Na-K ATPase Activity Research Articles

7342 Thyrotoxic Periodic Paralysis

Abstract Disclosure: G. Arora: None. Case presentation: A 40 year old male with no significant past medical history presented to the emergency department with diffuse body weakness for one day. On the night prior to admission, his thighs started getting sore which in a matter of a few hours, progressed to weakness involving his entire body. He was unable to flex his hips, shoulders, and neck. He was unable to get up or move without assistance. He was found to have an irregular heartbeat two weeks prior to admission and had undergone a thyroid ultrasound which showed inflammation of the thyroid gland. He was not started on any new medications. He endorsed fatigue, palpitations, neck swelling, and 45 lbs unintentional weight loss over 6 months. No fever, chills, vision changes, congestion, chest pain, shortness of breath, cough, tingling, numbness or weakness. He denied alcohol or drug use. Physical examination was notable for blood pressure 151/80 mm Hg, heart rate 120 bpm, enlarged thyroid gland tender to palpation, and muscle weakness (motor strength 3/5 in flexors and extensors of hips and knees). Labs were notable for K 2.3 (3.5 - 5.1 mmol/L), ALP 218 (32 - 91 U/L), TSH <0.010 (0.400 - 4.300 mcIU/mL), Free T4 3.5 (0.6 - 1.6 ng/dL), CK 608 (49 - 397 U/L). Urine toxicology was negative. MRI spine did not show any acute abnormality except for thyromegaly. He received potassium supplementation which led to symptomatic improvement and was admitted to medical floor. Further tests revealed: TSI 39.50 (<=0.54 IU/L), TSH receptor antibody 26 (<=1.75 IU/L), thyroid microsomal antibody 773.3 (0.0 - 9.0 IU/mL), anti-thyroglobulin antibody 4 (<4 IU/mL). Thyroid ultrasound showed an enlarged, heterogenous, hypervascular thyroid without any nodules. He was started on Propranolol and Propylthiouracil, which led to symptom resolution and he was discharged. Discussion: Thyrotoxic periodic paralysis (TPP) is a rare muscle disorder, belonging to the family of diseases called channelopathies. Thyroid hormone increases tissue responsiveness to beta-adrenergic stimulation, which increases Na-K ATPase activity on the skeletal muscle membrane driving potassium into cells. This leads to hyperpolarization of the muscle membrane and relative inexcitability of the muscle fibers, especially in the presence of a trigger such as provocation with exercise, fasting state or a high carbohydrate meal (insulin stimulates Na-K ATPase pump). Furthermore, more than 95% of TPP cases occur in males. This predominance is due to androgen-induced Na-K ATPase sensitization, more pronounced hyperinsulinemia, comparatively higher sympathetic and catecholamine-induced Na-K ATPase activity and higher muscle mass in males. Given the life-threatening association of severe hypokalemia, TPP should be considered in all patients presenting with acute painless muscle weakness. Presentation: 6/3/2024

Photobiomodulation of Na,K‐ATPase in native membrane fraction and reconstituted in DPPC:DPPE‐liposome

AbstractStudies focusing on how photobiomodulation (PBM) can affect the structure and function of proteins are scarce in the literature. Few previous studies have shown that the enzymatic activity of Na,K‐ATPAse (NKA) can be photo‐modulated. However, the variability of sample preparation and light irradiation wavelengths have not allowed for an unequivocal conclusion about the PBM of NKA. Here, we investigate minimal membrane models containing NKA, namely, native membrane fraction and DPPC:DPPE proteoliposome upon laser irradiation at wavelengths 532, 650, and 780 nm. Interestingly, we show that the PBM on the NKA enzymatic activity has a bell‐shaped profile with a stimulation peak (~15% increase) at around 20 J.cm−2 and 6 J.cm−2 for the membrane‐bound and the proteoliposome samples, respectively, and are practically wavelength independent. Further, by normalizing the enzymatic activity by the NKA enzyme concentration, we show that the PBM response is related to the protein amount with small influence due to protein's environment. The stimulation decays over time reaching the basal level around 6 h after the irradiation for the three lasers and both NKA samples. Our results demonstrate the potential of using low‐level laser therapy to modulate NKA activity, which may have therapeutic implications and benefits.

