ATPase Α1 Research Articles

Inflammatory alveolar macrophage-derived microvesicles damage lung epithelial cells and induce lung injury

Emerging evidence has demonstrated that several microvesicles (MVs) are secreted in bronchoalveolar lavage fluid (BALF) during the pathogenesis of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). However, the impact of alveolar macrophage (AM)-derived MVs on epithelial cells and their in vivo effects on ALI/ARDS require further exploration. In this study, MVs were isolated from BALF of mice or mouse alveolar macrophage (MHS) cells by sequential centrifugation and then delivered to epithelial cells or mice. Enzyme-linked immunosorbent assay revealed that BALF-derived MVs (BALF-MVs) and MHS-derived MVs (AM-MVs) were rich in tumor necrosis factor-α (TNF-α) at the early stage of lung injury. In vitro, both inflammatory BALF-MVs and AM-MVs decreased the expression of α subunit of epithelial sodium channel (α-ENaC), γ-ENaC, and Na+,K+-ATPase α1 and β1 in lung epithelial cells. However, antibodies against TNF-α inhibited the effects of inflammatory AM-MVs in epithelial cells. In vivo, the inflammatory AM-MVs, delivered intratracheally to mice, impaired lung tissues and increased the injury score. They also resulted in decreased alveolar fluid clearance and increased lung wet weight/dry weight ratio. Furthermore, inflammatory AM-MVs downregulated the α-ENaC, γ-ENaC, and Na+,K+-ATPase α1 and β1 levels in lung tissues. According to our results, inflammatory AM-derived MVs may potentially contribute to lung injury and pulmonary edema, thereby indicating a potential novel therapeutic approach against ALI/ARDS based on AM-MVs.

Downregulation of ATP1A1 Expression by Panax notoginseng (Burk.) F.H. Chen Saponins: A Potential Mechanism of Antitumor Effects in HepG2 Cells and In Vivo.

The Na+/K+-ATPase α1 subunit (ATP1A1) is a potential target for hepatic carcinoma (HCC) treatment, which plays a key role in Na+/K+ exchange, metabolism, signal transduction, etc. In vivo, we found that Panax notoginseng saponins (PNS) could inhibit tumor growth and significantly downregulate the expression and phosphorylation of ATP1A1/AKT/ERK in tumor-bearing mice. Our study aims to explore the potential effects of PNS on the regulation of ATP1A1 and the possible mechanisms of antitumor activity. The effects of PNS on HepG2 cell viability, migration, and apoptosis were examined in vitro. Fluorescence, Western blot, and RT-PCR analyses were used to examine the protein and gene expression. Further analysis was assessed with a Na+/K+-ATPase inhibitor (digitonin) and sorafenib in vitro. We found that the ATP1A1 expression was markedly higher in HepG2 cells than in L02 cells and PNS exhibited a dose-dependent effect on the expression of ATP1A and the regulation of AKT/ERK signaling pathways. Digitonin did not affect the expression of ATP1A1 but attenuated the effects of PNS on the regulation of ATP1A1/AKT/ERK signaling pathways and enhanced the antitumor effect of PNS by promoting nuclear fragmentation. Taken together, PNS inhibited the proliferation of HepG2 cells via downregulation of ATP1A1 and signal transduction. Our findings will aid a data basis for the clinical use of PNS.

Comparative description of the mRNA expression profile of Na+ /K+ -ATPase isoforms in adult mouse nervous system.

Mutations in genes encoding Na+/K+‐ATPase α1, α2, and α3 subunits cause a wide range of disabling neurological disorders, and dysfunction of Na+/K+‐ATPase may contribute to neuronal injury in stroke and dementia. To better understand the pathogenesis of these diseases, it is important to determine the expression patterns of the different Na+/K+‐ATPase subunits within the brain and among specific cell types. Using two available scRNA‐Seq databases from the adult mouse nervous system, we examined the mRNA expression patterns of the different isoforms of the Na+/K+‐ATPase α, β and Fxyd subunits at the single‐cell level among brain regions and various neuronal populations. We subsequently identified specific types of neurons enriched with transcripts for α1 and α3 isoforms and elaborated how α3‐expressing neuronal populations govern cerebellar neuronal circuits. We further analyzed the co‐expression network for α1 and α3 isoforms, highlighting the genes that positively correlated with α1 and α3 expression. The top 10 genes for α1 were Chn2, Hpcal1, Nrgn, Neurod1, Selm, Kcnc1, Snrk, Snap25, Ckb and Ccndbp1 and for α3 were Sorcs3, Eml5, Neurod2, Ckb, Tbc1d4, Ptprz1, Pvrl1, Kirrel3, Pvalb, and Asic2.

