Analysis of Signaling Pathways of Necroptotic and Pyroptotic Cell Death in the Hearts of Rats With Type 2 Diabetes Mellitus.

Diabetes mellitus is known to produce various cell-damaging events and thereby underlie heart dysfunction and remodeling. However, very little is known about its inflammation-associated pathomechanisms due to necrosis-like cell death. For this purpose, we aimed to investigate signaling pathways of necroptosis and pyroptosis, known to produce plasma membrane rupture with the resultant promotion of inflammation. One-year old Zucker diabetic fatty (ZDF) rats did not exhibit significant heart dysfunction as revealed by echocardiographic measurement. On the other hand, there was a decrease in heart rate due to diabetes. Immunoblotting analysis showed that the left ventricles of ZDF rats overexpress neither the main necroptotic proteins including receptor-interacting protein kinase 3 (RIP3) and mixed lineage domain kinase-like pseudokinase (MLKL), nor the pyroptotic regulators including NLR family pyrin domain containing 3 protein (NLRP3), caspase-1, interleukin-1 beta (IL-1beta and the N-terminal gasdermin D (GSDMD-N). On the other hand, the increased activation of the RIP3 kinase due to phosphorylation was found in such hearts. In summary, we showed for the first time that the activation of cardiac RIP3 is upregulated due to disturbances in glucose metabolism which, however, did not proceed to necrosis-like cell death. These data can indicate that the activated RIP3 might also underlie other pleiotropic, non-necroptotic signaling pathways under basal conditions.

Effect of sex and localization dependent differences of Na,K-ATPase properties in brain of rat.

Na,K-ATPase is the main system effectively excluding the superfluous sodium out of the cells on the expense of energy derived from hydrolysis of ATP. In brain 3 different isoforms of the catalytic α-subunit are known. The present study was focused to energy utilization and ability to bind sodium by the Na,K-ATPase as well as expression of all 3 isoforms of the catalytic α-subunit concerning its sex specificity in two selected regions of the brain, in cortex and in cerebellum of rats. Western blot analysis showed higher expression of all 3 catalytic α-subunits of Na,K-ATPase in cerebellum when compared to cortex which was not followed by higher activity. On contrary the total activity of the enzyme was lower in cerebellum comparing with cortex in females with no significant localization dependent differences of activities in males. Concerning sex dependence only the expression of α3 isoform was higher in cortex of male rats with no differences in the levels of α1 and α2 isoforms. However, the total activity of Na,K-ATPase in cortex was similar in male and female groups. On the other hand in cerebellum of females the total activity of Na,K-ATPase was significantly lower as compared with males. The obtained data indicate localization and sex dependent variations in maintenance of sodium homeostasis in the brain.

Localization dependent sensitivity of cerebral Na,K-ATPase to irradiation induced oxidative imbalance in rats.

Na,K-ATPase represents the key enzyme maintaining the ionic gradient across plasma membrane. It was documented that in directly irradiated organs the activity of this enzyme is decreased. The aim of present study was to clarify the remote effect of irradiation in mediastinal area on the activity of the Na,K-ATPase in selected brain regions in rats. Ionizing radiation in single dose 25 Gy induced alterations in oxidative status of blood plasma. Irradiation also decreased the activity of the Na,K-ATPase in cerebral cortex. Measurements of kinetic properties of the enzyme dependently on the concentration of energy substrate ATP or cofactor Na+ indicated that the lowered enzyme activity is probably a consequence of decreased number of active molecules of the enzyme, as suggested by lowered Vmax values (by 13 - 14%). Immunoblot analysis revealed that this effect is connected namely to decreased presence of α2 and α3 subunits (by 25% and 30% respectively). Considering the current concepts about involvement of the malfunction of α2 α3 subunits in development of primary brain dysfunctions, it may be hypothesized that the lowered functionality of those subunits of Na,K-ATPase may represent a predisposition to neurodegenerative disorders after irradiation. The observed effect seems to be localization dependent as the enzyme in cerebellum resisted to irradiation.

Sex-specific response of renal Na,K-ATPase to prenatal angiotensin 2 exposure and increased salt intake in offspring.

In rodents, increased angiotensin 2 (Ang2) during pregnancy increases blood pressure and decreases salt sensitivity in the offspring. To explore the underlying mechanisms, this study evaluated the effects of prenatal Ang2 exposure on the activity of renal Na,K-ATPase, which is one of the main systems that maintains sodium ion homeostasis in an organism. Moreover, this study also investigated the impact of a higher-salt diet on the enzyme activity in the offspring in a sex-dependent manner. Pregnant Wistar rats were implanted with osmotic minipumps that continuously released Ang2 (2 µg/kg/h) for 2 weeks. Male and female offspring of treated and control females were allocated to groups fed with normal or high-salt diets. In the offspring prenatally treated with Ang2, a significant Vmax increase (23 - 36%) was observed in females fed with both a normal and high-salt diet. In addition, a significant increase in Km (20 - 26%) was also observed in the female groups, compared to respective male groups, independently of their diet. Evaluation of KNa showed significantly lower values (13 - 17%) in female offspring fed with a high-salt diet, independent of the prenatal treatment. In conclusion, these data suggest that increased prenatal Ang2 has a predominant impact on the properties of renal Na,K-ATPase in both sexes. Moreover, the enzyme is resistant to higher salt intake in offspring prenatally exposed to Ang2.

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.

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.