Abstract PS03-08: Spatial biomechanics determines fate in breast cancer

Abstract Background: With the recent advancements of spatial omics, it has become increasingly recognized that spatial analysis the of tumor architecture is key to decipher the heterogenous intratumoral relationships between different tumor components and provide better understanding of cancer therapy resistance, as well as help to identify potential targets for personalized therapy. The challenge of the discovery and implementation of such biomarkers still lays in the technological and methodological difficulties, and their translation into clinical practice. In parallel a major advancement in cancer research has been the recognition of the importance of cancer biomechanical properties in cancer progression, metastasis, and therapy response. Methods: We developed a new computational platform to identify biomechanical drivers of cancer outcome from spatial omics data. We used it to identify recurring spatial patterns of these markers within the tumor microenvironment, and define the spatial scale of heterogeneity of such patterns. Our dataset consists in 700 primary breast cancer baseline samples analyzed with two 30-marker imaging mass cytometry panels to identify cancer, immune, and stromal cells and structures and their biomechanical states. Our patient cohort includes 20+ years of follow up, with up to 6 samples per patient from 64 patients alive 12 years post diagnosis, and 49 patients lost to breast cancer deaths. Patients were treated with surgery, radiation, chemotherapy, endocrine therapy, or combinations of these post-surgery. Based on this approach, we derived a new three-parameter quantitative metric of preferential spatial co-localization between different cells or structures, and we use this metric to stratify patients into responders and non-responders with the aid of single and multivariate survival analysis. Finally, we include a correlation with Atomic Force Microscopy to identify the mechanical signature of these driving patterns in breast cancer. Results: Our work enabled the identification of tumor-immune-stromal spatial patterns that drive breast cancer outcome and their spatial scale. Among our results, we were able to further define spatial regions of hypoxia-driven EMT. We found these regions to be usually on the scale of 50 mm radius, and enriched in presence of unspecified immune cells. They are also more frequent in ER+ areas, with high NaKATPase activity. Our analysis also shed light on the controversy on the role of fibroblasts. We have found that the presence of a strong, spatially structured stroma, in fibroblast-rich regions is a strong predictor of positive outcome. Similarly, in our analysis collagen cross-linking emerges as a positive control factor in Vimentin-rich microenvironments, offering new interpretation to the role of the ECM structure. Importantly, our first principle approach allows for the identification of driving structures beyond heterogeneity, and thus enables easy extension to additional datasets. Finally, when correlating these patterns with Atomic force microscopy measurements, we can clearly see that patterns predictive of poor survival present a clear heterogeneous stiffness signature, as opposed to a very homogenous good survival pattern signature. Conclusion: These results confirm the importance of spatial distribution of biomechanical drivers in cancer, offering new avenues of physics-based therapeutic targets. Our results also offer a solid base to inform machine learning algorithms on how to significantly parametrize spatial patterns in breast cancer for clinical significance. Furthermore, we demonstrate for the first time the use of Atomic Force Microscopy as a single biomarker for these patterns in breast cancer with a direct correlation based on pathology matching. Citation Format: Sara Nizzero, Maria Pelaez Soni, Gregory Zaugg, Mariam Gachechiladze, Yitian Xu, Licheng Zhang, Junjun Zheng, Brian Menegaz, Lee B Jordan, Colin A Purdie, Philip R Quinlan, Chandandeep Nagi, Karla A Sepulveda, Philipp Oertle, Tobias A Appenzeller, Marko Loparic, Zhihui Wang, Shu-Hsia Chen, Vittorio Cristini, Marija Plodinec, Alastair Thompson. Spatial biomechanics determines fate in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS03-08.

