bluuebunny/crossref_metadata_2025_split huggingface dataset:
A truncation mutation in human SLC12A2 leads to abnormal goblet cell mucus secretion
Authors: Koumangoye Rainelli , Delpire Eric 
Published: June 2021

The basolateral Na+‐K+‐2Cl− cotransporter (NKCC1) plays a critical role in maintaining the intracellular concentrations of Na+, K+, and Cl− ions, and cell volume. The cotransporter also plays a key role in mediating epithelial fluid secretion. In previous studies, we have described the first known human SLC12A2 (NKCC1) mutation (Cold Spring Harbor Mol. Case Stud. 2(6): a001289, 2016). The now 16‐year‐old patient carries a carboxyl‐terminal truncation mutation (NKCC1‐DFX) in the transporter that leads to complex clinical features including complete gastrointestinal and bladder failure, endocrine insufficiencies, small intestinal dysmotility, seizure‐like episodes, and multi‐organ failure. The 11 bp deletion leads to truncation of the cytosolic COOH‐terminal domain of the cotransporter, making it non‐functional. In polarized epithelial MDCK cells and a mouse model that recapitulates the patient mutation we demonstrated that expression of the truncated Na‐K‐2Cl cotransporter causes mistargeting of the mutant and wild‐type cotransporter to the apical membrane of Cl− secreting epithelia and affects proper lumen formation in MDCK cysts (Am. J. Physiol. Cell Physiol. 315: C258–C276, 2018). Recently due to chronic infections, the patient's colon was surgically resected and lately, the patient has been battling chronic yeast infections in the small intestine. To investigate whether NKCC1‐DFX affect the integrity and function of the gut epithelia, we used MDCK cells expressing the mutant transporter and a mouse model recapitulating the patient mutation. We show that expression of the NKCC1‐DFX mutant impairs tight junction integrity, as indicated by a decrease in trans‐epithelial electrical resistance and inability of MDCK‐I cells expressing NKCC1‐DFX to re‐establish barrier function after a calcium‐switch challenge. Compared to wild‐type mice, NKCC1‐DFX have increased intestinal transit time and displayed an increase in unhealthy mitochondria characterize by dense inclusion bodies. Relative to wild‐type mice, NKCC1‐DFX mice showed decreased expression of Claudin‐2, a tight junction protein involved in paracellular Na+ and water transport. NKCC1‐DFX expression causes defective goblet cells mucus granules exocytosis leading to secretion of intact granules in the lumen of the large intestine. In addition, NKCC1‐DFX colon submucosal glands secrete mucus that remained attached to the epithelium. Taken together, these data indicate that NKCC1‐DFX likely impairs gut barrier function by affecting epithelia tight junction integrity and mucus secretion.Support or Funding InformationSupported by NIH grant GM118944.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcineurin Inhibition Impairs the Function of Neuronal Potassium‐Chloride Cotransporter 2
Authors: Zessin Michael , Strauss Ulf , Deisz Rudolf , Borschewski Aljona , Schindler Maximilian , Endlich Nicole , Loyola Sebastian , Pleinis John , Rodan Aylin , Giesecke Torsten , McCormick James , Mrowka Ralf , Mutig Kerim 
Published: June 2021

Calcineurin inhibitors (CnI) such as Cyclosporin A (CsA) are instrumental for immunosuppression after organ transplantation but may cause serious neurologic side effects, including seizures.Neuronal excitability depends on intact Cl− homeostasis. Generation of hyperpolarizing synaptic responses to GABA and glycine requires a Cl− gradient across the cell membrane with a low [Cl−]i, mainly established by K+‐Cl− cotransporter 2 (KCC2) and to a lesser extent Na+‐K+‐Clcotransporter 1 (NKCC1); both are cation‐coupled chloride cotransporters (CCCs). Calcineurin has been implicated in the regulation of CCCs, whereas impaired CCC function is a well‐known condition in human pharmacoresistant epilepsy. Therefore, we hypothesized that CsA affects KCC2 or NKCC1 functions, thus causing neuronal hyperexcitability.In ex vivo intracellular recordings with sharp microelectrodes, Wistar rat pyramidal neurons in neocortical slices responded to CsA (5 μM for 1h) with a less negative GABAA reversal potential (+7.2 mV) and prolonged Cl− extrusion time after iontophoretic Cl− loading (+3.9 s). 