Cytotoxic effect of carbohydrate derivatives of digitoxigenin involves modulation of plasma membrane Ca2+ -ATPase.

Cardiac glycosides, such as digoxin and digitoxin, are compounds that interact with Na+ /K+ -ATPase to induce anti-neoplastic effects; however, these cardiac glycosides have narrow therapeutic index. Thus, semi-synthetic analogs of digitoxin with modifications in the sugar moiety has been shown to be an interesting approach to obtain more selective and more effective analogs than the parent natural product. Therefore, the aim of this study was to assess the cytotoxic potential of novel digitoxigenin derivatives, digitoxigenin-α-L-rhamno-pyranoside (1) and digitoxigenin-α-L-amiceto-pyranoside (2), in cervical carcinoma cells (HeLa) and human diploid lung fibroblasts (Wi-26-VA4). In addition, we studied the anticancer mechanisms of action of these compounds by comparing its cytotoxic effects with the potential to modulate the activity of three P-type ATPases; Na+ /K+ -ATPase, sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA), and plasma membrane Ca2+ -ATPase (PMCA). Briefly, the results showed that compounds 1 and 2 were more cytotoxic and selectivity for HeLa tumor cells than the nontumor cells Wi-26-VA4. While the anticancer cytotoxicity in HeLa cells involves the modulation of Na+ /K+ -ATPase, PMCA and SERCA, the modulation of these P-type ATPases was completely absent in Wi-26-VA4 cells, which suggest the importance of their role in the cytotoxic effect of compounds 1 and 2 in HeLa cells. Furthermore, the compound 2 inhibited directly erythrocyte ghosts PMCA and both compounds were more cytotoxic than digitoxin in HeLa cells. These results provide a better understanding of the mode of action of the synthetic cardiac glycosides and highlights 1 and 2 as potential anticancer agents.

The γ-Benzylidene Digoxin Derivative BD-15 Increases the α3-Na, K-ATPase Activity in Rat Hippocampus and Prefrontal Cortex and no Change on Heart.

Our study aimed to investigate the effects of the new cardiotonic steroid BD-15 (γ-benzylidene derivatives) in the behavioral parameters, oxidative stress and the Na, K-ATPase activity in the hippocampus, prefrontal cortex and heart from rats to verify the safety and possible utilization in brain disorders. For this study, groups of male Wistar rats were used after intraperitoneal injection of 20, 100 and 200 µg/Kg with BD-15. The groups were treated for three consecutive days and the control group received 0.9% saline. BD-15 did not alter behavior of rats treated with different doses. An increase in the specific α2,3-Na, K-ATPase activity was observed for all doses of BD-15 tested in the hippocampus. However, in the prefrontal cortex, only the dose of 100 µg/Kg increased the activity of all Na, K-ATPase isoforms. BD-15 did not cause alteration in the lipid peroxidation levels in the hippocampus, but in the prefrontal cortex, a decrease of lipid peroxidation (~ 25%) was observed. In the hippocampus, GSH levels increased with all doses tested, while in the prefrontal cortex no changes were found. Subsequently, when the effect of BD-15 on cardiac tissue was analyzed, no changes were observed in the tested parameters. BD-15 at a dosage of 100 µg/Kg proved to be promising because it is considered therapeutic for brain disorders, since it increases the activity of the α3-Na, K-ATPase in the hippocampus and prefrontal cortex, as well as decreasing the oxidative stress in these brain regions. In addition, this drug did not cause changes in the tissues of the heart and kidneys, preferentially demonstrating specificity for the brain.

Implications of Synthetic Modifications of the Cardiotonic Steroid Lactone Ring on Cytotoxicity.

