CFTR Suppresses Neointimal Formation Through Attenuating Proliferation and Migration of Aortic Smooth Muscle Cells.

ABSTRACT: Cystic fibrosis transmembrane conductance regulator (CFTR) plays important roles in arterial functions and the fate of cells. To further understand its function in vascular remodeling, we examined whether CFTR directly regulates platelet-derived growth factor-BB (PDGF-BB)-stimulated vascular smooth muscle cells (VSMCs) proliferation and migration, as well as the balloon injury-induced neointimal formation. The CFTR adenoviral gene delivery was used to evaluate the effects of CFTR on neointimal formation in a rat model of carotid artery balloon injury. The roles of CFTR in PDGF-BB-stimulated VSMC proliferation and migration were detected by mitochondrial tetrazolium assay, wound healing assay, transwell chamber method, western blot, and qPCR. We found that CFTR expression was declined in injured rat carotid arteries, while adenoviral overexpression of CFTR in vivo attenuated neointimal formation in carotid arteries. CFTR overexpression inhibited PDGF-BB-induced VSMC proliferation and migration, whereas CFTR silencing caused the opposite results. Mechanistically, CFTR suppressed the phosphorylation of PDGF receptor ß, serum and glucocorticoid-inducible kinase 1, JNK, p38 and ERK induced by PDGF-BB, and the increased mRNA expression of matrix metalloproteinase-9 and MMP2 induced by PDGF-BB. In conclusion, our results indicated that CFTR may attenuate neointimal formation by suppressing PDGF-BB-induced activation of serum and glucocorticoid-inducible kinase 1 and the JNK/p38/ERK signaling pathway.

Free fatty acid-induced H2O2 activates TRPM2 to aggravate endothelial insulin resistance via Ca2+-dependent PERK/ATF4/TRB3 cascade in obese mice.

Transient Receptor Potential Melastatin-2 (TRPM2) is a nonselective cation channel mediating Ca2+ influx in response to oxidative stress. Given that insulin resistance-related endothelial dysfunction in obesity attributes to fatty-acid-induced reactive oxygen species (ROS) overproduction, in this study, we addressed the possible role of TRPM2 in obesity-related endothelial insulin resistance and the underlying mechanisms. Whole-cell patch clamp technique, intracellular Ca2+ concentration measurement, western blot, vasorelaxation assay, and high-fat diet (HFD)-induced obese model were employed to assess the relationship between TRPM2 and endothelial insulin response. We found that both the expression and activity of TRPM2 were higher in endothelial cells of obese mice. Palmitate rose a cationic current in endothelial cells which was inhibited or enlarged by TRPM2 knockdown or overexpression. Silencing of TRPM2 remarkably improved insulin-induced endothelial Akt activation, nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production, while TRPM2 overexpression resulted in the opposite effects. Furthermore, TRPM2-mediated Ca2+ entry, CaMKII activation and the following activation of PERK/ATF4/TRB3 cascade were involved in the mechanism of obesity or palmitate-induced endothelial insulin resistance. Notably, in vivo study, knockdown of TRPM2 with adeno-associated virus harboring short-hairpin RNA (shRNA) against TRPM2 alleviated endothelial insulin resistance and ameliorated endothelium-dependent vasodilatation in obese mice. Thus, these results suggest that TRPM2-activated Ca2+ signaling is necessary to induce insulin resistance-related endothelial dysfunction in obesity. Downregulation or pharmacological inhibition of TRPM2 channels may lead to the development of effective drugs for treatment of endothelial dysfunction associated with oxidative stress state.

Knockdown of Chloride Channel-3 Inhibits Breast Cancer Growth In Vitro and In Vivo.

