The recently identified proton-activated chloride (PAC) channel is ubiquitously expressed, and it regulates several proton-sensitive physiological and pathophysiological processes. While the PAC channel is activated by strong acids due to the binding of protons to extracellular binding sites, here, we describe the way in which weak acids inhibit the PAC channel by a mechanism involving a distinct extracellular binding site. Whole-cell patch clamp was performed on wildtype HEK293T cells, PAC-knockout HEK293 cells expressing human (h)PAC mutant constructs, and on hiPSC-derived cardiomyocytes. Proton-induced cytotoxicity was examined in HEK293T cells. Acetic acid inhibited endogenous PAC channels in HEK 293T cells in a reversible, concentration-dependent, and pH-dependent manner. The inhibition of PAC channels was also induced by lactic acid, propionic acid, itaconic acid, and β-hydroxybutyrate. Weak acids also inhibited recombinant wildtype hPAC channels and PAC-like currents in hiPSC-derived cardiomyocytes. Replacement of the extracellular arginine 93 by an alanine (hPAC–Arg93Ala) strongly reduced the inhibition by some weak acids, including arachidonic acid. Although lactic acid inhibited PAC, it did not reduce the proton-induced cytotoxicity examined in wildtype HEK 293 cells. To conclude, weak acids inhibit PAC via an extracellular mechanism involving Arg93. These data warrant further investigations into the regulation of the PAC channel by endogenous weak acids.

Transient receptor potential vanilloid 2 (TRPV2) is relevant for diseases like cancer, cardiac dysfunction, and infection, warranting drug development targeting TRPV2. However, this has been complicated by the lack of good modulators targeting TRPV2 and questions about species selectivity, so more detailed molecular insights into channel function and pharmacology are required. Two recent studies identified distinct binding sites on rat (r) and mouse (m) TRPV2 for activation by 2-aminoethoxydiphenyl borate (2-APB). Here we aimed to determine whether the mechanisms for 2-APB sensitivity of TRPV2 indeed differ among these closely related orthologs. Patch clamp electrophysiology revealed that mTRPV2 and human TRPV2 display similar sensitivities to 2-APB when compared with a considerably higher sensitivity of rTRPV2. For both mTRPV2 and rTRPV2, we observed that the exchange of putative 2-APB binding residues within the vanilloid binding pocket alters overall channel sensitivity to 3 TRPV2 pharmacological activators that bind at different sites: 2-APB, cannabidiol, and probenecid. By contrast, the exchange of putative 2-APB binding residues at the S5 binding pocket in both channels resulted in strongly reduced 2-APB sensitivies without reducing sensitivity to cannabidiol and probenecid. rTRPV2 mutants lacking key residues of both binding sites were almost completely 2-APB insensitive. These functional data suggest that the mechanisms accounting for 2-APB sensitivity are similar across mammalian TRPV2 orthologs. Except for serving as a binding site for 2-APB, the vanilloid binding pocket plays a key role in the overall function of TRPV2. These findings are relevant for the emerging framework toward an improved understanding of TRPV2. SIGNIFICANCE STATEMENT: This study resolves the conflict regarding how 2-aminoethoxydiphenyl borate binds to transient receptor potential vanilloid 2 (TRPV2), showing a shared mechanism despite sensitivity differences. These findings enhance TRPV2 modulation insights and highlight species considerations in drug design, aiding the development of selective TRPV2-targeted therapies.

AbstractTransient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO2 activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.

