Inhibition Of Sodium Pump Research Articles

A synthetic cardiac episode generator for explainable, pathology based action potential using heuristic polynomial signatures

Synthetic generation of biophysical signals caters to diverse applications of test vectors generation, exploration, data augmentation and machine learning training. Arrhythmiator, our contribution is a Python framework to generate synthetically, action potential for explain-ability and explore-ability of pathology (Arrhythmia) influenced cardiac episodes. In this work, we address the problem of large cardiac data-sets need, for deep learning models which are hindered by privacy laws by synthetic generation. We have addressed the problem of explainable computations, multi-scale, by virtue of novel stack sliced polynomial signatures, piece-wise linear and application context driven fitting/filtering in algorithmic framework. To account the problem of lack of standardised pathology specifications we developed PDL (Pathology descriptive language) portal which can be customised. We also addressed the challenge of high turnaround development cycle, inter-operate-ability computation issue by developing a single unified Python framework. To address computational costs, we leverage domain-specific heuristics through sliced state space control algorithms and a constant temporal resolution. We demonstrate the utility of our framework by generating two cases of Tachycardia, one influenced by sodium pump inhibition of spontaneous diastolic depolarisation (SDD) slope and other by ischaemic episode causing maximum diastolic depolarisation (MDP) shift. The concept of the pacemaker hierarchy is encompassed by developing pathology explainable computation as a structure (PECS). We showcase the flexibility and explore-ability of our framework through ease of filter connect profile plug-ins for action potential states upstroke and plateau. We validated the generated waveforms by developing trace and debug algorithms and a reference deviation score which marks the pathology influence. The validator module is a vectored attribute of action potential based on state space control algorithms.

Ceramide-1-Phosphate as a Potential Regulator of the Second Sodium Pump from Kidney Proximal Tubules by Triggering Distinct Protein Kinase Pathways in a Hierarchic Way.

Kidney proximal tubules are a key segment in the reabsorption of solutes and water from the glomerular ultrafiltrate, an essential process for maintaining homeostasis in body fluid compartments. The abundant content of Na+ in the extracellular fluid determines its importance in the regulation of extracellular fluid volume, which is particularly important for different physiological processes including blood pressure control. Basolateral membranes of proximal tubule cells have the classic Na+ + K+-ATPase and the ouabain-insensitive, K+-insensitive, and furosemide-sensitive Na+-ATPase, which participate in the active Na+ reabsorption. Here, we show that nanomolar concentrations of ceramide-1 phosphate (C1P), a bioactive sphingolipid derived in biological membranes from different metabolic pathways, promotes a strong inhibitory effect on the Na+-ATPase activity (C1P50 ≈ 10 nM), leading to a 72% inhibition of the second sodium pump in the basolateral membranes. Ceramide-1-phosphate directly modulates protein kinase A and protein kinase C, which are known to be involved in the modulation of ion transporters including the renal Na+-ATPase. Conversely, we did not observe any effect on the Na+ + K+-ATPase even at a broad C1P concentration range. The significant effect of ceramide-1-phosphate revealed a new potent physiological and pathophysiological modulator for the Na+-ATPase, participating in the regulatory network involving glycero- and sphingolipids present in the basolateral membranes of kidney tubule cells.

The 370-Da Inhibitor of the Sodium Pump in the Plasma of Haemodialysis Patients

Background: Previous studies have shown that a molecule of mass 370 Da that inhibits the sodium pump can be extracted from human placentas and from the concentrated plasma or ultrafiltrate of volume-expanded patients. Aim: This study aimed to study the abundance of the 370-Da molecule and its changes across dialysis in a population of patients with renal failure treated by haemodialysis. Methods: Four millilitres of pre- and post-dialysis blood samples (2 mL plasma) were taken from patients receiving intermittent haemodialysis and analysed by high-performance liquid chromatography coupled to high sensitivity mass spectrometry. Results: In over half of the study population, the 370-Da molecule was present in abundance that exceeded the limit of quantitation. Most patients experienced a marked fall in the abundance of the molecule over a haemodiafiltration session, though exceptions were seen in 2 individuals, both of whom showed clear evidence for the presence of 2 structural isomers of the 370-Da molecule. Conclusions: Advanced renal failure is frequently accompanied by an increased abundance of a 370-Da inhibitor of the sodium pump and that abundance is strongly impacted by haemodialysis. The technique described here could readily be applied to other clinical situations where sodium pump inhibition might be anticipated, such as hypertension, pregnancy, and foetal medicine, and thereby lead to a better understanding of the physiology and pathophysiology of these conditions.