2‐photon fluorescence lifetime imaging in presence of Cl− sensitive dye MQAE showed an increased [Cl−]i in layer V neurons (+6.9 mM), suggesting reduced KCC2 activity. Co‐immunoprecipitation studies in rodent brain tissue suggested interactions of calcineurin Aβ (CnAβ) with KCC2 and NKCC1.In vivo, CsA administration to rats (5–25 mg/kg i.p.) enhanced levels of inhibitory tyrosine KCC2 phosphorylation at short term (4h; +172%) and reduced levels of activating S940‐KCC2 phosphorylation in the long term (14d; −61%). Reduced phospho‐S940‐KCC2 levels were observed in further models of calcineurin inhibition such as genetic CnAβ‐deficiency in mice (−78%) or CsA treatment of zebrafish larvae (10 μM in water: −54% after 24h). In contrast, NKCC1 and its activating kinase SPAK were stimulated upon calcineurin inhibition in rodents. Similar data were obtained in Drosophila melanogaster.Our results provide evidence that CnI attenuate KCC2 function but may stimulate NKCC1 leading to elevated [Cl−]i. These effects may increase neuronal excitability and contribute to CnI neurological adverse effects. The benefits of KCC2 activators or SPAK inhibitors for enhancing chloride extrusion in patients experiencing CnI neurotoxicity deserve further characterization.Support or Funding InformationThis study was supported by Deutsche Forschergemeinschaft.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Adipogenesis by α1 Na+/K+‐ATPase via Its Conserved Caveolin Binding Motif
Authors: Huang Minqi , Cai Liquan , Pierre Sandrine , Xie Zijian 
Published: June 2021

In addition to its role as an ion pump, Na/K‐ATPase (NKA) α1 isoform forms a signal receptor complex with the non‐receptor tyrosine kinase Src and the scaffolding protein caveolin‐1. Stimulation of this receptor leads to an activation of Src/ERK cascade and an increase in generation of reactive oxygen species (ROS). Pharmacological inhibition of the NKA‐mediated signal transduction has been shown to inhibit adipogenesis and high fat diet‐induced obesity. However, there is no genetic evidence supporting a role of α1 NKA in the regulation of adipogenesis. To address this issue in a model with relevance to human phtsiology, we employed CRISPR/Cas9 system to mutate a conserved caveolin‐binding motif (F97A and F100A) in the gene coding for α1 NKA (ATP1A1), and measured the effects of this loss‐of‐function mutation on pathways relevant to adipogenesis during the differentiation of human induced pluripotent stem cells (iPSC) to adipocytes. We found that the CBM mutant human iPSC are capable of differentiation into adipocytes. However, Oil Red O staining indicated a significant reduction in lipid accumulation during adipocyte maturation in the mutant cells. This observation was confirmed by electron microscopy imaging and by changes in the expression of adipocyte marker genes. Mechanistically, the loss of CBM in ATP1A1 resulted in alteration in mitochondrial structure and function observed by electron microscopy imaging, mitochondrial ROS generation, membrane potential, biogenesis, and marker genes expression by RT‐qPCR. This was further validated by metabolic profiling by Seahorse Extracellular Flux Analysis and RNA sequencing followed by KEGG enrichment analysis of metabolic pathways. Taken together, these results indicate an important role of NKA‐mediated signal transduction in adipogenesis through a regulation of mitochondrial structure and function. Thus, we suggest that NKA‐mediated signal transduction may serve as a new target for developing therapeutic avenues in the management of obesity and metabolic syndromes.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Na+/K+‐ATPase α1 and Cardiac Resilience
Authors: Marck Pauline V. , Xu Yunhui , King Cierra , Perdoncin Maddie , Yan Yanling , Blanco Gustavo , Cai Liquan , Lingrel Jerry , Xie Zijian , Pierre Sandrine V. 
