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The prevalence of Parkinson’s disease places a significant burden on society; therefore, there is an urgent need to develop more effective drugs. However, the development of these drugs is both expensive and risky. Quercetin (QUE) has potent pharmacological effects on neurodegenerative diseases, but its low solubility in water and poor bioavailability limit its use in pharmaceutical applications. In this study, Quercetin nanocrystals (QNC) were synthesized and compared to standard QUE. A network-pharmacology-based methodology was applied, including target prediction, network construction, a gene ontology (GO) analysis, a KEGG pathway enrichment analysis, and molecular docking. This study aimed to identify the targets of QUE relevant to the treatment of Parkinson’s disease and investigate the associated pharmacological mechanisms. Most of the predicted targets are involved in dopamine uptake during synaptic transmission. QUE regulates the key targets DRD2 and DRD4, which significantly affect dopaminergic synapses. The molecular docking results showed that QUE had a better binding affinity than the standard drug l-Dopa. From these experiments, it can be concluded that QNC effectively reduced the adverse effects caused by rotenone-induced oxidative stress in biochemical, neurochemical, and histopathological alterations. Therefore, QNC can potentially treat Parkinson’s disease, and its effectiveness should be assessed in future clinical trials.

Zidovudine (AZT) is one of the most referred antiretroviral drug. In spite of its higher bioavailability (50-75%) the most important reason of its cessation are bone marrow suppression, anemia, neutropenia and various organs related toxicities. This study aims at the improvement of oral delivery of AZT through its encapsulation in lactoferrin nanoparticles (AZT-lactonano). The nanoparticles (NPs) are of 50-60 nm in size and exhibit 67% encapsulation of the AZT. They are stable in simulated gastric and intestinal fluids. Anti-HIV-1 activity of AZT remains unaltered in nanoformulation in acute infection. The bioavailability and tissue distribution of AZT is higher in blood followed by liver and kidney. AZT-lactonano causes the improvement of pharmacokinetic profile as compared to soluble AZT; a more than 4 fold increase in AUC and AUMC in male and female rats. The serum Cmax for AZT-lactonano was increased by 30%. Similarly there was nearly 2-fold increase in Tmax and t1/2. Our in vitro study confirms that, the endosomal pH is ideal for drug release from NPs and shows constant release from up to 96h. Bone marrow micronucleus assay show that nanoformulation exhibits approximately 2fold lower toxicity than soluble form. Histopathological and biochemical analysis further confirms that less or no significant organ toxicities when nanoparticles were used. AZT-lactonano has shown its higher efficacy, low organs related toxicities, improved pharmacokinetics parameter while keeping the antiviral activity intact. Thus, the nanoformulation are safe for the target specific drug delivery.

The essential Drosophila RNA-binding protein Brain Tumor (Brat) represses specific genes to control embryogenesis and differentiation of stem cells. In the brain, Brat functions as a tumor suppressor that diminishes neural stem cell proliferation while promoting differentiation. Though important Brat-regulated target mRNAs have been identified in these contexts, the full impact of Brat on gene expression remains to be discovered. Here, we identify the network of Brat-regulated mRNAs by performing RNA sequencing (RNA-seq) following depletion of Brat from cultured cells. We identify 158 mRNAs, with high confidence, that are repressed by Brat. De novo motif analysis identified a functionally enriched RNA motif in the 3' untranslated regions (UTRs) of Brat-repressed mRNAs that matches the biochemically defined Brat binding site. Integrative data analysis revealed a high-confidence list of Brat-repressed and Brat-bound mRNAs containing 3'UTR Brat binding motifs. Our RNA-seq and reporter assays show that multiple 3'UTR motifs promote the strength of Brat repression, whereas motifs in the 5'UTR are not functional. Strikingly, we find that Brat regulates expression of glycolytic enzymes and the vacuolar ATPase complex, providing new insight into its role as a tumor suppressor and the coordination of metabolism and intracellular pH., (Published by Oxford University Press on behalf of Nucleic Acids Research 2024.)