Disruption of Glutamate Release and Uptake-Related Protein Expression After Noise-Induced Synaptopathy in the Cochlea.

High-intensity noise can cause permanent hearing loss; however, short-duration medium-intensity noise only induces a temporary threshold shift (TTS) and damages synapses formed by inner hair cells (IHCs) and spiral ganglion nerves. Synaptopathy is generally thought to be caused by glutamate excitotoxicity. In this study, we investigated the expression levels of vesicle transporter protein 3 (Vglut3), responsible for the release of glutamate; glutamate/aspartate transporter protein (GLAST), responsible for the uptake of glutamate; and Na+/K+-ATPase α1 coupled with GLAST, in the process of synaptopathy in the cochlea. The results of the auditory brainstem response (ABR) and CtBP2 immunofluorescence revealed that synaptopathy was induced on day 30 after 100 dB SPL noise exposure in C57BL/6J mice. We found that GLAST and Na+/K+-ATPase α1 were co-localized in the cochlea, mainly in the stria vascularis, spiral ligament, and spiral ganglion cells. Furthermore, Vglut3, GLAST, and Na+/K+-ATPase α1 expression were disrupted after noise exposure. These results indicate that disruption of glutamate release and uptake-related protein expression may exacerbate the occurrence of synaptopathy.

Research advances on the role of Na+-K+-ATPase regulation in pulmonary edema clearance of acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a critical disease with high mortality, and currently there is no specific treatment. ARDS is characterized by refractory hypoxemia secondary to pulmonary edema, but the underlying mechanism is not yet fully understood. Alveolar edema fluid is mainly actively transported and reabsorbed by sodium-water transport system. The sodium pump (Na+-K+-ATPase-mediated Na+ transport) on the basal side of type II alveolar epithelial cells (AT II) is the main driving force for pulmonary edema clearance. Na+-K+-ATPase regulation is affected by many regulatory factors through a variety of ways, among which "long-term regulation" mechanism plays an important role, including positively regulating the gene transcription and protein expression of Na+-K+-ATPase. Na+-K+-ATPase can also be degraded by ubiquitin-proteasome pathway (UPP) and autophagy lysosome pathway to affect its abundance and enzyme activity, meanwhile, Na+-K+-ATPase α1 plays a key role in sodium water transport. We review the "long-term regulation" mechanism of Na+-K+-ATPase related pathways in pulmonary edema clearance and explore the possibility of new therapies for ARDS based on this mechanism, so as to provide new targets for the treatment of ARDS.

FXYD6 Regulates Chemosensitivity by Mediating the Expression of Na+/K+-ATPase α1 and Affecting Cell Autophagy and Apoptosis in Colorectal Cancer.

Purpose Chemoresistance is a challenge of improving chemotherapeutic efficacy and prolonging survival time for patients with colorectal cancer (CRC); it is the major cause of frequent recurrence, rapid metastasis, and poor prognosis for CRC patients. FXYD6 is a regulator of Na+/K+-ATPase which is depressed in chemoresistant CRC patients. However, the biological roles of FXYD6 on regulating chemoresistance in CRC are still unclear. Methods GSE3964 and GSE69657 from GEO DataSets were used to analyze the relationship of genes and chemoresistance. The FXYD6 expression level was detected by western blotting and real-time PCR and also analyzed from TCGA DataSet. To investigate the functional role of FXYD6 and ATP-α1, FXYD6 and ATP-α1 functional cell models were constructed. Drug sensitivity and cell proliferation were performed by MTT assay. Autophagy and apoptosis were conducted by autophagy fluorescence analysis and flow cytometric analysis, respectively. Autophagy and apoptosis markers were tested by western blotting. Results FXYD6 was downregulated in CRC resistant patients and irinotecan- (Iri-) resistant SW620 cells (SW620/Iri). FXYD6 silence inhibited cell apoptosis and enhanced prosurvival autophagy, whereas FXYD6 overexpression produced the opposite effect which alleviated the drug resistance to irinotecan and oxaliplatin of CRC cells. FXYD6 regulates chemosensitivity by mediating the expression of Na+/K+-ATPase α1 and affecting cell autophagy and apoptosis in colorectal cancer. Conclusion FXYD6 functions as a chemosensitivity regulator which may predict the curative effect of chemotherapy in colorectal cancer.

