Electrical Input Filters of Ganglion Cells in Wild Type and Degenerating rd10 Mouse Retina as a Template for Selective Electrical Stimulation.

Bionic vision systems are currently limited by indiscriminate activation of all retinal ganglion cells (RGCs)- despite the dozens of known RGC types which each encode a different visual message. Here, we use spike-triggered averaging to explore how electrical responsiveness varies across RGC types toward the goal of using this variation to create type-selective electrical stimuli. A battery of visual stimuli and a randomly distributed sequence of electrical pulses were delivered to healthy and degenerating (4-week-old rd10) mouse retinas. Ganglion cell spike trains were recorded during stimulation using a 60-channel microelectrode array. Hierarchical clustering divided the recorded RGC populations according to their visual and electrical response patterns. Novel electrical stimuli were presented to assess type-specific selectivity. In healthy retinas, responses fell into 35 visual patterns and 14 electrical patterns. In degenerating retinas, responses fell into 12 visual and 23 electrical patterns. Few correspondences between electrical and visual response patterns were found except for the known correspondence of ON visual type with upward deflecting electrical type and OFF cells with downward electrical profiles. Further refinement of the approach presented here may yet yield the elusive nuances necessary for type-selective stimulation. This study greatly deepens our understanding of electrical input filters in the context of detailed visual response characterization and includes the most complete examination yet of degenerating electrical input filters.

The protective effect of zinc oxide and selenium oxide nanoparticles on the functional parameters of rat sperm during vitrification.

OBJECTIVE: While sperm freezing (cryopreservation) is an effective method for preserving fertility, it can potentially harm the structure and function of sperm due to an increase in the production of reactive oxygen species. This study aimed to assess the impact of zinc oxide nanoparticles (ZnONPs) and selenium oxide nanoparticles (SeONPs) on various sperm functional parameters, including motility, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), acrosome membrane integrity (ACi), and malondialdehyde (MDA) levels. METHODS: Semen samples were collected from 20 Albino Wistar rats. These samples were then divided into six groups: fresh, cryopreservation control, and groups supplemented with SeONPs (1, 2, 5 µg/mL) and ZnONPs (0.1, 1, 10 µg/mL). RESULTS: Statistical analysis revealed that all concentrations of SeONPs increased total motility and progressive reduction of MDA levels compared to the cryopreservation control group (p<0.05). However, supplementation with ZnONPs did not affect these parameters (p>0.05). Conversely, supplements of 1 and 2 µg/mL SeONPs and 1 µg/mL ZnONPs contributed to the improvement of PMI and ACi (p<0.05). Yet, no significant change was observed in MMP with any concentration of SeONPs and ZnONPs compared to the cryopreservation control group (p>0.05). CONCLUSION: The findings suggest that optimal concentrations of SeONPs may enhance sperm parameters during the freezing process.

Probing the Contribution of Vertical Processing Layers of the Retina to White-Noise Electrical Stimulation Responses.

Optimal stimulus parameters for epiretinal prostheses have been investigated by analyzing retinal ganglion cell (RGC) spiking responses to white-noise electrical stimulation, through a spike-triggered average (STA) analysis technique. However, it is currently unknown as to activation of which retinal cells contribute to features of the STA. We conducted whole-cell patch clamping recordings in ON and OFF RGCs in response to white-noise epiretinal electrical stimulation by using different inhibitors of synaptic transmission in a healthy retina. An mGluR6 agonist, L-AP4, was firstly used to selectively block the output of photoreceptors (PRs) to ON bipolar cells (BCs). We subsequently fully blocked all synaptic inputs to RGCs using a combination of pharmacological agents. Our data shows that PRs dominate the ability of ON RGCs to integrate electrical pulses and form a unique STA shape, while BCs do not contribute in any way. In addition, our results demonstrate that the ability of OFF RGCs to integrate pulses is consistently impaired after blocking the PR to ON BC pathway. We hypothesise that the mechanisms underlying this co-effect are related to the narrow field AII amacrine cells connecting ON and OFF pathways.Clinical Relevance-Recent retinal studies recorded mirror-inverted STAs in ON and OFF retinal pathways, thus raising the possibility of designing a stimulation approach that can differentially activate ON and OFF pathways with electrical stimulation. However, the detailed contribution of three major retinal cell layers in forming characteristic STAs is still unclear. It is of great clinical relevance to investigate the isolated contribution of PRs to the electrically driven STA since PRs progressively degenerate in the course of retinal disease.