Background and PurposeItch is associated with several pathologies and is a common drug-induced side effect. Chloroquine (CQ) is reported to induce itch by activating the Mas-related G protein-coupled receptor MrgprA3 and subsequently TRPA1. In this study, we demonstrate that CQ employs at least two MrgprA3-independent mechanisms to activate or sensitize TRPA1 and TRPV1. Experimental ApproachPatch clamp and calcium imaging were utilized to examine effects of CQ on TRPA1 and TRPV1 expressed in HEK 293T cells. Key ResultsIn calcium imaging, CQ induces a concentration-dependent but MrgprA3-independent activation of TRPA1 and TRPV1. Although CQ itself inhibits TRPA1 and TRPV1 in patch clamp recordings, co-application of CQ and ultraviolet A (UVA) light evokes membrane currents through both channels. This effect is inhibited by the reducing agent dithiothreitol (DTT) and is reduced on mutants lacking cysteine residues accounting for reactive oxygen species (ROS) sensitivity. The combination of CQ and UVA light triggers an accumulation of intracellular ROS, removes fast inactivation of voltage-gated sodium currents and activates TRPV2. On the other hand, CQ is a weak base and induces intracellular alkalosis. Intracellular alkalosis can activate TRPA1 and TRPV1, and CQ applied at alkaline pH values indeed activates both channels. Conclusion and ImplicationsOur data reveal novel pharmacological properties of CQ, allowing activation of TRPA1 and TRPV1 via photosensitization as well as intracellular alkalosis. These findings add more complexity to the commonly accepted dogma that CQ-induced itch is specifically mediated by MrgprA3 coupling to TRPA1.

TRPV1 mediates pain occurring during sickling episodes in sickle cell disease (SCD). We examined if hemin, a porphyrin released during intravascular hemolysis modulates TRPV1. Calcium imaging and patch clamp were employed to examine effects of hemin on mouse dorsal root ganglion (DRG) neurons and HEK293t cells expressing TRPV1 and TRPA1. Hemin induced a concentration-dependent calcium influx in DRG neurons which was abolished by the unspecific TRP-channel inhibitor ruthenium red. The selective TRPV1-inhibitor BCTC or genetic deletion of TRPV1 only marginally impaired hemin-induced calcium influx in DRG neurons. While hTRPV1 expressed in HEK293 cells mediated a hemin-induced calcium influx which was blocked by BCTC, patch clamp recordings only showed potentiated proton- and heat-evoked currents. This effect was abolished by the PKC-inhibitor chelerythrine chloride and in protein kinase C (PKC)-insensitive TRPV1-mutants. Hemin-induced calcium influx through TRPV1 was only partly PKC-sensitive, but it was abolished by the reducing agent dithiothreitol (DTT). In contrast, hemin-induced potentiation of inward currents was not reduced by DTT. Hemin also induced a redox-dependent calcium influx, but not inward currents on hTRPA1. Our data suggest that hemin induces a PKC-mediated sensitization of TRPV1. However, it also acts as a photosensitizer when exposed to UVA-light used for calcium imaging. The resulting activation of redox-sensitive ion channels such as TRPV1 and TRPA1 may be an in vitro artifact with limited physiological relevance.

Objective Syndecan-4 (sdc4) is a cell-anchored proteoglycan that consists of a transmembrane core protein and glucosaminoglycan (GAG) side chains. Binding of soluble factors to the GAG chains of sdc4 may result in the dimerisation of sdc4 and the initiation of downstream signalling cascades. However, the question of how sdc4 dimerisation and signalling affects the response of cells to inflammatory stimuli is unknown. Methods Sdc4 immunostaining was performed on rheumatoid arthritis (RA) tissue sections. Interleukin (IL)-1 induced extracellular signal-regulated kinases (ERK) phosphorylation and matrix metalloproteinase-3 production was investigated. Il-1 binding to sdc4 was investigated using immunoprecipitation. IL-1 receptor (IL1R1) staining on wild-type, sdc4 and IL1R1 knockout fibroblasts was performed in fluorescence-activated cell sorting analyses. A blocking sdc4 antibody was used to investigate sdc4 dimerisation, IL1R1 expression and the histological paw destruction in the human tumour necrosis factor-alpha transgenic mouse. Results We show that in fibroblasts, the loss of sdc4 or the antibody-mediated inhibition of sdc4 dimerisation reduces the cell surface expression of the IL-1R and regulates the sensitivity of fibroblasts to IL-1. We demonstrate that IL-1 directly binds to sdc4 and in an IL-1R-independent manner leads to its dimerisation. IL-1-induced dimerisation of sdc4 regulates caveolin vesicle-mediated trafficking of the IL1R1, which in turn determines the responsiveness to IL-1. Administration of antibodies (Ab) against the dimerisation domain of sdc4, thus, strongly reduces the expression IL1R1 on arthritic fibroblasts both in vitro and an animal model of human RA. Conclusion Collectively, our data suggest that Ab that specifically inhibit sdc4 dimerisation may support anti-IL-1 strategies in diseases such as inflammatory arthritis.

