Occludin loss enhances paracellular macromolecular permeability (radius up to ∼62.5 Å) and is necessary for TNF-induced barrier loss. The latter requires the C-terminal OCEL domain, which stabilizes tight junction–associated occludin and regulates trafficking. Thus OCEL-mediated interactions are critical regulators of macromolecular paracellular flux.
In vitro and in vivo studies implicate occludin in the regulation of paracellular macromolecular flux at steady state and in response to tumor necrosis factor (TNF). To define the roles of occludin in these processes, we established intestinal epithelia with stable occludin knockdown. Knockdown monolayers had markedly enhanced tight junction permeability to large molecules that could be modeled by size-selective channels with radii of ∼62.5 Å. TNF increased paracellular flux of large molecules in occludin-sufficient, but not occludin-deficient, monolayers. Complementation using full-length or C-terminal coiled-coil occludin/ELL domain (OCEL)–deficient enhanced green fluorescent protein (EGFP)–occludin showed that TNF-induced occludin endocytosis and barrier regulation both required the OCEL domain. Either TNF treatment or OCEL deletion accelerated EGFP-occludin fluorescence recovery after photobleaching, but TNF treatment did not affect behavior of EGFP-occludinΔOCEL. Further, the free OCEL domain prevented TNF-induced acceleration of occludin fluorescence recovery, occludin endocytosis, and barrier loss. OCEL mutated within a recently proposed ZO-1–binding domain (K433) could not inhibit TNF effects, but OCEL mutated within the ZO-1 SH3-GuK–binding region (K485/K488) remained functional. We conclude that OCEL-mediated occludin interactions are essential for limiting paracellular macromolecular flux. Moreover, our data implicate interactions mediated by the OCEL K433 region as an effector of TNF-induced barrier regulation.
Inflammatory bowel disease (IBD) is commonly treated with thiopurines such as azathioprine and mercaptopurine for the maintenance of remission. Studies examining chemopreventive of these medications on colorectal neoplasm in IBD patients have yielded conflicting results. We performed a meta-analysis to assess the role of thiopurines for this indication.
We performed a systematic search of PubMed, Web of Science, EMBASE and Cochrane to identify studies reporting colorectal neoplasm from IBD patients treated with thiopurines and conducted a meta-analysis of pooled relative risk (RR) using the random effects model.
Nine case-control and ten cohort studies fulfilled the inclusion criteria. The use of thiopurines was associated with a statistically significant decreased incidence of colorectal neoplasm (summary RR=0.71, 95% CI=0.54–0.94, p=0.017), even after adjustment for duration and extent of the disease, but there was high heterogeneity among studies (I2=68.0%, p<0.001). The RR of advanced neoplasm (high-grade dysplasia and cancer) was 0.72 (95%CI=0.50–1.03, p=0.070) and that of cancer was 0.70 (95% CI=0.46–1.09, p=0.111) for thiopurine-treated patients. Heterogeneity of the studies was affected by the sample size (≥100 cases) and whether the patients had longstanding colitis (≥7 years).
The current meta-analysis revealed that thiopurines had a chemopreventive effect of colorectal neoplasms and a tendency of reducing advanced colorectal neoplasms in IBD. Due to the heterogeneity of included studies, these results should be interpreted with caution.
A principal role of tight junctions is to seal the apical intercellular space and limit paracellular flux of ions and molecules. Despite the fact that tight junctions form heavily cross-linked structures, functional studies have fostered the hypothesis that the tight junction barrier is dynamic and defined by opening and closing events. However, it has been impossible to directly measure tight junction barrier function with sufficient resolution to detect such events. Nevertheless, recent electrophysiological and sieving studies have provided tremendous insight into the presence of at least two pathways of trans-tight junction flux: a high-capacity ion-selective “pore” pathway and a low-capacity “leak” pathway that allows the passage of macromolecules. Furthermore, it is now known that the tight junction molecular structure is highly dynamic and that dynamics are correlated with barrier function. Taken together, these data support a dynamic model of tight junction conductance and suggest that regulation of tight junction openings and closings may provide sensitive means of barrier regulation.
tight junction; FRAP; claudin; occluding; ZO-1
Myosin II (MyoII) is required for insulin-responsive glucose transporter 4 (GLUT4)-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC) of MyoIIA via myosin light chain kinase (MLCK). The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid, (BAPTA) (in the presence of insulin) impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKIIδ did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.