Estimation of Na\K - ATPase Enzyme Activity and Endogenous Digitalis in Patients with Diabetic Neuropathy

Background: Among the most common complications of diabetes is diabetic neuropathy (DN). Diabetic neuropathy is a heterogeneous group of disorders, which involves a different part of somatic and autonomic nervous systems, with a gradual loss of neural conductivity. Some studies have shown that they reduce the activity of the Na/K ATPase, however, elevated levels of endogenous sodium pump inhibitor in diabetic individuals, including those with neuropathy. Changes in this transfer enzyme are believed to be due to several diabetes complications. Objective: The study had designed to evaluate the Na/K ATPase enzymatic activity in the erythrocyte-membrane among three groups. The first group had represented the patients with type 2 diabetes mellitus (DM2) and neuropathy. The second group is diabetics without neuropathy. The third group was a healthy subject. As well, the study had estimated the inhibitory activity of endogenous digitalis among patient groups. Furthermore, the aim of this research was to see whether there was a connection between red blood cell membrane Na-K ATPase activity and the medical facts of the analysis subjects. Design and Methods: One-hundred fifty subjects had enrolled in this case-control study; 80 patients complained of diabetic neuropathy of both sexes, the mean age 59.3 years with an age range of 40-81, 40 DM2 without neuropathy (53.9 years), (35 – 70), and 30 healthy controls (30 years, 25 to 45). Patients in the first group were selected carefully according to their clinical manifestations and the nerve conduction study results. The evaluations of both inhibitory activities of endogenous digitalis and Na/K ATPase had completed using a spectrophotometer. Enzyme activity had expressed in micrograms of phosphate concentration per grams of red cell ghost total protein concentration. Results: The mean enzyme activity of Na/K ATPase was significantly lower (p<0.001) in patients with diabetic neuropathy (381±17.9) compared with the diabetic group without neuropathy (498±22.9) and the normal controls (837±61.43). There was a significant inhibitory activity of endogenous digitals (17.87±2.15) in patients with DNP, compared with the diabetics without neuropathy (8.78±0.89) and healthy control (5.3±1.33). There was a significant association of enzyme activity with the following parameters: duration of diabetes, age, level of glycated hemoglobin and endogenous digitalis with the respective p-values (0.000, 0.000, 0.000 and 0.021). Gender showed no significant relationship with enzyme activity (p 0.43). Conclusions: In DM2 with neuropathy, hyperglycemia can much reduce the activity of erythrocyte Na/K ATPase. In addition, it may enhance the inhibitory activity of endogenous digitals. The timedependent increase in diabetic complications can be due to a strong association between diabetes duration and erythrocyte Na/K-ATPase activities.

Cardiac Glycosides in Human Physiology and Disease: Update for Entomologists.

Cardiac glycosides, cardenolides and bufadienolides, are elaborated by several plant or animal species to prevent grazing or predation. Entomologists have characterized several insect species that have evolved the ability to sequester these glycosides in their tissues to reduce their palatability and, thus, reduce predation. Cardiac glycosides are known to interact with the sodium- and potassium-activated adenosine triphosphatase, or sodium pump, through a specific receptor-binding site. Over the last couple of decades, and since entomologic studies, it has become clear that mammals synthesize endogenous cardenolides that closely resemble or are identical to compounds of plant origin and those sequestered by insects. The most important of these are ouabain-like compounds. These compounds are essential for the regulation of normal ionic physiology in mammals. Importantly, at physiologic picomolar or nanomolar concentrations, endogenous ouabain, a cardenolide, stimulates the sodium pump, activates second messengers, and may even function as a growth factor. This is in contrast to the pharmacologic or toxic micromolar or milimolar concentrations achieved after consumption of exogenous cardenolides (by consuming medications, plants, or insects), which inhibit the pump and result in either a desired medical outcome, or the toxic consequence of sodium pump inhibition.