Published: June 2021

Systemic levels of endogenous cardiotonic steroids (CTS) vary in response to physiological and pathophysiological stresses such as exercise, pregnancy, hypertension, and heart failure. Ample experimental evidence has shown that exposure to CTS modulates cardiac Na/K‐ATPase (NKA) α1 ion‐pumping and non‐ion‐pumping functions, which profoundly affects cardiac contractility and structure. However, the physiological role of CTS as a group of hormones with distinct but related modulatory effects on cardiovascular equipoise and dysfunction remains poorly understood. We used 2 genetically engineered mouse models to test the hypothesis that the endogenous CTS/NKA α1 interaction confers cardiac resilience during a sustained hemodynamic challenge.Wild type mice expressing the relatively CTS‐insensitive NKA α1 and the CTS‐sensitive NKA α2 (α1r/rα2s/s), mice expressing NKA α1 mutated to CTS‐sensitivity (α1s/sα2s/s), and mice with cardiomyocyte‐specific NKA α1 knockout (α1−/−α2s/s) were treated with a pressor dose of Angiotensin II (Ang II) of 1.5 mg.kg−1. d−1 for 14 days via osmotic minipumps to induce adverse cardiac remodeling.All mice were born with the expected Mendelian frequency and survived to adulthood. In the α1s/sα2s/s mouse, total cardiac expression of NKA α1 and α2 assessed by western blotting and ouabain‐sensitive ATPase activity were unchanged, and the expected increased affinity for the CTS ouabain was detected. NKA expression and activity in the α1−/−α2s/s heart was consistent with a nearly complete ablation of NKA α1, partially compensated by an upregulation of NKA α2. Ang II or vehicle treatment was administered to 3 month‐old males (n=6–8/group), and cardiac remodeling was assessed by gravimetry, histology, and qPCR. In contrast to wild‐type α1r/rα2s/s, the Ang II treatment did not induce any significant cardiac hypertrophy or fibrosis in α1s/sα2s/s hearts. Conversely, the Ang II treatment was lethal in α1−/−α2s/s mice, with a 70% mortality rate at 8 days. A shorter (7 days) Ang II treatment revealed massive cardiac fibrosis (50% increase compared to α1r/rα2s/s). Taken together, these results support a physiological role of endogenous CTS in cardiac resilience through an interaction with the signaling cardiac NKA α1 isoform.Support or Funding InformationN/AThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylations in the Termini of Mitochondrial Calcium Uniporter Regulate Mitochondrial Calcium Uptake
Authors: O‐Uchi Jin , Cao Jessica L , Adaniya Stephanie M , Jhun Bong Sook , Sheu Shey Shing 
Published: June 2021

Mitochondrial Ca2+ (mtCa2+) uptake via the mtCa2+ uniporter (MCU) mediates cell survival and death. Our group previously showed that activation and mitochondrial translocation of proline‐rich tyrosine kinase 2 (Pyk2), tyrosine phosphorylation (P‐Tyr) of MCU, and enhancement of mtCa2+ uptake concomitantly occur in cardiomyocytes under Gq protein–coupled receptor (GqPCR) stimulation, which activates apoptotic myocyte death. However, it is still unclear how Pyk2 access to MCU structure, phosphorylate MCU, and modulate its channel function as well as mitochondrial Ca2+ uptake profile. Here, we determined Pyk2‐specific P‐Tyr site(s) in MCU and investigated the detailed molecular mechanism underling the regulation of mtCa2+ uptake via P‐Tyr of MCU. Through computational prediction programs for phosphorylation sites, we identified three Tyr residues (Y158, Y289, and Y317) in human MCU as candidates for general PTK phosphorylation sites, which are highly conserved across all eukaryotic species. P‐Tyr levels at these candidate sites were biochemically determined before and after the stimulation of a GqPCR in HEK293T cells stably overexpressing wild type (WT)‐ or dephosphomimetic mutants of