposure to TAA had no significant effect on Na⁺,K⁺-ATPase, but inhibited Mg²⁺-ATPase (−20% vs Control, p < 0.05). When administered to CD rats, TAA caused a significant decrease in Na⁺,K⁺-ATPase activity (−41% vs Control, p < 0.001), but Mg²⁺-ATPase activity was maintained into control levels. Our data revealed that an adult-onset 30-day dietary-induced CD had no effect on AChE activity. Treatment with TAA not only reversed the stimulatory effect of CD on adult rat brain Na⁺,K⁺-ATPase, but caused a dramatic decrease in its activity (−41%). Previous studies have linked this inhibition with metabolic phenomena related to TAA-induced fulminant hepatic failure and encephalopathy. Our data suggest that CD (at least under the examined 30-day period) is an unfavorable background for the effect of TAA-induced hepatic damage on Na⁺,K⁺-ATPase activity (an enzyme involved in neuronal excitability, metabolic energy production and neurotransmission).     

Effects of hyper- and hypothyroidism on acetylcholinesterase, (Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>)- and Mg <Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-ATPase activities of adult rat hypothalamus and cerebellum

Thyroid hormones (THs) are recognized as key metabolic hormones, and the metabolic rate increases in hyperthyroidism, while it decreases in hypothyroidism. The aim of this work was to investigate how changes in metabolism induced by THs could affect the activities of acetylcholinesterase (AChE), (Na⁺, K⁺)- and Mg²⁺-ATPase in the hypothalamus and the cerebellum of adult rats. Hyperthyroidism was induced by subcutaneous administration of thyroxine (25μg/100 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. All enzyme activities were evaluated spectrophotometrically in the homogenated brain regions of 10 three-animal pools. Neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE (−23%, p < 0.001) and the Na⁺, K⁺-ATPase activities (−26%, p < 0.001). Moreover, hypothyroidism had a similar effect on the examined enzyme activities: AChE (−17%, p < 0.001) and Na⁺, K⁺-ATPase (−27%, p < 0.001). Mg²⁺-ATPase activity was found unaltered in both the hyper- and the hypothyroid brain regions. In conclusion: neither hyper-, nor hypothyroidism had any effect on the examined hypothalamic enzyme activities. In the cerebellum, hyperthyroidism provoked a significant decrease in both the AChE and the Na⁺, K⁺-ATPase activities. The decreased (by the THs) Na⁺, K⁺-ATPase activities may increase the synaptic acetylcholine release, and thus, could result in a decrease in the cerebellar AChE activity. Moreover, the above TH-induced changes may affect the monoamine neurotransmitter systems.     

Changes in Antioxidant Status,Protein Concentration,Acetylcholinesterase, (Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>)-, and Mg<Superscript>2+</Superscript>-ATPase Activities in the Brain of Hyper- and Hypothyroid Adult Rats

It is a common knowledge that metabolic reactions increase in hyperthyroidism and decrease in hypothyroidism. The aim of this work was to investigate how the metabolic reactions could affect the total antioxidant status (TAS), protein concentration (PC) and the activities of acetylcholinesterase (AChE), (Na⁺,K⁺)-ATPase and Mg²⁺-ATPase in the brain of hyper- and hypothyroid adult male rats. Hyperthyroidism was induced in rats by subcutaneous administration of thyroxine (25 g/100 g body weight) once daily for 14 days, while hypothyroidism was induced by oral administration of propylthiouracil (0.05%) for 21 days. TAS, PC, and enzyme activities were evaluated spectrophotometrically in the homogenated brain of each animal. TAS, PC, and Mg²⁺-ATPase activity were found unaffected in hyperthyroidism, while AChE and Na⁺,K⁺-ATPase activities were reduced by 25% (p < 0.01). In contrast, TAS, (Na⁺,K⁺)-ATPase and Mg²⁺-ATPase activities were found to be increased (approx. 23–30%, p < 0.001) in the hypothyroid brain, while AChE activity and PC were shown to be inhibited (approx. 23–30%, p < 0.001). These changes on brain enzyme activities may reflect the different metabolic effects of hyper- and hypothyroidism. Such changes of the enzyme activities may differentially modulate the brain intracellular Mg²⁺, neural excitability, as well as the uptake and release of biogenic amines.     