Inhibition of altered Orai1 channels in Müller cells protects photoreceptors in retinal degeneration.

The expressions of ion channels by Müller glial cells (MGCs) may change in response to various retinal pathophysiological conditions. There remains a gap in our understanding of MGCs' responses to photoreceptor degeneration towards finding therapies. The study explores how an inhibition of store-operated Ca2+ entry (SOCE) and its major component, Orai1 channel, in MGCs protects photoreceptors from degeneration. The study revealed increased Orai1 expression in the MGCs of retinal degeneration 10 (rd10) mice. Enhanced expression of oxidative stress markers was confirmed as a crucial pathological mechanism in rd10 retina. Inducing oxidative stress in rat MGCs resulted in increasing SOCE and Ca2+ release-activated Ca2+ (CRAC) currents. SOCE inhibition by 2-Aminoethoxydiphenyl borate (2-APB) protected photoreceptors in degenerated retinas. Finally, molecular simulations proved the structural and dynamical features of 2-APB to the target structure Orai1. Our results provide new insights into the physiology of MGCs regarding retinal degeneration and shed a light on SOCE and Orai1 as new therapeutic targets.

Underpinning Learning Theories of Medical Educational Games: A Scoping Review.

Background: Gamification is the process of game thinking and game mechanics to attract learners and solve problems. It is a unique growing phenomenon in education and training programs. Educational games, by application of game design and game elements in learning environments, motivate students to learn and improve the teaching and learning process. Herein, this scoping review presents an overview of the theoretical underpinnings of gamification that is crucial in understanding the theoretical pillar of successful educational games. Methods: This scoping review follows Arksey and O'Malley's stages of scoping review. In this review, the gamification in medical education articles that implicitly or explicitly presented underpinning learning theories of gamification in medical education was retrieved. So, keywords such as gamification, learning theories, higher education, and medical education were searched in Scopus, PubMed, WEB OF SCIENCE, EMBASE, ERIC, and Cochrane Library from 1998 to March 2019. Results: The search indicated 5416 articles which were narrowed down by title and abstract relatedness. 464 articles entered the second phase of the study and after reviewing their full text, finally, 10 articles which were explicitly and implicitly reported the underpinning learning theories remained. Conclusion: Gamification is a strategy using game design techniques for non-game experiences for more effective learning and provides a more attractive environment for teaching and learning. Designing gamification based on learning theories (behavioral, cognitive, and constructivist), makes them more efficient, and the application of learning theories in designing gamification is recommended.

Functional 3-Dimensional Retinal Organoids: Technological Progress and Existing Challenges.

Stem cell scientists have developed methods for the self-formation of artificial organs, often referred to as organoids. Organoids can be used as model systems for research in multiple biological disciplines. Yoshiki Sasai's innovation for deriving mammalian retinal tissue from in vitro stem cells has had a large impact on the study of the biology of vision. New developments in retinal organoid technology provide avenues for in vitro models of human retinal diseases, studies of pathological mechanisms, and development of therapies for retinal degeneration, including electronic retinal implants and gene therapy. Moreover, these innovations have played key roles in establishing models for large-scale drug screening, studying the stages of retinal development, and providing a human model for personalized therapeutic approaches, like cell transplants to replace degenerated retinal cells. Here, we first discuss the importance of human retinal organoids to the biomedical sciences. Then, we review various functional features of retinal organoids that have been developed. Finally, we highlight the current limitations of retinal organoid technologies.

Decreased Na+/K+ ATPase Expression and Depolarized Cell Membrane in Neurons Differentiated from Chorea-Acanthocytosis Patients.

Loss of function mutations of the chorein-encoding gene VPS13A lead to chorea-acanthocytosis (ChAc), a neurodegenerative disorder with accelerated suicidal neuronal cell death, which could be reversed by lithium. Chorein upregulates the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the Na+/K+-ATPase, a pump required for cell survival. To explore whether chorein-deficiency affects Na+/K+ pump capacity, cortical neurons were differentiated from iPSCs generated from fibroblasts of ChAc patients and healthy volunteers. Na+/K+ pump capacity was estimated from K+-induced whole cell outward current (pump capacity). As a result, the pump capacity was completely abolished in the presence of Na+/K+ pump-inhibitor ouabain (100 µM), was significantly smaller in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (24 hours 2 mM). The effect of lithium was reversed by SGK1-inhibitor GSK650394 (24 h 10 µM). Transmembrane potential (Vm) was significantly less negative in ChAc neurons than in control neurons, and was significantly increased in ChAc neurons by lithium treatment (2 mM, 24 hours). The effect of lithium on Vm was virtually abrogated by ouabain. Na+/K+ α1-subunit transcript levels and protein abundance were significantly lower in ChAc neurons than in control neurons, an effect reversed by lithium treatment (2 mM, 24 hours). In conclusion, consequences of chorein deficiency in ChAc include impaired Na+/K+ pump capacity.