Altered mRNA and Protein Expression of Monocarboxylate Transporter MCT1 in the Cerebral Cortex and Cerebellum of Prion Protein Knockout Mice.

The effect of a cellular prion protein (PrPc) deficiency on neuroenergetics was primarily analyzed via surveying the expression of genes specifically involved in lactate/pyruvate metabolism, such as monocarboxylate transporters (MCT1, MCT2, MCT4). The aim of the present study was to elucidate a potential involvement of PrPc in the regulation of energy metabolism in different brain regions. By using quantitative real-time polymerase chain reaction (qRT-PCR), we observed a marked reduction in MCT1 mRNA expression in the cortex of symptomatic Zürich I Prnp−/− mice, as compared to their wild-type (WT) counterparts. MCT1 downregulation in the cortex was accompanied with significantly decreased expression of the MCT1 functional interplayer, the Na+/K+ ATPase α2 subunit. Conversely, the MCT1 mRNA level was significantly raised in the cerebellum of Prnp−/− vs. WT control group, without a substantial change in the Na+/K+ ATPase α2 subunit expression. To validate the observed mRNA findings, we confirmed the observed change in MCT1 mRNA expression level in the cortex at the protein level. MCT4, highly expressed in tissues that rely on glycolysis as an energy source, exhibited a significant reduction in the hippocampus of Prnp−/− vs. WT mice. The present study demonstrates that a lack of PrPc leads to altered MCT1 and MCT4 mRNA/protein expression in different brain regions of Prnp−/− vs. WT mice. Our findings provide evidence that PrPc might affect the monocarboxylate intercellular transport, which needs to be confirmed in further studies.

Extreme winter cold-induced osmoregulatory, metabolic, and physiological responses in European seabass (Dicentrarchus labrax) acclimatized at different salinities

Despite climate-change challenges, for most aquaculture species, physiological responses to different salinities during ambient extreme cold events remain unknown. Here, European seabass acclimatized at 3, 6, 12, and 30 PSU were subjected to 20 days of an ambient extreme winter cold event (8 °C), and monitored for growth and physiological performance. Growth performance decreased significantly (p < 0.05) in fish exposed at 3 and 30 PSU compared to 6 and 12 PSU. During cold stress exposure, serum Na+, Cl−, and K+ concentrations were significantly (p < 0.05) increased in fish exposed at 30 PSU. Serum cortisol, glucose, and blood urea nitrogen (BUN) were increased significantly (p < 0.05) in fish exposed at 3 and 30 PSU. In contrast, opposite trends were observed for serum protein, lactate, and triglycerides content during cold exposure. Transaminase activities [glutamic-pyruvate transaminase (GPT), glutamic oxaloacetic transaminase (GOT), lactic acid dehydrogenase (LDH), gamma-glutamyl-transaminase (γGGT)] were significantly higher in fish exposed at 3 and 30 PSU on days 10 and 20. The abundance of heat shock protein 70 (HSP70), tumor necrosis factor-α (TNF-α), cystic fibrosis transmembrane conductance (CFTR) were significantly (p < 0.05) increased in fish exposed at 3 and 30 PSU during cold shock exposure. In contrast, insulin-like growth factor 1 (Igf1) expression was significantly lower in fish exposed at 3 and 30 PSU. Whereas, on day 20, Na+/K+ ATPase α1 and Na+/K+/Cl− cotransporter-1 (NKCC1) were significantly upregulated in fish exposed at 30 PSU, followed by 12, 6, and 3 PSU. Results demonstrated that ambient extreme winter cold events induce metabolic and physiological stress responses and provide a conceivable mechanism by which growth and physiological fitness are limited at cold thermal events. However, during ambient extreme cold (8 °C) exposure, European seabass exhibited better physiological fitness at 12 and 6 PSU water, providing possible insight into future aquaculture management options.