Meloxicam-loaded nanocapsules as an alternative to improve memory decline in an Alzheimer’s disease model in mice: involvement of Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase

The objective of this study was to investigate the effect of meloxicam-loaded nanocapsules (M-NC) on the treatment of the memory impairment induced by amyloid β-peptide (aβ) in mice. The involvement of Na⁺, K⁺-ATPase and cyclooxygenase-2 (COX-2) activities in the hippocampus and cerebral cortex was also evaluated. Mice received aβ (3 nmol/ 3 μl/ per site, intracerebroventricular) or vehicle (3 μl/ per site, i.c.v.). The next day, the animals were treated with blank nanocapsules (17 mL/kg) or M-NC (5 mg/kg) or free meloxicam (M-F) (5 mg/kg). Treatments were performed every other day, until the twelfth day. Animals were submitted to the behavioral tasks (open-field, object recognition, Y-maze and step-down inhibitory avoidance tasks) from the twelfth day. Na⁺, K⁺-ATPase and COX-2 activities were performed in hippocampus and cerebral cortex. aβ caused a memory deficit, an inhibition of the hippocampal Na⁺, K⁺-ATPase activity and an increase in the hippocampal COX-2 activity. M-NC were effective against all behavioral and biochemical alterations, while M-F restored only the COX-2 activity. In conclusion, M-NC were able to reverse the memory impairment induced by aβ, and Na⁺, K⁺-ATPase is involved in the effect of M-NC.     

Neuro-pharmacological evaluation of anticonvulsant and neuroprotective activity of Cocculus laurifolius leaves in wistar rats

The aim of the study was to evaluate the anticonvulsant and neuroprotective activity of Cocculus laurifolius D.C leaves in albino wistar rats against strychnine induced convulsions. Initially the extract was investigated for acute oral toxicity testing in order to examine any signs of toxicity and mortality. For anticonvulsant activity, the ethanolic extract was evaluated at doses 200 and 400 mg/kg, p.o. against strychnine induced convulsions model, at 1, 7, 15 and 30th day of treatment. Meanwhile, the neuroprotective effect of the extract was investigated via histopathological assessment. Cocculus laurifolius (200 and 400 mg/kg, p.o.) exhibited anticonvulsant activity as indicated by significant delay in the onset of convulsions and time to death after strychnine induced convulsions. Similarly, significant reduction in the duration of convulsions and percentage of mortality was observed by ethanolic extract (200 and 400 mg/kg p.o.) at 1, 7, 15 and 30th day of test sessions. Furthermore, Cocculus laurifolius leaves (200 and 400 mg/kg p.o.) also exhibited neuroprotective effect with considerable preserved neuronal structures and significant decrease in neuronal apoptosis, in comparison with control. The results obtained from the present study indicate that ethanolic extract of Cocculus laurifolius leaves possess potential anticonvulsant and neuroprotective effect against strychnine induced convulsions. Therefore, it can be concluded that Cocculus laurifolius leaves may be a valuable in management of epilepsy, however further studies are required on large number of animals to confirm these findings.

     

Regulation of Renal Ouabain-Resistant Na+-ATPase by Leptin,Nitric Oxide,Reactive Oxygen Species,and Cyclic Nucleotides: Implications for Obesity-Associated Hypertension

This study examined the effect of leptin on renal ouabain-resistant Na⁺-ATPase, which drives the reabsorption of about 10% of sodium transported in the proximal tubule. Chronic leptin administration (0.25 mg/kg s.c. twice daily for seven days) increased Na⁺-ATPase activity by 62.9%. This effect was prevented by the coadministration of superoxide dismutase mimetic, tempol, or the NADPH oxidase inhibitor, apocynin (2 mM in the drinking water). Acutely administered NO donors decreased Na⁺-ATPase activity. This effect was abolished by soluble guanylate cyclase inhibitor, ODQ, but not by protein kinase G inhibitors. Exogenous cGMP reduced Na⁺-ATPase activity, but its synthetic analogues, 8-bromo-cGMP and 8-pCPT-cGMP, were ineffective. The inhibitory effect of NO donors and cGMP was abolished by EHNA, an inhibitor of cGMP‐stimulated phosphodiesterase (PDE2). Exogenous cAMP analogue and dibutyryl-cAMP increased Na⁺-ATPase activity and abolished the inhibitory effect of cGMP. Finally, the administration of superoxide-generating mixture (xanthine oxidase+hypoxanthine) increased Na⁺-ATPase activity. The results suggest that nitric oxide decreases renal Na⁺-ATPase activity by stimulating cGMP, which in turn activates PDE2 and decreases cAMP concentration. Increased production of reactive oxygen species may lead to the elevation of Na⁺-ATPase activity by scavenging NO and limiting its inhibitory effect. Chronic hyperleptinemia is associated with increased Na⁺-ATPase activity due to excessive oxidative stress.     