The exact mechanism by which Entamoeba histolytica disrupts the human colonic epithelium and invades the mucosa has yet to be clearly elucidated. E. histolytica produces a diverse array of putative virulent factors such as glycosidase, cysteine proteinases and amebapore that can modulate and/or disrupt epithelial barrier functions. However, it is currently thought that E. histolytica produces numerous other molecules and strategies to disrupt colonic mucosal defenses. In this study, we document a putative mechanism whereby the parasite alters the integrity of human epithelium by expressing a cognate tight junction protein of the host. We detected this protein as “occludin-like” as revealed by immunoblotting and immunoprecipitation studies and visualization by confocal microscopy using antibodies highly specific for human occludin. We propose that E. histolytica occludin-like protein might displace mucosal epithelial occludin-occludin tight junction interactions resulting in epithelial disruption analogous to sub mucosal human dendritic cells sampling luminal contents. These results indicate that E. histolytica occludin is a putative virulent component that can play a role in the pathogenesis of intestinal amebiasis.
Infections from enteric bacteria such as enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are a public health threat worldwide. EPEC and EHEC are extracellular pathogens, and their interaction with host surface receptors is critical to the infection process. We previously demonstrated that polyethylene glycol (PEG) downregulates surface receptors in intestinal cells. Here we show that PEG decreases β1-integrin, the surface receptor in intestinal cells that is critical for EPEC and EHEC attachment. We hypothesized that PEG would inhibit the attachment of these enteric pathogens to host cells and improve clinical signs of infection. We found that attachment of the mouse enteric pathogen Citrobacter rodentium, which belongs to the same group of pathogens as EPEC and EHEC, was attenuated by the concurrent presence of PEG. Pretreatment with PEG, without concurrent presence during infection, also reduced bacterial attachment. This finding was further supported in vivo such as that PEG administered by gavage daily during infection as well as prior to infection significantly decreased C. rodentium in the colon and improved the appearance of the infected colon in mice. In addition, PEG decreased the β1-integrin in colonic mucosa and reduced the C. rodentium-induced activation of epidermal growth factor receptors. PEG also significantly reduced infection-induced colonic inflammation. Finally, PEG efficiently reduced C. rodentium shedding from the colon during infection. In conclusion, PEG can be an efficient and safe preventive agent against EPEC and EHEC infections.
PEG; prophylaxis; enteropathogenic bacteria; C. rodentium; infection
Occludin S408 phosphorylation regulates interactions between occludin, ZO-1, and select claudins to define tight junction molecular structure and barrier function.
Although the C-terminal cytoplasmic tail of the tight junction protein occludin is heavily phosphorylated, the functional impact of most individual sites is undefined. Here, we show that inhibition of CK2-mediated occludin S408 phosphorylation elevates transepithelial resistance by reducing paracellular cation flux. This regulation requires occludin, claudin-1, claudin-2, and ZO-1. S408 dephosphorylation reduces occludin exchange, but increases exchange of ZO-1, claudin-1, and claudin-2, thereby causing the mobile fractions of these proteins to converge. Claudin-4 exchange is not affected. ZO-1 domains that mediate interactions with occludin and claudins are required for increases in claudin-2 exchange, suggesting assembly of a phosphorylation-sensitive protein complex. Consistent with this, binding of claudin-1 and claudin-2, but not claudin-4, to S408A occludin tail is increased relative to S408D. Finally, CK2 inhibition reversed IL-13–induced, claudin-2–dependent barrier loss. Thus, occludin S408 dephosphorylation regulates paracellular permeability by remodeling tight junction protein dynamic behavior and intermolecular interactions between occludin, ZO-1, and select claudins, and may have therapeutic potential in inflammation-associated barrier dysfunction.
Background & Aims
Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that Akt signaling cooperates with Wnt to activate β-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-β-catenin552). We investigated whether phosphoinositide 3- kinase (PI3K) is required for Akt-mediated activation of β-catenin during intestinal inflammation.
The class IA subunit of PI3K was conditionally deleted from intestinal epithelial cells in mice. Acute inflammation was induced in these mice (I-pik3r1KO) and their intestines were analyzed by biochemical and histological methods. The effects of chemically blocking PI3K in colitic IL-10−/− mice were examined. Biopsy samples from patients were examined.
Compared to wild type mice, I-pik3r1KO mice had reduced T-cell–mediated Akt and β-catenin signaling in intestinal stem and progenitor cells and limited crypt epithelial proliferation. Biochemical analyses indicated that PI3K–Akt signaling increased nuclear total β-catenin and P-β-catenin552 levels and reduced phosphorylation of N-terminal β-catenin, which is associated with degradation. PI3K inhibition in IL-10−/− mice impaired colitis-induced epithelial Akt and β-catenin activation, reduced progenitor cell expansion, and prevented dysplasia. Human samples had increased numbers of progenitor cells with P-β-catenin552 throughout expanded crypts and increased mRNA expression of β-catenin target genes in CUC, colitis-associated cancer, tubular adenomas, and sporadic colorectal cancer, compared with control samples.