Cancer cell-specific functional relation between Na,K-ATPase and volume-regulated anion channel

Digitoxin and digoxin are plant-derived cardiac glycosides. They are Na+,K+-ATPase (sodium pump) inhibitors, and have been used clinically for treatment and prevention of heart failure and various tachycardia. On the other hand, some epidemiological studies showed that digoxin users have a lower cancer risk compared to the non-users, and that cancer patients who had been treated with digoxin face on improvement of their survival. In various in vitro studies, cardiac glycosides at sub-μM concentrations, which have no significant effect on enzymatic and ion-transporting activities of Na+,K+-ATPase, show anti-cancer effects. Na+,K+-ATPase is ubiquitously expressed, so it remains unclear why low concentrations of cardiac glycosides have cancer-specific effects. Recently, we found that the receptor-type Na+,K+-ATPase, which has no pumping activity, is associated with leucine-rich repeat-containing 8 family, member A(LRRC8A), one of the components of volume-regulated anion channel (VRAC), in the membrane microdomains of plasma membrane of cancer cells, and that this crosstalk contributes to the inhibition of the cancer cell growth by sub-μM cardiac glycosides. In this mechanism, cardiac glycosides bind to the receptor-type Na+,K+-ATPase, and then stimulate the production of reactive oxygen species (ROS) via NADPH oxidase. The ROS activate VRAC within the membrane microdomains, thus eliciting anti-proliferative effects. VRAC is ubiquitously expressed, and it is normally activated by cell swelling. However, VRAC is activated by cardiac glycoside without cell swelling. On the other hand, the cardiac glycosides-induced effects were not observed in non-cancer cells. Our findings can partly explain why cardiac glycosides elicit selective effects in cancer cells.

Cancer cell-specific functional relation between Na+,K+-ATPase and volume-regulated anion channel

Digitoxin and digoxin are plant-derived cardiac glycosides. They are Na+,K+-ATPase (sodium pump) inhibitors, and have been used clinically for treatment and prevention of heart failure and various tachycardia. On the other hand, some epidemiological studies showed that digoxin users have a lower cancer risk compared to the non-users, and that cancer patients who had been treated with digoxin face on improvement of their survival. In various in vitro studies, cardiac glycosides at sub-μM concentrations, which have no significant effect on enzymatic and ion-transporting activities of Na+,K+-ATPase, show anti-cancer effects. Na+,K+-ATPase is ubiquitously expressed, so it remains unclear why low concentrations of cardiac glycosides have cancer-specific effects. Recently, we found that the receptor-type Na+,K+-ATPase, which has no pumping activity, is associated with leucine-rich repeat-containing 8 family, member A(LRRC8A), one of the components of volume-regulated anion channel (VRAC), in the membrane microdomains of plasma membrane of cancer cells, and that this crosstalk contributes to the inhibition of the cancer cell growth by sub-μM cardiac glycosides. In this mechanism, cardiac glycosides bind to the receptor-type Na+,K+-ATPase, and then stimulate the production of reactive oxygen species (ROS) via NADPH oxidase. The ROS activate VRAC within the membrane microdomains, thus eliciting anti-proliferative effects. VRAC is ubiquitously expressed, and it is normally activated by cell swelling. However, VRAC is activated by cardiac glycoside without cell swelling. On the other hand, the cardiac glycosides-induced effects were not observed in non-cancer cells. Our findings can partly explain why cardiac glycosides elicit selective effects in cancer cells.

Marinobufagenin inhibits glioma growth through sodium pump α1 subunit and ERK signaling-mediated mitochondrial apoptotic pathway.

