Intestinal Dysbiosis: A Possible Mechanism of Alcohol-Induced Endotoxemia and Alcoholic Steatohepatitis in Rats

Background:  Clinical and animal data indicate that gut-derived endotoxin and other luminal bacterial products are necessary cofactors for development of alcoholic liver disease (ALD). Although gut leakiness is clearly an important cause of endotoxemia in ALD, it cannot fully explain endotoxemia in all ALD subjects and thus other factors may be involved. One possible factor is a change in gut microbiota composition (dysbiosis). Thus, the aim of our study was to interrogate the gut bacterial microbiota in alcohol-fed rats to see if chronic alcohol consumption affects gut bacteria composition.
Method:  Male Sprague–Dawley rats were given either alcohol or dextrose intragastrically by gavage twice daily for up to 10 weeks. A subgroup of rats was also given either a probiotic (lactobacillus GG) or a prebiotic (oats) by gavage. Ileal and colonic mucosal-attached microbiota composition were interrogated by Length Heterogeneity PCR (LH-PCR) fingerprinting.
Results:  Bacterial microbiota composition in alcohol-fed rats is not different from dextrose-fed rats at weeks 4 and 6. Mucosa-associated microbiota composition in the colon is altered at 10 weeks of daily alcohol gavage. Both LGG and oats prevented alcohol-induced dysbiosis up to 10 weeks of alcohol treatment.
Conclusion:  Daily alcohol consumption for 10 weeks alters colonic mucosa-associated bacterial microbiota composition in rats. Our data showed, for the first time, that daily alcohol consumption can affect colonic microbiome composition and suggest that dysbiosis may be an important mechanism of alcohol-induced endotoxemia. Further studies are needed to determine how dysbiotic microbiota contributes to development of ALD and whether therapeutic interventions targeted towards dysbiotic microbiota can prevent complications of alcoholism like ALD.     

清肝活血方对肠黏膜上皮细胞紧密连接相关蛋白ZO-1的影响

目的:探讨清肝活血方对肠黏膜上皮细胞紧密连接蛋白ZO-1的影响,明确清肝活血方干预肠源性内毒素移位的作用位点。方法:应用Caco-2细胞株,建立肠上皮细胞模型,筛选酒精作用的剂量、时间,以Western方法检测该方药物对Caco-2表达ZO-1蛋白的影响。结果:低浓度酒精即可明显增加Caco-2细胞模型的通透性,减少ZO-1蛋白的表达;清肝活血方药物可以提高ZO-1蛋白的表达,且呈一定的量效关系。结论:清肝活血方可以通过调控ZO-1蛋白的表达改善酒精引起的Caco-2细胞的通透性。     

The type of dietary fat modulates intestinal tight junction integrity, gut permeability, and hepatic toll-like receptor expression in a mouse model of alcoholic liver disease

Background: Interactions between the gut, immune system, and the liver, as well as the type of fat in the diet, are critical components of alcoholic liver disease (ALD). The goal of the present study was to determine the effects of saturated fat (SF) and unsaturated fat (USF) on ethanol (EtOH)‐induced gut‐liver interactions in a mouse model of ALD. Methods: C57BL/6N mice were fed Lieber–DeCarli liquid diets containing EtOH and enriched in USF (corn oil) or SF (medium chain triglycerides:beef tallow). Control mice were pair‐fed on an isocaloric basis. Liver injury and steatosis, blood endotoxin levels, intestinal permeability, and tight junction (TJ) integrity, as well as hepatic Toll‐like receptor (TLR) gene expression, were evaluated. Results: After 8 weeks of EtOH feeding, liver injury and steatosis were observed in USF + EtOH group compared with control and SF + EtOH. Significantly increased intestinal permeability in conjunction with elevated blood endotoxin levels were observed in the ileal segments of the mice fed USF + EtOH. USF diet alone resulted in down‐regulation of intestinal TJ protein mRNA expression compared with SF. Importantly, alcohol further suppressed TJ proteins in USF + EtOH, but did not affect intestinal TJ in SF + EtOH group. The type of fat in the diet alone did not affect hepatic TLR expression. Compared with control animals, hepatic TLR (TLR 1, 2, 3, 4, 7, 8, 9) mRNA expression was significantly (p < 0.05) increased in USF + EtOH, but not in SF + EtOH group. Notably, TLR5 was the only up‐regulated TLR in both SF + EtOH and USF + EtOH groups. Conclusions: Dietary fat is an important cofactor in alcohol‐associated liver injury. We demonstrate that USF (corn oil/linoleic acid) by itself results in dysregulation of intestinal TJ integrity leading to increased gut permeability, and alcohol further exacerbates these alterations. We postulate that elevated blood endotoxin levels in response to USF and alcohol in conjunction with up‐regulation of hepatic TLRs combine to cause hepatic injury in ALD.     