Sensitivity to ketamine, alone or in combination with ethanol, is altered in mice selectively bred for sensitivity to ethanol's locomotor effects

BACKGROUND: Sensitivity to erthanol's locomotor activating and reinforcing effects may be influenced by some common neural mechanisms. Mice selectively bred in replicate for increased (FAST-1 and FAST-2) and decreased (SLOW-1 and SLOW-2) sensitivity to ethanol's locomotor stimulant effects are useful for investigating the neural substrates of ethanol's effects. Previous studies have suggested that differences in N-methyl-d-aspartate (NMDA) receptors may underlie differences in ethanol-induced locomotion in these mice. This study examined the responses of FAST and SLOW mice to ketamine, a fast-acting NMDA antagonist. In addition, reverse-selected lines (r-FAST-1, r-FAST-2, r-SLOW-1, and r-SLOW-2) were tested as a means of verifying correlations detected in the forward-selected lines. Two initial studies characterized ketamine-induced locomotion in DBA/2J (D2) mice, an inbred strain chosen for its high sensitivity to ethanol-induced locomotion. METHODS: After a 2- to 3-day period of habituation to test procedures, mice were given intraperitoneal injections of ketamine alone (0, 5, 10, 20, 30, and 60 mg/kg) or in combination with 1 or 2 g/kg ethanol. Locomotor activity was measured for 20 to 30 min in automated activity monitors. RESULTS: When administered alone, ketamine dose-dependently stimulated the locomotor activity of D2 mice and also reduced the amount of ethanol-induced stimulation. Ketamine stimulated locomotion more in FAST mice than in SLOW mice. Reverse selection abolished these differences, because r-FAST and r-SLOW mice did not differ in their responses to ketamine. Ketamine potentiated ethanol's locomotor effects within FAST mice and potentiated ethanol's locomotor depressant effect within one replicate of SLOW mice. CONCLUSIONS: We propose that sensitivities to ethanol- and ketamine-induced locomotion are genetically correlated and that the combined effects of ethanol and ketamine in FAST mice reflect a leftward shift in ethanol's biphasic dose-response curve.     

Effects of Ranitidine on Gastric Vesicles Containing H+, K+-Adenosine Triphosphatase in Rats

Background: To ascertain the mechanism for rebound acid hypersecretion after treatment with an H₂-receptor blocker, we investigated the effects of ranitidine on gastric H⁺, K⁺-adenosine triphosphatase (ATPase) in rats, Methods: Male Wistar rats received ranitidine (1-50mg/kg body weight intraperito-neally twice a day for 5 days). The rats were starved for 15 h after the last treatment and then killed, and gastric vesicles containing H⁺, K⁺-ATPase were prepared. Results: Treatment with ranitidine dose-dependently increased protein content in the gastric vesicular fraction purified from the gastric mucosa without changing total protein content. Ranitidine also increased the content of a 94,000-dalton protein, the catalytic subunit of H⁺, K⁺-ATPase. On the other hand, ranitidine did not affect the specific activity of the enzyme (μmol/min/mg of the gastric vesicular protein). Since gastric vesicles in the fasting state mainly consist of the tubulovesicular membrane, these results suggest that ranitidine administration increases total tubulovesicular H⁺, K⁺-ATPase content (μmol/min/rat) by increasing the number of tubulovesicles per parietal cell. The ranitidine-induced increase in total tubulovesicular H⁺, K⁺-ATPase activity was still evident 1 week after treatment and returned to control level 1 month later. Conclusions: All these findings suggest that the increased content and total activity of tubulovesicular H⁺, K⁺-ATPase after ranitidine treatment may contribute to the mechanism for acid rebound after H₂-blocker therapy.     

