Na-H Exchanger Isoform-2 (NHE2) Mediates Butyrate-dependent Na+ Absorption in Dextran Sulfate Sodium (DSS)-induced Colitis

Diarrhea associated with ulcerative colitis (UC) occurs primarily as a result of reduced Na⁺ absorption. Although colonic Na⁺ absorption is mediated by both epithelial Na⁺ channels (ENaC) and Na-H exchangers (NHE), inhibition of NHE-mediated Na⁺ absorption is the primary cause of diarrhea in UC. As there are conflicting observations reported on NHE expression in human UC, the present study was initiated to identify whether NHE isoforms (NHE2 and NHE3) expression is altered and how Na⁺ absorption is regulated in DSS-induced inflammation in rat colon, a model that has been used to study UC. Western blot analyses indicate that neither NHE2 nor NHE3 expression is altered in apical membranes of inflamed colon. Na⁺ fluxes measured in vitro under voltage clamp conditions in controls demonstrate that both HCO₃⁻-dependent and butyrate-dependent Na⁺ absorption are inhibited by S3226 (NHE3-inhibitor), but not by HOE694 (NHE2-inhibitor) in normal animals. In contrast, in DSS-induced inflammation, butyrate-, but not HCO₃⁻-dependent Na⁺ absorption is present and is inhibited by HOE694, but not by S3226. These observations indicate that in normal colon NHE3 mediates both HCO₃⁻-dependent and butyrate-dependent Na⁺ absorption, whereas DSS-induced inflammation activates NHE2, which mediates butyrate-dependent (but not HCO₃⁻-dependent) Na⁺ absorption. In in vivo loop studies HCO₃⁻-Ringer and butyrate-Ringer exhibit similar rates of water absorption in normal rats, whereas in DSS-induced inflammation luminal butyrate-Ringer reversed water secretion observed with HCO₃⁻-Ringer to fluid absorption. Lumen butyrate-Ringer incubation activated NHE3-mediated Na⁺ absorption in DSS-induced colitis. These observations suggest that the butyrate activation of NHE2 would be a potential target to control UC-associated diarrhea.     

Sorting nexin 27 regulates basal and stimulated brush border trafficking of NHE3

Sorting nexin 27 (SNX27) contains a PDZ domain that is phylogenetically related to the PDZ domains of the NHERF proteins. Studies on nonepithelial cells have shown that this protein is located in endosomes, where it regulates trafficking of cargo proteins in a PDZ domain–dependent manner. However, the role of SNX27 in trafficking of cargo proteins in epithelial cells has not been adequately explored. Here we show that SNX27 directly interacts with NHE3 (C-terminus) primarily through the SNX27 PDZ domain. A combination of knockdown and reconstitution experiments with wild type and a PDZ domain mutant (GYGF → GAGA) of SNX27 demonstrate that the PDZ domain of SNX27 is required to maintain basal NHE3 activity and surface expression of NHE3 in polarized epithelial cells. Biotinylation-based recycling and degradation studies in intestinal epithelial cells show that SNX27 is required for the exocytosis (not endocytosis) of NHE3 from early endosome to plasma membrane. SNX27 is also required to regulate the retention of NHE3 on the plasma membrane. The findings of the present study extend our understanding of PDZ-mediated recycling of cargo proteins from endosome to plasma membrane in epithelial cells.     

Isotope-reinforced polyunsaturated fatty acids protect yeast cells from oxidative stress

The facile abstraction of bis-allylic hydrogens from polyunsaturated fatty acids (PUFAs) is the hallmark chemistry responsible for initiation and propagation of autoxidation reactions. The products of these autoxidation reactions can form cross-links to other membrane components and damage proteins and nucleic acids. We report that PUFAs deuterated at bis-allylic sites are much more resistant to autoxidation reactions, because of the isotope effect. This is shown using coenzyme Q-deficient Saccharomyces cerevisiae coq mutants with defects in the biosynthesis of coenzyme Q (Q). Q functions in respiratory energy metabolism and also functions as a lipid-soluble antioxidant. Yeast coq mutants incubated in the presence of the PUFA α-linolenic or linoleic acid exhibit 99% loss of colony formation after 4 h, demonstrating a profound loss of viability. In contrast, coq mutants treated with monounsaturated oleic acid or with one of the deuterated PUFAs, 11,11-D₂-linoleic or 11,11,14,14-D₄-α-linolenic acid, retain viability similar to wild-type yeast. Deuterated PUFAs also confer protection to wild-type yeast subjected to heat stress. These results indicate that isotope-reinforced PUFAs are stabilized compared to standard PUFAs, and they protect coq mutants and wild-type yeast cells against the toxic effects of lipid autoxidation products. These findings suggest new approaches to controlling ROS-inflicted cellular damage and oxidative stress.     

Quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spots using liquid chromatography-tandem mass spectrometry