Overdosing of the analgesic acetaminophen (APAP) is one of the most common causes for acute liver failure in modern countries. Although the exact molecular mechanisms mediating hepatocellular necrosis are still elusive, it is preceded by oxidative stress triggered by excessive levels of the metabolite N-acetyl-para-benzoquinone imine (NAPQI). Here, we describe the role of the redox-sensitive transient receptor potential (TRP) ion channel TRP vanilloid 4 (TRPV4) for APAP-induced hepatoxicity. Both pharmacological inhibition and genetic deletion of TRPV4 ameliorate APAP-induced necrosis in mouse and human hepatocytes in vitro. Liver injury caused by a systemic overdose of APAP is reduced in TRPV4-deficient mice and in wild-type mice treated with a TRPV4 inhibitor. The reduction of hepatotoxicity accomplished by systemic TRPV4 inhibition is comparable to the protective effects of the antioxidant N-acetyl-cysteine. Although TRPV4 does not modulate intrahepatic levels of glutathione, both its inhibition and genetic deletion attenuate APAP-induced oxidative and nitrosative stress as well as mitochondrial membrane depolarization. NAPQI evokes a calcium influx by activating heterologously expressed TRPV4 channels and endogenous TRPV4 channels in hepatoma cells but not in primary mouse hepatocytes. Taken together, our data suggest that TRPV4 mediates APAP-induced hepatotoxicity and thus may be a suitable target for treatment of this critical side effect.-Echtermeyer, F., Eberhardt, M., Risser, L., Herzog, C., Gueler, F., Khalil, M., Engel, M., Vondran, F., Leffler, A. Acetaminophen-induced liver injury is mediated by the ion channel TRPV4.

Significance Redox sensitivity is a common property of temperature-sensitive transient receptor potential (TRP) ion channels. Here we show that oxidation sensitizes and activates rodent TRPV2 orthologs known to have a high temperature threshold (>50 °C), but also the heat-insensitive human TRPV2. This oxidation-induced channel gating is intact in cell-free membrane patches, cysteine-independent but reduced upon replacement of the methionine residues M528 and M607. Blocking of TRPV2 and the reducing agent DTT reduce phagocytosis in macrophages which also generate heat-induced membrane currents following oxidation. These data reveal a methionine-dependent redox sensitivity of TRPV2 which may resemble a decisive endogenous mechanism enabling channel activation.

Severe ischemia reperfusion injury (IRI) results in rapid complement activation, acute kidney injury and progressive renal fibrosis. Little is known about the roles of the C5aR1 and C5aR2 complement receptors in IRI. In this study C5aR1-/- and C5aR2-/- mice were compared to the wild type in a renal IRI model leading to renal fibrosis. C5a receptor expression, kidney morphology, inflammation, and fibrosis were measured in different mouse strains one, seven and 21 days after IRI. Renal perfusion was evaluated by functional magnetic resonance imaging. Protein abundance and phosphorylation were assessed with high content antibody microarrays and Western blotting. C5aR1 and C5aR2 were increased in damaged tubuli and even more in infiltrating leukocytes after IRI in kidneys of wild-type mice. C5aR1-/- and C5aR2-/- animals developed less IRI-induced inflammation and showed better renal perfusion than wild-type mice following IRI. C5aR2-/- mice, in particular, had enhanced tubular and capillary regeneration with less renal fibrosis. Anti-inflammatory IL-10 and the survival/growth kinase AKT levels were especially high in kidneys of C5aR2-/- mice following IRI. LPS caused bone marrow-derived macrophages from C5aR2-/- mice to release IL-10 and to express the stress response enzyme heme oxygenase-1. Thus, C5aR1 and C5aR2 have overlapping actions in which the kidneys of C5aR2-/- mice regenerate better than those in C5aR1-/- mice following IRI. This is mediated, at least in part, by differential production of IL-10, heme oxygenase-1 and AKT.