Malignant glioma is one of the most challenging central nervous system diseases to treat and has high rates of recurrence and mortality. Current therapies often fail to control tumor progression or improve patient survival. Marinobufagenin (MBG) is an endogenous mammalian cardiotonic steroid involved in sodium pump inhibition. Currently, various studies have indicated the potential of MBG in cancer treatments; however, the precise mechanisms are poorly understood. The functions of MBG were examined using colony formation, migration, cell cycle, and apoptosis assays in glioma cells. A mitochondrial membrane potential assay was performed to determine the mitochondrial transmembrane potential change, and cytochrome c release from mitochondria was assayed by fluorescence microscopy. An immunofluorescence assay was performed, and the nuclear translocation of NF‐κB in glioma cells was confirmed by confocal microscopy. Western blotting and RT‐qPCR were used to detect the protein and gene expression levels, respectively. In addition, transfection experiment of ATP1A1‐siRNA was further carried out to confirm the role of sodium pump α1 subunit in the anticancer effect of MBG in human glioma. The apoptosis‐promoting and anti‐inflammatory effects of MBG were further investigated, and the sodium pump α1 subunit and the ERK signaling pathway were found to be involved in the anticancer effect of MBG. The in vivo anticancer efficacy of MBG was also tested in xenografts in nude mice. Thus, therapies targeting the ERK signaling‐mediated mitochondrial apoptotic pathways regulated by MBG might represent potential treatments for human glioma, and this study could accelerate the finding of newer therapeutic approaches for malignant glioma treatment.

Cardiac glycoside bufalin blocks cancer cell growth by inhibition of Aurora A and Aurora B activation via PI3K-Akt pathway.

In our previous study, cardiac glycosides including bufalin, a group of sodium pump (Na+/K+-ATPase) inhibitors widely used to treat heart failure for many years, have been demonstrated to induce a delay of mitotic entry and mitotic arrest in many cancer cells. However, the underlying mechanism remains poorly understood. Here, we reported for the first time that cardiac glycoside bufalin induced mitotic entry delay and prometaphase arrest by inhibition of activation of Aurora A/B. Furthermore, cardiac glycoside bufalin prevented Aurora A recruitment to mitotic centrosomes and Aurora B recruitment to unattached kinetochores. Mechanistically, bufalin and knockdown of sodium pump inhibited PI3K-Akt pathway, which in turn inhibit the activation of Aurora A/B, followed by a delay in mitotic entry and mitotic arrest. These actions were reversed by overexpression of Akt. In addition, ERK, mTOR, and ROS are not involved in bufalin-mediated downregulation of active form of Aurora A/B. Taken together, cardiac glycoside bufalin induces mitotic entry delay and mitotic arrest in cancer cells through inhibition of Aurora A/B activation via PI3K-Akt pathway. Based on this novel finding we could suggest that targeting PI3K-Akt pathway may have therapeutic value for the treatment of cancers associated with sodium pump overexpression.

Endogenous sodium pump inhibitors: common pathogenetic mechanisms of impaired glucose tolerance and preeclampsia

Aim. Study of the influence of sodium chloride overconsumption by pregnant rats with induced diabetes mellitus on the synthesis of marinobufagenin and Na/K-ATPase activity. Methods. The study was performed on 72 female Sprague Dawley rats. Type 2 diabetes mellitus was induced by administration of 65 mg/kg streptozotocin on day 4 after birth. Increased intake of salt was achieved by replacement of drinking water with 1.8% sodium chloride solution on days 12 to 19 of pregnancy. Results. In intact rats, pregnancy was associated with a two-fold increase of marinobufagenin level in the blood and mild impairment of glucose tolerance. Pregnant rats with induced diabetes mellitus apart from fetal macrosomia exhibited greater impairment of glucose tolerance and higher levels of marinobufagenin compared to those in intact pregnant animals. Compared to healthy pregnant rats, salt supplementation for laboratory animals with diabetes was associated with an increase in systolic blood pressure, decreased fetal and placental weight, five-fold elevation of marinobufagenin excretion, and in 42% - with inhibition of Na/K-ATPase activity in erythrocytes. In nonpregnant rats, pre-treatment with anti-marinobufagenin antibodies produced an exaggerated increase of blood levels of glucose and insulin in oral glucose tolerance test. Conclusion. Marinobufagenin is an important factor of pathogenesis of both preeclampsia and diabetes mellitus, and regulation of glucose tolerance may be one of the physiological functions of endogenous cardiotonic steroids.