Oral treatment with plecanatide or dolcanatide attenuates visceral hypersensitivity via activation of guanylate cyclase-C in rat models

AIM To investigate the effects of plecanatide and dolcanatide on maintenance of paracellular permeability, integrity of tight junctions and on suppression of visceral hypersensitivity. METHODS Transport of fluorescein isothiocyanate(FITC)-dextran was measured to assess permeability across cell monolayers and rat colon tissues. Effects of plecanatide and dolcanatide on the integrity of tight junctions in Caco-2 and T84 monolayers and on the expression and localization of occludin and zonula occludens-1(ZO-1) were examined by immunofluorescence microscopy. Anti-nociceptive activity of these agonists was evaluated in trinitrobenzene sulfonic acid(TNBS)-induced inflammatory as well as in non-inflammatory partial restraint stress(PRS) rat models. Statistical significance between the treatment groups in the permeability studies were evaluated using unpaired t-tests.RESULTS Treatment of T84 and Caco-2 monolayers with lipopolysaccharide(LPS) rapidly increased permeability, which was effectively suppressed when monolayers were also treated with plecanatide or dolcanatide. Similarly, when T84 and Caco-2 monolayers were treated with LPS, cell surface localization of tight junction proteins occludin and ZO-1 was severely disrupted. When cell monolayers were treated with LPS in the presence of plecanatide or dolcanatide, occludin and ZO-1 were localized at the cell surface of adjoining cells, similar to that observed for vehicle treated cells. Treatment of cell monolayers with plecanatide or dolcanatide without LPS did not alter permeability, integrity of tight junctions and cell surface localization of either of the tight junction proteins. In rat visceral hypersensitivity models, both agonists suppressed the TNBS-induced increase in abdominal contractions in response to colorectal distension without affecting the colonic wall elasticity, and both agonists also reduced colonic hypersensitivity in the PRS model. CONCLUSION Our results suggest that activation of GC-C signaling might be involved in maintenance of barrier function, possibly through regulating normal localization of tight junction proteins. Consistent with these findings, plecanatide and dolcanatide showed potent antinociceptive activity in rat visceral hypersensitivity models. These results imply that activation of GC-C signaling may be an attractive therapeutic approach to treat functional constipation disorders and inflammatory gastrointestinal conditions.     

Epidermal growth factor prevents acetaldehyde-induced paracellular permeability in Caco-2 cell monolayer

BACKGROUND: Intestinal permeability and endotoxemia play a crucial role in the pathogenesis of alcoholic liver disease. Previous studies showed that acetaldehyde disrupts intestinal epithelial barrier function and increases paracellular permeability by a tyrosine kinase-dependent mechanism. In the present study, the role of epidermal growth factor (EGF) in protection of epithelial barrier function from acetaldehyde was evaluated in Caco-2 intestinal epithelial cell monolayer. METHODS: Caco-2 cells on Transwell inserts were exposed to acetaldehyde in the absence or presence of EGF, and the paracellular permeability was evaluated by measuring transepithelial electrical resistance and unidirectional flux of inulin. Integrity of epithelial tight junctions and adherens junctions was analyzed by confocal immunofluorescence microscopy and immunoblot analysis of occludin, zonula occludens (ZO)-1, E-cadherin, and beta-catenin in the actin cytoskeleton. Reorganization of actin cytoskeletal architecture was examined by confocal microscopy. RESULTS: Acetaldehyde increased paracellular permeability to inulin and lipopolysaccharide, and EGF significantly reduced these effects of acetaldehyde in a time- and dose-dependent manner. EGF prevented acetaldehyde-induced reorganization of occludin, ZO-1, E-cadherin, and beta-catenin from the cellular junctions to the intracellular compartments. Acetaldehyde treatment induced a reorganization of actin cytoskeletal network and reduced the levels of occludin, ZO-1, E-cadherin, and beta-catenin associated with the actin cytoskeleton. EGF effectively prevented acetaldehyde-induced reorganization of actin cytoskeleton and the interaction of occludin, ZO-1, E-cadherin, and beta-catenin with the actin cytoskeleton. CONCLUSION: These results indicate that EGF attenuates acetaldehyde-induced disruption of tight junctions and adherens junctions and prevents acetaldehyde-induced reorganization of actin cytoskeleton and its interaction with occludin, ZO-1, E-cadherin, and beta-catenin.     