Controllable Drug Delivery by Na+/K+ ATPase α1 Targeting Peptide Conjugated DSPE-PEG Nanocarriers for Breast Cancer.

Although Epirubicin (EPI) is a commonly used anthracycline for the treatment of breast cancer in clinic, the serious side effects limit its long-term administration including myelosuppression and cardiomyopathy. Nanomedicines have been widely utilized as drug delivery vehicles to achieve precise targeting of breast cancer cells. Herein, we prepared a DSPE-PEG nanocarrier conjugated a peptide, which targeted the breast cancer overexpression protein Na+/K+ ATPase α1 (NKA-α1). The nanocarrier encapsulated the EPI and grafted with the NKA-α1 targeting peptide through the click reaction between maleimide and thiol groups. The EPI was slowly released from the nanocarrier after entering the breast cancer cells with the guidance of the targeting NKA-α1 peptide. The precise and controllable delivery and release of the EPI into the breast cancer cells dramatically inhibited the cells proliferation and migration in vitro and suppressed the tumor volume in vivo. These results demonstrate significant prospects for this nanocarrier as a promising platform for numerous chemotherapy drugs.

The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells

The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells

Immunohistochemical location of Na+, K+-ATPase α1 subunit in the human inner ear

Na+, K+-ATPase (Na,K-ATPase) is an ubiquitous enzyme in the inner ear and a key factor in the maintenance of the osmotic gradient of the endolymph. This study uses Na,K-ATPase α1 subunit immunoreactivity (IR) to identify cellular structures in the normal and disease human cochlea. Formalin-fixed celloidin-embedded (FFCE) human temporal bone sections were immunoreacted with mouse monoclonal antibodies against Na,K-ATPase α1 subunit. Na,K-ATPase α1 IR was examined in the cochlea of 30 patients: four with normal hearing, 5 with Meniere's disease, and 21 with other inner ear diseases: 11 male, 19 female; ages 42 to 96 years-old (yo), average age of 77 yo. Na,K-ATPase α1 IR area was quantified using the ImageJ software program. Na,K-ATPase α1 IR was located in the stria vascularis, and in type I, II and IV fibrocytes of the spiral ligament in the cochlea from patients with normal hearing. Na,K-ATPase α1 IR was seen in Deiters's cells and inner phalangeal cells of the organ of Corti. Na,K-ATPase α1 IR was present in satellite cells that surround the neurons of the spiral ganglia. In the inner ear of pathological specimens, Na,K-ATPase IR area was decreased (compared to the normal) in the stria vascularis, supporting cells in the organ of Corti and satellite cells of the spiral ganglia. These results show that Na,K-ATPase α1 IR is a good marker to identify cellular structures of the human inner ear and may be used to study cellular changes in the cochlea associated with aging and disease. The ubiquitous localization of Na,K-ATPase α1 in the human cochlea is consistent with the Na,K-ATPase role in ionic homeostasis and osmolarity, similar to that seen in animal models.

Migration and fate of vestibular melanocytes during the development of the human inner ear.

Melanocytes are present in various parts of the inner ear, including the stria vascularis in the cochlea and the dark cell areas in the vestibular organs, where they contribute to endolymph homeostasis. Developmental studies describing the distribution of vestibular melanocytes are scarce, especially in humans. In this study, we investigated the distribution and maturation of the vestibular melanocytes in relation to the developing dark cell epithelium in inner ear specimens from week 5 to week 14 of development and in surgical specimens of the adult ampulla. Vestibular melanocytes were located around the utricle and the ampullae of the semicircular canals before week 7 and were first seen underneath the transitional zones and dark cell areas between week 8 and week 10. At week 10, melanocytes made intimate contact with epithelial cells, interrupting the local basement membrane with their dendritic processes. At week 11, most melanocytes were positioned under the dark cell epithelia. No melanocytes were seen around or in the saccule during all investigated developmental stages. The dark cell areas gradually matured and showed an adult immunohistochemical profile of the characteristic ion transporter protein Na+/K+‐ATPase α1 by week 14. Furthermore, we investigated the expression of the migration‐related proteins ECAD, PCAD, KIT, and KITLG in melanocytes and dark cell epithelium. This is the first study to describe the spatiotemporal distribution of vestibular melanocytes during the human development and thereby contributes to understanding normal vestibular function and pathophysiological mechanisms underlying vestibular disorders.