Na,K-ATPase (NKA) and cardiotonic steroids (CTS) have shown potent cytotoxic and anticancer effects. Here, we have synthesized a series of CTS digoxin derivatives (γ-benzylidene) with substitutions in the lactone ring and evaluated the cytotoxicity caused by digoxin derivatives in tumor and non-tumor cells lines, as well as their effects on NKA. The cytotoxicity assay was determined in HeLa, A549, and WI-26 VA4 after they were treated for 48 h with increased concentrations of CTS. The effects of CTS on NKA activity and immunoblotting of α1 and ß1 isoforms were evaluated at IC50 concentrations in A549 cell membrane. NKA activity from mouse brain cortex was also measured. The majority of CTS exhibited low cytotoxicity in tumor and non-tumor cells, presenting IC50 values at micromolar concentrations, while digoxin showed cytotoxicity at nanomolar concentrations. BD-15 presented the lowest IC50 value (8 µM) in A549 and reduced its NKA activity in 28%. In contrast, BD-7 was the compound that most inhibited NKA (56% inhibition) and presented high IC50 value for A549. In mouse cortex, only BD-15 modulated the enzyme activity in a concentration-dependent inhibition curve. These results demonstrate that the cytotoxicity of these compounds is not related to NKA inhibition. The substitutions in the lactone ring of digoxin led to an increase in the cytotoxic concentration in tumor cells, which may not be interesting for cancer, but it has the advantage of increasing the therapeutic margin of these molecules when compared to classic CTS, and can be used safely in research for other diseases.

Cytotoxicity of glucoevatromonoside alone and in combination with chemotherapy drugs and their effects on Na+,K+-ATPase and ion channels on lung cancer cells.

Cardiac glycosides (CGs) are useful drugs to treat cardiac illnesses and have potent cytotoxic and anticancer effects in cultured cells and animal models. Their receptor is the Na+,K+ ATPase, but other plasma membrane proteins might bind CGs as well. Herein, we evaluated the short- and long-lasting cytotoxic effects of the natural cardenolide glucoevatromonoside (GEV) on non-small-cell lung cancer H460 cells. We also tested GEV effects on Na+,K+ -ATPase activity and membrane currents, alone or in combination with selected chemotherapy drugs. GEV reduced viability, migration, and invasion of H460 cells spheroids. It also induced cell cycle arrest and death and reduced the clonogenic survival and cumulative population doubling. GEV inhibited Na+,K+-ATPase activity on A549 and H460 cells and purified pig kidney cells membrane. However, it showed no activity on the human red blood cell plasma membrane. Additionally, GEV triggered a Cl-mediated conductance on H460 cells without affecting the transient voltage-gated sodium current. The administration of GEV in combination with the chemotherapeutic drugs paclitaxel (PAC), cisplatin (CIS), irinotecan (IRI), and etoposide (ETO) showed synergistic antiproliferative effects, especially when combined with GEV + CIS and GEV + PAC. Taken together, our results demonstrate that GEV is a potential drug for cancer therapy because it reduces lung cancer H460 cell viability, migration, and invasion. Our results also reveal a link between the Na+,K+-ATPase and Cl- ion channels.

Antagonistic anticancer effect of paclitaxel and digoxin combination.

Despite the growing interest in the antitumor effect of cardiotonic steroids, combination treatments with well-established chemotherapy drugs like paclitaxel have been rarely investigated. Moreover, paclitaxel has been suggested as a Na+ /K+ -ATPase inhibitor. Here we investigated the effect of paclitaxel and digoxin alone or in combination on the viability of human lung (A549) and cervical cancer (HeLa) cell lines and the inhibitory effect of paclitaxel on several mammalian Na+ /K+ -ATPases. Although the viability of both tumor cell lines was concentration-dependently affected by digoxin treatment after 48 hours (A549 IC50 = 31 nM and HeLa IC50 = 151 nM), a partial effect was observed for paclitaxel, with a maximal inhibitory effect of 45% at 1000 nM with A549 and around 70% with HeLa cells (IC50 = 1 nM). Although the two drugs were cytotoxic, their combined effect in HeLa cells was revealed to be antagonistic, as estimated by the combination index. No direct inhibitory effect of paclitaxel was detected in human, pig, rat, and mouse Na+ /K+ -ATPase enzymes, but high concentrations of paclitaxel decreased the Na+ /K+ -ATPase activity in HeLa cells after 48 hours without affecting protein expression. Our findings demonstrate that, under our conditions, paclitaxel and digoxin cotreatment produce antagonistic cytotoxic effects in HeLa cells, and the mechanism of action of paclitaxel does not involve a direct inhibition of Na+ /K+ -ATPase. More studies shall be designed to evaluate the consequences of the interaction of cardiotonic steroids and chemotherapy drugs.