PURPOSE: Chloride channel-3 (ClC-3) is a member of the chloride channel family and plays a critical role in a variety of cellular activities. The aim of the present study is to explore the molecular mechanisms underlying the antitumor effect of silencing ClC-3 in breast cancer. METHODS: Human breast cancer cell lines MDA-MB-231 and MCF-7 were used in the experiments. Messenger RNA and protein expression were examined by quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation was measured by the bromodeoxyuridine method, and the cell cycle was evaluated using fluorescence-activated cell sorting. Protein interaction in cells was analyzed by co-immunoprecipitation. Tumor tissues were stained with hematoxylin-eosin and tumor burden was measured using the Metamorph software. RESULTS: Breast cancer tissues collected from patients showed an increase in ClC-3 expression. Knockdown of ClC-3 inhibited the secretion of insulin-like growth factor (IGF)-1, cell proliferation, and G1/S transition in breast cancer cells. In the mouse xenograft model of human breast carcinoma, tumor growth was significantly slower in animals injected with ClC-3-deficient cells compared with the growth of normal human breast cancer cells. In addition, silencing of ClC-3 attenuated the expression of proliferating cell nuclear antigen, Ki-67, cyclin D1, and cyclin E, as well as the activation of extracellular signalregulated protein kinases (ERK) 1/2, both in vitro and in vivo. CONCLUSION: Together, our data suggest that upregulation of ClC-3 by IGF-1 contributes to cell proliferation and tumor growth in breast cancer, and ClC-3 deficiency suppresses cell proliferation and tumor growth via the IGF/IGF receptor/ERK pathway.

Requirement of ClC-3 in G0/G1 to S Phase Transition Induced by IGF-1 via ERK1/2-Cyclins Cascade in Multiple Myeloma Cells.

BACKGROUND: ClC-3 is involved in the proliferation and migration of several cancer cells. However, ClC-3 expression and its role of cell-cycle control in multiple myeloma (MM) has not yet been investigated. METHODS: MM cells were treated with different concentrations of IGF (30, 100, 300 ng/mL), and their proliferation was examined by CCK-8. The effects of ClC-3 on cell cycle progression was detected by flow cytometry. Western blot was used to analyze the relative levels of ClC3, CD138, P21, P27, CDK, p-Erk1/2, and t-Erk1/2 protein expression. Transfection of RPMI8226 with gpClC-3 cDNA and siRNA alters the expression of ClC-3. RESULTS: We compared the expression of ClC-3 in primary myeloma cells and in MM cell lines (U266 and RPMI8266) with that in normal plasma cells (PCs) from normal subjects and found that myeloma cells from patients and MM cell lines had significantly higher expression of ClC-3. Additionally, silencing of ClC-3 with the small interfering RNA (siRNA) that targets human ClC-3 decreased proliferation of RPMI8226 after IGF-1 treatment and slowed cell cycle progression from G0/G1 to S phase, which was associated with diminished phosphorylation of ERK1/2, down-expression of cyclin E, cyclin D1 and up-regulation of p27 and p21. By contrast, overexpression of ClC-3 potentiated cell proliferation induced by IGF-1, raised the percentage of S phase cells, enhanced phosphorylation of ERK1/2, downregulated p27 and p21 and upregulated cyclin E and cyclin D1. CONCLUSIONS: ClC-3 accelerated G0/G1 to S phase transition in the cell cycle by modulating ERK1/2 kinase activity and expression of G1/S transition related proteins, making ClC-3 an attractive therapeutic target in MM.

Redox signal-mediated TRPM2 promotes Ang II-induced adipocyte insulin resistance via Ca2+-dependent CaMKII/JNK cascade.

