Properties of Na,K-ATPase in cerebellum of male and female rats: effects of acute and prolonged diabetes.

The present study was oriented to gender specificity of Na,K-ATPase in cerebellum, the crucial enzyme maintaining the intracellular homeostasis of Na ions in healthy and diabetic Wistar rats. The effects of diabetes on properties of the Na,K-ATPase in cerebellum derived from normal and streptozotocin (STZ)-diabetic rats of both genders were investigated. The samples were excised at different time intervals of diabetes induced by STZ (65 mg kg-1) for 8 days and 16 weeks. In acute 8-day-lasting model of diabetes, Western blot analysis showed significant depression of α1 isoform of Na,K-ATPase in males only. On the other hand, concerning the activity, the enzyme seems to be resistant to the acute model of diabetes in both genders. Prolongation of diabetes to 16 weeks was followed by increasing the number of active molecules of Na,K-ATPase exclusively in females as indicated by enzyme kinetic studies. Gender specificity was observed also in nondiabetic animals revealing higher Na,K-ATPase activity in control males probably caused by higher number of active enzyme molecules as indicated by increased value of V max when comparing to control female group. This difference seems to be age dependent: at the age of 16 weeks, the V max value in females was higher by more than 90%, whereas at the age of 24 weeks, this difference amounted to only 28%. These data indicate that the properties of Na,K-ATPase in cerebellum, playing crucial role in maintaining the Na+ and K+ gradients, depend on gender, age, and duration of diabetic impact.

Effect of duration of diabetes mellitus type 1 on properties of Na, K-ATPase in cerebral cortex.

Time sequence study was performed to characterize the effects of diabetes mellitus type 1 on properties of the Na, K-ATPase in cerebral cortex derived from normal and streptozotocin (STZ)-diabetic rats of both genders. The samples were excised at varying time intervals of diabetes induced by STZ (65 mg kg(-1)) for 8 days, and 8 and 16 weeks. Expression of α1-3 isoforms of Na, K-ATPase was not altered in statistically significant level during all stages of diabetes neither in female nor in male rats as revealed from Western blot analysis. Studies of kinetic properties of the enzyme resulted in variations in active number of Na, K-ATPase molecules as well as its qualitative properties. Sixteen-week-old control male rats showed better affinity to substrate as indicated by 13 % decrease of K m value. The effect persisted also in males subjected to 8 days lasting diabetes; however, in males subjected to 8 weeks lasting diabetes, the effect was lost. In 25-week-old rats, the Na, K-ATPase revealed again altered properties in males and females but the mechanism of the variation was different. In females, the number of active molecules of Na, K-ATPase was higher by 32 % in controls and by 17 % in rats with chronic diabetes when comparing to respective male groups as suggested by increased value of V max. So the properties of Na, K-ATPase in cerebral cortex, playing crucial role in maintaining intracellular homeostasis of Na(+) ions, depend on gender, age, and duration of diabetic insult.

The effect of omega-3 fatty acids on expression of connexin-40 in Wistar rat aorta after lipopolysaccharide administration.

Connexin (Cx)-channels can represent one of targets of omega-3 fatty acids (n-3 PUFA) in protection of cardiovascular system against injury. We investigated the anti-inflammatory effect of 10-day n-3 PUFA intake (30 mg/kg/day for 10 days) on expression of Cx40 isoform in the aorta of Wistar rats injected with a single dose of lipopolysaccharide (LPS, 1 mg/kg, i.p.). LPS resulted in up-regulation of Cx40 expression in the aorta associated with reduced endothelium-dependent relaxation. LPS increased levels of inflammatory markers C-reactive protein and malondialdehyde in circulation as well as NOS activity and CD68 expression in aortic tissue indicating presence of moderate inflammation. N-3 PUFA supplementation decreased expression of both Cx40 and CD68 in aortic tissue and suppressed concentrations of C-reactive protein and malondialdehyde of endotoxemic rats. N-3 PUFA did not improve NO-dependent relaxation of aorta and NOS activity in LPS rats. The results indicate the involvement of Cx40 in development of LPS-induced endothelium-dependent functional impairment of the aorta and partial health benefits of n-3 PUFA diet associated with improved Cx40 expression.

Effects of γ-irradiation on Na,K-ATPase in cardiac sarcolemma.