Mode of Action of the Natural Insecticide, Decaleside Involves Sodium Pump Inhibition.

Decalesides are a new class of natural insecticides which are toxic to insects by contact via the tarsal gustatory chemosensilla. The symptoms of their toxicity to insects and the rapid knockdown effect suggest neurotoxic action, but the precise mode of action and the molecular targets for decaleside action are not known. We have presented experimental evidence for the involvement of sodium pump inhibition in the insecticidal action of decaleside in the cockroach and housefly. The knockdown effect of decaleside is concomitant with the in vivo inhibition of Na+, K+ -ATPase in the head and thorax. The lack of insecticidal action by experimental ablation of tarsi or blocking the tarsal sites with paraffin correlated with lack of inhibition of Na+- K+ ATPase in vivo. Maltotriose, a trisaccharide, partially rescued the toxic action of decaleside as well as inhibition of the enzyme, suggesting the possible involvement of gustatory sugar receptors. In vitro studies with crude insect enzyme preparation and purified porcine Na+, K+ -ATPase showed that decaleside competitively inhibited the enzyme involving the ATP binding site. Our study shows that the insecticidal action of decaleside via the tarsal gustatory sites is causally linked to the inhibition of sodium pump which represents a unique mode of action. The precise target(s) for decaleside in the tarsal chemosensilla and the pathway linked to inhibition of sodium pump and the insecticidal action remain to be understood.

Sodium Pumps Mediate Activity-Dependent Changes in Mammalian Motor Networks.

Ubiquitously expressed sodium pumps are best known for maintaining the ionic gradients and resting membrane potential required for generating action potentials. However, activity- and state-dependent changes in pump activity can also influence neuronal firing and regulate rhythmic network output. Here we demonstrate that changes in sodium pump activity regulate locomotor networks in the spinal cord of neonatal mice. The sodium pump inhibitor, ouabain, increased the frequency and decreased the amplitude of drug-induced locomotor bursting, effects that were dependent on the presence of the neuromodulator dopamine. Conversely, activating the pump with the sodium ionophore monensin decreased burst frequency. When more “natural” locomotor output was evoked using dorsal-root stimulation, ouabain increased burst frequency and extended locomotor episode duration, whereas monensin slowed and shortened episodes. Decreasing the time between dorsal-root stimulation, and therefore interepisode interval, also shortened and slowed activity, suggesting that pump activity encodes information about past network output and contributes to feedforward control of subsequent locomotor bouts. Using whole-cell patch-clamp recordings from spinal motoneurons and interneurons, we describe a long-duration (∼60 s), activity-dependent, TTX- and ouabain-sensitive, hyperpolarization (∼5 mV), which is mediated by spike-dependent increases in pump activity. The duration of this dynamic pump potential is enhanced by dopamine. Our results therefore reveal sodium pumps as dynamic regulators of mammalian spinal motor networks that can also be affected by neuromodulatory systems. Given the involvement of sodium pumps in movement disorders, such as amyotrophic lateral sclerosis and rapid-onset dystonia parkinsonism, knowledge of their contribution to motor network regulation also has considerable clinical importance.SIGNIFICANCE STATEMENT The sodium pump is ubiquitously expressed and responsible for at least half of total brain energy consumption. The pumps maintain ionic gradients and the resting membrane potential of neurons, but increasing evidence suggests that activity- and state-dependent changes in pump activity also influence neuronal firing. Here we demonstrate that changes in sodium pump activity regulate locomotor output in the spinal cord of neonatal mice. We describe a sodium pump-mediated afterhyperpolarization in spinal neurons, mediated by spike-dependent increases in pump activity, which is affected by dopamine. Understanding how sodium pumps contribute to network regulation and are targeted by neuromodulators, including dopamine, has clinical relevance due to the role of the sodium pump in diseases, including amyotrophic lateral sclerosis, parkinsonism, epilepsy, and hemiplegic migraine.

Flavonolignans As a Novel Class of Sodium Pump Inhibitors.