Regulation of cadherin-mediated cell adhesion is crucial not only for maintaining tissue integrity and barrier function in the endothelium and epithelium but also for electromechanical coupling within the myocardium. Therefore, loss of cadherin-mediated adhesion causes various disorders, including vascular inflammation and desmosome-related diseases such as the autoimmune blistering skin dermatosis pemphigus and arrhythmogenic cardiomyopathy. Mechanisms regulating cadherin-mediated binding contribute to the pathogenesis of diseases and may also be used as therapeutic targets. Over the last 30 years, cyclic adenosine 3',5'-monophosphate (cAMP) has emerged as one of the master regulators of cell adhesion in endothelium and, more recently, also in epithelial cells as well as in cardiomyocytes. A broad spectrum of experimental models from vascular physiology and cell biology applied by different generations of researchers provided evidence that not only cadherins of endothelial adherens junctions (AJ) but also desmosomal contacts in keratinocytes and the cardiomyocyte intercalated discs are central targets in this scenario. The molecular mechanisms involve protein kinase A- and exchange protein directly activated by cAMP-mediated regulation of Rho family GTPases and S665 phosphorylation of the AJ and desmosome adaptor protein plakoglobin. In line with this, phosphodiesterase 4 inhibitors such as apremilast have been proposed as a therapeutic strategy to stabilize cadherin-mediated adhesion in pemphigus and may also be effective to treat other disorders where cadherin-mediated binding is compromised., (© 2023 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Aims: Arrhythmogenic cardiomyopathy (AC) is a severe heart disease predisposing to ventricular arrhythmias and sudden cardiac death caused by mutations affecting intercalated disc (ICD) proteins and aggravated by physical exercise. Recently, autoantibodies targeting ICD proteins, including the desmosomal cadherin desmoglein 2 (DSG2), were reported in AC patients and were considered relevant for disease development and progression, particularly in patients without underlying pathogenic mutations. However, it is unclear at present whether these autoantibodies are pathogenic and by which mechanisms show specificity for DSG2 and thus can be used as a diagnostic tool., Methods and Results: IgG fractions were purified from 15 AC patients and 4 healthy controls. Immunostainings dissociation assays, atomic force microscopy (AFM), Western blot analysis and Triton X-100 assays were performed utilizing human heart left ventricle tissue, HL-1 cells and murine cardiac slices. Immunostainings revealed that autoantibodies against ICD proteins are prevalent in AC and most autoantibody fractions have catalytic properties and cleave the ICD adhesion molecules DSG2 and N-cadherin, thereby reducing cadherin interactions as revealed by AFM. Furthermore, most of the AC-IgG fractions causing loss of cardiomyocyte cohesion activated p38MAPK, which is known to contribute to a loss of desmosomal adhesion in different cell types, including cardiomyocytes. In addition, p38MAPK inhibition rescued the loss of cardiomyocyte cohesion induced by AC-IgGs., Conclusion: Our study demonstrates that catalytic autoantibodies play a pathogenic role by cleaving ICD cadherins and thereby reducing cardiomyocyte cohesion by a mechanism involving p38MAPK activation. Finally, we conclude that DSG2 cleavage by autoantibodies could be used as a diagnostic tool for AC., (© 2023. The Author(s).)

Arrhythmogenic cardiomyopathy (AC) is a familial heart disease partly caused by impaired desmosome turnover. Thus, stabilization of desmosome integrity may provide new treatment options. Desmosomes, apart from cellular cohesion, provide the structural framework of a signaling hub. Here, we investigated the role of the epidermal growth factor receptor (EGFR) in cardiomyocyte cohesion. We inhibited EGFR under physiological and pathophysiological conditions using the murine plakoglobin-KO AC model, in which EGFR was upregulated. EGFR inhibition enhanced cardiomyocyte cohesion. Immunoprecipitation showed an interaction of EGFR and desmoglein 2 (DSG2). Immunostaining and atomic force microscopy (AFM) revealed enhanced DSG2 localization and binding at cell borders upon EGFR inhibition. Enhanced area composita length and desmosome assembly were observed upon EGFR inhibition, confirmed by enhanced DSG2 and desmoplakin (DP) recruitment to cell borders. PamGene Kinase assay performed in HL-1 cardiomyocytes treated with erlotinib, an EGFR inhibitor, revealed upregulation of Rho-associated protein kinase (ROCK). Erlotinib-mediated desmosome assembly and cardiomyocyte cohesion were abolished upon ROCK inhibition. Thus, inhibiting EGFR and, thereby, stabilizing desmosome integrity via ROCK might provide treatment options for AC.