BACKGROUND:Voltage-gated sodium channels generate action potentials in excitable cells, but they have also been attributed noncanonical roles in nonexcitable cells. We hypothesize that voltage-gated sodium channels play a functional role during extravasation of neutrophils.METHODS:Expression of voltage-gated sodium channels was analyzed by polymerase chain reaction. Distribution of Nav1.3 was determined by immunofluorescence and flow cytometry in mouse models of ischemic heart and kidney injury. Adhesion, transmigration, and chemotaxis of neutrophils to endothelial cells and collagen were investigated with voltage-gated sodium channel inhibitors and lidocaine in vitro. Sodium currents were examined with a whole cell patch clamp.RESULTS:Mouse and human neutrophils express multiple voltage-gated sodium channels. Only Nav1.3 was detected in neutrophils recruited to ischemic mouse heart (25 ± 7%, n = 14) and kidney (19 ± 2%, n = 6) in vivo. Endothelial adhesion of mouse neutrophils was reduced by tetrodotoxin (56 ± 9%, unselective Nav-inhibitor), ICA121431 (53 ± 10%), and Pterinotoxin-2 (55 ± 9%; preferential inhibitors of Nav1.3, n = 10). Tetrodotoxin (56 ± 19%), ICA121431 (62 ± 22%), and Pterinotoxin-2 (59 ± 22%) reduced transmigration of human neutrophils through endothelial cells, and also prevented chemotactic migration (n = 60, 3 × 20 cells). Lidocaine reduced neutrophil adhesion to 60 ± 9% (n = 10) and transmigration to 54 ± 8% (n = 9). The effect of lidocaine was not increased by ICA121431 or Pterinotoxin-2.CONCLUSIONS:Nav1.3 is expressed in neutrophils in vivo; regulates attachment, transmigration, and chemotaxis in vitro; and may serve as a relevant target for antiinflammatory effects of lidocaine.

Background: The transmembrane heparan sulfate proteoglycan Syndecan-4 (Sdc4) plays an important role in the regulation of various inflammatory disorders. However, the involvement of Sdc4 in intestinal inflammation remains unknown. Therefore, we assessed the impact of Sdc4 deficiency on experimental colitis and epithelial wound healing in vitro and in vivo. Methods: Dextran sulfate sodium (DSS)-induced colitis was monitored in wild type and Sdc4-deficient (Sdc4-/-) mice by assessment of body weight, histology, inflammatory cellular infiltration, and colon length. Syndecan-4 expression was measured by immunohistochemistry, Western blot, and quantitative real-time PCR. Epithelial permeability was evaluated by Evans blue measurements, Western blot, and immunohistological analysis of tight junction protein expression. Impact of Sdc4 on epithelial wound healing was determined by scratch assay in vitro and by colonoscopy following mechanical wounding in vivo. Results: In Sdc4(-/-) mice, colitis-like symptoms including severe weight loss, shortened colon length, histological damage, and invasion of macrophages and granulocytes were markedly aggravated compared with wild type (WT) animals. Moreover, colonic epithelial permeability in Sdc4(-/-) mice was enhanced, while tight junction protein expression decreased. Furthermore, Sdc4(-/-) colonic epithelial cells had lower cell proliferation and migration rates which presented in vivo as a prolonged intestinal wound healing phenotype. Strikingly, in WT animals, Sdc4 expression was reduced during colitis and was elevated during recovery. Conclusions: The loss of Sdc4 aggravates the course of experimental colitis, potentially through impaired epithelial cell integrity and regeneration. In view of the development of current treatment approaches involving Sdc4 inhibition for inflammatory disorders like arthritis, particular caution should be taken in case of adverse gastrointestinal side-effects.