We examined the inhibitory effects of three flavonolignans and their dehydro- derivatives, taxifolin and quercetin on the activity of the Na+/K+-ATPase (NKA). The flavonolignans silychristin, dehydrosilychristin and dehydrosilydianin inhibited NKA with IC50 of 110 ± 40 μM, 38 ± 8 μM, and 36 ± 14 μM, respectively. Using the methods of molecular modeling, we identified several possible binding sites for these species on NKA and proposed the possible mechanisms of inhibition. The binding to the extracellular- or cytoplasmic C-terminal sites can block the transport of cations through the plasma membrane, while the binding on the interface of cytoplasmic domains can inhibit the enzyme allosterically. Fluorescence spectroscopy experiments confirmed the interaction of these three species with the large cytoplasmic segment connecting transmembrane helices 4 and 5 (C45). The flavonolignans are distinct from the cardiac glycosides that are currently used in NKA treatment. Because their binding sites are different, the mechanism of inhibition is different as well as the range of active concentrations, one can expect that these new NKA inhibitors would exhibit also a different biomedical actions than cardiac glycosides.

Fast Nongenomic Effect of Aldosterone on the Volume of Principal Cells in Collecting Tube and Genetic Heterogeneity of Epithelial Sodium Channel in the Postnatal Ontogenesis of Rat Kidney.

The effects of amiloride, epithelial sodium pump inhibitor, on the fast nongenomic effect of aldosterone in principal cells of an isolated segment of the distal portion of renal collecting tubes were studied in 10-day-old and adult rats. Fluorescent staining with Calcein AM showed various effects of amiloride (10(-5) M) on the stabilizing effect of aldosterone (10 nM) in hypotonic shock (280/140 mOsm/kg). Amiloride attenuated by 30% the effect of aldosterone on the amplitude of principal cell swelling in adult animals and almost completely abolished this effect in 10-day rats (p<0.05). These age-specific differences in the contribution of the distal portion of the collecting tube to the nongenomic effect of aldosterone did not depend on genetic heterogeneity of its α-subunit.

Cariprazine delays ouabain-evoked epileptiform spikes and loss of activity in rat hippocampal slices

In the only bipolar cycling in vitro model, rat hippocampal slices are treated with the sodium pump inhibitor ouabain, which induces epileptiform activity, followed by refractory activity loss that recovers and cycles back to epileptiform activity. Thus, clinical cycling seen in patients with bipolar disorder is modeled on a cellular level as alternating hyperactivity and hypoactivity interspersed with normal activity. In this study, we tested the ability of cariprazine a new antipsychotic candidate to block ouabain-induced changes in rat hippocampal slices. Cycling of population spikes and epileptiform bursts was evoked using an extracellular stimulation electrode located in the Schaeffer collaterals of 400-µm-thick rat hippocampal slices treated with ouabain (3.3μM) alone or in combination with cariprazine (1, 5, 25, and 50µM). Responses were recorded using an extracellular electrode placed in the cell body layer of the CA1 region. Cariprazine 25 and 50µM delayed ouabain-induced epileptiform burst onset and subsequent activity loss. Lower cariprazine concentrations were ineffective. Cariprazine delays the onset of ouabain-induced epileptiform bursts and the loss of spiking activity similarly to that previously demonstrated with the mood stabilizer lithium. These results suggest that cariprazine may have therapeutic potential for treatment of bipolar disorder.

Cardiotonic Steroids Inhibit NHE3 in Renal Proximal Tubules by a Src Kinase Dependent Pathway