A Disintegrin And Metalloprotease (ADAM) family proteins are involved in several cardiac diseases, and some ADAMs have been associated with cardiomyopathies. ADAM17 is known to cleave desmoglein 2 (DSG2), one of the proteins involved in the pathogenesis of arrhythmogenic cardiomyopathy (AC). Desmosomal stability is impaired in AC, an inheritable genetic disease, the underlying causes of which can be mutations in genes coding for proteins of the desmosome, such as DSG2, desmoplakin (DP), plakoglobin (PG), plakophilin 2 or desmocollin 2. Stabilizing desmosomal contacts can therefore be a treatment option. In the heart of the murine Jup -/- AC model, ( Jup being the gene coding for PG) mice, elevated levels of p38MAPK, an activator of ADAM17, were found. However, ADAM17 levels were unaltered in Jup -/- mice hearts. Nonetheless, inhibition of ADAM17 led to enhanced cardiomyocyte cohesion in both Jup +/+ and Jup -/- mice, and in HL-1 cardiomyocytes. Further, enhanced cohesion in HL-1 cardiomyocytes after acute inhibition of ADAM17 was paralleled by enhanced localization of DSG2 and DP at the membrane, whereas no changes in desmosomal assembly or the desmosomal complex were observed. In conclusion, acute inhibition of ADAM17 might lead to reduced cleavage of DSG2, thereby stabilizing the desmosomal adhesion, evidenced by increased DSG2 and DP localization at cell borders and eventually cardiomyocyte cohesion. We believe that similar mechanisms exist in AC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shoykhet, Waschke and Yeruva.)

Pemphigus vulgaris is a life-threatening blistering skin disease caused by autoantibodies destabilizing desmosomal adhesion. Current therapies focus on suppression of autoantibody formation and thus treatments directly stabilizing keratinocyte adhesion would fulfill an unmet medical need. We here demonstrate that apremilast, a phosphodiesterase 4 inhibitor used in psoriasis, prevents skin blistering in pemphigus vulgaris. Apremilast abrogates pemphigus autoantibody-induced loss of keratinocyte cohesion in ex-vivo human epidermis, cultured keratinocytes in vitro and in vivo in mice. In parallel, apremilast inhibits keratin retraction as well as desmosome splitting, induces phosphorylation of plakoglobin at serine 665 and desmoplakin assembly into desmosomal plaques. We established a plakoglobin phospho-deficient mouse model that reveals fragile epidermis with altered organization of keratin filaments and desmosomal cadherins. In keratinocytes derived from these mice, intercellular adhesion is impaired and not rescued by apremilast. These data identify an unreported mechanism of desmosome regulation and propose that apremilast stabilizes keratinocyte adhesion and is protective in pemphigus., (© 2023. The Author(s).)