Sirtuin 1 alleviates endoplasmic reticulum stress-mediated apoptosis of intestinal epithelial cells in ulcerative colitis

BACKGROUND Sirtuin 1(SIRT1) is a nicotinamide adenine dinucleotide(NAD~+)-dependent protein deacetylase that is involved in various diseases,including cancers,metabolic diseases,and inflammation-associated diseases.However,the role of SIRT1 in ulcerative colitis(UC) is still confusing.AIM To investigate the role of SIRT1 in intestinal epithelial cells(IECs) in UC and further explore the underlying mechanisms.METHODS We developed a coculture model using macrophages and Caco-2 cells.After treatment with the SIRT1 activator SRT1720 or inhibitor nicotinamide(NAM),the expression of occludin and zona occludens 1(ZO-1) was assessed by Western blot analysis.Annexin V-APC/7-AAD assays were performed to evaluate Caco-2 apoptosis.Dextran sodium sulfate(DSS)-induced colitis mice were exposed to SRT1720 or NAM for 7 d.Transferase-mediated dUTP nick-end labeling(TUNEL) assays were conducted to assess apoptosis in colon tissues.The expression levels of glucose-regulated protein 78(GRP78),CCAAT/enhancerbinding protein homologous protein(CHOP),caspase-12,caspase-9,and caspase-3 in Caco-2 cells and the colon tissues of treated mice were examined by quantitative real-time PCR and Western blot RESULTS SRT1720 treatment increased the protein levels of occludin and ZO-1 and inhibited Caco-2 apoptosis,whereas NAM administration caused the opposite effects.DSS-induced colitis mice treated with SRT1720 had a lower disease activity index(P 0.01),histological score(P 0.001),inflammatory cytokine levels(P 0.01),and apoptotic cell rate(P 0.01),while exposure to NAM caused the opposite effects.Moreover,SIRT1 activation reduced the expression levels of GRP78,CHOP,cleaved caspase-12,cleaved caspase-9,and cleaved caspase-3 in Caco-2 cells and the colon tissues of treated mice.CONCLUSION SIRT1 activation reduces apoptosis of IECs via the suppression of endoplasmic reticulum stress-mediated apoptosis-associated molecules CHOP and caspase-12 SIRT1 activation may be a potential therapeutic strategy for UC.     

Hydrogen peroxide-induced alterations of tight junction proteins in bovine brain microvascular endothelial cells

Occludin and zonular occludens (ZO)-1 in tight junctions (TJs) and actin play an important role in maintaining blood-brain barrier (BBB) endothelial ion and solute barriers. Malfunction of BBB by reactive oxygen species (ROS) has been attributed to the disruption of TJs. This study examined H2O2 effects on changes of paracellular permeability, actin, and TJ proteins (occludin and ZO-1) using primary culture of bovine brain microvessel endothelial cells. The BBB permeability, measured as transendothelial electrical resistance (TER), decreased in a dose- and time-dependent manner when treated with H2O2. Cytotoxicity test revealed that H2O2 did not cause cell death at 0.01, 0.1, and 1.0 mM H2O2. H2O2 caused increased protein expression of occludin (1.17- to 1.29-fold) and actin (1.2- to 1.3-fold). ZO-1 maintained steady state levels of expression. H2O2 caused rearrangement of occludin and ZO-1 at tight junctions and formation of actin stress fiber. Although ZO-1 did not show significant change in protein expression, permeability changes shown in the current study correlate with alterations in expression and localization of occludin, actin, and ZO-1. These data suggest that H2O2 induces increased paracellular permeability of BBB that is accompanied with redistribution of occludin and ZO-1 and increased protein expression of occludin and actin.     