Ouabain attenuates oxidative stress and modulates lipid composition in hippocampus of rats in lipopolysaccharide-induced hypocampal neuroinflammation in rats.

Our study aimed to analyze the effect of ouabain (OUA) administration on lipopolysaccharide (LPS)-induced changes in hippocampus of rats. Oxidative parameters were analyzed in Wistar rats after intraperitoneal injection of OUA (1.8 µg/kg), LPS (200 µg/kg), or OUA plus LPS or saline. To reach our goal, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), in addition to levels of reduced glutathione (GSH), protein carbonyl (PCO) and lipid peroxidation (LPO) were evaluated. We also analyzed the membrane lipid profile and some important lipids for the nervous system, such as phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid and sphingomyelin. The group that received only LPS showed increased oxidative stress, as evidenced by an increase in LPO (about twice), PCO (about three times) levels, and CAT activity (80%). Conversely, administration of LPS decreased GSH levels (55%), and GPx activity (30%), besides a reduction in the amount of PI (60%) and PC (45%). By other side, OUA alone increased the amount of PI (45%), PE (85%), and PC (70%). All harmful effects recorded were attenuated by OUA, suggesting a protective effect against LPS-induced oxidative stress. The relevance of our results extends beyond changes in oxidative parameters induced by LPS, because nanomolar doses of OUA may be useful in neurodegenerative models. Other studies on other cardenolides and substances related issues, as well as the development of new molecules derived from OUA, could also be useful in general oxidative and/or cellular stress, a condition favoring the appearance of neuronal pathologies.

Evaluation of the effect of cafeteria diet on the kidney Na,K-ATPase activity, and oxidative stress.

In this study, renal tissue, subdivided into the cortex and medulla of Wistar rats subjected to a cafeteria diet (CAF) for 24 days or to normal diet, was used to analyze whether the renal enzyme Na,K-ATPase activity was modified by CAF diet, as well as to analyze the α1 subunit of renal Na,K-ATPase expression levels. The lipid profile of the renal plasma membrane and oxidative stress were verified. In the Na,K-ATPase activity evaluation, no alteration was found, but a significant decrease of 30% in the cortex was detected in the α1 subunit expression of the enzyme. There was a 24% decrease in phospholipids in the cortex of rats submitted to CAF, a 17% increase in cholesterol levels in the cortex, and a 23% decrease in the medulla. Lipid peroxidation was significantly increased in the groups submitted to CAF, both in the cortical region, 29%, and in the medulla, 35%. Also, a reduction of 45% in the glutathione levels was observed in the cortex and medulla with CAF. CAF showed a nearly two-fold increase in glutathione peroxidase (GPX) activity in relation to the control group in the cortex and a 59% increase in the GPx activity in the medulla. In conclusion, although the diet was administered for a short period of time, important results were found, especially those related to the lipid profile and oxidative stress, which may directly affect renal function.

The effect of gamma radiation on the lipid profile of irradiated red blood cells.