Renal Function and Transporters in the Na/K-ATPase α1S/S Mouse Model of High Affnity for Cardiotonic Steroids

Genetic ablation of ATP1A1 in the mouse renal proximal tubule (RPT) revealed that Na/K-ATPase (NKA) α1 tonically inhibits sodium and water reabsorption. Mechanistically, findings in KO mouse RPT and renal epithelial cells were consistent with a prevalent role of a NKA/Src-dependent downregulation of Na+/H+ exchanger 3 (NHE3). Pharmacologically, functional activation of this pathway has been obtained in vitro and ex vivo using low concentrations of NKA archetypal ligands cardiotonic steroids (CTS). However, the physiological implication of an activation of the endogenous CTS (eCTS)/NKA/Src on renal transporters including NHE3 has not been explored. We assessed the renal impact of CTS/NKA α1 receptor activation by comparing mice expressing the naturally resistant NKA α1 and age-matched mice genetically engineered to express a mutated NKA α1 with high affnity for CTS (R111Q/D122N double substitution). Mice obtained from established colonies at the University of Cincinnati were a gift from Dr. Jerry Lingrel. CTS-sensitive (α1S/S) male mice aged 3-6 months on a C57Bl6 background and age-matched controls (α1R/R) were used. Mice were born with a normal Mendelian ratio, with no observable difference between α1S/S and α1R/R. Renal Na/K-ATPase inhibitory dose-response curve to the CTS ouabain was shifted to the left, as expected, and occurred without detectable change of total renal NKA activity, NKA α1 or β1 protein contents. Metabolic cage studies revealed that increased α1 sensitivity to CTS resulted in a significant increase in urine output in α1S/S mice compared to age-matched α1R/R controls (3.9±0.3 vs. 2.4±0.2 ml/24h; n=6, p<0.01). Consistent with an activation of the NKA/Src receptor by eCTS, western blot analyses of renal cortical preparation revealed an increased phosphorylation of NKA α1 at Y260 (Src target residue) and decreased NHE3 content (43.6 %; n=5, p<0.01). RNA-seq analysis of whole kidneys from α1S/S and α1R/R littermate mice using the Illumina NovaSeq 6000 system (Novogene) and Volcano Plot (DEG data using Python) further showed differential expression of 296 genes (147 upregulated and 149 downregulated). Gene Set Enrichment Analysis (GSEA) using the WebGestalt toolkit showed that the altered pathways were mostly involved in solute transport. Taken together, these results provide genetic evidence of the physiological role of eCTS/NKAα1 on renal function in the intact mouse. Increased affnity to eCTS is suffcient to lead to an activation of NKAα1/Src, a downregulation of NHE3, and transcriptional regulation of renal transporters associated with increased urine output. This work was supported by AHA, Predoctoral fellowship #19PRE34450095, NIH DK129937, and NIH Grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Noradrenaline enhances Na-K ATPase subunit expression by HuR-induced mRNA stabilization and their transportation to the cell surface through PLC and PKC mediated pathway: Implications with REMS-loss associated disorders.

Rapid eye movement sleep (REMS) maintains brain excitability at least by regulating Na-K ATPase activity. Although REMS deprivation (REMSD)-associated elevated noradrenaline (NA) increases Na-K ATPase protein expression, its mRNA transcription did not increase. We hypothesized and confirmed both invivo as well as invitro that elevated mRNA stability explains the apparent puzzle. The mRNA stability was measured in control and REMSD rat brain with or without invivo treatment with α1-adrenoceptor (AR) antagonist, prazosin (PRZ). Upon REMSD, Na-K ATPase α1-, and α2-mRNA stability increased significantly, which was prevented by PRZ. To decipher the molecular mechanism of action, we estimated NA-induced Na-K ATPase mRNA stability in Neuro-2a cells under controlled conditions and by transcription blockage using Actinomycin D (Act-D). NA increased Na-K ATPase mRNA stability, which was prevented by PRZ and propranolol (PRP, β-AR antagonist). The knockdown assay confirmed that the increased mRNA stabilization was induced by elevated cytoplasmic abundance of Human antigen R (HuR) and involving (Phospholipase C) PLC-mediated activation of Protein Kinase C (PKC). Additionally, using cell-impermeable Enz-link sulfo NHS-SS-Biotin, we observed that NA increased Na-K ATPase α1-subunits on the Neuro-2a cell surface. We conclude that REMSD-associated elevated NA, acting on α1- and β-AR, increases nucleocytoplasmic translocation of HuR and increases Na-K ATPase mRNA stability, resulting in increased Na-K ATPase protein expression. The latter then gets translocated to the neuronal membrane surface involving both PKC and (Protein Kinase A) PKA-mediated pathways. These findings may be exploited for the amelioration of REMSD-associated chronic disorders and symptoms.