A customizable microfluidic platform for medium-throughput modeling of neuromuscular circuits.

Neuromuscular circuits (NMCs) are vital for voluntary movement, and effective models of NMCs are needed to understand the pathogenesis of, as well as to identify effective treatments for, multiple diseases, including Duchenne's muscular dystrophy and amyotrophic lateral sclerosis. Microfluidics are ideal for recapitulating the central and peripheral compartments of NMCs, but myotubes often detach before functional NMCs are formed. In addition, microfluidic systems are often limited to a single experimental unit, which significantly limits their application in disease modeling and drug discovery. Here, we developed a microfluidic platform (MFP) containing over 100 experimental units, making it suitable for medium-throughput applications. To overcome detachment, we incorporated a reactive polymer surface allowing customization of the environment to culture different cell types. Using this approach, we identified conditions that enable long-term co-culture of human motor neurons and myotubes differentiated from human induced pluripotent stem cells inside our MFP. Optogenetics demonstrated the formation of functional NMCs. Furthermore, we developed a novel application of the rabies tracing assay to efficiently identify NMCs in our MFP. Therefore, our MFP enables large-scale generation and quantification of functional NMCs for disease modeling and pharmacological drug targeting.

FUS pathology in ALS is linked to alterations in multiple ALS-associated proteins and rescued by drugs stimulating autophagy.

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and associated with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS' tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small molecules reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a critical role in FUS-ALS and can be treated by stimulating autophagy.

Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin.

BACKGROUND/AIMS: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. METHODS: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. RESULTS: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). CONCLUSION: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition.

Trifluoperazine-Induced Suicidal Erythrocyte Death and S-Nitrosylation Inhibition, Reversed by the Nitric Oxide Donor Sodium Nitroprusside.

BACKGROUND AND PURPOSE: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl)-propyl]-2-(trifluoromethyl)-(10)H-phenothiazine dihydrochloride; TFP) may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i) and inhibited by nitric oxide (NO). We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. RESULTS: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP), an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. CONCLUSIONS: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.

P38 Kinase, SGK1 and NF-κB Dependent Up-Regulation of Na+/Ca2+ Exchanger Expression and Activity Following TGFß1 Treatment of Megakaryocytes.

BACKGROUND: TGFß1, a decisive regulator of megakaryocyte maturation and platelet formation, has previously been shown to up-regulate both, store operated Ca2+ entry (SOCE) and Ca2+ extrusion by Na+/Ca2+ exchange. The growth factor thus augments the increase of cytosolic Ca2+ activity ([Ca2+]i) following release of Ca2+ from intracellular stores and accelerates the subsequent decline of [Ca2+]i. The effect on SOCE is dependent on a signaling cascade including p38 kinase, serum & glucocorticoid inducible kinase SGK1, and nuclear factor NFκB. The specific Na+/Ca2+ exchanger isoforms involved and the signalling regulating the Na+/Ca2+ exchangers remained, however elusive. The present study explored, whether TGFß1 influences the expression and function of K+ insensitive (NCX) and K+ sensitive (NCKX) Na+/Ca2+ exchangers, and aimed to shed light on the signalling involved. METHODS: In human megakaryocytic cells (MEG01) RT-PCR was performed to quantify NCX/NCKX isoform transcript levels, [Ca2+]i was determined by Fura-2 fluorescence, and Na+/Ca2+ exchanger activity was estimated from the increase of [Ca2+]i following switch from an extracellular solution with 130 or 90 mM Na+ and 0 mM Ca2+ to an extracellular solution with 0 Na+ and 2 mM Ca2+. K+ concentration was 0 mM for analysis of NCX and 40 mM for analysis of NCKX. RESULTS: TGFß1 (60 ng/ml, 24 h) significantly increased the transcript levels of NCX1, NCKX1, NCKX2 and NCKX5. Moreover, TGFß1 (60 ng/ml, 24 h) significantly increased the activity of both, NCX and NCKX. The effect of TGFß1 on NCX and NCKX transcript levels and activity was significantly blunted by p38 kinase inhibitor Skepinone-L (1 µM), the effect on NCX and NCKX activity further by SGK1 inhibitor GSK-650394 (10 µM) and NFκB inhibitor Wogonin (100 µM). CONCLUSIONS: TGFß1 markedly up-regulates transcription of NCX1, NCKX1, NCKX2, and NCKX5 and thus Na+/Ca2+ exchanger activity, an effect requiring p38 kinase, SGK1 and NFκB.