For electromechanical coupling of cardiomyocytes, intercalated discs (ICDs) are pivotal as highly specialized intercellular contact areas. ICD consists of adhesive contacts, such as desmosomes and adherens junctions (AJs) that are partially intermingled and thereby form an area composita to provide mechanical strength, as well as gap junctions (GJ) and sodium channels for excitation propagation. In contrast, in epithelia, mixed junctions with features of desmosomes and AJs are regarded as transitory primarily during the formation of desmosomes. The anatomy of desmosomes is defined by a typical ultrastructure with dense intracellular plaques anchoring the cadherin-type adhesion molecules to the intermediate filament cytoskeleton. Desmosomal diseases characterized by impaired adhesive and signalling functions of desmosomal contacts lead to arrhythmogenic cardiomyopathy when affecting cardiomyocytes and cause pemphigus when manifesting in keratinocytes or present as cardiocutaneous syndromes when both cell types are targeted by the disease, which underscores the high biomedical relevance of these cell contacts. Therefore, comparative analyses regarding the structure and regulation of desmosomal contacts in cardiomyocytes and epithelial cells are helpful to better understand disease pathogenesis. In this brief review, we describe the structural properties of ICD compared to epithelial desmosomes and suggest that mechanisms regulating adhesion may at least in part be comparable. Also, we discuss whether phenomena such as hyperadhesion or the bidirectional regulation of desmosomes to serve as signalling hubs in epithelial cells may also be relevant for ICD., (© 2022 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Aim: Cardiac autonomic nervous system (ANS) dysregulation is a hallmark of several cardiovascular diseases. Adrenergic signaling enhanced cardiomyocyte cohesion via PKA-mediated plakoglobin phosphorylation at serine 665, referred to as positive adhesiotropy. This study investigated cholinergic regulation of cardiomyocyte cohesion using muscarinic receptor agonist carbachol (CCH)., Methods: Dissociation assays, Western blot analysis, immunostaining, atomic force microscopy (AFM), immunoprecipitation, transmission electron microscopy (TEM), triton assays, and siRNA knockdown of genes were performed in either HL-1 cells or plakoglobin (PG) wild type (Jup +/+ ) and knockout (Jup -/- ) mice, which served as a model for arrhythmogenic cardiomyopathy., Results: In HL-1 cells grown in norepinephrine (NE)-containing medium for baseline adrenergic stimulation, and murine cardiac slice cultures from Jup +/+ and Jup -/- mice CCH treatment impaired cardiomyocyte cohesion. Immunostainings and AFM experiments revealed that CCH reduced desmoglein 2 (DSG2) localization and binding at cell borders. Furthermore, CCH reduced intercalated disc plaque thickness in both Jup +/+ and Jup -/- mice, evidenced by TEM analysis. Immunoprecipitation experiments in HL-1 cells revealed no changes in DSG2 interaction with desmoplakin (DP), plakophilin 2 (PKP2), PG, and desmin (DES) after CCH treatment. However, knockdown of any of the above proteins abolished CCH-mediated loss of cardiomyocyte cohesion. Furthermore, in HL-1 cells, CCH inhibited adrenergic-stimulated ERK phosphorylation but not PG phosphorylation at serine 665. In addition, CCH activated the AKT/GSK-3β axis in the presence of NE., Conclusion: Our results demonstrate that cholinergic signaling antagonizes the positive effect of adrenergic signaling on cardiomyocyte cohesion and thus causes negative adhesiotropy independent of PG phosphorylation., (© 2022 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Arrhythmogenic cardiomyopathy (AC) is a heart disease often caused by mutations in genes coding for desmosomal proteins, including desmoglein-2 (DSG2), plakoglobin (PG), and desmoplakin (DP). Therapy is based on symptoms and limiting arrhythmia, because the mechanisms by which desmosomal components control cardiomyocyte function are largely unknown. A new paradigm could be to stabilize desmosomal cardiomyocyte adhesion and hyperadhesion, which renders desmosomal adhesion independent from Ca2+. Here, we further characterized the mechanisms behind enhanced cardiomyocyte adhesion and hyperadhesion. Dissociation assays performed in HL-1 cells and murine ventricular cardiac slice cultures allowed us to define a set of signaling pathways regulating cardiomyocyte adhesion under basal and hyperadhesive conditions. Adrenergic signaling, activation of PKC, and inhibition of p38MAPK enhanced cardiomyocyte adhesion, referred to as positive adhesiotropy, and induced hyperadhesion. Activation of ERK1/2 paralleled positive adhesiotropy, whereas adrenergic signaling induced PG phosphorylation at S665 under both basal and hyperadhesive conditions. Adrenergic signaling and p38MAPK inhibition recruited DSG2 to cell junctions. In PG-deficient mice with an AC phenotype, only PKC activation and p38MAPK inhibition enhanced cardiomyocyte adhesion. Our results demonstrate that cardiomyocyte adhesion can be stabilized by different signaling mechanisms, which are in part offset in PG-deficient AC.

Desmosomal proteins are components of the intercalated disc and mediate cardiac myocyte adhesion. Enhancement of cardiac myocyte cohesion, referred to as "positive adhesiotropy", was demonstrated to be a function of sympathetic signaling and to be relevant for a sufficient inotropic response. We used the inotropic agent digitoxin to investigate the link between inotropy and adhesiotropy. In contrast to wild-type hearts, digitoxin failed to enhance pulse pressure in perfused mice hearts lacking the desmosomal protein plakoglobin which was paralleled with abrogation of plaque thickening indicating that positive inotropic response requires intact desmosomal adhesion. Atomic force microscopy revealed that digitoxin increased the binding force of the adhesion molecule desmoglein-2 at cell-cell contact areas. This was paralleled by enhanced cardiac myocyte cohesion in both HL-1 cardiac myocytes and murine cardiac slices as determined by dissociation assays as well as by accumulation of desmosomal proteins at cell-cell contact areas. However, total protein levels or cytoskeletal anchorage were not affected. siRNA-mediated depletion of desmosomal proteins abrogated increase of cell cohesion demonstrating that intact desmosomal adhesion is required for positive adhesiotropy. Mechanistically, digitoxin caused activation of ERK1/2. In line with this, inhibition of ERK1/2 signaling abrogated the effects of digitoxin on cell-cell adhesion and desmosomal reorganization. These results show that the positive inotropic agent digitoxin enhances cardiac myocyte cohesion with reorganization of desmosomal proteins in an ERK1/2-dependent manner. Desmosomal adhesion seems to be important for a sufficient positive inotropic response of digitoxin treatment, which can be of medical relevance for the treatment of heart failure.