Ginkgo biloba supplement modulates mTOR/ERK1/2 activities to mediate cardio-protection in cyclosporin-A-induced cardiotoxicity in Wistar rats

BackgroundCyclosporine-A (Cs-A), an immunosuppressant drug indicated for organ transplant patients has been associated with toxicities arising from activation of pathological signalling. Meanwhile Ginkgo biloba supplement (GBS) is an antioxidant-enhancing herbal product widely used for treating neurological and vascular dysfunctions. ObjectivesThis study investigated the ameliorative effect of GBS on cyclosporine-A induced cardiotoxicity in Wistar rats. MethodsThe study included 20 Wistar rats (n = 5) and they were treated as follows: group 1 received distilled water (10 mL/kg), group 2 received Cs-A (25 mg/kg), group 3 received GBS (50 mg/kg) and group 4 received Cs-A (25 mg/kg) and GBS (50 mg/kg). All treatment were done intraperitoneal for 14 days and thereafter, animals were euthanised and heart tissues were harvested for biochemical assays as well as immunohistochemical studies. Blood was also collected for serum biochemical studies. ResultsOur results revealed that GBS reduces Cs-A induced increase of AST, ALT, ALP, LDH, and GGT levels in serum relative to Cs-A group. Also, increased oxido-nitrergic markers like MDA, NO, together with elevated concentrations of cholesterol, triglycerides, LDL, creatine kinase-MB, troponin-I, as well as pro-inflammatory cytokines (IL-6, and TNF-α) and heart weight index were reduced by GBS. The Cs-A induced suppression of low-density lipoprotein, antioxidant (GSH, SOD, CAT), Na-K ATPase activities and antiapoptotic element, Bcl-2 were increased by GBS. Additionally, signalling pathways including ERK1/2, and mTOR as well as ventricular thickness were significantly alleviated by GBS treatment. ConclusionThe findings suggest that GBS ameliorated Cs-A-induced cardiotoxicity by activating mTOR/ERK1/2 signalling pathways.

The Na/K-ATPase role as a signal transducer in lung inflammation.

Acute respiratory distress syndrome (ARDS) is marked by damage to the capillary endothelium and alveolar epithelium following edema formation and cell infiltration. Currently, there are no effective treatments for severe ARDS. Pathologies such as sepsis, pneumonia, fat embolism, and severe trauma may cause ARDS with respiratory failure. The primary mechanism of edema clearance is the epithelial cells' Na/K-ATPase (NKA) activity. NKA is an enzyme that maintains the electrochemical gradient and cell homeostasis by transporting Na+ and K+ ions across the cell membrane. Direct injury on alveolar cells or changes in ion transport caused by infections decreases the NKA activity, loosening tight junctions in epithelial cells and causing edema formation. In addition, NKA acts as a receptor triggering signal transduction in response to the binding of cardiac glycosides. The ouabain (a cardiac glycoside) and oleic acid induce lung injury by targeting NKA. Besides enzymatic inhibition, the NKA triggers intracellular signal transduction, fostering proinflammatory cytokines production and contributing to lung injury. Herein, we reviewed and discussed the crucial role of NKA in edema clearance, lung injury, and intracellular signaling pathway activation leading to lung inflammation, thus putting the NKA as a protagonist in lung injury pathology.