Protection by microRNA-7a-5p Antagomir Against Intestinal Mucosal Injury Related to the JNK Pathway in TNBS-Induced Experimental Colitis

Background: Increasing evidence shows that microRNA-7a-5p (miR-7a-5p) plays an important role in regulating the inflammatory process in inflammatory bowel disease (IBD). How miR-7a-5p contributes to this process is poorly defined. The purpose of this study was to examine whether miR-7a-5p regulates 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced inflammatory responses via the JNK pathway.MethodsColitis was induced in male mice by intracolonic administration of TNBS; mice were divided into 3 groups: normal control (NC), TNBS, and miR-7a-5p antagomir-treated group. Inflammatory responses were estimated by disease activity index (DAI) and histological scores. The relative expressions of miR-7a-5p and tight junction protein, ZO-1, were detected by RT-qPCR. Western blot assays were used to estimate the level of JNK pathway proteins and ZO-1. After miRNA-antagomir injection, the extent of colonic tissue injury and expression levels of ZO-1 and JNK in intestinal tissue were compared.ResultsmiR-7a-5p and p-JNK expression were higher in the intestinal tissue of the TNBS group as compared to NC. Inhibition of the expression of miR-7a-5p resulted in significantly decreased expression of p-JNK but increased expression of ZO-1 and promoted the recovery of intestinal mucosa.ConclusionThis work demonstrates a correlation between the JNK pathway and miR-7a-5p in TNBS-induced experimental colitis in mice, which may provide a new research direction for the treatment of IBD.     

水通道蛋白3与紧密连接的相关性

目的:探讨水通道蛋白3(aquaporin 3,AQP3)对肠黏膜上皮细胞间紧密连接(tight junction,TJ)的影响,并探讨其可能的作用机制.方法:应用Caco-2细胞系在体外构建肠黏膜上皮屏障,构建沉默AQP3的shRNA慢病毒载体,建立稳定转染细胞系.实验分为3组:空白对照组(BLANK)、阴性对照组(NC)、AQP3干扰组(AQP3 shRNA).Western blot验证TJ相关蛋白Occludin以及Claudin-1的表达情况;并且采用免疫细胞化学法观察TJ相关蛋白的分布和结构变化.结果:RT-PCR及Western blot结果显示在Caco-2细胞系中成功沉默AQP3的表达.干扰组与对照组相比下降约75%.Western blot结果显示AQP3干扰组TJ相关蛋白Occludin以及Claudin-1的表达明显降低.免疫细胞化学结果显示Caco-2细胞间Occludin以及Claudin-1主要表达在细胞膜和/或胞浆中,Occludin和Claudin-1细胞间棕褐色颗粒减少,结构变模糊.相邻Caco-2细胞间TJ结构遭到破坏.结论:靶向AQP3的shRNA技术可以引起TJ的结构变化和相关蛋白的表达分布的异常.     

Lactobacillus acidophilus protects tight junctions from aspirin damage in HT-29 cells