BACKGROUND AND OBJECTIVE: Redox-sensitive transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable, nonselective cation channel which plays a crucial role in various physiological processes. However, little is known whether TRPM2 is involved in adipocyte dysfunction during hypertension. In the present study, we determined the role of TRPM2 in angiotensin II (Ang II)-induced insulin resistance in adipocytes and the underlying mechanisms. METHODS: Ang II-induced adipocyte insulin resistant model was conducted. Data from Ang II-induced hypertensive mice were used to measure the effects of TRPM2 inhibitor on insulin resistance in vivo. Whole-cell patch clamp technique, intracellular Ca2+ concentration measurement, glucose uptake assay, western blot, cDNA and siRNA transfection were employed to investigate the TRPM2/Ca2+/CaMKII/JNK signaling. RESULTS: Ang II rose a cation current similar to that activated by hydrogen peroxide (H2O2) or ADP-ribose (ADPR), which was blocked by TRPM2 inhibitor or TRPM2 siRNA in adipocytes. Knockdown of TRPM2 significantly improved the lowered insulin sensitivity induced by Ang II, including insulin stimulated glucose uptake, phosphorylation of IRS1 and Akt, interaction between IR and IRS1 and the membrane translocation of GLUT4, whereas overexpression of TRPM2 resulted in the opposite effects. These results were related to the potentiated effects of TRPM2 on Ca2+ influx and CaMKII/JNK cascade activation upon Ang II-induced challenge. Notably, the pharmacological TRPM2 inhibitor, N-(p-amylcinnamoyl)anthranilic acid (ACA), was proved to improve insulin sensitivity in adipose tissue during Ang II-induced hypertension progress. CONCLUSIONS: These data suggested that TRPM2 is a positive regulator of Ang II-induced adipocyte insulin resistance via Ca2+/CaMKII/JNK-dependent signaling pathway. Targeting TRPM2 may be a novel therapeutic strategy to treat hypertension-associated insulin resistance.

Shear stress-mediated upregulation of GTP cyclohydrolase/tetrahydrobiopterin pathway ameliorates hypertension-related decline in reendothelialization capacity of endothelial progenitor cells.

OBJECTIVES: Guanosine triphosphate cyclohydrolase/tetrahydrobiopterin (GTPCH)/(BH4) pathway has been proved to regulate the function of endothelial progenitor cells (EPCs) in deoxycorticosterone acetate-salt hypertensive mice, indicating that GTPCH/BH4 pathway may be an important repair target for hypertension-related endothelial injury. Shear stress is an important nonpharmacologic strategy to modulate the function of EPCs. Here, we investigated the effects of laminar shear stress on the GTPCH/BH4 pathway and endothelial repair capacity of circulating EPCs in hypertension. METHOD: Laminar shear stress was loaded on the human EPCs from hypertensive patients and normotensive patients. The in-vitro function, in-vivo reendothelialization capacity and GTPCH/BH4 pathway of human EPCs were evaluated. RESULTS: Both in-vitro function and reendothelialization capacity of EPCs were lower in hypertensive patients than that in normotensive patients. The GTPCH/BH4 pathway of EPCs was downregulated in hypertensive patients. Shear stress increased in-vitro function and reendothelialization capacity of EPCs from hypertensive patients and normotensive patients. Furthermore, shear stress upregulated the expression of GTPCH I and levels of BH4, nitric oxide, and cGMP of EPCs, and reduced thrombospondin-1 expression. With treatment of GTPCH knockdown or nitroarginine methyl ester inhibition, shear stress-induced increased levels of BH4, nitric oxide and cGMP of EPCs was suppressed. When GTPCH/BH4 pathway of EPCs was blocked, the effects of shear stress on in-vitro function and reendothelialization capacity of EPCs were inhibited. CONCLUSION: The study demonstrates for the first time that shear stress-induced upregulation of the GTPCH/BH4 pathway ameliorates hypertension-related decline in endothelial repair capacity of EPCs. These findings provide novel nonpharmacologic therapeutic approach for hypertension-related endothelial repair.

Endophilin-A2-mediated increase in scavenger receptor expression contributes to macrophage-derived foam cell formation.