Protein kinase C effects on nerve function, perfusion, Na+,K+-ATPase activity and glutathione content in diabetic rats

Abstract Aims//hypothesis. Increased protein kinase C activity has been linked to diabetic vascular complications in the retina and kidney, which were attenuated by protein kinase C antagonist treatment. Neuropathy has a vascular component, therefore, the aim was to assess whether treatment with WAY151 003 or chelerythrine, inhibitors of protein kinase C regulatory and catalytic domains respectively, could correct nerve blood flow, conduction velocity, Na⁺,K⁺-ATPase, and glutathione deficits in diabetic rats. Methods. Diabetes was induced by streptozotocin. Sciatic nerve conduction velocity was measured in vivo and sciatic endoneurial perfusion was monitored by microelectrode polarography and hydrogen clearance. Glutathione content and Na⁺,K⁺-ATPase activity were measured in extracts from homogenised sciatic nerves. Results. After 8 weeks of diabetes, sciatic blood flow was 50 % reduced. Two weeks of WAY151 003 (3 or 100 mg/kg) treatment completely corrected this deficit and chelerythrine dose-dependently improved nerve perfusion. The inhibitors dose-dependently corrected a 20 % diabetic motor conduction deficit, however, at high doses ( > 3.0 mg/kg WAY151003; > 0.1 mg/kg chelerythrine) conduction velocity was reduced towards the diabetic level. Sciatic Na⁺,K⁺-ATPase activity, 42 % reduced by diabetes, was partially corrected by low but not high dose WAY151 003. In contrast, only a very high dose of chelerythrine partially restored Na⁺,K⁺-ATPase activity. A 30 % diabetic deficit in sciatic glutathione content was unchanged by protein kinase C inhibition. The benefits of WAY151 003 on blood flow and conduction velocity were blocked by nitric oxide synthase inhibitor co-treatment. Conclusion/interpretation. Protein kinase C contributes to experimental diabetic neuropathy by a neurovascular mechanism rather than through Na⁺,K⁺-ATPase defects. [Diabetologia (1999) 42: 1120–1130] Received: 21 December 1998 and in final revised form: 23 April 1999     

Hepatoprotective effects of parsley (Petroselinum Crispum) extract in rats with bile duct ligation

Background and study aimsThis study aimed to investigate the possible protective effects of parsley extract (Petroselinum Crispum; PC) against oxidative liver damage caused by bile obstruction in rats.Material and methodsBile duct ligation (BDL) method was used to induce liver injury in rats. The rats were divided into the three groups each consisting of 8 rats; Sham-operated control (C), bile duct ligated + saline treated (BDL), and BDL + PC treated groups. PC extract was given at a dose of 2 g/kg orally for 28 days. Aspartate amino transferase (AST), alanin amino transferase (ALT), and bilirubin levels were analyzed in sera. In order to determine free radicals in liver injury, luminol and lucigenin chemiluminescence tests used. Oxidative stress was evaluated through superoxide dismutase, glutathione, malondialdehyde, Na⁺/K⁺-ATPase and 8-hydroxy guanosine levels. Furthermore, inflammation marker myeloperoxidase, apoptosis marker caspase-3, and fibrosis markers TGF- β and hydoxyproline were investigated. The liver tissues were also examined for histological evaluations.ResultsWhile PC treatment decreased AST and ALT levels which increased with BDL, oxidant damage parameters also decreased with this treatment.ConclusionThe present study, which is the first research for PC extract on cholestasis induced liver damage, demonstrated that PC extract could be a potential therapeutic agent against liver fibrosis and need further studies.     

Investigation of dose-dependent effects of berberine against renal ischemia/reperfusion injury in experimental diabetic rats