BACKGROUND/AIMS: Non-steroidal anti-inflammatory drugs cause enterocyte damage inducing an increase of intestinal permeability. Tight junctions are the key structures in the permeability of the intestinal mucosa. ZO-1 is a tight junction associated protein considered a good marker of their integrity. It has been suggested that probiotics could play a protective role in the intestinal barrier function. We determined, in vitro, whether the heat-killed Lactobacillus acidophilus strain LB (LaLB) with its spent culture supernatant protects tight junctions of HT-29 cells from aspirin (ASA) damage. METHODS: HT-29 cells were treated with ASA alone or ASA and LaLB with its spent culture supernatant together. Morphological alterations of tight junctions were evaluated by immunofluorescence using an anti-ZO-1 antibody. Moreover, a semiquantitative assay for ZO-1 was performed by Western blot. RESULTS: Immunofluorescence analysis showed a fragmented and granulous ZO-1 staining, after ASA treatment. Using both ASA and LaLB with its spent culture supernatant together, we found a fine continuous linear web at cell-cell contacts similarly to control. Western blot revealed that ASA inhibited ZO-1 expression and LaLB with its spent culture supernatant counteracted this effect. CONCLUSIONS: This pilot study shows, for the first time, the protective effect of LaLB with its spent culture supernatant on tight junctions from ASA damage. These results suggest that probiotics could play a role in the prevention of ASA-induced alterations of intestinal permeability.     

灭活血吸虫卵对实验性结肠炎肠黏膜的影响

目的 研究灭活血吸虫卵对2,4,6一三硝基苯磺酸(tinitrobenzene sulfonic acid,TNBS)诱导小鼠结肠炎肠黏膜紧密连接蛋白ZO-1和Occludin基因及蛋白表达影响及其机制.方法 清洁级BALB/C雌性小鼠50只分成对照组(10只)、TNBS+0.9%氯化钠溶液组(20只)和TNBS+血吸虫卵组(20只).TNBS+血吸虫卵组在造模前第3、14天分别给予腹腔注射冰冻灭活血吸虫卵10 000个(1 ml冰0.9%氯化钠溶液混悬液);TNBS+0.9%氯化钠溶液组给予相同体积的冰0.9%氯化钠溶液腹腔注射.后两组予TNBS溶液灌肠(100 mg/kg)建立结肠炎模型,建模后第7天处死存活小鼠,观察各组小鼠结肠的大体形态和HE染色光镜下病理特征;荧光实时定量PCR法测定结肠组织的Occludin和ZO-1基因表达;Western印迹法检测蛋白表达;免疫组化法测定结肠组织紧密连接蛋白表达分布.结果 TNBS+血吸虫卵组小鼠死亡率较TNBS+0.9%氯化钠溶液组明显下降(15%比30%).TNBS+0.9%氯化钠溶液组组织学评分为(4.21±0.40)分,较TNBS+血吸虫卵组和对照组高[(1.74±0.10)和(1.06±0.20)分,P<0.05].TNBS+0.9%氯化钠溶液组ZO-1和Occludin mRNA表达量较对照组显著下降(P<0.01),而TNBS+血吸虫卵组较TNBS+0.9%氯化钠溶液组显著增加(P<0.05).TNBs+0.9%氯化钠溶液组ZO-1蛋白相对灰度值较正常对照组降低50.3%(P<0.05),而TNBS+血吸虫卵组较TNBS+0.9%氯化钠溶液增加41.1%(P<0.05);TNBS+0.9%氯化钠溶液组Occludin相对灰度值较对照组下降48.7%(P<0.05),而血吸虫卵组较TNBS+0.9%氯化钠溶液组增加23.6%(P<0.05).ZO-1、Occludin蛋白染色强度TNBS+0.9%氯化钠溶液组分布均较对照组间增强(P<0.01),而TNBS+血吸虫卵组染色强度信号分布较TNBS+0.9%氯化钠溶液组显著增加(P<0.05).结论 灭活血吸虫卵能在细胞水平加强紧密连接蛋白ZO-1、Occludin聚集及表达,通过稳定紧密连接蛋白,增加肠道黏膜屏障功能,显著改善实验性结肠炎的肠道炎症反应.     

中药清肠栓对溃疡性结肠炎大鼠结肠黏膜紧密连接蛋白ZO-1、occludin的影响

目的:探讨复方中药清肠栓对三硝基苯磺酸(TNBS)诱导的UC大鼠结肠黏膜上皮紧密连接相关蛋白-l(ZO-1)、闭锁蛋白(occludin)的修复作用.方法:清洁级♂SD大鼠36只,随机分为正常组、模型组、SASP组、清肠栓高、中、低剂量组,每组6只.选用TNBS诱导的UC大鼠模型.采用免疫荧光的方法观察各组大鼠结肠黏膜...     