Fibulin-6, an essential component of extracellular matrix determines the architecture of cellular junctions in tissues undergoing strain. Increased expression and deposition of fibulin-6 facilitates fibroblast migration in response to TGF-β, following myocardial infarction in mouse heart. The underlying mechanism still remains elusive. In conjunction with our previous study, we have now demonstrated that in fibulin-6 knockdown (KD) fibroblasts, not only TGF-β dependent migration, but also stress fiber formation, cellular networking and subsequently fibroblast wound contraction is almost abrogated. SMAD dependent TGF-β pathway shows ~75% decreased translocation of R-SMAD and co-SMAD into the nucleus upon fibulin-6 KD. Consequently, SMAD dependent pro-fibrotic gene expression is considerably down regulated to basal levels both in mRNA and protein. Also, investigating the non-SMAD pathways we observed a constitutive increase in pERK-levels in fibulin-6 KD fibroblast compared to control, but no change was seen in pAKT. Immunoprecipitation studies revealed 60% reduced interaction of TGF-β receptor II and I (TGFRII and I) accompanied by diminished phosphorylation of TGFRI at serin165 in fibulin-6 KD cells. In conclusion, fibulin-6 plays an important role in regulating TGF-β mediated responses, by modulating TGF-β receptor dimerization and activation to further trigger downstream pathways.

Tissue engineering provides the potential for unlimited bone substitute material. So far clinical applications of scaffold-based tissue engineering of bone exceeding a critical size failed. It has been hypothesized that uneven oxygen and nutrient supply are the limiting factors for cell survival within cell-seeded three dimensional (3D) constructs. Therefore we continuously measured the oxygen concentration within the centre of cell-seeded scaffolds using needle type oxygen microsensors and analysed the effect on cell survival under static and dynamic cell culture conditions. The oxygen measurements were used to optimize dynamic 3D cell culture conditions regarding the perfusion speeds. Our results show that under static conditions central oxygen concentrations drop to 0% after as little as 5 days leading to cell death in the centre of the scaffold. Dynamic cell culture conditions significantly increase central oxygen concentrations thus promoting cell survival in central areas of the constructs. We conclude that oxygen is a key factor when optimizing three dimensional culture conditions. These limitations have to be overcome in order to guarantee an effective (pre-) clinical use of tissue engineered bone grafts.

Background The ubiquitously expressed transmembrane heparan sulfate proteoglycan Syndecan-4 (Scd4) is crucial in inflammatory diseases, like rheumatoid arthritis. Depending on the tissue, it can either protect or promote an inflammatory process. By its binding of molecules, such as cytokines and growth factors, it can initiate signalling pathways and it has been implicated in cell-matrix adhesion, cell migration, differentiation as well as proliferation. However, the involvement of Sdc4 in intestinal inflammation is unknown so far. Our group revealed a protective function of Sdc4 in experimental intestinal inflammation. Material and methods We monitored the course of DSS-induced colitis in Scd4-/- and C57BL/6 WT mice and analysed the changes in body weight, colon length, histology and inflammatory cellular infiltrate. We also evaluated Sdc4 protein- and mRNA-level by immunofluorescence staining (IF) and quantitative real-time PCR. Colon-permeability was examined in vivo by using the Evans Blue method and measuring the clearance for Citrobacter rodentium in vivo. Wound healing effects of Scd4 were analysed in vitro by scratch assay analysis with human epithelial colon cell line (T-84) and in vivo by mechanically induced wounds in colonoscopies of Scd4-/- compared to WT mice. Results The expression of Scd4 is decreased upon the course of colitis and increased during remission. The course of colitis was markedly aggravated in Scd4-/- mice, reflected by dramatically loss of body weight, increased mortality rates and histological damage, emphasised by increased invasion of macrophages and granulocytes into the colon. Also colonic epithelial permeability of DSS-treated Scd4-/- mice was enhanced associated with an altered expression of tight junction proteins. Furthermore, Sdc4 deficiency resulted in a prolonged intestinal wound healing in vitro and in vivo due to reduced proliferation rates in vitro. Conclusions Our data indicates that Scd4 is crucial in experimental intestinal inflammation. It exerts protective effects by maintaining epithelial barrier integrity and regeneration. Further studies are needed to explore the mechanisms of Sdc4-signalling in colitis.