BACKGROUND AND AIMS: Macrophage-derived foam cell formation (MFCF) is a crucial step in the pathogenesis of atherosclerosis. Uptake of oxidized low-density lipoprotein (oxLDL) by scavenger receptors is indispensable for MFCF. Endophilin-A2 has been reported to regulate clathrin-mediated endocytosis (CME). In this study, we tested the hypothesis that endophilin-A2 regulates oxLDL uptake and MFCF by mediating CME of oxLDL-scavenger receptor complexes. METHODS: In vitro MFCF was induced by oxLDL treatment. Involvement of endophilin-A2 in oxLDL cytomembrane binding, cellular uptake, and MFCF was evaluated by manipulation of endophilin-A2. RESULTS: Endophilin-A2 was involved in MFCF via scavenger receptor CD36 and scavenger receptor-A (SR-A)-mediated positive feedback pathways. We observed that oxLDL triggered interaction of endophilin-A2 with CD36 or SR-A, and induced an endophilin-A2-dependent activation of the apoptosis signal-regulating kinase-1 (ASK1)/Jun N-terminal kinase (JNK)/p38 signaling pathway. The activation of ASK1-JNK/p38 signal increased expression of both CD36 and SR-A, which promoted oxLDL cytomembrane binding, cellular uptake, and MFCF. In the absence of oxLDL, endophilin-A2 up-regulated the expression of receptors and Dil-oxLDL binding and uptake, but not the intracellular accumulation of lipids. In the presence of oxLDL, the CME inhibitors pitstop2 and ikarugamycin mimicked the inhibiting effect of endophilin-A2 knockdown and eliminated the elevating effect of endophilin-A2 overexpression on oxLDL uptake and MFCF. CONCLUSIONS: Endophilin-A2 was identified as a novel molecule regulating MFCF by mechanisms attributable to CME and beyond CME.

Endophilin A2 protects H2O2-induced apoptosis by blockade of Bax translocation in rat basilar artery smooth muscle cells.

BACKGROUND: Apoptosis plays a central role in maintaining the normal cell number and tissue homeostasis. Endophilins are a family of evolutionarily conserved proteins that have the critical role in endocytosis. Here, we determined whether endophilin A2 (EndoII) contributes to hydrogen peroxide (H2O2)-induced apoptosis in rat basilar artery smooth muscle cells (BASMCs) and the underlying mechanisms. METHODS AND RESULTS: By using small interference RNA (siRNA) and EndoII overexpression strategy, we found that EndoII siRNA knockdown reduced cell viability and promoted H2O2-induced cell apoptosis, evidenced by loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-9, 3 and poly (ADP-ribose) polymerase (PARP). In contrast, EndoII overexpression showed opposite effects and inhibited H2O2-induced BASMCs apoptosis. Further studies revealed that there was a direct interaction between EndoII and Bax. Upon H2O2-induced apoptosis, the association of EndoII with Bax were significantly decreased, while the interaction of Bax/tBid were increased, accompanied by a translocation of Bax from cytosol to mitochondria. Knockdown of EndoII did not affect the expression of Bax, but further promoted the binding of Bax with tBid and favored the accumulation of Bax to mitochondria as well as Bax activation; whereas EndoII overexpression produced the opposite effects. In addition, EndoII siRNA aggravated, but EndoII overexpression alleviated, the reduction of Bcl-2 expression in H2O2-treated cells. CONCLUSIONS: These data suggested a role of EndoII in protecting BASMCs apoptosis induced by H2O2, possibly by inhibiting the addressing of Bax to mitochondria. Targeting on EndoII may be a new strategy to treat apoptosis-associated diseases.

Threonine532 phosphorylation in ClC-3 channels is required for angiotensin II-induced Cl(-) current and migration in cultured vascular smooth muscle cells.