An investigation into the effects of irradiation and of the storage time on aging and quality are a relevant issue to ensure the safety and the efficiency of irradiation in the prevention of transfusion-associated graft-versus-host disease (TA-GVHD). In this work, the biochemical properties and alterations presented by erythrocyte membranes, up to 28-days post-irradiation, with a dose of 25 Gy, were studied as a function of storage and post-irradiation time. There was a considerable variation in the total of phospholipid content, when comparing the control and irradiated samples, mostly from the third day onwards; and at the same time, the effect occurred as a function on the storage time of blood bags. The levels of total cholesterol decreased 3-9 days after irradiation. TBARS levels were increased after irradiation and 7 days of storage, but no increment of catalase activity was observed after the irradiation. Furthermore, the protein profile was maintained throughout the irradiation and storage time, until the 21st day, with the presence of a protein fragmentation band of around 28 kDa on the 28th day. In conclusion, although gamma irradiation is the main agent for the prevention of TA-GVHD, a better understanding of the physical and biochemical properties of erythrocytes are necessary to better assess their viability, and to be able to issue more secure recommendations on the shelf life of blood bags, and the safe use of the irradiated red cells therein.

The effect of γ-radiation on the hemoglobin of stored red blood cells: the involvement of oxidative stress in hemoglobin conformation.

The aim of the present work was to evaluate the redox and oligomeric effects associated with the human hemoglobin of stored red blood cells that had been previously submitted to gamma radiation. Whole blood was collected from healthy donors and irradiated with 25 Gy of γ-radiation within 24 h of collection. At days 3, 5, 7, 9, 11, 14, and 28 postirradiation, fractions were removed and centrifuged, and the levels of methehemoglobin and oxyhemoglobin were measured. Hb was isolated to measure the denaturation and UV-vis spectra. The results from electrophoresis demonstrated that there was no fragmentation or cross-linking of the hemoglobin. However, ferrous center oxidation was identified as a very significant process. This mechanism is likely through an autoxidation process of the ferrous heme center, which has a maximal intensity between 5 and 7 days of storage. Interestingly, a subsequent reduction of the oxidized heme species was observed, and after 9 days of storage, the difference between the ferric species present in the control and irradiated samples was not representative. This interesting fact suggests a type of "protective action" by the blood to control the oxidative stress generated by the gamma irradiation. The UV-vis measurements demonstrated that the oxidized species was predominantly formed by hemichrome species (bis-histidine ferric heme species), which are usually associated with Heinz bodies. After 28 days of storage, evidence from the UV-vis measurements indicated that the oxidation of the irradiated sample was much higher than that observed in the control sample. These results demonstrate that despite the minimal polypeptide changes observed in the hemoglobin of stored red blood cells after gamma irradiation, the oxidation of the heme metallic center is not irrelevant and must be controlled to improve the hematological clinical procedures associated with the storage of red blood cells.

Effect of Ouabain on Glutamate Transport in the Hippocampus of Rats with LPS-Induced Neuroinflammation.

A lipopolysaccharide (LPS)-induced neuroinflammation rat model was used to study the effects of ouabain (OUA) at low concentrations, which can interact with the Na,K-ATPase, causing the modulation of intracellular signalling pathways in the Central Nervous System. Our study aimed to analyse the effects of OUA on glutamate transport in the hippocampus of rats with LPS-induced neuroinflammation. Adult male Wistar rats were divided into four groups: OUA (1.8 µg/kg), saline (CTR), LPS (200 µg/kg), and OUA + LPS (OUA 20 min before LPS). The animals were sacrificed after 2 h, and the hippocampus was collected for analysis. After treatment, we determined the activities of Na,K-ATPase and glutamine synthetase (GS). In addition, expression of the α1, α2, and α3 isoforms of Na,K-ATPase and the glutamate transporters, EAAT1 and EAAT2, were also analysed. Treatment with OUA caused a specific increase in the α2 isoform expression (~20%), whereas LPS decreased its expression (~22%), and treatment with OUA before LPS prevented the effects of LPS. Moreover, LPS caused a decrease of approximately 50% in GS activity compared with that in the CTR group; however, OUA pre-treatment attenuated this effect of LPS. Notably, it was found that treatment with OUA caused an increase in the expression of EAAT1 (~30%) and EAAT2 (~25%), whereas LPS caused a decrease in the expression of EAAT1 (~23%) and EAAT2 (~25%) compared with that in the CTR group. When treated with OUA, the effects of LPS were abrogated. In conclusion, the OUA pre-treatment abolished the effect caused by LPS, suggesting that this finding may be related to the restoration of the interaction between FXYD2 and the studied membrane proteins.