The Spatial Extent of Epiretinal Electrical Stimulation in the Healthy Mouse Retina.

BACKGROUND/AIMS: Retinal prostheses use electrical stimulation to restore functional vision to patients blinded by retinitis pigmentosa. A key detail is the spatial pattern of ganglion cells activated by stimulation. Therefore, we characterized the spatial extent of network-mediated electrical activation of retinal ganglion cells (RGCs) in the epiretinal monopolar electrode configuration. METHODS: Healthy mouse RGC activities were recorded with a micro-electrode array (MEA). The stimuli consisted of monophasic rectangular cathodic voltage pulses and cycling full-field light flashes. RESULTS: Voltage tuning curves exhibited significant hysteresis, reflecting adaptation to electrical stimulation on the time scale of seconds. Responses decreased from 0 to 300 µm, and were also dependent on the strength of stimulation. Applying the Rayleigh criterion to the half-width at half-maximum of the electrical point spread function suggests a visual acuity limit of no better than 20/946. Threshold voltage showed only a modest increase across these distances. CONCLUSION: The existence of significant hysteresis requires that future investigations of electrical retinal stimulation control for such long-memory adaptation. The spread of electrical activation beyond 200 µm suggests that neighbouring electrodes in epiretinal implants based on indirect stimulation of RGCs may be indiscriminable at interelectrode spacings as large as 400 µm.

Role of Na+/Ca2+ Exchangers in Therapy Resistance of Medulloblastoma Cells.

BACKGROUND/AIMS: Alterations of cytosolic Ca2+-activity ([Ca2+]i) are decisive in the regulation of tumor cell proliferation, migration and survival. Transport processes participating in the regulation of [Ca2+]i include Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether medulloblastoma cells express Na+/Ca2+-exchangers, whether expression differs between therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells, and whether Na+/Ca2+-exchangers participate in the regulation of cell survival. METHODS: In therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells transcript levels were estimated by RT-PCR, protein abundance by Western blotting, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/ Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free by Na+ free and Ca2+ containing (2 mM) extracellular perfusate as well as cell death from PI -staining and annexin-V binding in flow cytometry. RESULTS: The transcript levels of NCX3, NCKX2, and NCKX5, protein abundance of NCX3, slope and peak of Δ[Ca2+]i as well as Ica were significantly lower in therapy sensitive D283 than in therapy resistant UW228-3 medulloblastoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i, and augmented the ionizing radiation-induced apoptosis but did not significantly modify clonogenicity of medulloblastoma cells. Apoptosis was further enhanced by NCX3 silencing. CONCLUSIONS: Na+/Ca2+-exchanger activity significantly counteracts apoptosis but does not significantly affect clonogenicity after radiation of medulloblastoma cells.

Leucine-Rich Repeat Kinase 2 (Lrrk2)-Sensitive Na+/K+ ATPase Activity in Dendritic Cells.

Leucine-rich repeat kinase 2 (Lrrk2) has been implicated in the pathophysiology of Parkinson's disease. Lrrk2 is expressed in diverse cells including neurons and dendritic cells (DCs). In DCs Lrrk2 was shown to up-regulate Na+/Ca2+-exchanger activity. The elimination of Ca2+ by Na+/Ca2+ -exchangers requires maintenance of the Na+ gradient by the Na+/K+ -ATPase. The present study thus explored whether Lrrk2 impacts on Na+/K+ -ATPase expression and function. To this end DCs were isolated from gene-targeted mice lacking Lrrk2 (Lrrk2-/-) and their wild-type littermates (Lrrk2+/+). Na+/K+ -ATPase activity was estimated from K+ induced, ouabain sensitive, current determined by whole cell patch clamp. Na+/K+ -ATPase α1 subunit transcript and protein levels were determined by RT-qPCR and flow cytometry. As a result, the K+ induced current was significantly smaller in Lrrk2-/- than in Lrrk2+/+ DCs and was completely abolished by ouabain (100 µM) in both genotypes. The K+ induced, ouabain sensitive, current in Lrrk2+/+ DCs was significantly blunted by Lrrk2 inhibitor GSK2578215A (1 µM, 24 hours). The Na+/K+ -ATPase α1 subunit transcript and protein levels were significantly lower in Lrrk2-/- than in Lrrk2+/+ DCs and significantly decreased by Lrrk2 inhibitor GSK2578215A (1 µM, 24 hours). In conclusion, Lrrk2 is a powerful regulator of Na+/K+ -ATPase expression and activity in dendritic cells.