BackgroundIschemia–reperfusion injury causes various severe morphological and functional changes in diabetic patients. To date, numerous antidiabetic and antioxidant agents have been used for treatment of the disease-related changes.ObjectivesWe aimed to examine effective therapeutic doses or doses of berberine against renal ischemia/reperfusion injury (IRI) in a streptozotocin (STZ)-induced diabetic rat model by histopathological and biochemical analysis.MethodsThirty male Sprague Dawley rats were treated with STZ injection for the development of diabetes, and divided into the following groups: STZ-induced diabetic group (STZ); IRI-induced diabetic group (STZ + IRI); 50 mg/kg berberine (BRB) treated diabetic group after inducing IRI (STZ + IRI + BRB₁); 100 mg/kg BRB treated diabetic group after IRI (STZ + IRI + BRB₂); 150 mg/kg BRB treated diabetic group after IRI (STZ + IRI + BRB₃). Bilateral renal ischemia model was applied for 45 min, then reperfusion was allowed for 14 days in STZ-induced diabetic rats. Renal injury was detected histopathologically. Blood urea nitrogen (BUN), creatinine and lactate dehydrogenase (LDH) levels were measured in serum using the ELISA method. Total antioxidant status (TAS) and total oxidant status (TOS) of renal tissue was studied by spectrophotometric assay. Oxidative stress index (OSI) was calculated as TOS-to-TAS ratio. Tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP), Na⁺/K⁺-ATPase (sodium pump), and Ca²⁺-ATPase (calcium ATPase) enzyme levels were measured in tissues using the ELISA method. Anti-apoptotic Bax and pro-apoptotic Bcl-2 protein levels were detected by Western blot analysis. All data were evaluated statistically.ResultsThe highest histopathological score was detected in the STZ + IRI group compared to the other group. BRB administration at the doses of 100 mg/kg and 150 mg/kg markedly improved renal injury. BUN and creatinine levels significantly increased in the STZ + IRI group compared to the STZ group (p < 0.001). 100 mg/kg and 150 mg/kg BRB administration significantly decreased those levels (p < 0.01). The highest TOS and the lowest TAS levels were detected in the STZ + IRI group (p < 0.001). IRI markedly aggravated inflammation via increasing levels of TNF-α and CRP (<0.001), and caused apoptosis via inducing Bcl-2 protein, and suppressing Bax protein (p < 0.001). BRB administration at the doses of 100 mg/kg and 150 mg/kg showed anti-oxidant, anti-inflammatory and anti-apoptotic effects (p < 0.01). The LDH enzyme, was used as a necrosis marker, was higher in the STZ + IRI group than other groups. BRB administration at all of the doses, resulted in the decline of LDH enzyme level (p < 0.001). Ca²⁺-ATPase and Na⁺/K⁺-ATPase enzyme activities decreased in the STZ + IRI group compared to the STZ group (p < 0.001), while BRB administration at the doses of 100 mg/kg and 150 mg/kg significantly increased those of enzyme activities, respectively (p < 0.05).ConclusionIschemia with diabetes caused severe histopathological and biochemical damage in renal tissue. The high doses of berberine markedly improved histopathological findings, regulated kidney function via decreasing BUN and creatinine levels, and rearranged intercellular ion concentration via increasing Na⁺/K⁺-ATPase and Ca^2+? ATPase levels. Berberine showed anti-oxidant, anti-apoptotic, and anti-inflammatory effects. According to these data, we suggest that berberine at the doses of 100 and 150 mg may be used as a potential therapeutic agent to prevent renal ischemic injury.     

Insulin does not regulate vascular smooth muscle Na+, K+-ATPase activity in rabbit aorta

Summary To determine whether insulin regulates vascular smooth muscle Na⁺, K⁺-ATPase activity and if impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity could be a cause of increased vascular reactivity to norepinephrine and angiotensin II in diabetic states, the effects of insulin on Na⁺, K⁺-ATPase activity were examined in normal rabbit aortic intima-media incubated with normal plasma glucose and myo-inositol levels for 30 min. Insulin at 100 U/ml (600 pmol/l) had no effect on Na⁺, K⁺-ATPase activity. At 250 U/ml it caused a 4.2±0.8% increase, and at 500 U/ml insulin caused a 17.7±1.4% increase in Na⁺, K⁺-ATPase activity that was completely inhibited by amiloride (1 mmol/l). Human insulin-like growth factor I (600 pmol/l) caused an 18.0±1.0% increase in Na⁺, K⁺-ATPase activity that was inhibited by amiloride. Insulin does not regulate (stimulate) aortic vascular smooth muscle Na⁺, K⁺-ATPase activity. Supraphysiological insulin concentrations, probably acting through an insulin-like growth factor I receptor, stimulate Na⁺/H⁺ exchange in aortic vascular smooth muscle and cause small secondary increases in Na⁺, K⁺-ATPase activity. In aortic intima-media incubated with normal plasma glucose and myo-inositol levels, endogenously released adenosine stimulates and maintains a component of resting Na⁺, K⁺-ATPase activity and stimulates acute increases in activity when norepinephrine (1 mol/l) or angiotensin II (100 nmol/l) is added. These adenosine-stimulated components of Na⁺, K⁺-ATPase activity are selectively inhibited when the medium glucose is raised to 30 mmol/l during a 30-min equilibration and 30-min incubation. Insulin (100 U/ml) added during the incubation had no effect on the alterations in Na⁺, K⁺-ATPase activity induced by glucose at an elevated plasma level. Impaired insulin stimulation of vascular smooth muscle Na⁺, K⁺-ATPase activity is not a possible cause for alterations in vascular reactivity in diabetes.     