Y-27632 Promotes the Repair Effect of Umbilical Cord Blood-Derived Endothelial Progenitor Cells on Corneal Endothelial Wound Healing.

To investigate the proliferation of umbilical cord blood-derived endothelial progenitor cells (UCB EPCs) and the differentiation efficiency toward corneal endothelial cell (CEC)-like cells induced by rho-associated protein kinase (ROCK) inhibitor Y-27632 and to determine the most effective strategy for repairing corneal endothelium injuries in rabbits. UCB EPCs were cultured in Endothelial Cell Growth Medium-2 (EGM-2) media or conditioned media (CM) from human CECs, with and without the addition of Y-27632. Bromo-deoxyuridine (BrdU) immunocytochemistry and cell counting kit-8 assays were used to examine the proliferation of the cells. Real-time polymerase chain reaction, western blot, and immunocytochemistry were used to detect the CEC markers. Nd:YAG laser was used to establish an appropriate endothelium injury model based on rabbit corneas. The following intracameral injections were then performed to repair the model: 100 μL Opti-MEM I reduced serum medium (model group), 2 × 105 UCB EPCs diluted in 100 μL Opti-MEM I reduced serum medium (EPC group), 100 μM Y-27632 diluted in 100 μL Opti-MEM I reduced serum medium (Y-27632 group), and 2 × 105 UCB EPCs supplemented with 100 μM Y-27632 (final volume 100 μL, EPC/Y-27632 group). The follow-up tests focused on corneal transparency, central corneal thickness, intraocular pressure, and in vivo confocal microscopy, which were performed to evaluate the healing of the wounds. Culturing UCB EPCs in CM supplemented with 10 μM Y-27632 resulted in higher proliferation rates compared with EGM-2 media and CM. There were significantly improved protein levels of Zona Occludens 1, N-cadherin, Na+-K+-ATPase α1, Na+-K+-ATPase β1, and Pax6 and improved mRNA levels of collagen type IV and VIII and AQP1. The combined intracameral injection of Y-27632 and UCB EPCs accelerated the recovery of corneal transparency, regression of corneal edema, and healing of the corneal endothelium compared with the injections of Y-27632 and UCB EPCs on their own. Y-27632 not only promotes the proliferation of UCB EPCs but also contributes to differentiation of UCB EPCs toward CECs in the presence of CM. The intracameral injection of Y-27632 itself promotes the healing of corneal endothelium wounds. On this basis, supplementing UCB EPCs with Y-27632 accelerates the healing of corneal endothelium wounds.

Type 1 Diabetes Induces Hearing Loss: Functional and Histological Findings in An Akita Mouse Model.

The relationship between type 1 diabetes and hearing loss is not well known, although based on many pathological studies, type 2 diabetes induced hearing loss is associated with microcirculation problems in the inner ear. The purpose of this study was to investigate the correlation between type 1 diabetes and hearing loss through hearing function and immunohistochemical analyses using type 1 diabetic Akita or wild-type (WT) mice. The Akita mice had a significant increase in hearing thresholds, blood glucose, and insulin tolerance compared to WT mice. Histological analysis showed that the loss of cells and damage to mitochondria in the spiral ganglion neurons of Akita mice were significantly increased compared to WT. Also, the stria vascularis showed decreased thickness, loss of intermediate cells, and disturbance in blood capillary shape in the Akita mice. Moreover, a reduction in type I, II, and IV fibrocytes and Na+/K+-ATPase α1 expression in spiral ligament was also observed. Cleaved caspase-3 expression was highly expressed in spiral ganglion neurons. In conclusion, hearing loss in type 1 diabetes is caused not only by ion imbalance and blood flow disorders of cochlear endolymph, but through the degenerative nervous system via apoptosis-mediated cell death.