HIF-2α Controls Expression and Intracellular Trafficking of the α2-Subunit of Na,K-ATPase in Hypoxic H9c2 Cardiomyocytes.

The Na,K-ATPase (NKA) pump plays essential roles for optimal function of the heart. NKA activity decreases in necropsy materials from ischemic heart disease, heart failure and in experimental models. Cellular adaptation to hypoxia is regulated by hypoxia-induced transcription factors (HIF); we tested whether HIFs are involved in regulating the expression and intracellular dynamics of the α2-isoform of NKA (α2-NKA). HIF-1α and HIF-2α expression was suppressed in H9c2 cardiomyocytes by adenoviral infection, where cells were kept in 1% O2 for 24 h. The silencing efficiency of HIFs was tested on the mRNA and protein expression. We measured the mRNA expression of α2-NKA in HIF-silenced and hypoxia-exposed cells. The membrane and intracellular expression of α2-NKA was measured after labelling the cell surface with NHS-SS-biotin, immunoprecipitation and Western blotting. Hypoxia increased the mRNA expression of α2-NKA 5-fold compared to normoxic cells in an HIF-2α-sensitive manner. The plasma membrane expression of α2-NKA increased in hypoxia by 2-fold and was fully prevented by HIF-2α silencing. Intracellular expression of α2-NKA was not affected. These results showed for the first time that in hypoxic cardiomyocytes α2-NKA is transcriptionally and translationally regulated by HIF-2α. The molecular mechanism behind this regulation needs further investigation.

Hypoxia and HIF-1α Regulate the Activity and Expression of Na,K-ATPase Subunits in H9c2 Cardiomyoblasts.

The optimal function of the Na,K-ATPase (NKA) pump is essential for the heart. In ischemic heart disease, NKA activity decreases due to the decreased expression of the pump subunits. Here, we tested whether the hypoxia-inducible transcription factor (HIF-1α), the key signaling molecule regulating the adaptation of cells to hypoxia, is involved in controlling the expression and cellular dynamics of α1- and β1-NKA isoforms and of NKA activity in in-vitro hypoxic H9c2 cardiomyoblasts. HIF-1α was silenced through adenoviral infection, and cells were kept in normoxia (19% O2) or hypoxia (1% O2) for 24 h. We investigated the mRNA and protein expression of α1-, β1-NKA using RT-qPCR and Western blot in whole-cell lysates, cell membranes, and cytoplasmic fractions after labeling the cell surface with NHS-SS-biotin and immunoprecipitation. NKA activity and intracellular ATP levels were also measured. We found that in hypoxia, silencing HIF-1α prevented the decreased mRNA expression of α1-NKA but not of β1-NKA. Hypoxia decreased the plasma membrane expression of α1-NKA and β1- NKA compared to normoxic cells. In hypoxic cells, HIF-1α silencing prevented this effect by inhibiting the internalization of α1-NKA. Total protein expression was not affected. The decreased activity of NKA in hypoxic cells was fully prevented by silencing HIF-1α independent of cellular ATP levels. This study is the first to show that in hypoxic H9c2 cardiomyoblasts, HIF-1α controls the internalization and membrane insertion of α1-NKA subunit and of NKA activity. The mechanism behind this regulation needs further investigation.

FRI550 Case Of Thyrotoxic Periodic Paralysis In African American Man With Cannabis Use