MCU (MCU‐YFs). Only two Tyr sites (Y517F at the N‐ and Y317 C‐termini of MCU) exhibited increased P‐Tyr levels in response to GqPCR stimulation. Overexpression of MCU‐Y317F failed to increase mtCa2+ uptake in response to cytosolic Ca2+ elevation, although the overexpression of WT‐ and the other two MCU‐YFs significantly augmented mtCa2+ uptake compared to non‐transfected cells. Furthermore, we also found that MCU‐Y317F‐flag exhibits less association with WT‐MCU‐GFP compared to those observed in WT‐MCU‐flag, indicating reduced subunit oligomerization by this point mutation. Finally, we demonstrated that increased in P‐Tyr of MCU levels occurs in the ventricle tissues from transgenic (TG) mice with cardiac‐specific overexpression of a constitutively active Gαq protein (Gαq‐Q209L TG mice) compared to those from WT, concomitant with higher Pyk2 activation and apoptotic signaling. In summary, we conclude that MCU contains Pyk2‐specific phosphorylation site(s) and Pyk2‐dependent P‐Tyr of MCU at these site(s) increases mitochondrial Ca2+ uptake possibly via enhanced subunit oligomerization.Support or Funding InformationJ.O.‐U. was supported by NIH/NHLBI R01HL136757, AHA 16SDG27260248, Rhode Island Foundation #20164376 Medical Research Grant, and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award. J.L.C. and S.M.A were supported by Undergraduate Teaching & Research Award (UTRA) from Brown University. B.S.J was supported by 18CDA34110091. S.S.S was supported by NIH/NHLBI R01HL093671 and R01HL122124.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reveal the Role of Store‐Operated Calcium Entry in Epirubicin‐induced Acute Oxidative Stress in Cardiomyocytes
Authors: Liu Xian , Pan Zui 
Published: June 2021

Epirubicin (EPI) is a widely used anthracycline chemotherapy drug, whose use is limited by its side effects including cardiomyopathy, hypertrophy and chronic heart failure. The cardiotoxicity of EPI has been linked to the generation of reactive oxygen species (ROS) in mitochondria and disruption of cardiac ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum membrane. As Store‐Operated Calcium Entry (SOCE) is important in regulating intracellular Ca2+ signaling, we tested whether dysregulated SOCE was also involved in EPI‐induced acute cardiotoxicity. This study compared intracellular ROS levels using fluorescent probe dihydroethidium (DHE) in EPI‐treated HL‐1, an adult mouse atrial cardiomyocyte cell line. Treatment of EPI at 1uM for 30min significantly increased intracellular ROS levels in HL‐1 compared with vehicle‐treated cells (3.8fold, p<0.0001). HL‐1 cells were then treated with 20uM BTP2, a potent SOCE inhibitor, for 30min. However, BTP2 failed to significantly decrease the EPI‐induced intracellular ROS (p=0.9987, n=8 replicates). In addition, 50uM ethylene glycol‐bis (β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester (EGTA‐AM) failed to prevent EPI‐induced ROS increase (p=0.9768, n=8 replicates). These results might be limited by the technical challenge such as DHE not able to quantitatively distinguish intracellular ROS levels and EGTA‐AM might not be fast enough to chelate Ca2+ from Ca2+ influx. Ongoing project will seek to overcome the technical limitation to further test the role of SOCE in EPI‐induced acute cardiotoxicity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of a new Dent Disease mouse model carrying a pathogenic mutation of ClC‐5
Authors: Sakhi Imene , Bignon Yohan , Frachon Nadia , Lourdel Stephane 
Published: June 2021