Abstract 6393: Targeting the increased endocytosis of Na/K ATPase α1 subunit as a new therapeutic approach to prostate cancer

Abstract Prostate cancer (PCa) is the second leading cause of cancer-related death in males and there is no effective therapeutics to treat aggressive metastatic PCa (~70% mortality rate). We previously reported that the α1 Na/K-ATPase (α1 NKA), the product of ATP1A1 gene, is a tumor suppressor protein whose expression was significantly reduced in primary tumor and further reduced in the bone metastatic lesions (≤10% of normal tissue). Here we show that loss of cell surface expression of ATP1A1 induces Epithelial Mesenchymal Transition (EMT), and promotes metastatic potential and tumor growth of PCa by decreasing E-cadherin expression and upregulating c-Myc expression via Src-dependent signaling. The progressive loss of α1 NKA in PCa is because of increased endocytosis by activating the α1 NKA/Src receptor complex. Using a unique α1 NKA screening platform, we have discovered MB5, an inverse agonist of receptor α1 NKA that can block ouabain, an agonist-induced signaling and endocytosis of α1 NKA. Functionally, MB5 treatment increased the expression of α1 NKA and E cadherin-which reversed EMT and decreased the invasion and growth of tumor xenografts. Thus, we have revealed a hitherto unrecognized mechanism that regulates the EMT and invasiveness, and identified α1 NKA inverse agonists such as MB5 as a potential drug candidate for the treatment of aggressive PCa. Citation Format: Moumita Banerjee, Zhichuan Li, Yingnyu Gao, Minqi Huang, Liquan Cai, Tianyan Gao, Xie Zijian. Targeting the increased endocytosis of Na/K ATPase α1 subunit as a new therapeutic approach to prostate cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6393.

Expression of Neuronal Na+/K+-ATPase α Subunit Isoforms in the Mouse Brain Following Genetically Programmed or Behaviourally-induced Oxidative Stress

The Na+/K+-ATPase is a transmembrane ion pump that has a critical homeostatic role within every mammalian cell; however, it is vulnerable to the effects of increased oxidative stress. Understanding how expression of this transporter is influenced by oxidative stress may yield insight into its role in the pathophysiology of neurological and neuropsychiatric diseases. In this study we investigated whether increased oxidative stress could influence Na+/K+-ATPase expression in various brain regions of mice. We utilized two different models of oxidative stress: a behavioural chronic unpredictable stress protocol and the Aldh2−/− mouse model of oxidative stress-based and age-related cognitive impairment. We identified distinct regional baseline mRNA and protein expression patterns of the Na+/K+-ATPase α1 and α3 isoforms within the neocortex, hippocampus, and brainstem of wildtype mice. Consistent with previous studies, there was a higher proportion of α3 expression relative to α1 in the brainstem versus neocortex, but a higher proportion of α1 expression relative to α3 in the neocortex versus the brainstem. The hippocampus had similar expression levels of both α1 and α3. Despite increased staining for oxidative stress in higher brain, no differences in α1 or α3 expression were noted in Aldh2−/− mice versus wildtype, or in mice exposed to a 28-day chronic unpredictable stress protocol. In both models of oxidative stress, gene and protein expression of Na+/K+-ATPase α1 and α3 isoforms within the higher and lower brain was remarkably stable. Thus, Na+/K+-ATPase function previously reported as altered by oxidative stress is not through induced changes in the expression of pump isoforms.

Transcription of branchial ion transporter genes in response to high salinity in the amphibious, inter-tidal mudskipper Periophthalmus waltoni

ABSTRACT In this study, we explored the gill mRNA expression ofNa+/K+-ATPase α1 (NKAα1) and Na+/K+/2Cl− cotransporter 1a(NKCC1a) in an amphibious mudskipper species, Periophthalmus waltoniat high salinities of mangrove tidal zones. Fish were exposed to four salinities including 35 (control), 45, 55, and 70 g kg−1. The branchial transcripts of NKAα1 were elevated in fish kept at 55 and 70 g kg−1 salinities with a strong elevation after medium-term (11 days) of exposure. No obvious changes were observed in NKCC1a expression among different salinity groups after short-term (3 days) and long-term (21 days) of exposure, except for a medium-term strong upregulation of NKCC1agene at 55 g kg−1. The NKAα1 and NKCC1a expressions returned to baseline levels after the long term exposure salinity period, suggesting complete molecular and physiological acclimation.These results demonstrate the osmoregulatory role of ion transporter enzymes in acclimation of amphibious fish to high salinities.