BACKGROUND AND PURPOSE: Angiotensin II (AngII) induces migration and growth of vascular smooth muscle cell (VSMC), which is responsible for vascular remodelling in some cardiovascular diseases. Ang II also activates a Cl(-) current, but the underlying mechanism is not clear. EXPERIMENTAL APPROACH: The A10 cell line and primary cultures of VSMC from control, ClC-3 channel null mice and WT mice made hypertensive with AngII infusions were used. Techniques employed included whole-cell patch clamp, co-immunoprecipitation, site-specific mutagenesis and Western blotting, KEY RESULTS: In VSMC, AngII induced Cl(-) currents was carried by the chloride ion channel ClC-3. This current was absent in VSMC from ClC-3 channel null mice. The AngII-induced Cl(-) current involved interactions between ClC-3 channels and Rho-kinase 2 (ROCK2), shown by N- or C-terminal truncation of ClC-3 protein, ROCK2 siRNA and co-immunoprecipitation assays. Phosphorylation of ClC-3 channels at Thr(532) by ROCK2 was critical for AngII-induced Cl(-) current and VSMC migration. The ClC-3 T532D mutant (mutation of Thr(532) to aspartate), mimicking phosphorylated ClC-3 protein, significantly potentiated AngII-induced Cl(-) current and VSMC migration, while ClC-3 T532A (mutation of Thr(532) to alanine) had the opposite effects. AngII-induced cell migration was markedly decreased in VSMC from ClC-3 channel null mice that was insensitive to Y27632, an inhibitor of ROCK2. In addition, AngII-induced cerebrovascular remodelling was decreased in ClC-3 null mice, possibly by the ROCK2 pathway. CONCLUSIONS AND IMPLICATIONS: ClC-3 protein phosphorylation at Thr(532) by ROCK2 is required for AngII-induced Cl(-) current and VSMC migration that are involved in AngII-induced vascular remodelling in hypertension.

TRPC3 channel confers cerebrovascular remodelling during hypertension via transactivation of EGF receptor signalling.

AIMS: Ionic perturbation in vascular smooth muscle cells contributes to cerebrovascular remodelling in the setting of hypertension, but the role of transient receptor potential (TRP) channel superfamily remains unknown. The present study was conducted to define the contribution of TRP channels to cerebrovascular remodelling. METHODS AND RESULTS: By integrating quantitative PCR, western blotting, patch clamping, and Ca(2+) imaging, we identified TRP channel, subfamily canonical, member 3 (TRPC3) as the channel subtype most considerably elevated in basilar arteries of two-kidney, two-clip stroke-prone hypertensive rats. Importantly, administration of pyrazole 3 (Pyr3), a TRPC3 channel blocker, attenuated cerebrovascular remodelling. During hypertension, epidermal growth factor receptor (EGFR) was transactivated, as evidenced by marked EGFR phosphorylation, increased pro-HB-EGF shedding, and elevated activity of ADAM17 (HB-EGF sheddase). ADAM17 activity was increased owing to enhanced activation rather than elevated expression. Remarkably, Pyr3 treatment suppressed EGFR transactivation in hypertension. In proliferating basilar artery smooth muscle cells or basilar arteries of hypertensive rats, co-immunoprecipitation assay revealed an interaction between TRPC3 and ADAM17 upon Ang II stimulation. CONCLUSION: Collectively, we demonstrated that enhanced EGFR transactivation, due to increased TRPC3 expression and functional coupling of TRPC3/ADAM17, resulted in cerebrovascular remodelling. Therefore, TRPC3-induced EGFR transactivation may be therapeutically exploited to prevent hypertension-induced cerebrovascular remodelling.

Liver Transplantation in Antituberculosis Drugs-Induced Fulminant Hepatic Failure: A Case Report and Review of the Literature.

The antituberculosis drugs isoniazid (INH), rifampicin (RMP), pyrazinamide (PZA), and ethambutol (EMB) usually expose patients to the risk of fulminant hepatic failure (FHF). This report presents a case of liver transplantation in antituberculosis drugs-induced FHF and reviews the relevant literature. A 39-year-old woman with pelvic and salpinx tuberculosis experienced complex pelvic exenteration. After the operation, she was administrated INH, RMP, PZA, and EMB to prevent tuberculosis. Two months later, examination revealed severe FHF and the antituberculosis therapy regimen was changed to ciprofloxacin and streptomycin. Subsequently, urgent orthotopic liver transplantation was performed. Posttransplantation, her serum transaminases improved gradually, but her total bilirubin level and direct bilirubin level continued to worsen, which may have been related to the rejection. However, irreversible damage from antituberulosis drugs was note excluded. Two liver biopsies and histological examinations were performed. One year after transplantation, she died as a consequence of ischemic cholangitis and pulmonary infection. A literature review revealed 9 other published cases of antituberculosis drugs-associated FHF with liver transplantation.This report suggests that, in most cases of antituberculosis drugs-induced FHF, discontinuation of toxic drugs and orthotopic liver transplantation are always sufficient treatment.