Intercalated discs (ICDs), which connect adjacent cardiomyocytes, are composed of desmosomes, adherens junctions (AJs) and gap junctions (GJs). Previous data demonstrated that adrenergic signaling enhances cardiac myocyte cohesion, referred to as positive adhesiotropy, via PKA-mediated phosphorylation of plakoglobin (PG). However, it was unclear whether positive adhesiotropy caused ultrastructural modifications of ICDs. Therefore, we further investigated the role of PG in adrenergic signaling-mediated ultrastructural changes in the ICD of cardiomyocytes. Quantitative transmission electron microscopy (TEM) analysis of ICD demonstrated that cAMP elevation caused significant elongation of area composita and thickening of the ICD plaque, paralleled by enhanced cardiomyocyte cohesion, in WT but not PG-deficient cardiomyocytes. STED microscopy analysis supported that cAMP elevation ex vivo enhanced overlap of desmoglein-2 (Dsg2) and N-cadherin (N-cad) staining in ICDs of WT but not PG-deficient cardiomyocytes. For dynamic analyses, we utilized HL-1 cardiomyocytes, in which cAMP elevation induced translocation of Dsg2 and PG but not of N-cad to cell junctions. Nevertheless, depletion of N-cad but not of Dsg2 resulted in a decrease in basal cell cohesion whereas positive adhesiotropy was abrogated in monolayers depleted for either Dsg2 or N-cad. In the WT mice, ultrastrutural changes observed after cAMP elevation were paralleled by phosphorylation of PG at serine 665. Our data demonstrate that in murine hearts adrenergic signaling enhanced N-cad and Dsg2 in the ICD paralleled by ultrastrutural strengthening of ICDs and that effects induced by positive adhesiotropy were strictly dependent on Pg., (Copyright © 2020 Yeruva, Kempf, Egu, Flaswinkel, Kugelmann and Waschke.)

Arrhythmogenic cardiomyopathy (AC) is a genetic disease causing arrhythmia and sudden cardiac death with only symptomatic therapy available at present. Mutations of desmosomal proteins, including desmoglein-2 (Dsg2) and plakoglobin (Pg), are the major cause of AC and have been shown to lead to impaired gap junction function. Recent data indicated the involvement of anti-Dsg2 autoantibodies in AC pathogenesis. We applied a peptide to stabilize Dsg2 binding similar to a translational approach to pemphigus, which is caused by anti-desmoglein autoantibodies. We provide evidence that stabilization of Dsg2 binding by a linking peptide (Dsg2-LP) is efficient to rescue arrhythmia in an AC mouse model immediately upon perfusion. Dsg2-LP, designed to cross-link Dsg2 molecules in proximity to the known binding pocket, stabilized Dsg2-mediated interactions on the surface of living cardiomyocytes as revealed by atomic force microscopy and induced Dsg2 oligomerization. Moreover, Dsg2-LP rescued disrupted cohesion induced by siRNA-mediated Pg or Dsg2 depletion or l-tryptophan, which was applied to impair overall cadherin binding. Dsg2-LP rescued connexin-43 mislocalization and conduction irregularities in response to impaired cardiomyocyte cohesion. These results demonstrate that stabilization of Dsg2 binding by Dsg2-LP can serve as a novel approach to treat arrhythmia in patients with AC.

Intestinal Na + -nutrient cotransport depends on claudin-2 and claudin-15 mediated Na + recycling. Expression of these proteins is coordinately regulated during postnatal development. While expression of claudin-2 and claudin-15 has been studied in inflammatory bowel disease (IBD) and celiac disease (CD), it has not been assessed in other malabsorptive diseases, and no reports have compared expression in children and adults. We used quantitative immunofluorescence microscopy to assess claudin-2 and claudin-15 expression in duodenal biopsies from children and adults with malabsorptive disease and healthy controls. Consistent with previous work in rodents, claudin-2 expression in healthy children was markedly greater, and claudin-15 expression was less, than that in adults. Claudin-2 expression was increased in adults with CD and downregulated in children with graft-versus-host disease (GVHD). In contrast, claudin-15 expression was reduced in adults with GVHD and common variable immunodeficiency (CVID). These data show that one of the two Na + /water pore-forming claudins is upregulated in CD and downregulated in GVHD and CVID. The specific claudin whose expression changes, however, reflects the age of the patient (child or adult). We conclude that contributions of claudin-2 and claudin-15 to pathophysiology of and responses to diarrhea in children and adults with GVHD and CVID differ from those in CD and IBD.