Regulatory phosphorylation of FXYD2 by PKC and cross interactions between FXYD2, plasmalemmal Ca-ATPase and Na,K-ATPase.

FXYD2 is a regulatory peptide associated with the α-subunit of the kidney Na,K-ATPase. FXYD2 can be phosphorylated by PKA, and its phosphorylation activates Na,K-ATPase. Here we show that FXYD2 is phosphorylated by PKC (PKC-FXYD2-P), by PKA (PKA-FXYD2-P) or by PKA and PKC simultaneously (FXYD2-P(2)) modulating both the erythrocyte Na,K-ATPase and the plasma membrane Ca(2+)-ATPase (PMCA). In erythrocyte ghosts, the addition of PKA-FXYD2-P activated Na,K-ATPase by 80%, while non-phosphorylated FXYD2 (np) activated only 55%. The addition of np FXYD2 did not affect PMCA basal activity, but FXYD2-P(2) increased the basal PMCA activity by up to 200%. Calmodulin-activated PMCA activity was increased by np FXYD2 (3-fold) or FXYD2-P(2) (2.5-fold). However, PKC-FXYD2-P increased PMCA activity only by 50%. In contrast, when PMCA was treated with PKA-FXYD2-P, the ATPase activity was inhibited by 50%. The effect of all forms of FXYD2-P on calcium uptake from PMCA resembled the pattern observed in ATP hydrolysis. Our results suggest that the FXYD2 anchoring site could be conserved among the P-ATPase family permitting cross regulation. The effects of FXYD2 on calcium uptake and calcium-stimulated ATP hydrolysis suggest a novel role for FXYD2 on PMCA.

Biochemical Evaluation of the Effects of Hydroxyurea in Vitro on Red Blood Cells.

Hydroxyurea (HU) is a low-cost, low-toxicity drug that is often used in diseases, such as sickle cell anemia and different types of cancer. Its effects on the red blood cells (RBC) are still not fully understood. The in vitro effects of HU were evaluated on the biochemical parameters of the RBC from healthy individuals that were treated with 0.6 mM or 0.8 mM HU for 30 min and 1 h. After 30 min, there was a significant increase in almost all of the parameters analyzed in the two concentrations of HU, except for the pyruvate kinase (PK) activity. A treatment with 0.8 mM HU for 1 h resulted in a reduction of the levels of lipid peroxidation, Fe3+, and in the activities of some of the enzymes, such as glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD), and PK. After the incubation for 1 h, the levels of H2O2, lipid peroxidation, reduced glutathione (GSH), enzymatic activity (hexokinase, G6PD, and superoxide dismutase (SOD) were reduced with the treatment of 0.8 mM HU when compared with 0.6 mM. The results have suggested that a treatment with HU at a concentration of 0.8 mM seemed to be more efficient in protecting against the free radicals, as well as in treating diseases, such as sickle cell anemia. HU appears to preferentially stimulate the pentose pathway over the glycolytic pathway. Although this study was carried out with the RBC from healthy individuals, the changes described in this study may help to elucidate the mechanisms of action of HU when administered for therapeutic purposes.

Cytotoxic and cytostatic effects of digitoxigenin monodigitoxoside (DGX) in human lung cancer cells and its link to Na,K-ATPase.