Measurement of Magic Wavelengths for the

We demonstrate experimentally the existence of magic wavelengths and determine the ratio of oscillator strengths for a single trapped ion. For the first time, two magic wavelengths near 396 nm for the ^{40}Ca^{+} clock transition are measured simultaneously with high precision. By tuning the applied laser to an intermediate wavelength between transitions 4s_{1/2}→4p_{1/2} and 4s_{1/2}→4p_{3/2}, the sensitivity of the clock transition Stark shift to the oscillator strengths is greatly enhanced. Furthermore, with the measured magic wavelengths, we determine the ratio of the oscillator strengths with a deviation of less than 0.5%. Our experimental method may be applied to measure magic wavelengths for other ion clock transitions. Promisingly, the measurement of these magic wavelengths paves the way to building all-optical trapped ion clocks.

Increased endothelial progenitor cells and nitric oxide in young prehypertensive women.

This study investigated the effect of sex differences on circulating endothelial progenitor cells (EPCs) in prehypertension and its underlying mechanism. The authors found that premenopausal women show increased number and activity of circulating EPCs when compared with men, which was similar to enhanced nitric oxide (NO) level in plasma or culture medium. There was no difference in the number and activity of circulating EPCs and NO level between normotensive and prehypertensive premenopausal women. There was also no difference seen in levels of vascular endothelial growth factor and granulocyte macrophage colony-stimulating factor. Both number and activity of circulating EPCs were correlated with the level of NO. The present study firstly demonstrated that the number and activity of circulating EPCs were preserved in prehypertensive premenopausal women, which was related to the restoration of NO production. The sex differences in EPCs in prehypertension may be involved in the mechanism underlying vascular protection in premenopausal women.

A randomized controlled trial of combination treatment with ketoconazole 2% cream and adapalene 0.1% gel in pityriasis versicolor.

BACKGROUND: Ketoconazole cream and adapalene gel are effective drugs against pityriasis versicolor. However, there are no reports on combination treatment with both compounds in pityriasis versicolor. OBJECTIVE: To evaluate the efficacy and safety of combination therapy with adapalene 0.1% gel and ketoconazole 2% cream against pityriasis versicolor. METHODS: Participants with pityriasis versicolor were randomly assigned to two groups: the combination group was treated with adapalene 0.1% gel and ketoconazole 2% cream once daily, and the monotherapy group received ketoconazole 2% cream twice daily. The treatment lasted 2 weeks in both groups. Outcomes were assessed at baseline and 1, 2 and 4 weeks after the initiation of treatment. RESULTS: We noted clinically significant differences in total improvement rates between groups Weeks 1 and 2. A statistically significant difference was obtained Week 4. The treatment was well tolerated by all participants. CONCLUSIONS: The combination of adapalene 0.1% gel and ketoconazole 2% cream is effective and safe in the treatment of pityriasis versicolor. This therapeutic regimen was rapid, providing a valuable option for patients with pityriasis versicolor.

Combination treatment of oral terbinafine with topical terbinafine and 10% urea ointment in hyperkeratotic type tinea pedis.