Natural cardenolides suppress coronaviral replication by downregulating JAK1 via a Na+/K+-ATPase independent proteolysis

An unprecedented biological function of natural cardenolides independent of their membrane target Na+/K+-ATPase is disclosed. Previously, we reported that cardenolides impart anti-transmissible gastroenteritis coronavirus (anti-TGEV) activity through the targeting of Na+/K+-ATPase and its associated PI3K_PDK1_RSK2 signaling. Swine testis cells with Na+/K+-ATPase α1 knocked down exhibited decreased susceptibility to TGEV infectivity and attenuated PI3K_PDK1_RSK2 signaling. Herein, we further explored a Na+/K+-ATPase-independent signaling axis induced by natural cardenolides that also afforded significant anti-coronaviral activity for porcine TGEV and human HCoV-OC43. Using pharmacological inhibition and gene silencing techniques, we found that this anti-TGEV or anti-HCoV-OC43 activity was caused by JAK1 proteolysis and mediated through upstream activation of Ndfip1/2 and its effector NEDD4. This study provides novel insights into the pharmacological effects of natural cardenolides, and is expected to inform their future development as antiviral agents.

Intravenous Bone Marrow Mononuclear Cells Transplantation in Aged Mice Increases Transcription of Glucose Transporter 1 and Na+/K+-ATPase at Hippocampus Followed by Restored Neurological Functions.

We recently reported that intravenous bone marrow mononuclear cell (BM-MNC) transplantation in stroke improves neurological function through improvement of cerebral metabolism. Cerebral metabolism is known to diminish with aging, and the reduction of metabolism is one of the presumed causes of neurological decline in the elderly. We report herein that transcription of glucose transporters, monocarboxylate transporters, and Na+/K+-ATPase is downregulated in the hippocampus of aged mice with impaired neurological functions. Intravenous BM-MNC transplantation in aged mice stimulated the transcription of glucose transporter 1 and Na+/K+-ATPase α1 followed by restoration of neurological function. As glucose transporters and Na+/K+-ATPases are closely related to cerebral metabolism and neurological function, our data indicate that BM-MNC transplantation in aged mice has the potential to restore neurological function by activating transcription of glucose transporter and Na+/K+-ATPase. Furthermore, our data indicate that changes in transcription of glucose transporter and Na+/K+-ATPase could be surrogate biomarkers for age-related neurological impairment as well as quantifying the efficacy of therapies.

The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells.

Increased metabolic acid production and upregulation of net acid extrusion render pH homeostasis profoundly dysregulated in many cancers. Plasma membrane activity of vacuolar H+ ATPases (V-ATPases) has been implicated in acid extrusion and invasiveness of some cancers, yet often on the basis of unspecific inhibitors. Serving as a membrane anchor directing V-ATPase localization, the a subunit of the V0 domain of the V-ATPase (ATP6V0a1-4) is particularly interesting in this regard. Here, we map the regulation and roles of ATP6V0a3 in migration, invasion, and growth in pancreatic ductal adenocarcinoma (PDAC) cells. a3 mRNA and protein levels were upregulated in PDAC cell lines compared to non-cancer pancreatic epithelial cells. Under control conditions, a3 localization was mainly endo-/lysosomal, and its knockdown had no detectable effect on pHi regulation after acid loading. V-ATPase inhibition, but not a3 knockdown, increased HIF-1α expression and decreased proliferation and autophagic flux under both starved and non-starved conditions, and spheroid growth of PDAC cells was also unaffected by a3 knockdown. Strikingly, a3 knockdown increased migration and transwell invasion of Panc-1 and BxPC-3 PDAC cells, and increased gelatin degradation in BxPC-3 cells yet decreased it in Panc-1 cells. We conclude that in these PDAC cells, a3 is upregulated and negatively regulates migration and invasion, likely in part via effects on extracellular matrix degradation.