Cardiac glycosides (CGs) are natural compounds widely used to treat several cardiac conditions and more recently have been recognized as potential antitumor agents. They are known as Na,K-ATPases ligands, which is a promising drug target in cancer. In this study, the short and long-lasting cytotoxic effects of the natural cardenolide digitoxigenin monodigitoxoside (DGX) were evaluated against two non-small cell lung cancer lines (A549 and H460 cells). It was found that DGX induced cytotoxic effects in both cells and the apoptotic effects were more pronounced on H460 cells. In long-term analysis, using the clonogenic and the cumulative population doubling (CPD) assays, DGX showed a reduction of cell survival, after 15days without re-treatment. To better understand DGX effects in A549 cells, several assays were conducted. In cell cycle analysis, DGX caused an arrest in S and G2/M phases. This compound also increased the number of cells in subG1 phase in a concentration- and time-dependent manner. The presence of ß-galactosidase positive cells, large nucleus and flattened cells indicated senescence. Additionally, DGX inhibited Na,K-ATPase activity in A549 cells, as well as in purified pig kidney and in human red blood cell membrane preparations, at nanomolar range. Moreover, results of molecular docking showed that DGX binds with high efficiency (-11.4Kcal/mol) to the Na,K-ATPase (PDB:4HYT). Taken together, our results highlight the potent effects of DGX both in A549 and H460 cells, and disclose its link with Na,K-ATPase inhibition.

Performance of point of care assays for hepatitis B and C viruses in chronic kidney disease patients.

AIMS: Point of care testing (POCT) has been used for hepatitis B and C diagnosis in general population, but little is known about the influence of clinical conditions in the accuracy of these assays. This study aims to evaluate the performance of POCTs for detection of hepatitis B virus surface antigen (HBsAg) and antibodies to Hepatitis C Virus (anti-HCV) in Chronic Kidney Disease (CKD) patients. METHODS: A total of 286 subjects were included in this study. HBsAg and anti-HCV were detected using commercial EIAs and four POCTs: HBsAg (WAMA Imuno-Rápido HBsAg and VIKIA HBsAg) and anti-HCV (DOLES HCV teste rápido and WAMA Imuno-Rápido anti-HCV) in serum and whole blood. RESULTS: Using EIA, HBsAg and anti-HCV prevalence was 4.5% and 16.1% in CKD patients. HBsAg and anti-HCV POCTs had sensitivities from 92.3% to 100% and 84.8% to 89.1% while specificities were 99.3% to 100% and 99.2% to 99.6%, respectively. POCT using serum samples performed well compared with whole blood samples and true positive samples of POCTs had high optical density to cut-off (OD/CO) values compared with EIA. CONCLUSIONS: This study demonstrates good performance of HBsAg and anti-HCV POCTs in CKD patients, especially in serum samples indicating low interference of this disease in the performance of these assays. POCTs could be an important tool for HBV and HCV screening in high-risk populations.

Prevalence of Hepatitis B and C virus infection among alcoholic individuals: importance of screening and vaccination.

Drug users have been reported to have an increased risk for acquisition of viral hepatitis. This study aims to evaluate the prevalence of HBV and HCV infection and usefulness of saliva for HBsAg and anti-HCV detection in alcoholic patients.A total of 90 alcoholic patients were recruited in 2013. HBsAg and anti-HCV were tested in serum and saliva, anti-HBc and anti-HBs were tested in serum using commercial enzyme immunoassays (EIA).Using serum samples, anti-HCV, HBsAg, anti-HBc and anti-HBs prevalences were 5.6%, 0%, 15.7%, and 29.2%. HBsAg detection in saliva showed 100% of specificity and anti-HCV detection demonstrated 100% of sensitivity and 94.7% of specificity. Low prevalence of HBV and high prevalence of anti-HCV were found and reinforced the recommendation of HBV vaccination to avoid the acute and chronic cases and HCV screening in this group to identify cases for antiviral therapy. Saliva samples could be used for anti-HCV detection in this population, what could increase the diagnosis access.

Performance of rapid diagnostic tests for detection of Hepatitis B and C markers in HIV infected patients.