Hyperkeratotic-type tinea pedis is chronic and recalcitrant to topical antifungal agents. Some topical antifungal agents are effective; however, long duration of therapy is required, which often reduce the treatment compliance of patients. To seek for short period therapy of hyperkeratotic type tinea pedis, in this study, we observed the efficacy and safety of treatment of topical terbinafine and 10% urea ointment combined oral terbinafine. Participants with hyperkeratotic type tinea pedis were randomly assigned to two groups. Patients in group I were treated with oral terbinafine for 2 weeks and topical terbinafine and 10% urea ointment for 4 weeks, whereas in group II, only the above topical agents were applied for 12 weeks. Clinical improvement rates and fungal eradication rates were compared between the two groups at 24 weeks after the initiation of treatment. The group I had stopped the topical therapy 8 weeks earlier than group II. There were no significant differences in mycological eradication rates and clinical improvement rates between the two groups, besides, no major side effects were noted in both groups. The short combination therapy with oral terbinafine was effective and safe; it should be a valuable option for patients with hyperkeratotic type tinea pedis.

ClC-3 chloride channel/antiporter defect contributes to inflammatory bowel disease in humans and mice.

BACKGROUND: ClC-3 channel/antiporter plays a critical role in a variety of cellular activities. ClC-3 has been detected in the ileum and colon. OBJECTIVE: To determine the functions of ClC-3 in the gastrointestinal tract. DESIGN: After administration of dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS), intestines from ClC-3-/- and wild-type mice were examined by histological, cellular, molecular and biochemical approaches. ClC-3 expression was determined by western blot and immunostaining. RESULTS: ClC-3 expression was reduced in intestinal tissues from patients with UC or Crohn's disease and from mice treated with DSS. Genetic deletion of ClC-3 increased the susceptibility of mice to DSS- or TNBS-induced experimental colitis and prevented intestinal recovery. ClC-3 deficiency promoted DSS-induced apoptosis of intestinal epithelial cells through the mitochondria pathway. ClC-3 interacts with voltage-dependent anion channel 1, a key player in regulation of mitochondria cytochrome c release, but DSS treatment decreased this interaction. In addition, lack of ClC-3 reduced the numbers of Paneth cells and impaired the expression of antimicrobial peptides. These alterations led to dysfunction of the epithelial barrier and invasion of commensal bacteria into the mucosa. CONCLUSIONS: A defect in ClC-3 may contribute to the pathogenesis of IBD by promoting intestinal epithelial cell apoptosis and Paneth cell loss, suggesting that modulation of ClC-3 expression might be a new strategy for the treatment of IBD.

Solution of the Dirac Coulomb equation for helium-like ions in the Poet-Temkin model.

The Dirac-Coulomb equation for the helium atom is studied under the restrictions of the Poet-Temkin model which replaces the 1/r12 interaction by the simplified 1/r> form. The effective reduction in the dimensionality made it possible to obtain binding energies for the singlet and triplet states in this model problem with a relative precision from 10(-8) to 10(-10). The energies for the singlet state were consistent with a previous configuration interaction calculation [H. Tatewaki and Y. Watanabe, Chem. Phys. 389, 58 (2011)]. Manifestations of Brown-Ravenhall disease were noted at higher values of nuclear charge and ultimately limited the accuracy of the Poet-Temkin model energy. The energies from a no-pair configuration interaction (CI) calculation (the negative-energy states for the appropriate hydrogen-like ion were excluded from the CI expansion) were found to be different from the unrestricted B-spline calculation.

Deficiency of volume-regulated ClC-3 chloride channel attenuates cerebrovascular remodelling in DOCA-salt hypertension.