Activation of SGK1 in Endometrial Epithelial Cells in Response to PI3K/AKT Inhibition Impairs Embryo Implantation.

BACKGROUND: Serum & Glucocorticoid Regulated Kinase 1 (SGK1) plays a fundamental role in ion and solute transport processes in epithelia. In the endometrium, down-regulation of SGK1 during the window of receptivity facilitates embryo implantation whereas expression of a constitutively active mutant in the murine uterus blocks implantation. METHODS/RESULTS: Here, we report that treatment of endometrial epithelial cells with specific inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT activity pathway results in reciprocal activation of SGK1. Flushing of the uterine lumen of mice with a cell permeable, substrate competitive phosphatidylinositol analogue that inhibits AKT activation (AKT inhibitor III) resulted in Sgk1 phosphorylation, down-regulation of the E3 ubiquitin-protein ligase Nedd4-2, and increased expression of epithelial Na+ channels (ENaC). Furthermore, exposure of the uterine lumen to AKT inhibitor III prior to embryo transfer induced a spectrum of early pregnancy defects, ranging from implantation failure to aberrant spacing of implantation sites. CONCLUSION: Taken together, our data indicate that the balanced activities of two related serine/threonine kinases, AKT and SGK1, critically govern the implantation process.

LEFTYA Activates the Epithelial Na+ Channel (ENaC) in Endometrial Cells via Serum and Glucocorticoid Inducible Kinase SGK1.

BACKGROUND: Serum & glucocorticoid inducible kinase (SGK1) regulates several ion channels, including amiloride sensitive epithelial Na+ channel (ENaC). SGK1 and ENaC in the luminal endometrium epithelium, are critically involved in embryo implantation, although little is known about their regulation. The present study explored whether SGK1 and ENaC are modulated by LEFTYA, a negative regulator of uterine receptivity. METHODS: Expression levels were determined by qRT-PCR and Western blotting, ENaC channel activity by whole cell patch clamp and transepithelial current by Ussing chamber experiments. RESULTS: Treatment of Ishikawa cells, an endometrial adenocarcinoma model cell line of endometrial epithelial cells, with LEFTYA rapidly up-regulated SGK1 and ENaC transcript and protein levels. Induction of ENaC in response to LEFTYA was blunted upon co-treatment with the SGK1 inhibitor EMD638683. ENaC levels also significantly upregulated upon expression of a constitutively active, but not a kinase dead, SGK1 mutant in Ishikawa cells. LEFTYA increased amiloride sensitive Na+-currents in Ishikawa cells and amiloride sensitive transepithelial current across the murine endometrium. Furthermore, LEFTYA induced the expression of ENaC in the endometrium of wild-type but not of Sgk1-deficient mice. CONCLUSIONS: LEFTYA regulates the expression and activity of ENaC in endometrial epithelial cells via SGK1. Aberrant regulation of SGK1 and ENaC by LEFTYA could contribute to the pathogenesis of unexplained infertility.

Expression of JAK3 Sensitive Na+ Coupled Glucose Carrier SGLT1 in Activated Cytotoxic T Lymphocytes.