BACKGROUND: Hemorrhage is the most important complication of antithrombotic therapy with P2Y(12) receptor blockers. The administration of platelet concentrates (PCs) and von Willebrand factor (vWF) concentrates are common procedures to normalize impaired primary hemostasis in bleeding patients. We tested whether this strategy reverses the effect of clopidogrel using a parallel plate flow chamber model.METHODS: Whole blood from patients, who received a loading dose of clopidogrel with 600 mg and an ongoing dual antiplatelet therapy with 75 mg/d clopidogrel and 100 mg/d acetyl salicylic acid, compared with blood from healthy volunteers was examined in a collagen-coated parallel plate flow chamber. Blood was perfused by suction at a shear rate of 300/s, which is equivalent to 14 dynes/cm(2) to resemble shear stress in conduit arteries. Platelet-covered area, individual thrombus size, and the average thrombus size were assessed morphometrically. The equivalent of 2 or 5 units of PC and/or 2 U/mL of vWF concentrate were used in an attempt to restore coagulation capacity in blood samples of clopidogrel-treated patients.RESULTS: In this model, clopidogrel reduced the increase of thrombus size. The equivalent of 2 U of PC or 2 U/mL of vWF alone did not show any significant changes in thrombus size. 5 U of PC increased thrombus size in clopidogrel-treated patients (P < .05). Thrombus size in clopidogrel blood was increased by combined PC and vWF treatment (by 50%, P < .05), but this increase did not reach control levels (P < .05).CONCLUSIONS: This flow chamber model is suitable for detection of the antiplatelet effect of clopidogrel. Ex vivo addition of PC or vWF does not overcome the effects of clopidogrel in this model, but the combination of both shows a mild and significant improvement in thrombus size.

The irritant receptor TRPA1 was suggested to mediate analgesic, antipyretic but also pro-inflammatory effects of the non-opioid analgesic acetaminophen, presumably due to channel activation by the reactive metabolites parabenzoquinone (pBQ) and N-acetyl-parabenzoquinonimine (NAPQI). Here we explored the effects of these metabolites on the capsaicin receptor TRPV1, another redox-sensitive ion channel expressed in sensory neurons. Both pBQ and NAPQI, but not acetaminophen irreversibly activated and sensitized recombinant human and rodent TRPV1 channels expressed in HEK 293 cells. The reducing agents dithiothreitol and N-acetylcysteine abolished these effects when co-applied with the metabolites, and both pBQ and NAPQI failed to gate TRPV1 following substitution of the intracellular cysteines 158, 391 and 767. NAPQI evoked a TRPV1-dependent increase in intracellular calcium and a potentiation of heat-evoked currents in mouse spinal sensory neurons. Although TRPV1 is expressed in mouse hepatocytes, inhibition of TRPV1 did not alleviate acetaminophen-induced hepatotoxicity. Finally, intracutaneously applied NAPQI evoked burning pain and neurogenic inflammation in human volunteers. Our data demonstrate that pBQ and NAQPI activate and sensitize TRPV1 by interacting with intracellular cysteines. While TRPV1 does not seem to mediate acetaminophen-induced hepatotoxicity, our data identify TRPV1 as a target of acetaminophen with a potential relevance for acetaminophen-induced analgesia, antipyresia and inflammation.

Background and objectives The ubiquitously expressed transmembrane heparan sulfate proteoglycan Syndecan-4 (Scd4) is able to act in different ways. By binding a variety of molecules, such as cytokines, it can function either as a decoy receptor or it can initiate signalling pathways. It has been also implicated in cell-matrix adhesion, cell migration, differentiation, proliferation and plays an important role during inflammation in rheumatoid arthritis. Our group showed that the loss of Sdc4 has a protective effect in cartilage damage, but in osteopontin-mediated liver damage, in which syndecan-deficiency increased the pathology. Because of these dual effects, we investigate the role of Sdc4 in murine experimental colitis. Material and methods We performed DSS-induced colitis in Scd4-/- and C57BL/6 WT mice at different time points. First, we analysed the Scd4 protein- and mRNA-level by immunofluorescence staining (IF) and quantitative real-time PCR, followed by measuring the course of colitis by weight loss, colon length, histological scoring of colonic modifications and IF of inflammatory marker cells. Colon-permeability was examined in vitro by using the Evans Blue method. Wound healing effects of Scd4 were analysed in vitro by performing scratch assay analyses withhuman epithelial colon cell line (T-84) and in vivo by colonoscopy of Scd4-/- compared to WT mice. Results The expression of Scd4 was downregulated in the course of colitis and was elevated during the recovery phase. DSS-treated Scd4-/- mice lost dramatically more body weight compared to WT mice and the histological damage according to the Dieleman-Score was markedly elevated in the Scd4-/- mice. Also, we found increased invasion of macrophages and granulocytes into the colon. In DSS-treated Scd4-/- mice colon-permeability was higher compared to WT mice. The knockdown of Scd4 as well as treatment of T-84 cells with the anti-Scd4 antibody led to a delayed cell migration. Furthermore, colonoscopy experiments showed a decelerated wound healing effect in the Scd4-/- mice. Conclusions Our data indicate that Scd4 is a major molecule in the homeostasis of the colon epithelium. It exerts protective effects in intestinal inflammation with Scd4 deficiency leading to a higher permeability of the colon as well as a delayed cell migration and diminished wound healing.