There is little information describing the influence of HIV infection upon the performance of rapid diagnostic tests (RDTs) for hepatitis B and C virus diagnosis. This study aims to evaluate the performance of RDTs for HBsAg and anti-HCV detection among HIV-infected individuals. A total of 362 HIV infected individuals were recruited from clinics between January 2013 to November 2014 in the southeast and northeast of Brazil. HBsAg and anti-HCV were detected using commercial EIAs and four RDTs: HBV (Vikia HBsAg® and Wama Imuno-Rapido HBV®) and HCV (Bioeasy Teste Rápido HCV® and Wama Imuno-Rapido HCV®). Reactive HBsAg and anti-HCV serum samples were tested for HBV DNA and HCV RNA. Sensitivity, specificity and kappa statistic were determined. Using EIA, HBsAg and anti-HCV were detected in 14 (3.9%) and 37 (10.2%) serum samples respectively. Using serum only, HBsAg RDTs demonstrated sensitivities and specificities above 92.0% and Kappa values above 89.0%. Anti-HCV RDTs demonstrated sensitivity and specificities above 82.0% and Kappa higher than 89.0%. Using whole blood samples, Vikia HBsAg® and Wama Imuno-Rapido HCV® showed sensitivity and specificity above 99.0% with Kappa of 66.4% and 100%, respectively. HIV viral load was higher among discordant results for anti-HCV RDT. RDTs demonstrated good performance in HIV infected individuals showing the usefulness of assays in this population.

The gamma subunit of Na+, K+-ATPase: role on ATPase activity and regulatory phosphorylation by PKA.

In kidney, Na+, K+-ATPase is an oligomer (alphabeta gamma) with equimolar amounts of essential alpha and beta subunits and one small hydrophobic FXYD protein (gamma subunit). This report describes gamma subunit as an activator of pig kidney outer medulla Na+, K+-ATPase in aqueous medium. The effects of gamma subunit on Na+, K+-ATPase were dose-dependent and preincubation-dependent. Changes in alphabeta/gamma stoichiometry did not alter Km1 for ATP, and slightly increased Km2, but Vmax was increased at both catalytic and regulatory sites. Hydroxylamine treatment of enzyme phosphorylated by ATP (E-P), in the presence of additional gamma subunit, revealed that 52% of the E-P accumulation was not via acyl-phosphate formation. The gamma subunit was phosphorylated by endogenous kinases and by commercial catalytic subunit of protein kinase A (PKA). Additionally, we demonstrated that PKA phosphorylation of gamma subunit increased its capacity to stimulate ATP hydrolysis. These results suggest that gamma subunit can act as an intrinsic Na+, K+-ATPase regulator in kidney.

Could Na,K-ATPase play a role in potassium leakage from irradiated erythrocytes?

BACKGROUND: In developing countries, the access to red blood cell (RBC) irradiators is restricted. Thus, it is a common practice in blood banks to stock irradiated RBC units until they expire. The aim of this work is to elucidate the involvement of Na,K-ATPase in potassium leakage from prophylactically irradiated RBCs. METHODS: Whole blood was collected from healthy donors, and blood concentrates were irradiated with 25Gy of γ-radiation within 24h of collection. At days 3, 5, 7, 9, 11, 14 and 28 post-irradiation, fractions were removed and centrifuged and Na,K-ATPase activity from ghost membranes was determined. RESULTS: The inhibition of Na,K-ATPase activity in RBCs reached 12.6% by day 7 of storage and up to 50% by day 14 of storage. The addition of vitamin C prevented the irradiation-induced loss of Na,K-ATPase activity. The irradiation of RBCs provoked an increase in potassium plasma levels and a decrease in sodium plasma levels. The incubation of RBCs with ouabain did not change the sodium or potassium levels in the plasma, and the addition of vitamin C only partially prevented a decrease in sodium levels caused by irradiation. CONCLUSION: Because the inhibition of Na,K-ATPase by ouabain did not cause potassium accumulation in the plasma, we conclude that the irradiation-induced inhibition of the pump is not a key factor driving this effect.