Previous studies showed that adverse effect of ionizing radiation on the cardiovascular system is beside other factors mostly mediated by reactive oxygen and nitrogen species, which deplete antioxidant stores. One of the structures highly sensitive to radicals is the Na,K-ATPase the main system responsible for extrusion of superfluous Na(+) out of the cell which utilizes the energy derived from ATP. The aim of present study was the investigation of functional properties of cardiac Na,K-ATPase in 20-week-old male rats 6 weeks after γ-irradiation by a dose 25 Gy (IR). Irradiation induced decrease of systolic blood pressure from 133 in controls to 85 mmHg in IR group together with hypertrophy of right ventricle (RV) and hypotrophy of left ventricle (LV). When activating the cardiac Na,K-ATPase with substrate, its activity was lower in IR in the whole concentration range of ATP. Evaluation of kinetic parameters revealed a decrease of the maximum velocity (V max) by 40 % with no changes in the value of Michaelis-Menten constant (K m). During activation with Na(+), we observed a decrease of the enzyme activity in hearts from IR at all tested Na(+) concentrations. The value of V max decreased by 38 %, and the concentration of Na(+) that gives half maximal reaction velocity (K Na) increased by 62 %. This impairment in the affinity of the Na(+)-binding site together with decreased number of active Na,K-ATPase molecules, as indicated by lowered V max values, are probably responsible for the deteriorated efflux of the excessive Na(+) from the intracellular space in hearts of irradiated rats.

Gender specific influence of fish oil or atorvastatin on functional properties of renal Na,K-ATPase in healthy Wistar and hypertriglyceridemic rats.

For better understanding of pathophysiological processes leading to increased retention of sodium as a consequence of hyperlipidemia, the properties of renal Na,K-ATPase, a key enzyme involved in maintaining sodium homeostasis in the organism, were studied. Enzyme kinetics of renal Na,K-ATPase were used for characterization of ATP- and Na(+)-binding sites after administration of fish oil (FO) (30 mg·day(-1)) or atorvastatin (0.5 mg·100 g(-1)·day(-1)) to healthy Wistar rats and rats with hereditary hypertriglyceridemia of both genders. Untreated healthy Wistar and also hypertriglyceridemic female rats revealed higher Na,K-ATPase activity as compared to respective untreated male groups. Hypertriglyceridemia itself was accompanied with higher Na,K-ATPase activity in both genders. Fish oil improved the enzyme affinity to ATP and Na(+), as indicated by lowered values of K(m) and K(Na) in Wistar female rats. In Wistar male rats FO deteriorated the enzyme in the vicinity of the Na(+)-binding site as revealed from the increased K(Na) value. In hypertriglyceridemic rats FO induced a significant effect only in females in the vicinity of the sodium binding sites resulting in improved affinity as documented by the lower value of K(Na). Atorvastatin aggravated the properties of Na,K-ATPase in both genders of Wistar rats. In hypertriglyceridemic rats protection of Na,K-ATPase was observed, but this effect was bound to females only. Both treatments protected renal Na,K-ATPase in a gender specific mode, resulting probably in improved extrusion of excessive intracellular sodium out of the cell affecting thus the retention of sodium in hHTG females only.

Effect of quercetin on kinetic properties of renal Na,K-ATPase in normotensive and hypertensive rats.

The effect of quercetin, a plant-derived bioflavonoid with documented positive effect on the cardiovascular system, was examined after 4-week supplementation in the dose of 20 mg kg(-1) x day(-1) to young male normotensive control (C) and to spontaneously hypertensive rats (SHR) over the period of their 5(th)-8(th) week of age. The study was focused on the influence of quercetin on properties of the renal Na,K-ATPase, a key system in maintaining the homeostasis of sodium in the organism. Spontaneous hypertension by itself enhanced the activity of Na,K-ATPase probably as a consequence of a higher number of active enzyme molecules, as suggested by the 15% increase of V(max), along with improved affinity to ATP, as indicated by the 30% decrease in the value of Michaelis-Menten constant K(m) in untreated SHR vs. untreated normotensive rats. Quercetin induced a decrease of Na,K-ATPase activity in the presence of all ATP and Na(+) concentrations investigated. Evaluation of kinetic parameters resulted in a constant V(max) value. The ATP-binding properties of the enzyme were not influenced by quercetin, as suggested by statistically insignificant changes in the value of K(m) both in controls and in SHR. On the other hand, the affinity to sodium decreased, as suggested by an increase in the K(Na) value by 22% and 31% in normotensive and hypertensive groups, respectively. This impairment in the affinity of the Na(+)-binding site of Na,K-ATPase molecules was probably responsible for the deteriorated enzyme function in the kidneys of quercetin treated animals.

Regulatory role of nitric oxide on the cardiac Na, K-ATPase in hypertension.