Dent Disease is a rare X‐linked inherited renal proximal disorder characterized by low molecular weight (LMW) proteinuria, hypercalciuria, kidney stones and progressive renal failure. Inactivating mutations of the CLCN5 gene encoding the 2Cl−/H+ exchanger ClC‐5 have been identified in approximately two‐thirds of patients with Dent Disease. ClC‐5 is predominantly expressed in early endosomes of proximal tubules where it optimizes the function of a vacuolar H+ pump to ensure an efficient endocytosis of LMW proteins.We have previously investigated the functional consequences of CLCN5 mutations in heterologous expression systems and shown that 60 % of the mutations lead to a defect in protein folding and processing. As a consequence, the misfolded ClC‐5 are retained within the endoplasmic reticulum (ER) where they could induce ER stress response. Such ER stress might in turn induce oxidative stress in proximal tubule cells. To investigate the consequences of intracellular retention of a misfolded ClC‐5, we created a mouse model carrying the N340K pathogenic mutation.ClC‐5 expression and localization in WT and N340K male mice were studied by Western blot and on kidney sections by immunofluorescence. Daily food, water intake, body weight and urine excretion data were collected in metabolic cages for N340K and WT mice. Urine as well as blood samples were analysed for ions, protein, glucose and creatinine to detect kidney dysfunction as observed in patients with Dent Disease.Mice carrying the N340K mutation displayed ClC‐5 intracellular retention in proximal tubules cells. They showed an increased urinary calcium and glucose excretion and a decreased urinary pH. They also had a severe LMW proteinuria, recapitulating common features of Dent Disease.The present study validates a new Dent Disease mouse model carrying a pathogenic Clcn5 mutation retained in the ER. The potential link between ClC‐5 retention in the ER, ER stress and oxidative stress responses has yet to be demonstrated.Support or Funding InformationPrix “Jeune Chercheur”, Fondation du Rein 2017This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological Inhibition of Glucose‐6‐phosphate Dehydrogenase Produces Inhibition of L‐type Calcium Channel Current in Arterial Smooth Muscle Cells
Authors: Ochi Rikuo A. , Chettimada Sukrutha , Kizub Igor , Gupte Sachin A. 
Published: June 2021

Glucose‐6‐phosphate dehydrogenase (G6PD) is the first enzyme in pentose phosphate pathway (PPP). G6PD is involved in cellular redox regulation by reducing NADP+ to NADPH, a primary cellular reducing agent, and PPP produces ribose 5‐phosphate, a substrate necessary for nucleotide synthesis. We used two inhibitors of G6PD, 6‐aminonicotinamide (6‐AN) and dehydroepiandrosterone (DHEA), to clarify involvement of G6PD in the regulation of L‐type Ca2+ channel current (ICa,L) in arterial smooth muscle cells (ASMCs). 6‐AN is converted to 6‐ANADP in the cell to competitively inhibit NADP+ binding to G6PD. DHEA is an adrenal steroid hormone with the highest serum concentration among steroid hormones with its sulfate ester DHEAS. DHEA inhibits G6PD in a non‐competitive manner. The potency of DHEAS to inhibit G6PD is about 1/100 of DHEA. DHEA induced relaxation of high K+‐induced contraction in arterial strips excised from carotid artery and aorta of rat in a dose‐dependent manner. In contrast, DHEAS did not produce the relaxation. In previous meetings we reported that DHEA inhibits ICa,L in voltage‐dependent and ‐independent mechanisms in ASMCs. 6‐AN produced voltage‐independent inhibition of ICa,L recorded with 10 mM Ba2+ as the charge carrier in A7r5 ASMCs. When constant depolarization step was repetitively applied from hyperpolarized HP, 6‐AN (3 mM) gradually induced inhibition of ICa,L decreasing the peak amplitude of ICa,L (ICa,L, peak) by 25% in 5 min and 45% in 10 min. The inhibition was not reversible by washout of 6‐AN. This was in contrast to DHEA‐induced inhibition of ICa,L which exhibits rapid onset and rapid recovery by washout. 