Choline-deprivation alters crucial brain enzyme activities in a rat model of diabetic encephalopathy

Diabetic encephalopathy describes the moderate cognitive deficits, neurophysiological and structural central nervous system changes associated with untreated diabetes. It involves neurotoxic effects such as the generation of oxidative stress, the enhanced formation of advanced glycation end-products, as well as the disturbance of calcium homeostasis. Due to the direct connection of choline (Ch) with acetylcholine availability and signal transduction, a background of Ch-deficiency might be unfavorable for the pathology and subsequently for the treatment of several metabolic brain diseases, including that of diabetic encephalopathy. The aim of this study was to shed more light on the effects of adult-onset streptozotocin (STZ)-induced diabetes and/or Ch-deprivation on the activities of acetylcholinesterase (AChE) and two important adenosinetriphosphatases, namely Na⁺,K⁺-ATPase and Mg²⁺-ATPase. Male adult Wistar rats were divided into four main groups, as follows: control (C), diabetic (D), Ch-deprived (CD), and Ch-deprived diabetic (D+CD). Deprivation of Ch was provoked through the administration of Ch-deficient diet. Both the induction of diabetes and the beginning of dietary-mediated provoking of Ch-deprivation occurred at the same day, and rats were killed by decapitation after 30 days (1 month; groups C1, D1, CD1 and D1+CD1) and 60 days (2 months; groups C2, D2, CD2 and D2+CD2, respectively). The adult rat brain AChE activity was found to be significantly increased by both diabetes (+10%, p < 0.001 and +11%, p < 0.01) and Ch-deprivation (+19%, p < 0.001 and +14%, p < 0.001) when compared to the control group by the end of the first (C1) and the second month (C2), respectively. However, the Ch-deprived diabetic rats’ brain AChE activity was significantly altered only after a 60-day period of exposure, resulting in a +27% increase (D2+CD2 vs. C2, p < 0.001). Although the only significant change recorded in the brain Na⁺,K⁺-ATPase activity after the end of the first month is attributed to Ch-deprivation (+21%, p < 0.05, CD1 vs. C1), all groups of the second month exhibited a statistically significant decrease in brain Na⁺,K⁺-ATPase activity (−24%, p < 0.01, D2 vs. C2; −21%, p < 0.01, CD2 vs. C2; −22%, p < 0.01, D2+CD2 vs. C2). As concerns Mg²⁺-ATPase, the enzyme’s activity demonstrates no significant changes, with the sole exception of the D2+CD2 group (+21%, p < 0.05, D2+CD2 vs. C2). In addition, statistically significant time-dependent changes concerning the brain Mg²⁺-ATPase activity were recorded within the diabetic (p < 0.05, D2 vs. D1) and the Ch-deprived (p < 0.05, CD2 vs. CD1) rat groups. Our data indicate that Ch-deprivation seems to be an undesirable background for the above-mentioned enzymatic activities under untreated diabetes, in a time-evolving way. Further studies on the issue should focus on a region-specific reevaluation of these crucial enzymes’ activities as well as on the possible oxidative mechanisms involved.     