The coxsackievirus and adenovirus receptor is a transmembrane component of the tight junction

The coxsackievirus and adenovirus receptor (CAR) mediates viral attachment and infection, but its physiologic functions have not been described. In nonpolarized cells, CAR localized to homotypic intercellular contacts, mediated homotypic cell aggregation, and recruited the tight junction protein ZO-1 to sites of cell-cell contact. In polarized epithelial cells, CAR and ZO-1 colocalized to tight junctions and could be coprecipitated from cell lysates. CAR expression led to reduced passage of macromolecules and ions across cell monolayers, and soluble CAR inhibited the formation of functional tight junctions. Virus entry into polarized epithelium required disruption of tight junctions. These results indicate that CAR is a component of the tight junction and of the functional barrier to paracellular solute movement. Sequestration of CAR in tight junctions may limit virus infection across epithelial surfaces.     

Regulated assembly of tight junctions by protein kinase C.

We have previously shown that protein phosphorylation plays an important role in the sorting and assembly of tight junctions. We have now examined in detail the role of protein kinases in intercellular junction biogenesis by using a combination of highly specific and broad-spectrum inhibitors that act by independent mechanisms. Our data indicate that protein kinase C (PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibited development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, was paralleled by a delay in the sorting of the tight-junction protein, zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction were not detectably affected, as determined by immunocytochemical analysis. In addition, ZO-1 was phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevented approximately 85% of the phosphorylation increase. Furthermore, in vitro measurements indicate that ZO-1 may be a direct target of PKC. Moreover, membrane-associated PKC activity more than doubled during junction assembly, and immunocytochemical analysis revealed a pool of PKC zeta that appeared to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins was detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may be occludin. The mass of this complex and the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were not PKC dependent.     

Fatty acids and epithelial permeability: effect of conjugated linoleic acid in Caco-2 cells

Conjugated linoleic acid (CLA) is a collective term referring to the positional and geometric isomers of linoleic acid. This novel fatty acid has been shown to have a number of beneficial actions, including immunomodulatory, anticarcinogenic, and antiatherogenic effects. Tight junctions of epithelial cells determine epithelial membrane integrity and selective paracellular permeability to ions and macromolecules. Occludin and ZO-1 are integral structural components of the tight junction, which are involved in the biogenesis and functional integrity of the epithelial monolayer. This study investigated the effects of two isomers of CLA (cis-9 and trans-10 isomers) on Caco-2 cell transepithelial resistance (TER) development, paracellular epithelial permeability, and occludin and ZO-1 expression. Caco-2 cells were grown in media supplemented with 0.05 mM linoleic acid, cis-9 CLA, or trans-10 CLA for 21 days. The trans-10 CLA isomer delayed Caco-2 cell TER development, which is an in vitro measure of epithelial cell integrity, and increased paracellular epithelial permeability. Immunofluorescent staining of Caco-2 cell epithelial monolayers grown in media supplemented trans-10 CLA showed that the trans-10 CLA isomer altered distribution of occludin and ZO-1. The trans-10 CLA isomer delayed the acquisition of transepithelial resistance and altered the cellular distribution of occludin, which have important implications in relation to epithelial permeability.     

The tight junction protein ZO-1 is homologous to the Drosophila discs-large tumor suppressor protein of septate junctions.

Tight junctions form an intercellular barrier between epithelial cells, serve to separate tissue compartments, and maintain cellular polarity. Paracellular sealing properties vary among cell types and are regulated by undefined mechanisms. Sequence of the full-length cDNA for human ZO-1, the first identified tight junction component, predicts a protein of 1736 aa. The N-terminal 793 aa are homologous to the product of the lethal(1)discs-large-1 (dlg) tumor suppressor gene of Drosophila, located in septate junctions, and to a 95-kDa protein located in the postsynaptic densities of rat brain, PSD-95. All three proteins contain both a src homology region 3 (SH3 domain), previously identified in membrane proteins involved in signal transduction, and a region homologous to guanylate kinase. ZO-1 contains an additional 943-aa C-terminal domain that is proline-rich (14.1%) and contains an alternatively spliced domain, whose expression was previously shown to correlate with variable properties of tight junctions. dlg mutations result in loss of apical-basolateral epithelial cell polarity and in neoplastic growth. These results suggest a protein family specialized for signal transduction on the cytoplasmic surface of intercellular junctions. These results also provide biochemical evidence for similarity between invertebrate septate and vertebrate tight junctions. The C-terminal domain of ZO-1, and its alternatively spliced region, appears to confer variable properties unique to tight junctions.     