Abstract Disclosure: H. Al Jumaili: None. A. Mahaldar: None. S. Campbell: None. A. Gosmanova: None. Introduction: Recent changes in cannabis use (CU) laws led to growing interest in its use for recreational and therapeutic purposes. may have serious health problems such as hypokalemia, paralysis and rhabdomyolysis. Thyrotoxic Periodic Paralysis (TPP) is a rare and life-threatening complication of the thyrotoxicosis characterized by acute reversable painless muscle weakness or paralysis and hypokalemia due to massive intracellular shift of potassium. This condition is mainly seen males of Asian descent. Case: A 31-year-old African American man with Graves’ disease admitted to the hospital for upper and lower extremities weakness after Thanksgiving Day. He woke up with sudden inability to move any of his extremities with. He had high carbohydrate meal, on the evening. Patient reported having similar periodic episodes of paralysis, usually worse in the lower extremities for the last 2 years. This episode lasted longer than usual. On exam, he had enlarged thyroid gland and tachycardia. motor weakness, worse on lower extremities, hyporeflexia and intact sensation. Potassium was 1.8 mmol/l, magnesium 1.5 mg/dl, TSH <0.01 mIU/ml, FT4 4.3 ng/dl, FT3 18.3 ng/dl, anti-thyroid antibodies were elevated at 18 IU/L, and urine screen was positive for cannabinoid. Potassium was replaced, he was given propranolol and continued on methimazole. Patient's paralysis had improved in all four limbs, with serum potassium of 4.1 mmol/L. Discussion: TPP is a rarely seen condition in US but is known in an eastern Asian population, and in US the incidence rate was reported to be around 0.1 to 0.2%. American Indian are thought to be the higher risk. Underlying mechanism is imbalance in potassium homeostasis due to increased cellular Na-K ATPase pump activity with shift of potassium to the intracellular space. There are many theories regarding the cause of exaggerated intracellular potassium influx in TPP. Na-K ATPase activity is directly stimulated by thyroid hormone and insulin response to carbohydrate ingestion which promotes potassium uptake into muscle correlating with clinical observation that paralysis seen after a high carbohydrate meal. KCNJ18 gene mutations which alter the function of an inwardly rectifying potassium channel named Kir2.6 have been detected in some patients. In our patient, additional factor such as cannabis use potentiated intracellular potassium shift and hypokalemia. Recent evidence suggests that the CB1 receptor is expressed not only in the brain, but also found in lungs, heart, and skeletal muscle. Stimulation of (CB1) and activation of G protein-coupled inwardly rectifying potassium (GIRK) channels through intra-cellular signal cascades promotes hypokalemia and paralysis. Conclusion: This case raises awareness of TPP in non-Asian population and highlights the importance to consider cannabis as additional trigger for hypokalemia and paralysis. Presentation: Friday, June 16, 2023

Hypoxic Stress-Dependent Regulation of Na,K-ATPase in Ischemic Heart Disease.

In cardiomyocytes, regular activity of the Na,K-ATPase (NKA) and its Na/K pump activity is essential for maintaining ion gradients, excitability, propagation of action potentials, electro-mechanical coupling, trans-membrane Na+ and Ca2+ gradients and, thus, contractility. The activity of NKA is impaired in ischemic heart disease and heart failure, which has been attributed to decreased expression of the NKA subunits. Decreased NKA activity leads to intracellular Na+ and Ca2+ overload, diastolic dysfunction and arrhythmias. One signal likely related to these events is hypoxia, where hypoxia-inducible factors (HIF) play a critical role in the adaptation of cells to low oxygen tension. HIF activity increases in ischemic heart, hypertension, heart failure and cardiac fibrosis; thus, it might contribute to the impaired function of NKA. This review will mainly focus on the regulation of NKA in ischemic heart disease in the context of stressed myocardium and the hypoxia-HIF axis and argue on possible consequences of treatment.

Assessment of the Effect of C-Peptide Level on Na-K ATPase Activity In Individuals with Type II Diabetic Peripheral Neuropathy