Effects of high fat diet on kidney lipid content and the Na, K-ATPase activity

ABSTRACT It is widely known that high fat diet (HFD) can contribute to the advent of health problems. Recent studies have indicated that obesity imposes a hemodynamic overload to the kidneys. In order to further investigate such injuries, two groups of six Swiss mice each were fed with a controlled AIN93G diet or a high fat (AIN93G modified) diet for eight weeks. Blood samples were collected to determine the hormonal, lipid profile, glucose, urea, and creatinine levels. Histopathological and immunohistochemical analysis were carried out to analysis the kidney damage. Fractions of renal membranes were prepared to assess the Na,K-ATPase activity, lipid peroxidation, total cholesterol, and phospholipid content. The results indicated that the blood lipid profile, urea and creatinine was not altered by the HFD. On the other hand, it was observed in HFD diet mice elevated glucose blood levels along with an augment on insulin and a decrease on corticosterone release. HFD provoked a reduction in the diameter of the convoluted tubules and cell volume in Bowman's capsule and an increased number of positive cells with Na,K-ATPase, but reduced the Na,K-ATPase activity and the cholesterol content in the kidney cell membrane but favored the lipid peroxidation.

Bioética e doação de órgãos no Brasil: aspectos éticos na abordagem à família do potencial doador / Bioética y donación de órganos en el Brasil: enfoque ético a la familia del posible donante / Bioethics and organ donation in Brazil: ethical aspects in approaching the potential donor's family

Este artigo consiste em reflexão teórica que pretende identificar questões éticas que poderiam influenciar nas taxas de consentimento de familiares em relação à doação de órgãos. Identificou-se como principal questão ética a falta de esclarecimentos durante o processo de obtenção do consentimento junto ao familiar, sendo identificadas como deficientes as informações acerca do diagnóstico de morte encefálica, a aparência e a desfiguração do corpo. Também se constatou que outros aspectos éticos podem influenciar neste processo, tais como o respeito ao momento que o familiar vivencia, às suas crenças e valores, bem como o acolhimento e a disponibilidade por parte do entrevistador e a garantia de que o familiar tenha escolhido a alternativa mais adequada para a situação. Portanto, ressalta-se a importância de profissionais de saúde capacitados para a abordagem ao familiar, visando, com isto, maior aceitação e uma assistência pautada na ética.

Este artículo es una reflexión teórica que trata de identificar las cuestiones éticas que podrían influir en las tasas de consentimiento de la familia respecto a la donación de órganos. Se identificó como la principal cuestión ética la falta de esclarecimiento en el proceso de obtención del consentimiento de la familia y se identifican como deficientes las informaciones sobre el diagnóstico de muerte cerebral, la apariencia y la desfiguración del cuerpo. También se encontró que otras cuestiones éticas también pueden influir en este proceso, tales como el respeto al momento que está viviendo la familia, sus creencias y valores, así como la acogida y el hecho de que el entrevistador esté disponible y la garantía de que el familiar haya escogido la alternativa más adecuada para la situación. Por lo tanto, debe hacerse hincapié en la importancia de los profesionales de salud capacitados para el acercamiento a la familia, visando así una mayor aceptación y una asistencia pautada en la ética.

This article is a theoretical reflection aiming at identifying the ethical issues that might influence on family?s consent rates regarding organ donation. The lack of clarification during the process of obtaining consent from the family was identified as the main ethical issue and information on brain death diagnosis, corpse?s appearance and disfigurement were identified as deficient. It was found that other ethical issues may also influence this process, such as respect for the moment lived by family member, to his beliefs and values, as well as interviewer?s reception and availability, in addition to the assurance that the family has chosen the most appropriate alternative for the situation. Therefore, one emphasizes the importance of health professionals trained to approach the family, aiming, thus, at greater acceptance and an assistance guided by ethics.