Cardiotonic steroids (CE) are known as sodium‐pump inhibitors. The natriuretic property of these compounds is related to the inhibition of the α1 isoform of Na+/K+ATPase (NKA) in renal tubules. NHE3, the main route for Na+ and bicarbonate reabsorption in proximal tubules, depends on the Na+ gradient generated by the NKA. The present study aimed to investigate and compare the effects and mechanisms of 50 nM bufalin (BUF), telocinobufagenin (TBG), marinobufagenin (MBG) and ouabain (OUA) on NHE3 activity by the stationary renal microperfusion technique. By this method we are able to measure the H+ secretion in nephron segments through H+ sensitive microelectrodes. We observed that all compounds studied promoted a inhibition of proximal H+ secretion / bicarbonate reabsorption when compared to the control, being BUF the most potent compound (JHCO3‐,nmol.cm‐2.s‐1–control ethanol ‐2,11±0,16(10); BUF 1.04 ± 0.07 (8)*; TBG 0.077 ± 1.36(10)*; OUA 1.48 ± 0.10 (11)* and MBG 1.63 ± 0.08 (10)*. Our data also showed that this effect involved the inhibition of NHE3, since in the presence of NHE3 inhibitor (S3226), BUF did not promote additional inhibition. Furthermore, we demonstrated the involvement of NKA‐Src kinase pathway in the action signaling mechanism of these steroids on NHE3 activity, since in the presence of Src kinase specific inhibitor (SU6656), the inhibitory effect promoted by the CE was abolished.

Oxidative inhibition of erythrocyte sodium pump: A functionally relevant circulating marker of oxidative stress

O stress plays critical roles in the pathogenesis of diabetes and heart disease. The activity of Na pump has been shown to be depressed from the membrane of erythrocyte preparations of these patients coupled with alterations in membrane protein composition. We had previously discovered that the β1 subunit of the Na pump undergoes oxidative modification by oxidative stress in vitro (β1-GSS) and this mediates Na pump inhibition. This study aims to develop and validate erythrocyte β1-GSS as an oxidative stress biomarker in the premier emerging tool for prognosis of pathophysiological oxidative stress in patients with or at risk of cardiovascular disease (CVD) and diabetes. The marker is quickly and easily tested from blood using ELISA; and could be packaged and marketed as a simple kit. The eβ1-GSS biomarker is modified in heart attack, heart failure and diabetes, and exhibits potential to predict disease progression. With further investment, the eβ1-GSS biomarker could be developed initially as a versatile CVD prognosis tool for universal pre-hospital diagnostics, CVD-severity risk-stratification, and as a Companion Diagnostic (Dx) for CVD medications. Subsequently it could be adapted for diabetes.

Abstract 14650: A Monoclonal Antibody to a Sodium Pump Inhibitor Marinobufagenin Reversed Aortic Remodeling and Stiffness in Normotensive Rats on a High Salt Intake

Background: Marinobufagenin (MBG), an endogenous cardiotonic steroid, is a Na/K-ATPase inhibitor and a vasoconstrictor. Previously it was demonstrated, that administration of 3E9 anti-MBG-antibody (mAb) reduced blood pressure (BP) and reversed left ventricular fibrosis in animal models of salt-sensitive hypertension and nephropathy. In the present study we investigated whether mAb alleviates BP and vascular remodeling in normotensive rats on a high salt intake. Methods: Wistar rats (5 months old) received normal salt diet (CTRL; n=8) or high salt intake (2% NaCl in drinking water) for 4 weeks. Rats on a high salt were administered vehicle (SALT; n=8) or mAb (50 ug/kg) (SALT-AB; n=8) 3 times during the last week of a high salt diet. BP was measured at baseline, after 3 and 4 weeks of experiment. Na/K-ATPase activity was measured in erythrocytes. Aortas were weighed, and were used to study sensitivity to the vasorelaxant effect of sodium nitroprusside (SNP), and for the histochemistry analysis of collagen deposition. Renal 24-hr MBG excretion was measured at week 4. Results: In SALT vs. CTRL, in the absence of BP changes, elevated levels of MBG (14.1±1.1 vs. 9.0±1.6 pmol/24hr, p&lt;0.05) were associated with inhibition of erythrocyte Na/K-ATPase (12.6±0.3 vs. 14.2±0.35 μmol Pi/ml/hr, p&lt;0.05), increased aortic weights (217±15 vs. 158±9 mg/kg BW, p&lt;0.01), increased levels of collagen in aorta (2.5-fold; p&lt;0.05), and compromised SNP vasorelaxant effect in aortic explants (EC50=167±19.3 nM vs. 99±2.0 nM; P&lt;0.01). Antibody treatment in SALT-AB vs. SALT increased Na/K-ATPase activity (13.93±0.54 μmol Pi/ml/hr, p&lt;0.05), reduced the aortic weight (180±12 mg/kg; P&lt;0.05) and collagen deposition 3-fold (P&lt;0.05), and restored the vasorelaxation of aortic rings by SNP to the levels in CTRL (70±1.5 nM, p&lt;0.01). Conclusion: These findings for the first time demonstrated that in normotensive rats on a high salt intake heightened MBG levels induced vascular fibrosis and impairment of vasorelaxation in the absence of blood pressure changes. Immunoneutralization of MBG reversed these changes. Thus, high dietary NaCl intake in normotensive animals can stimulate vascular fibrosis via pressure-independent/ MBG-dependent mechanisms, and this remodeling is reversible.