6‐AN little affected the time course of inactivation of ICa,L during depolarization steps, while DHEA markedly accelerated the time course. When I‐V relationship was obtained with 5 min interval before and during application of 6‐AN, ICa,L did not significantly change in control, but decreased in 5 min in the presence of 6‐AN and the inhibition was increased by another 5 min application of 6‐AN. In the presence of 6‐AN, 0.1 mM DHEA rapidly decreased ICa,L,peak and markedly accelerated the time course of inactivation in a reversible manner. Inhibition of G6PD by 6‐AN and DHEA could inhibit ICa,L,peak by thiol‐oxidation of CaV1.2 channel proteins via decrease of NADPH. DHEA additionally augmented voltage‐dependent inactivation of ICa,L in a mode of open channel block produced by Ca2+ channel blockers.Support or Funding InformationNational Heart, Lung, and Blood Institute Grant RO1‐HL‐085352 and ROI‐HL‐132574 to S.A.G.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial Upregulation of Mechanosensitive Channel Piezo1 in Pulmonary Hypertension
Authors: Wang Ziyi , Babicheva Alexandra , Wu Linda , Yuan Jason X.‐J. , Wang Jian 
Published: June 2021

RationaleThe Piezo channel family (Piezo1 and Piezo2), one of the most important family of mechanosensing ion channels. Surprisingly, there are no studies on Piezo in the pulmonary hypertension. We focus on Piezo1 because of its imprtance in vascular deveopment, especially in endothelial cells. Our working hypothesis is that upregulated Piezo1 may be a critical pathogenic mechanism that contributes to augmented Ca2+ influx and excessive PAEC proliferation or apoptosis in patients with IPAH.MethodsRNA levels of Piezo1 and Piezo2 was quantified by RT‐PCR Analysis. Protein levels of Piezo1, bax, bcl‐2, Calpain‐1 and Calpain‐2 was quantified by Western blot analysis. adult Male C57BL/6 mice (6–8 weeks) were exposed to persistent chronic hypoxia (10% O2) for 21 days to establish hypoxia‐induced PH. For Sugen 5416/hypoxia (SuHx) induced‐PH rat model, SD rats (age, 6–8 weeks old) were either injected s.c. with 20 mg·kg−1 SU5416 (Sigma‐Aldrich, St. Louis, MO, USA) or vehicle, exposed to hypoxic conditions for 3 weeks (10% O2) and returned to normoxic condition (21% O2) for 2 weeks. In the MCT group, a single intraperitoneal injection of MCT (60 mg/kg, Sigma Aldrich) was given at day 1 to establish the MCT‐induced PH.ResultsPiezo1 and Piezo2 have high expression in rat lung tissue and human PAEC, and also Piezo1 has significantly higher mRNA levels in both human PASMC and PAEC than Piezo2 (n=5, p<0.01). Upregulated Piezo1 in whole‐lung tissue (mainly containing ECs) from MCT‐mediated PH (MCT‐PH) compared to normal control rats (1.00±0.07; 4.74±0.41, n>5, p<0.001). Compared to controls, Piezo1 was increased in whole‐lung tissues in hypoxic mice (1.00±0.07; 4.74±0.41, n>5, p<0.01) and SuHx rats (1.00±0.15; 1.99±0.31, n>5, p<0.01). Also, compared to normoxia condition, increased Piezo1 was associated with a increased Calpain‐1 and Calpain‐2 treated with hypoxia (3% O2 for 72 hours) (1.00±0.16; 4.14±0.75; 1.00±0.14, 2.32±0.35; 1.00±0.07; 1.74±0.14, n>5, p<0.01). Moreover, PAECs from IPAH patients exhibited significant elevation of Piezo1 compared to normal control subjects (1.00±0.09; 6.20±1.78, n=5, p<0.01).ConclusionsThese results are the first to implicate that Piezo1 as a mechanosensor may play an important role in the development of pulmonary hypertension via, at least in part, induction of PAEC apoptosis and enhancement of Ca2+ in PAECs. Piezo1 may be involved in the transcriptional regulation under hypoxia and play critical roles in the development of PH. Therefore, further studies are needed to identify the potential contribution of Piezo1 in PH.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.