Diversity among tight junctions in rat kidney: glomerular slit diaphragms and endothelial junctions express only one isoform of the tight junction protein ZO-1.

ZO-1 is a 225-kDa peripheral membrane protein present in all tight junctions. It was recently shown to consist of two isoforms that differ in the presence of an internal 80-amino acid domain termed motif-alpha. To obtain information on their distribution and potential functional significance we have localized the two isoforms in rat kidney by using antibodies that recognize either both ZO-1 isoforms or the larger, motif-alpha-containing isoform. By immunofluorescence, staining with both antibodies was demonstrated at all tight junctions of tubular epithelial cells and the epithelial cells of Bowman's capsule. In contrast, the motif-alpha-containing isoform was absent from the slit diaphragms of the glomerular epithelium and the tight junctions of glomerular and peritubular capillary endothelial cells. This restricted isoform expression was confirmed by immunoblot analysis comparing proteins from purified glomeruli with those from kidney cortex or medulla. Thus, while both isoforms are expressed in typical epithelial tight junctions, only a single isoform, lacking motif-alpha, is expressed in the highly specialized slit diaphragms, where the intercellular spaces are normally open, and in endothelial junctions, which are readily opened by physiologic signals. The differential expression of ZO-1 isoforms in structurally and functionally distinct junctions in the kidney suggests that they may contribute to defining the variable functional properties, in particular the lability of these intercellular junctions.     

Nitric Oxide-Mediated Intestinal Injury Is Required for Alcohol-Induced Gut Leakiness and Liver Damage

Background: Alcoholic liver disease (ALD) requires endotoxemia and is commonly associated with intestinal barrier leakiness. Using monolayers of intestinal epithelial cells as an in vitro barrier model, we showed that ethanol‐induced intestinal barrier disruption is mediated by inducible nitric oxide synthase (iNOS) upregulation, nitric oxide (NO) overproduction, and oxidation/nitration of cytoskeletal proteins. We hypothesized that iNOS inhibitors [NG‐nitro‐l ‐arginine methyl ester (l ‐NAME), l ‐N⁶‐(1‐iminoethyl)‐lysine (l ‐NIL)] in vivo will inhibit the above cascade and liver injury in an animal model of alcoholic steatohepatitis (ASH). Methods: Male Sprague–Dawley rats were gavaged daily with alcohol (6 g/kg/d) or dextrose for 10 weeks ± l ‐NAME, l ‐NIL, or vehicle. Systemic and intestinal NO levels were measured by nitrites and nitrates in urine and tissue samples, oxidative damage to the intestinal mucosa by protein carbonyl and nitrotyrosine, intestinal permeability by urinary sugar tests, and liver injury by histological inflammation scores, liver fat, and myeloperoxidase activity. Results: Alcohol caused tissue oxidation, gut leakiness, endotoxemia, and ASH. l ‐NIL and l ‐NAME, but not the d ‐enantiomers, attenuated all steps in the alcohol‐induced cascade including NO overproduction, oxidative tissue damage, gut leakiness, endotoxemia, hepatic inflammation, and liver injury. Conclusions: The mechanism we reported for alcohol‐induced intestinal barrier disruption in vitro — NO overproduction, oxidative tissue damage, leaky gut, endotoxemia, and liver injury — appears to be relevant in vivo in an animal model of alcohol‐induced liver injury. That iNOS inhibitors attenuated all steps of this cascade suggests that prevention of this cascade in alcoholics will protect the liver against the injurious effects of chronic alcohol and that iNOS may be a useful target for prevention of ALD.