AIMS: We have previously demonstrated that ClC-3 chloride channel activity and expression are significantly increased in remodelled cerebral vessels of hypertensive rats. This study aims to examine whether this channel directly regulates cerebrovascular remodelling during hypertension by using ClC-3(-/-) mice. METHODS AND RESULTS: After DOCA-salt treatment, medial cross-sectional area, media thickness, and media-lumen ratio of the basilar artery of ClC-3(+/+) mice were significantly increased, accompanied by reduced lumen diameter, indicating apparent vascular remodelling. The vascular ultrastructure of ClC-3(+/+) hypertensive mice by electron microscopy revealed obvious disarray of SMCs and extracellular matrix accumulation. Immunofluorescence analysis showed that fibronectin was overexpressed in ClC-3(+/+) DOCA-salt mice. All of these vascular structure alterations were prevented in ClC-3(-/-) mice despite DOCA-salt treatment. However, propranolol, which reduced blood pressure as effectively as ClC-3 deficiency, failed to prevent basilar artery from remodelling. The vascular structure injury in ClC-3(+/+) hypertensive mice was accompanied by significantly increased expression of matrix metalloproteinase (MMP)-2, membrane-type (MT)1-MMP, and tissue inhibitor of metalloproteinase (TIMP)-2, which was inhibited by ClC-3 knockout. Additionally, the increase in transforming growth factor (TGF)-ß1 level in serum, as well as phosphorylation of Smad3 at serine 423/425 in basilar artery, induced by DOCA-salt, was markedly prevented in ClC-3(-/-) mice. CONCLUSION: Our findings suggest that ClC-3 deficiency attenuates cerebrovascular remodelling possibly via the suppression of MMPs/TIMP expression and TGF-ß1/Smad3 signalling pathway in this hypertension.

Mitochondria dependent pathway is involved in the protective effect of bestrophin-3 on hydrogen peroxide-induced apoptosis in basilar artery smooth muscle cells.

Bestrophin 3 (Best-3) is expressed in a variety of tissues, such as cardiac, smooth muscle and renal tissues, and it is highly expressed in rat basilar arterial smooth muscle cells (BASMCs). Lee et al. (Biochim Biophys Acta 1823:1864-1876, 2012) reported that Best-3 prevented apoptotic cell death induced by endoplasmic reticulum stress. In the present study, we used small interference RNA (siRNA) and bestrophin 3 cDNA transfection strategy to investigate whether Best-3 can provide a protective effect on apoptosis induced by hydrogen peroxide (H2O2) in BASMCs and studied the underlying mechanisms. We found that silencing of Best-3 with siRNA resulted in an increased H2O2-induced apoptosis and a decreased cell viability, whereas overexpression of Best-3 significantly prevented the apoptotic cell death and increased the cell viability. Overexpression of Best-3 could stabilize the mitochondrial membrane potential, increase the ratio of Bcl-2/Bax, and decrease cytochrome c release and caspase-3 activation. In contrast, silencing of Best-3 produced the opposite effects. Our present data strongly suggest that Best-3 inhibits apoptosis induced by H2O2 in BASMCs through mitochondria dependent pathway.

Tyrosine 284 phosphorylation is required for ClC-3 chloride channel activation in vascular smooth muscle cells.

AIMS: The ClC-3 chloride channel (and current, ICl,ClC-3) plays an important role in cell volume regulation, proliferation, and apoptosis in vascular smooth muscle cells, and is a potential target for prevention of vascular remodelling and stroke. However, modulation of ICl,ClC-3 by intercellular signalling is not fully understood. Although it has been suggested that tyrosine phosphorylation is required for ICl,ClC-3 activation, the potential tyrosine residues in the ClC-3 protein are not clear. In the present study, the critical tyrosine residues in ClC-3 protein were investigated. METHODS AND RESULTS: Site-specific mutagenesis, immunoprecipitation, patch clamp, and Cl(-) transport imaging techniques were employed. We found that activation of ICl,ClC-3 was associated with tyrosine phosphorylation of the ClC-3 protein. Three potential tyrosine residues, Y284, Y572, and Y631, were mutated to phenylalanine, and only mutation, at Y284 within a consensus Src-phosphorylation site, completely blocked ICl,ClC-3. Phosphomimetic mutation Y284D increased the Cl(-) current and Cl(-) efflux mediated by ClC-3. The Y284F mutation completely abolished the protective effect of ClC-3 on apoptosis, whereas the Y284D mutation potentiated it. There was an interaction between Src kinase and ClC-3 protein, and the Y284D mutation abrogated the inhibitory effect of SU6656, a Src family kinase inhibitor, on ClC-3 Cl(-) current. CONCLUSION: Tyrosine 284 phosphorylation in the ClC-3 channel targeted by Src kinase is an important molecular mechanism for ClC-3 channel activation.