Summary Background Acetate‐containing balanced electrolyte solutions are frequently used for fluid therapy in pediatric anesthesia, but no studies investigating the compatibility with common anesthetic drugs are available. Aim To reveal possible incompatibilities between common anesthetic drugs and the acetate‐containing balanced electrolyte solutions BS (Sterofundin ISO ; B.Braun Melsungen AG , Melsungen, Germany) and BS ‐G1 (E148G1 Päd; Serumwerk Bernburg AG , Bernburg, Germany), with normal saline ( NS ) as control. Methods All tested infusion solutions were mixed 1 : 1 with 28 common anesthetic drugs in concentrations used in daily clinical practice. Electrical conductivity, pH , and turbidimetric light diffusion at 405 nm were measured. Macroscopic changes such as gross precipitation, change in color, or bubble formation were also assessed. All measurements were performed immediately after mixing as well as 30 and 60 min after. Results The vast majority of drugs showed no significant change in pH , electric conductivity, turbidimetric detectable light diffusion, or macroscopic appearance after mixing with BS , BS ‐G1, and NS . Phenytoin immediately precipitated in response to all tested solutions as did diazepam. Thiopental precipitated after mixing with BS only. Conclusions Most of the tested drugs did not show any signs or evidence of incompatibility reactions. However, phenytoin and diazepam should not be in contact with the three tested solutions, including NS . Thiopental should be used with caution because it can precipitate in solutions with a low pH (e.g., BS ).

The iron-sulfur cluster containing protein mitoNEET is known to modulate the oxidative capacity of cardiac mitochondria but its function during myocardial reperfusion injury after transient ischemia is unknown. The purpose of this study was to analyze the impact of mitoNEET on oxidative stress induced cell death and its relation to the glutathione-redox system in cardiomyocytes in an in vitro model of hypoxia and reoxygenation (H/R). Our results show that siRNA knockdown (KD) of mitoNEET caused an 1.9-fold increase in H/R induced apoptosis compared to H/R control while overexpression of mitoNEET caused a 53% decrease in apoptosis. Necrosis was not affected. Apoptosis of both, mitoNEET-KD and control cells was diminished to comparable levels by using the antioxidants Tiron and glutathione compound glutathione reduced ethyl ester (GSH-MEE), indicating that mitoNEET-dependent apoptosis is mediated by oxidative stress. The interplay between mitoNEET and glutathione redox system was assessed by treating cardiomyocytes with 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthio-carbonylamino) phenylthiocarbamoylsulfanyl] propionic acid (2-AAPA), known to effectively inhibit glutathione reductase (GSR) and to decrease the GSH/GSSG ratio. Surprisingly, inhibition of GSR-activity to 20% by 2-AAPA decreased apoptosis of control and mitoNEET-KD cells to 23% and 25% respectively, while at the same time mitoNEET-protein was increased 4-fold. This effect on mitoNEET-protein was not accessible by mitoNEET-KD but was reversed by GSH-MEE. In conclusion we show that mitoNEET protects cardiomyocytes from oxidative stress-induced apoptosis during H/R. Inhibition of GSH-recycling, GSR-activity by 2-AAPA increased mitoNEET-protein, accompanied by reduced apoptosis. Addition of GSH reversed these effects suggesting that mitoNEET can in part compensate for imbalances in the antioxidative glutathione-system and therefore could serve as a potential therapeutic approach for the oxidatively stressed myocardium.