BACKGROUND: Similar to tumor cells, activated T-lymphocytes generate ATP mainly by glycolytic degradation of glucose. Lymphocyte glucose uptake involves non-concentrative glucose carriers of the GLUT family. In contrast to GLUT isoforms, Na+-coupled glucose-carrier SGLT1 accumulates glucose against glucose gradients and is effective at low extracellular glucose concentrations. The present study explored expression and regulation of SGLT1 in activated murine splenic cytotoxic T cells (CTLs) and human Jurkat T cells. METHODS: FACS analysis, immunofluorescence, confocal microscopy, chemiluminescence and Western blotting were employed to estimate SGLT1 expression, function and regulation in lymphocytes, as well as dual electrode voltage clamp in SGLT1 ± JAK3 expressing Xenopus oocytes to quantify the effect of janus kinase3 (JAK3) on SGLT1 function. RESULTS: SGLT1 is expressed in murine CTLs and also in human Jurkat T cells. 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose uptake was significantly decreased by SGLT1-blocker phloridzin (0.2 mM) and by pharmacological inhibition of JAK3 with WHI-P131 (156 µM), WHI-P154 (11.2 µM) and JAK3 inhibitor VI (0.5 µM). Electrogenic glucose transport (Iglucose) in Xenopus oocytes expressing human SGLT1 was increased by additional expression of human wild type JAK3, active A568VJAK3 but not inactive K851AJAK3. Coexpression of JAK3 enhanced the maximal transport rate without significantly modifying affinity of the carrier. Iglucose in SGLT1+JAK3 expressing oocytes was significantly decreased by WHI-P154 (11.2 µM). JAK3 increased the SGLT1 protein abundance in the cell membrane. Inhibition of carrier insertion by brefeldin A (5 µM) in SGLT1+JAK3 expressing oocytes resulted in a decline of Iglucose, which was similar in presence and absence of JAK3. CONCLUSIONS: SGLT1 is expressed in murine cytotoxic T cells and human Jurkat T cells and significantly contributes to glucose uptake in those cells post activation. JAK3 up-regulates SGLT1 activity by increasing the carrier protein abundance in the cell membrane, an effect enforcing cellular glucose uptake into activated lymphocytes and thus contributing to the immune response.

Up-Regulation of the Large-Conductance Ca2+-Activated K+ Channel by Glycogen Synthase Kinase GSK3ß.

BACKGROUND/AIMS: The pleotropic functions of the large conductance Ca2+-activated K+ channels (maxi K+ channel or BK channels) include regulation of neuronal excitation and cell volume. Kinases participating in those functions include the glycogen synthase kinase GSK3 ß which is under negative control of protein kinase B (PKB/Akt). GSK3ß is inhibited by the antidepressant Lithium. The present study thus explored whether GSK3ß modifies the activity of BK channels. METHODS: cRNA encoding the Ca2+ insensitive BK channel mutant BKM513I+Δ899-903 was injected into Xenopus laevis oocytes without or with additional injection of cRNA encoding wild-type GSK3ß, inactive K85RGSK3ß, or wild-type GSK3ß with wild-type PKB. K+ channel activity was measured utilizing dual electrode voltage clamp. RESULTS: BK channel activity in BKM513I+Δ899-903 expressing oocytes was significantly increased by co-expression of GSK3ß, but not by co-expression of K85RGSK3ß. The effect of wild type GSK3ß was abrogated by additional co-expression of wild-type PKB and by 24 hours incubation with Lithium (1 mM). Disruption of channel insertion into the cell membrane by brefeldin A (5 µM) was followed by a decline of the current to a similar extent in oocytes expressing BK and GSK3ß and in oocytes expressing BK alone. CONCLUSION: GSK3ß may up-regulate BK channels, an effect disrupted by Lithium or additional expression of PKB and possibly participating in the regulation of cell volume and excitability.

Up-Regulation of Na+/Ca2+ Exchange in Megakaryocytes Following TGFß1 Treatment.

BACKGROUND: Blood platelets are activated by increase of cytosolic Ca2+ activity ([Ca2+]i). Ca2+ entry is accomplished in part by store operated Ca2+ entry (SOCE) involving Ca2+ release activated Ca2+-channel (CRAC) moiety Orai1 and its regulator STIM1, which are stimulated by depletion of intracellular Ca2+ stores. An increase of [Ca2+]i is terminated by Na+/Ca2+-exchange. The expression of both, Orai1 and STIM1 in megakaryocytes is up-regulated by tumor growth factor TGFß1, a powerful regulator of megakaryocyte differentiation. The present study explored whether TGFß1 similarly modifies megakaryocyte Na+/Ca2+-exchanger activity. METHODS: [Ca2+]i was determined utlizing Fura-2 fluorescence, SOCE from increase of [Ca2+]i, following readdition of extracellular Ca2+ after store depletion, and Na+/Ca2+-exchanger activity from increase of [Ca2+]i and whole cell currents following removal of extracellular Na+. RESULTS: TGFß1 treatment not only augments the increase of [Ca2+]i following store depletion and SOCE, but significantly up-regulates Na+/Ca2+-exchanger activity as apparent from [Ca2+]i measurements and whole cell currents. CONCLUSIONS: TGFß1 is a powerful stimulator of both, SOCE and Na+/Ca2+-exchanger activity in megakaryocytes.