Порівняльний аналіз механокінетики скоротливої активності гладеньких м’язів міометрія щурів за дії каліксарену С-99 та уабаїну

It is known that calix[4]arene with cipher C-99 selectively and with high affinity capable of inhibiting the Na + , K + -ATPase (compared with a known inhibitor of this enzyme – ouabain) of smooth muscle cells plasma membrane preparations. Therefore, the work was carried out comparative study of influence calixarene C-99 and ouabain (both compounds at a concentration of 10 µM) on spontaneous and caused (high-potassium solution, acetylcholine and oxytocin) contractions of the rat uterus longitudinal smooth muscles. Contractile activity studied tensometrically in isometric mode, analysis of the kinetic properties of contractions performed the calculation of the normalized maximal velocity of contraction ( V n с ) and relaxation ( V nr ) phases. Ouabain and calixarene C-99 unidirectional changed the spontaneous contractile activity, causing a significant increase in the amplitude and frequency of contractions. Both compounds are equally causing decrease in maximum force oxytocin-evocked (0.1 IU) contractions, also cau­sing acceleration the growth of force (this is reflected in the increasing parameter V nc ). Also calixarene C-99 and ouabain not change the maximum force of acetylcholine- (10 µM) and K + -induced (80 mM) contractions and do not affect the character of growth of contractile responses ( V nc parameter remains unchanged). The responsiveness of relaxation velocity for the actions ouabain and calixarene C-99 depend on the nature of stimulation: in the case of oxytocin-evocked contractions it decreased and for K + -induced contractions – remained unchanged. Thus, ouabain and calixarene C-99 unidirectional changed the mechanokinetical parameters of spontaneous, agonist- and depolarization-induced contractions of rat myometrium. These results suggest that the effect of calixarene C-99 on the rat uterus smooth muscles is similar to the action of ouabain and is caused by inhibition of plasma membrane sodium pump. Keywords : smooth muscle, uterus, contraction, kinetical parameters, calixarene C-99, plasma membrane Na + , K + -ATPase.

Regulation of neuronal bioenergy homeostasis by glutamate

Bioenergy homeostasis is crucial in maintaining normal cell function and survival and it is thus important to understand cellular mechanisms underlying its regulation. Neurons use a large amount of ATP to maintain membrane potential and synaptic communication, making the brain the most energy consuming organ in the body. Glutamate mediates a large majority of synaptic transmission which is responsible for the expression of neural plasticity and higher brain functions. Most of the energy cost is attributable to the glutamatergic system; under pathological conditions such as stroke and brain ischemia, neural energy depletion is accompanied by a massive release of glutamate. However, the specific cellular processes implicated in glutamate-dependent bioenergy dynamics are not well understood. We find that glutamate induces a rapid and dramatic reduction of ATP levels in neurons, through reduced ATP genesis and elevated consumption. ATP reduction depends on NMDA receptor activity, but is not a result of neuronal firing, gap junction-mediated leaking or intracellular signaling. Similar changes in ATP levels are also induced by synaptic glutamate accumulation following suppression of glutamate transporter activity. Furthermore, the glutamate-induced ATP down-regulation is blocked by the sodium pump inhibitor ouabain, suggesting the sodium pump as the primary energy consumer during glutamate stimulation. These data suggest the important role of glutamate in the control of cellular ATP homeostasis.