The present study was focused on regulatory role of nitric oxide on functional properties of the cardiac Na, K-ATPase in three various animal models of hypertension: spontaneously hypertensive male rats (SHR) with increased activity of nitric oxide synthase (NOS) by 60 % (Sh1), SHR with decreased activity of NOS by 40 % (Sh2) and rats with hypertension induced by L-NAME (40 mg/kg/day) with depressed activity of NOS by 72 % (LN). Studying the utilization of energy substrate we observed higher Na, K-ATPase activity in the whole concentration range of ATP in Sh1 and decreased activity in Sh2 and LN. Evaluation of kinetic parameters revealed an increase of Vmax value by 37 % in Sh1 and decrease by 30 % in Sh2 and 17 % in LN. The KM value remained unchanged in Sh2 and LN, but was lower by 38 % in Sh1 indicating increased affinity of the ATP binding site, as compared to controls. During the activation with Na+ we observed increased Vmax by 64 % and increased KNa by 106 % in Sh1. In Sh2 we found decreased Vmax by 40 % and increased KNa by 38 %. In LN, the enzyme showed unchanged Vmax with increased KNa by 50 %. The above data indicate a positive role of increased activity of NOS in improved utilization of ATP as well as enhanced binding of Na+ by the cardiac Na, K-ATPase.

Effect of the pyridoindole antioxidant stobadine on ATP-utilisation by renal Na,K-ATPase in rats with streptozotocin-induced diabetes.

The effect of the pyridoindole antioxidant stobadine on diabetes-induced changes of Na,K-ATPase, especially those concerning the utilisation of its substrate ATP, was investigated. Sixteen weeks of streptozotocin-induced diabetes (single i.v. dose of streptozotocin; 55 mg/kg) was followed by decrease in the enzyme activity. This effect was emphasised in the presence of higher concentrations of substrate and in the presence of 8 mmol x l(-1) ATP it represented 20%. It might be a consequence of altered functional properties of Na,K-ATPase as suggested by 20% decrease in the V(max) value along with decrease in the K(m) value by 20%. Administration of 0.05% (w/w) stobadine in the diet to diabetic rats improved the function of renal Na,K-ATPase with respect to utilisation of ATP as suggested by significant increase in the enzyme activity in the whole concentration range of ATP investigated as a consequence of V(max) elevation to the level comparable to absolute controls. In conclusion, stobadine may play a positive role in restoring the functional properties of renal Na,K-ATPase, especially concerning the utilisation of energy derived from hydrolysis of ATP, improving thus the maintenance of ionic homeostasis during diabetes.

Effect of the pyridoindole antioxidant stobadine on the cardiac Na(+),K(+)-ATPase in rats with streptozotocin-induced diabetes.

In the present study we examined the effect of dietary supplementation with the pyridoindole antioxidant stobadine on functional properties of the cardiac Na(+),K(+)-ATPase in diabetic rats. Diabetes lasting sixteen weeks which was induced by a single i.v. dose of streptozotocin (55 mg x kg(-1)) was followed by decrease in the enzyme activity. Evaluation of kinetic parameters revealed a statistically significant decrease in the maximum velocity (Vmax) (32% for ATP-activation, 33% for Na(+)-activation), indicating a diabetes-induced diminution of the number of active enzyme molecules in cardiac sarcolemma. The ATP-binding properties of the enzyme were not affected by diabetes as suggested by statistically insignificant changes in the value of Michaelis-Menten constant, K(M (ATP)). On the other hand, the affinity to sodium decreased as suggested by 54% increase in the K(M (Na+)) value. This impairment in the affinity of the Na(+)-binding site together with decreased number of active Na(+),K(+)-ATPase molecules are probably responsible for the deteriorated enzyme function in hearts of diabetic animals. Administration of stobadine to diabetic rats dramatically improved the function of cardiac Na(+),K(+)-ATPase with regard to Na(+)-handling, as documented by statistically significant elevation of Vmax by 66 and 47% decrease in K(M (Na+)). Our data suggest that stobadine may prevent the diabetes-induced deterioration of cardiac Na(+),K(+)-ATPase, thus enabling to preserve its normal function in regulation of intracellular homeostasis of Na(+) and K(+) ions.

In vitro inhibition of lens aldose reductase by (2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid in enzyme preparations isolated from diabetic rats.

(2-benzyl-2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-yl)-acetic acid (compound 1), a novel aldose reductase inhibitor, was assayed for efficacy and selectivity to inhibit rat lens aldose reductase under in vitro conditions by using enzyme preparations obtained from diabetic animals. The inhibitory efficiency was characterized by IC(50) in micromolar region. Enzyme kinetics analysis revealed uncompetitive type of inhibition, both in relation to the D,L-glyceraldehyde substrate and to the NADPH cofactor. In testing for selectivity, comparisons to rat kidney aldehyde reductase, an enzyme with the highest homology to aldose reductase, was used. The inhibition selectivity of the compound tested was characterized by selectivity factor around 20 and was even slightly improved under conditions of prolonged experimental diabetes. These findings were identical with those in the control rats. To conclude, the inhibitory mode, efficacy and selectivity of compound 1, a novel aldose reductase inhibitor, was preserved even under the conditions of prolonged STZ-induced experimental diabetes of rats.