A high-performance liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the quantitative analysis of NIM811, a cyclophilin inhibitor, in human dried blood spot (DBS) samples, which were produced by spotting 20 μl whole blood onto FTA cards. A 3mm disc was cut from the DBS samples and extracted using methanol, followed by liquid-liquid extraction with MTBE. The reconstituted extracts were chromatographed using a Halo C(18) column and gradient elution for MS/MS detection. The possible impact of hematocrit, blood sample volume and punching location on DBS sampling was investigated. The results showed that blood sample volume or punching location has no impact on assay performance, but the presence of a high hematocrit resulted in significantly increased analyte concentrations measured from the high QC samples. The current method was fully validated over the range of 10.0-5000 ng/ml with correlation coefficients (r(2)) for three validation batches equal to or better than 0.991. The accuracy and precision (CV) at the LLOQ were -0.7 to 6.0% bias of the nominal value (10.0 ng/ml) and 10.2-2.3%, respectively. For the balance of QC samples (20.0, 50.0, 750, 1500 and 3750 ng/ml), the precision (CV) ranged from 3.2 to 11.7% and from 5.6 to 10.2%, respectively, for the intra-day and inter-day evaluations. The accuracy ranged from -6.8 to 8.5% and -0.2% to 2.7% bias, respectively, for the intra-day and inter-day batches. NIM811 is stable in the DBS samples for at least 24h at room temperature and 4h at 60°C. Interestingly, the long term stability (LTS) assessment showed that the stability of the analyte is better when the DBS samples were stored at a lower storage temperature (e.g. ≤ -60°C) compared to storage at room temperature. This is probably due to the interaction of the additives and/or other materials (e.g. cellulose, etc) on the DBS card with NIM811, a cyclic peptide. The current methodology has been applied to determine the NIM811 levels in DBS samples prepared from a clinical study.     

ATR autophosphorylation as a molecular switch for checkpoint activation

The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response.     

IL-18 inhibits growth of murine orthotopic prostate carcinomas via both adaptive and innate immune mechanisms

Interleukin(IL)-18 is a pleiotrophic cytokine with functions in immune modulation, angiogenesis and bone metabolism. In this study, the potential of IL-18 as an immunotherapy for prostate cancer (PCa) was examined using the murine model of prostate carcinoma, RM1 and a bone metastatic variant RM1(BM)/B4H7-luc. RM1 and RM1(BM)/B4H7-luc cells were stably transfected to express bioactive IL-18. These cells were implanted into syngeneic immunocompetent mice, with or without an IL-18-neutralising antibody (αIL-18, SK113AE4). IL-18 significantly inhibited the growth of both subcutaneous and orthotopic RM1 tumors and the IL-18 neutralizing antibody abrogated the tumor growth-inhibition. In vivo neutralization of interferon-gamma (IFN-γ) completely eliminated the anti-tumor effects of IL-18 confirming an essential role of IFN-γ as a down-stream mediator of the anti-tumor activity of IL-18. Tumors from mice in which IL-18 and/or IFN-γ was neutralized contained significantly fewer CD4(+) and CD8(+) T cells than those with functional IL-18. The essential role of adaptive immunity was demonstrated as tumors grew more rapidly in RAG1(-/-) mice or in mice depleted of CD4(+) and/or CD8(+) cells than in normal mice. The tumors in RAG1(-/-) mice were also significantly smaller when IL-18 was present, indicating that innate immune mechanisms are involved. IL-18 also induced an increase in tumor infiltration of macrophages and neutrophils but not NK cells. In other experiments, direct injection of recombinant IL-18 into established tumors also inhibited tumor growth, which was associated with an increase in intratumoral macrophages, but not T cells. These results suggest that local IL-18 in the tumor environment can significantly potentiate anti-tumor immunity in the prostate and clearly demonstrate that this effect is mediated by innate and adaptive immune mechanisms.     

The spatial organization of proton and lactate transport in a rat brain tumor

Tumors create a heterogeneous acidic microenvironment which assists their growth and which must be taken into account in the design of drugs and their delivery. In addition, the acidic extracellular pH (pHe) is itself exploited in several experimental techniques for drug delivery. The way the acidity is created is not clear. We report here the spatial organization of key proton-handling proteins in C6 gliomas in rat brain. The mean profiles across the tumor rim of the Na+/H+ exchanger NHE1, and the lactate-H+ cotransporter MCT1, both showed peaks. NHE1, which is important for extension and migration of cells in vitro, showed a peak 1.55 times higher than in extratumoural tissue at 0.33 mm from the edge. MCT1 had a broader peak, further into the tumor (maximum 1.76 fold at 1.0 mm from the edge). In contrast, MCT4 and the carbonic anhydrase CAIX, which are associated with hypoxia, were not significantly upregulated in the rim. The spatial distribution of MCT4 was highly correlated with that of CAIX, suggesting that their expression is regulated by the same factors. Since protons extruded by NHE1 diffuse away through extracellular clefts, NHE1 requires a continuous source of intracellular protons. From the stoichiometries of metabolic pathways that produce or consume H+, and the greater availability of glucose compared to oxygen in most parts of a tumor, we support the classic view that most of the net proton efflux from C6 gliomas originates in glycolytic formation of lactate and H+ inside the tumor, but add that some lactate is taken up into cells in the rim on MCT1, and some lactate diffuses away, leaving its associated protons available to re-enter cells for extrusion on NHE1. Therapeutic inhibition of NHE1, MCT1 or CAIX is predicted to affect different parts of a tumor.     

Stiffness gradients mimicking in vivo tissue variation regulate mesenchymal stem cell fate

Mesenchymal stem cell (MSC) differentiation is regulated in part by tissue stiffness, yet MSCs can often encounter stiffness gradients within tissues caused by pathological, e.g., myocardial infarction ～8.7±1.5 kPa/mm, or normal tissue variation, e.g., myocardium ～0.6±0.9 kPa/mm; since migration predominantly occurs through physiological rather than pathological gradients, it is not clear whether MSC differentiate or migrate first. MSCs cultured up to 21 days on a hydrogel containing a physiological gradient of 1.0±0.1 kPa/mm undergo directed migration, or durotaxis, up stiffness gradients rather than remain stationary. Temporal assessment of morphology and differentiation markers indicates that MSCs migrate to stiffer matrix and then differentiate into a more contractile myogenic phenotype. In those cells migrating from soft to stiff regions however, phenotype is not completely determined by the stiff hydrogel as some cells retain expression of a neural marker. These data may indicate that stiffness variation, not just stiffness alone, can be an important regulator of MSC behavior.