Although absorption of di- and tripeptides into intestinal epithelial cells occurs via the peptide transporter 1 (PEPT1, also called solute carrier family 15 member 1 (SLC15A1)), the detailed regulatory mechanisms are not fully understood. We examined: (a) whether dipeptide absorption in villous enterocytes is associated with a rise in cytosolic Ca 2+ ([Ca 2+ ] cyt ), (b) whether the calcium sensing receptor (CaSR) is involved in dipeptide-elicited [Ca 2+ ] cyt signaling, and (c) what potential consequences of [Ca 2+ ] cyt signaling may enhance enterocyte dipeptide absorption. Dipeptide Gly-Sar and CaSR agonist spermine markedly raised [Ca 2+ ] cyt in villous enterocytes, which was abolished by NPS-2143, a selective CaSR antagonist and U73122, an phospholipase C (PLC) inhibitor. Apical application of Gly-Sar induced a jejunal short-circuit current (Isc), which was reduced by NPS-2143. CaSR expression was identified in the lamina propria and on the basal enterocyte membrane of mouse jejunal mucosa in both WT and Slc15a1 -/- animals, but Gly-Sar-induced [Ca 2+ ] cyt signaling was significantly decreased in Slc15a1 -/- villi. Clotrimazole and TRM-34, two selective blockers of the intermediate conductance Ca 2+ -activated K + channel (IK Ca ), but not iberiotoxin, a selective blocker of the large-conductance K + channel (BK Ca ) and apamin, a selective blocker of the small-conductance K + channel (SK Ca ), significantly inhibited Gly-Sar-induced Isc in native tissues. We reveal a novel CaSR-PLC-Ca 2+ -IK Ca pathway in the regulation of small intestinal dipeptide absorption, which may be exploited as a target for future drug development in human nutritional disorders., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Aims: Mutations in desmosomal proteins can induce arrhythmogenic cardiomyopathy with life-threatening arrhythmia. Previous data demonstrated adrenergic signalling to be important to regulate desmosomal cohesion in cardiac myocytes. Here, we investigated how signalling pathways including adrenergic signalling, PKC and SERCA regulate desmosomal adhesion and how this controls gap junctions (GJs) in cardiac myocytes., Methods: Immunostaining, Western blot, dissociation assay and multi-electrode array were applied in HL-1 cardiac myocytes to evaluate localization, expression and function of desmosomal and GJ components. cAMP levels were determined by ELISA., Results: Activation of PKC by PMA or adrenergic signalling increased cell cohesion and desmoglein-2 and desmoplakin localization at cell-cell junctions, whereas tryptophan (Trp) treatment to inhibit cadherin binding or inhibition of SERCA by thapsigargin reduced cell cohesion, while cAMP elevation rescued this effect. Despite no changes in protein expression, accumulation of GJ protein connexin-43 was detectable at cell-cell contacts in parallel to increased cohesion. Disruption of cell cohesion by Trp, PMA or thapsigargin impaired conduction of excitation comparable to GJ inhibition. cAMP elevation was effective to improve arrhythmia after Trp treatment. Weakened cell cohesion by Trp or depletion of desmoglein-2 or plakoglobin blocked signalling via the β1-adrenergic receptor. Moreover, silencing of desmosomal proteins increased arrhythmia and reduced conduction velocity, which were rescued by cAMP elevation., Conclusion: These data demonstrate the interplay of GJs, desmosomes and the β1-adrenergic receptor with regulation of their function by cell cohesion, adrenergic and PKC signalling or SERCA inhibition. These results support the identification of new targets to treat arrhythmogenic cardiomyopathy., (© 2018 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background/aims: Enterocytes express a number of NHE isoforms with presumed localization in the apical (NHE2, 3 and 8) or basolateral (NHE1) membrane. Functional activity and localization of enterocyte NHE isoforms were assessed using fully differentiated Caco-2BBe cells, whose genetic expression profile closely resembles mature enterocytes., Methods: The activity of the different NHEs was analyzed by fluorometric pH i -metry in a perfusion chamber with separate apical and basolateral perfusion, using specific inhibitors and shRNA knockdown of NHE2. The expression of the NHEs and of other relevant acid extrusion transporters was quantified by qPCR., Results: Quantitative comparison of the mRNA expression levels of the different NHE isoforms in 14 day-differentiated Caco-2BBe cells showed the following order: NHE2>NHE8>NHE3>NHE1. Acid-activated NHE exchange rates in the basolateral membrane were >6-fold higher than in the apical membrane. 79 ± 3 % of the acid-activated basolateral Na⁺/H⁺ exchange rate displayed a NHE1-typical inhibitor profile, and no NHE2/3/8 typical activity could be observed. Analysis of the apical Na⁺/H⁺ exchange rates revealed that approximately 51 ± 3 % of the total apical activity displayed a NHE2/8-typical inhibitor profile and 31 ± 6 % a NHE3-typical inhibitor profile. Because no selective NHE2 inhibitor is available, a stable NHE2 knockdown cell line (C2NHE2KD) was generated. C2NHE2KD displayed a reduced NHE2-typical apical Na⁺/H⁺ exchange rate and maintained a lower steady-state pH i , despite high expression levels of other acid extruders, in particular NBCn1 (Slc4a7)., Conclusion: Differentiated Caco-2BBe cells display particularly high mRNA expression levels of NHE2, which can be functionally identified in the apical membrane. Although at low intracellular pH, NHE2 transport rate was far lower than that of NHE1. NHE2 activity was nevertheless essential for the maintenance of the steady-state pH i of these cells., Competing Interests: The authors have no ethical conflicts to disclose. The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Despite nutrition interventions, stunting thought to be secondary to underlying environmental enteropathy (EE) remains pervasive among infants residing in resource-poor countries and remains poorly characterized. From a birth cohort of 380 children, 65 malnourished infants received 12 weeks of nutritional supplementation with ready-to-use therapeutic food (RUTF). Eleven children with insufficient response to RUTF underwent upper endoscopy with duodenal biopsies, which were compared with U.S., age-matched specimens for healthy, celiac disease, non-celiac villous atrophy, non-celiac intraepithelial lymphocytosis, and graft-versus-host disease patients. Of the 11 children biopsied, EE was found in 10 (91%) with one subject with celiac disease. Morphometry demonstrated decreased villus-to-crypt (V:C) ratios in EE relative to healthy and non-celiac lymphocytosis patients. Environmental enteropathy villus volumes were significantly decreased relative to healthy controls. In EE, average CD3 + cells per 100 epithelial cells and per 1,000 µm 2 of lamina propria and the number of lamina propria CD20 + B-cell aggregates were increased relative to all other groups. Our results indicate that V:C ratios are reduced in EE but are less severe than in celiac disease. Environmental enteropathy intraepithelial and lamina propria T lymphocytosis is of greater magnitude than that in celiac disease. The increases in lamina propria B and T lymphocytes suggest that non-cytolytic lymphocytic activation may be a more prominent feature of EE relative to celiac disease. These results provide new insights into shared yet distinct histological and immunological features of EE and celiac disease in children.