Background: Both Type 1 and Type 2 diabetes can cause neuropathy, which is a frequent and severe consequence. C-peptide depletion may be partly related to in the occurrence of certain diabetic complications. It has been demonstrated that even a little amount of residual C-peptide has a considerable metabolic advantage. Objective: The study’s objective was to predict the relation of plasma C-peptide levels in patients with diabetic neuropathy, and its effect on Na-K ATPase activity. Design and Methods: In this case-control study, 150 individuals have been included: 80 patients with diabetic neuropathy, 40 diabetics without neuropathy and 30 non-diabetic subjects as a control. Patients in the first group were carefully chosen based on their clinical symptoms and nerve conduction studies results. The assessment of plasma C-peptide was done by ELISA, Na-K ATPase enzyme activity by spectrophotometer, and HbA1C by HPLC. Results: Mean plasma C-peptide level and Erythrocyte Na-K ATPase activity were substantially lower in neuropathy type 2 DM patients compared to diabetes without neuropathy and control (p= 0.002, 0.000 respectively). The negative correlation between C-peptide with HbA1c, and diabetes period were all negligible (p= 0.447,0.098), Even though there was a notable negative correlation with age (p= 0.03). On the other hand, the relationship linking C-peptide and Na-K ATPase enzyme activity was shown to be insignificant (p=0.69). Conclusions: Diabetic neuropathy is related to a low C-peptide level. The association between C-peptide and Na-K ATPase enzyme activity, on the other hand, was shown to be insignificant. C-peptide HbA1c, and duration of diabetes all had minor negative associations.

Effects of long-term anti-ischemic drug treatment on Na,K-ATPase isoforms in cardiomyopathic hamsters.

We investigated the effects of long-term anti-ischemic therapy with trimetazidine on Na,K-ATPase (NKA) activity and protein expression in cardiomyopathy. NKA isoforms in membrane fractions from cardiomyopathic hamsters of the BIO 14.6 strain were studied and compared with those from healthy Syrian golden hamsters (F1B). Trimetazidine was orally administered to a subset of cardiomyopathic hamsters in the early stage of active disease (30 days) until the congestive stage (350 days). In the congestive stage of cardiac failure, the cardiomyopathic hamsters displayed altered NKA activity (-55 % vs. F1B; p<0.01), which was related to a specific decrease in abundance of the membrane NKA ?1 isoform (-27 % vs. F1B). Trimetazidine partially prevented the cardiomyopathy-induced changes in NKA activity (+38 %) and ?1 membrane expression (+ 66 %) without inducing changes in the expression of the ?2 isoform or 1 isoform of NKA. Cardiac hypertrophy and remodeling were reduced after trimetazidine treatment. Additionally, the abundance of NKA ?1 in membranes was negatively correlated with the ventricular weight/body weight ratio (an index of cardiac hypertrophy) (r2 = 0.99; p<0.0015). These findings suggest that some of the cardioprotective effect of trimetazidine during long-term cardiomyopathy may be achieved via regulation of cardiac remodeling and selective modulation cardiac NKA isoforms.

Inducible Slc4a11 Knockout Triggers Corneal Edema Through Perturbation of Corneal Endothelial Pump.

PurposeThe conventional Slc4a11 knockout (KO) shows significant corneal edema at eye opening, a fact that complicates the study of the initial events leading to edema. An inducible KO would provide opportunities to examine early events following loss of Slc4a11 activity.Methods Slc4a11 Flox (SF) mice were crossed with mice expressing the estrogen receptor Cre Recombinase fusion protein and fed tamoxifen (Tm) for two weeks. Corneal thickness (CT) was measured by OCT. At eight weeks endpoint, oxidative damage, tight junction integrity, stromal lactate concentration, endothelial permeability, differentially expressed transporters, and junction proteins were determined. Separately, a keratocyte only inducible Slc4a11 KO was also examined.ResultsAt four weeks post-Tm induction Slc4a11 transcript levels were 2% of control. Corneal thickness increased gradually and was 50% greater than Wild Type (WT) after eight weeks with significantly altered endothelial morphology, increased nitrotyrosine staining, significantly higher stromal lactate, decreased expression of lactate transporters and Na-K ATPase activity, higher ATP, altered expression of tight and adherens junctions, and increased fluorescein permeability. No significant differences in CT were found between WT and keratocyte only Slc4a11 KO.ConclusionsThe Slc4a11 inducible KO shows development of a similar phenotype as the conventional KO, thereby validating the model and providing a tool for further use in examining the sequence of cellular events by use of noninvasive in vivo physiological probes.

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 48h with increased concentrations of CTS. The effects of CTS on NKA activity and immunoblotting of α1 and β1 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 (8µM) 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.