Binding of [3H]ouabain to endothelial cells derived from various vascular beds

Summary Binding experiments were performed with [³H]ouabain on plasma membranes derived from several types of isolated and cultivated endothelial cells. Identical saturation curves for [³H]ouabain binding to endothelial cells form pig aorta, caval vein, and pulmonary artery were obtained with a dissociation constant (K_D) of 3.29±0.31 nmol/l and a binding capacity (B_max) of 5.22±0.12 pmol/mg protein. On guinea-pig coronary endothelial cells, saturation of [³H]ouabain revealed much lower affinity (K_D 95±15 nmol/l, B_max 2.08±0.09 pmol/mg protein). All Scatchard plots were linear, indicating a homogeneous class of binding sites. In competition experiments, cardiac glycosides and their aglycons displaced the radioligand with a structure-activity relationship typical for interaction with Na⁺/K⁺-ATPase (proscillaridin A>ouabain>digoxin>g-strophanthidin>digoxigenin>dihydrodigoxin); in particular, removal of the sugar moiety results in considerable reduction of affinity. Furthermore, K⁺ displayed a steep inhibition curve with a half-maximal inhibitory constant of 2 mmol/l. All these findings suggest the presence of endothelial ouabain receptors linked to Na⁺/K⁺-ATPase. However, direct measurement of this enzyme was not possible due to an extremely high Mg²⁺-ATPase activity.     

Ketamine suppresses intestinal NF-kappa B activation and proinflammatory cytokine in endotoxic rats

AIM: To investigate the protective effect of ketamine on the endotoxin-induced proinflammatory cytokines and NFkappa B activation in the intestine. ETHODS: Adult male Wistar rats were randomly divided into 6 groups: (a) normal saline control, (b) challenged with endotoxin (5 mg/kg) and treated by saline, (c) challenged with endotoxin (5 mg/kg) and treated by ketamine (0.5 mg/kg), (d) challenged with endotoxin (5 mg/kg) and treated by ketamine (5 mg/kg ), (e) challenged with endotoxin (5 mg/kg) and treated by ketamine (50 mg/kg), and (f) saline injected and treated by ketamine (50 mg/kg). After 1, 4 or 6 h, TNF-α and IL-6 mRNA were investigated in the tissues of the intestine (jejunum) by RT-PCR. TNF-α and IL-6 were measured by ELISA. We used electrophoretic mobility shift assay (EMSA) to investigate NF-kappa B activity in the intestine. RESULTS: NF-kappa B activity, the expression of TNF-α and IL-6 were enhanced in the intestine by endotoxin. Ketamine at a dose of 0.5 mg/kg could suppress endotoxininduced TNF-α mRNA and protein elevation and inhibit NFkappa B activation in the intestine. However the least dosage of ketamine to inhibit IL-6 was 5 mg/kg in our experiment. CONCLUSION: Ketamine can suppress endotoxin-induced production of proinflammatory cytokines such as TNF-α and IL-6 production in the intestine. This suppressive effect may act through inhibiting NF-kappa B.     

Curcumin counteracts the aluminium-induced ageing-related alterations in oxidative stress, Na+, K+ ATPase and protein kinase C in adult and old rat brain regions

This study investigated the effect of curcumin on aluminium-induced alterations in ageing-related parameters: lipid peroxidation, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-s-transferase (GST), protein kinase C (PKC), Na⁺, K⁺-adenosine triphosphatase (Na⁺, K⁺-ATPase) and acetylcholinesterase (AChE) in the cerebral cortex and hippocampus of the brain of 10- and 24-month-old rats. Measurements taken from aluminium-fed rats were compared with those from rats in which curcumin and aluminium were co-administered. In aluminium-treated rats the levels of lipid peroxidation, PKC and AChE were enhanced while the activities of SOD, GPx, GST and Na⁺, K⁺-ATPase were significantly decreased in both the brain regions of both age-groups. In animals co-administered with curcumin and aluminium, the levels of lipid peroxidation, activities of PKC and AChE were significantly lowered while the activities of SOD, GPx, GST and Na⁺, K⁺-ATPase were significantly enhanced in the two brain regions studied indicating curcumin’s protective effects against aluminium toxicity. Though the magnitudes of curcumin-induced alterations varied in young and old animals, the results of the present study also demonstrated that curcumin exerts a protective effect against aluminium-induced elevation of ageing-related changes by modulating the extent of oxidative stress (by upregulating the activities of antioxidant enzymes) and by regulating the activities of Na⁺, K⁺ ATPase, PKC and AChE. Therefore, it is suggested that curcumin counters aluminium-induced enhancement in ageing-related processes.