Epithelial cells are essential to the survival and homeostasis of complex organisms. These cells cover the surfaces of all mucosae, the skin, and other compartmentalized structures essential to physiological function. In addition to maintenance of barriers that separate internal and external compartments, epithelia display a variety of organ-specific differentiated functions. Function is reflected in overall epithelial structure and organization, shape of individual cells, and proteins expressed by these cells. More than one epithelial cell type is often present within a single organ and, in many cases, individual cells differentiate to change their functional behaviors as part of normal development or in response to extracellular stimuli. This article discusses the diversity of epithelial structure and function in general terms and explores representative tissues in greater depth to highlight organ specific functions and their contributions to physiology and disease. © 2017 American Physiological Society. Compr Physiol 7:1497-1518, 2017., (Copyright © 2017 John Wiley & Sons, Inc.)

A core function of epithelia is to form barriers that separate tissue compartments within complex organisms. These barriers also separate the internal milieu from the external environment and are, therefore, an essential component of host defense. However, in many cases, a perfect barrier would be improbable with life itself. Examples include the air spaces within the lungs, the renal tubules, and the intestines. Here, we focus on the mechanisms by which barriers are assembled, maintained, and regulated in the context of health and disease. Because of its unique challenges and extensive study, we focus on the gastrointestinal tract as an organ-specific example of the essential contributions of the paracellular barrier to life., (Copyright © 2017. Published by Elsevier Inc.)