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&lt;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.

NaKATPase activity regulated by glycemia level in patients with DM

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Flowerpot method for rapid eye movement sleep deprivation does not induce stress as defined by elevated serum corticosterone level in rats

Flowerpot method of rapid eye movement sleep (REMS) deprivation (REMSD) has been most extensively used in experiments to decipher the functions of REMS. The most common but serious criticism of this method has been presumed stress experienced by the experimental animals. The lack of systematic studies with appropriate controls to resolve this issue prompted this study. We have compared serum corticosterone levels as a marker of stress in male rats under REMSD by the flowerpot method and multiple types of control conditions. Additionally, to maintain consistency and uniformity of REMSD among groups, in the same rats, we estimated brain Na-K ATPase activity, which has been consistently reported to increase upon REMSD. The most effective method was one rat in single- or multiple-platforms set-up in a pool because it significantly increased Na-K ATPase activity without elevating serum corticosterone level. More than one rat in multiple platform set-up was ineffective and must be avoided. Also, large platform- and recovery-controls must be carried out simultaneously to rule out non-specific confounding effects.

Effects of Saint John's Wort extract on intestinal injury and apoptosis

Aim: Reactive oxygen species play an important role in the pathophysiology of intestinal ischemia/reperfusion (I/R) injury by causing apoptosis. The present study aimed at exploring the possible protective effect of Saint John’s Wort (SJW) extract in I/R injury.Materials and Methods: Twenty four healthy male Wistar rats of 6 to 8 weeks old weighting averagely 200 to 250 g were studied. They were fed on standard rat food and drinking water at room temperature with 12/12 hours periods of day and night. SJW (300 mg/kg, peroral) or saline was given by oral route for 3 days prior to ischemia, 30 minutes before the ischemia, and just before reperfusion. To the rats in the control group, sham operation and application of saline were done. Levels of TNF-α, IL-1β and LDH were measured in the serum samples. In the small bowel tissue, levels of malonaldehyde (MDA) and glutathione (GSH) and activity of myeloperoxidase (MPO) and Na-K ATPase and level of caspase-3/β-actine and Bcl-2/β-actine were measured.Results: I/R increased serum levels of LDH, TNF-α and IL-1β, small bowel level of MDA and MPO whereas it decreased level of GSH and activity of Na-K ATPase in the small bowel tissue. The level of caspase-3/β-actine increased while the level of Bcl-2/β-actine decreased in the I/R group compared to the controls. With the application of SJW, these values approached the control levels.Conclusion: These results indicate that SJW recovers small bowel function by reducing oxidation injury during I/R.

A previously undescribed phenylethanoid glycoside from Callicarpa kwangtungensis Chun acts as an agonist of the Na/K-ATPase signal transduction pathway

The new concept that Na/K-ATPase acts as a receptor prompted us to look for new ligands from Callicarpa kwangtungensis Chun. Using column chromatography, an undescribed phenethyl alcohol glycoside, callicarpanoside A, and an undescribed benzyl alcohol glycoside, callicarpanoside B, along with twelve known polyphenols were isolated from Callicarpa kwangtungensis Chun. All the isolated compounds were evaluated for their Na/K-ATPase (NKA) inhibitory activities. Using our NKA technology platform-based screening assay protocols, callicarpanoside B was identified as an undescribed Na/K-ATPase agonist. In particular, the newly identified benzyl alcohol glycoside was found to bind NKA and activate the receptor NKA/Src complex, resulting in the activation of protein kinase cascades. These cascades included extracellular signal-regulated kinases and protein kinase C epsilon, as well as NKA α1 endocytosis at nanomolar concentrations. Unlike the class of cardiotonic steroids, callicarpanoside B showed less inhibition of NKA activity and caused less cellular toxicity. Moreover, callicarpanoside B was found to bind NKA at a different site other than the cardiotonic steroids binding site. Thus, we have identified an undescribed NKA α1 agonist that may be used to enhance the physiological processes of NKA α1 signaling.