Intestinal damage in malnutrition constitutes a threat to the survival of many thousands of children globally. We studied children in Lusaka, Zambia, with severe acute malnutrition (SAM) and persistent diarrhea using endoscopy, biopsy and analysis of markers and protective proteins in blood and intestinal secretions. We carried out parallel investigations in apparently healthy adults, and analyzed biomarkers only in apparently healthy children. Villus height and crypt depth did not differ in children with SAM and adult controls, but epithelial surface was reduced in children with SAM (median 445, interquartile range (IQR) 388, 562μm per 100μm muscularis mucosae) compared to adults (578, IQR 465,709; P=0.004). Histological lesions and disruptions of claudin-4 and E-cadherin were most pronounced in children with SAM. Circulating lipopolysaccharide, a marker of bacterial translocation, was higher in malnourished children (251, IQR 110,460EU/ml) than in healthy children (51, IQR 0,111; P=0.0001). Other translocation markers showed similar patterns. Anti-Deamidated Gliadin Peptide IgG concentrations, although within the normal range, were higher in children with SAM (median 2.7U/ml, IQR 1.5-8.6) than in adults (1.6, 1.4-2.1; P=0.005), and were inversely correlated with villus height (ρ=-0.79, n=13, P=0.001). Malnutrition enteropathy is associated with intestinal barrier failure and immune dysregulation., (Copyright © 2017. Published by Elsevier B.V.)

Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na + /H + exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1-dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1-dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ∼10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady-state pH i and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process., (© 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.)

Diarrhea is a host response to enteric pathogens, but its impact on pathogenesis remains poorly defined. By infecting mice with the attaching and effacing bacteria Citrobacter rodentium, we defined the mechanisms and contributions of diarrhea and intestinal barrier loss to host defense. Increased permeability occurred within 2 days of infection and coincided with IL-22-dependent upregulation of the epithelial tight junction protein claudin-2. Permeability increases were limited to small molecules, as expected for the paracellular water and Na + channel formed by claudin-2. Relative to wild-type, claudin-2-deficient mice experienced severe disease, including increased mucosal colonization by C. rodentium, prolonged pathogen shedding, exaggerated cytokine responses, and greater tissue injury. Conversely, transgenic claudin-2 overexpression reduced disease severity. Chemically induced osmotic diarrhea reduced colitis severity and C. rodentium burden in claudin-2-deficient, but not transgenic, mice, demonstrating that claudin-2-mediated protection is the result of enhanced water efflux. Thus, IL-22-induced claudin-2 upregulation drives diarrhea and pathogen clearance., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The PDZ adaptor protein PDZK1 modulates the membrane expression and function of a variety of intestinal receptors and ion/nutrient transporters. Its expression is strongly decreased in inflamed intestinal mucosa of mice and IBD patients. Aim and Methods: We investigated whether the inflammation-associated PDZK1 downregulation is a direct consequence of proinflammatory cytokine release by treating intestinal Caco-2BBE cells with TNF-α, IFN-γ, and IL-1β, and analysing PDZK1 promotor activity, mRNA and protein expression. Results: IL-1β was found to significantly decrease PDZK1 promoter activity, mRNA and protein expression in Caco-2BBE cells. A distal region of the hPDZK1 promoter was identified to be important for basal expression and IL-1β-responsiveness. This region harbors the retinoid acid response element RARE as well as binding sites for transcription factors involved in IL-β downstream signaling. ERK1/2 inhibition by the specific MEK1/2 inhibitors PD98059/U0126 significantly attenuated the IL-1β mediated downregulation of PDZK1, while NF-κB, p38 MAPK, and JNK inhibition did not. Expression of the nuclear receptors RXRα and PPARα was decreased in inflamed colonic-mucosa of ulcerative colitis patients and in IL-1β-treated Caco2-BBE cells. Moreover, the RAR/RXR ligand 9-cis retinoic acid and the PPARα-agonist GW7647 stimulated PDZK1 mRNA and protein expression and attenuated IL-1β-mediated inhibition. Conclusions: The strong decrease in PDZK1 expression during intestinal inflammation may be in part a consequence of IL-1β-mediated RXRα and PPARα repression and can be attenuated by agonists for either nuclear receptor, or by ERK1/2 inhibition. The negative consequences of inflammation-induced PDZK1 downregulation on epithelial transport-function may thus be amenable to pharmacological therapy.

A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.