PI3K–Akt signaling cooperates with Wnt to increase β-catenin signaling during inflammation. PI3K-induced and Akt-mediated β-catenin signaling are required for progenitor cell activation during the progression from CUC to CAC; these factors might be used as biomarkers of dysplastic transformation in the colon.
PI3K; pik3r1; intestinal stem cells; intestinal progenitor cells; ulcerative colitis; inflammatory bowel disease; colon cancer; β-catenin; T-cell activation
Soy consumption is associated with a lower incidence of colon cancer which is believed to be mediated by one of its of components, genistein. Genistein may inhibit cancer progression by inducing apoptosis or inhibiting proliferation, but mechanisms are not well understood. Epidermal growth factor (EGF)-induced proliferation of colon cancer cells plays an important role in colon cancer progression and is mediated by loss of tumor suppressor FOXO3 activity. The aim of this study was to assess if genistein exerts anti-proliferative properties by attenuating the negative effect of EGF on FOXO3 activity.
The effect of genistein on proliferation stimulated by EGF-mediated loss of FOXO3 was examined in human colonic cancer HT-29 cells. EGF-induced FOXO3 phosphorylation and translocation were assessed in the presence of genistein. EGF-mediated loss of FOXO3 interactions with p53 (co-immunoprecipitation) and promoter of p27kip1 (ChIP assay) were examined in presence of genistein in cells with mutated p53 (HT-29) and wild type p53 (HCT116). Silencing of p53 determined activity of FOXO3 when it is bound to p53.
Genistein inhibited EGF-induced proliferation, while favoring dephosphorylation and nuclear retention of FOXO3 (active state) in colon cancer cells. Upstream of FOXO3, genistein acts via the PI3K/Akt pathway to inhibit EGF-stimulated FOXO3 phosphorylation (i.e. favors active state). Downstream, EGF-induced disassociation of FOXO3 from mutated tumor suppressor p53, but not wild type p53, is inhibited by genistein favoring FOXO3-p53(mut) interactions with the promoter of the cell cycle inhibitor p27kip1 in colon cancer cells. Thus, the FOXO3-p53(mut) complex leads to elevated p27kip1 expression and promotes cell cycle arrest.
These novel anti-proliferative mechanisms of genistein suggest a possible role of combining genistein with other chemoreceptive agents for the treatment of colon cancer.
Genistein; EGF; FOXO3; proliferation; colon cancer
Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is characterized by chronic mucosal injury and the infiltration of inflammatory cells. Tumor suppressor FOXO3 regulates gene expression and its translocation to the cytosol leads to the abrogation of its transcriptional function. We have previously shown that bacterial infection regulates FOXO3 in intestinal epithelial cells and increases cytokine levels. As TNFα is a major contributor in intestinal inflammation, the aim of this study was to assess its effect on FOXO3 and FOXO3's contribution to intestinal inflammation in vitro and in vivo. TNFα induces the translocation of nuclear FOXO3 into the cytosol where it undergoes proteasomal degradation in human intestinal HT-29 cells. Proximally, the PI3K and IKK pathways mediate TNFα-induced FOXO3 phosphorylation. In FOXO3-silenced HT-29 cells, TNFα-induced IL-8 expression is increased ∼83%. In vivo, Foxo3 is present in the nuclei and cytosol of colonic crypt epithelia. In DSS-induced colonic inflammation, Foxo3's nuclear localization is lost and it is only found in the cytosol. Consistent with a role for Foxo3 in colitis, Foxo3-deficient mice treated with DSS developed more severe colonic inflammation with an increased number of intraepithelial lymphocytes and PMNs infiltrated in the epithelia, than wild-type mice. In summary, TNFα inactivates FOXO3 in intestinal epithelia through the PI3K and IKK pathways and FOXO3 inactivation leads to the upregulation of IL-8 in vitro; in vivo Foxo3 is in the cytosol of inflamed colonic epithelia and Foxo3 deficiency leads to severe intestinal inflammation.
FOXO3; inflammation; intestinal epithelia; signaling; TNFα
Although tight junction morphology is not obviously affected by TNF, this proinflammatory cytokine promotes internalization of occludin, resulting in disrupted barrier function within the intestine.
Epithelial paracellular barrier function, determined primarily by tight junction permeability, is frequently disrupted in disease. In the intestine, barrier loss can be mediated by tumor necrosis factor (α) (TNF) signaling and epithelial myosin light chain kinase (MLCK) activation. However, TNF induces only limited alteration of tight junction morphology, and the events that couple structural reorganization to barrier regulation have not been defined. We have used in vivo imaging and transgenic mice expressing fluorescent-tagged occludin and ZO-1 fusion proteins to link occludin endocytosis to TNF-induced tight junction regulation. This endocytosis requires caveolin-1 and is essential for structural and functional tight junction regulation. These data demonstrate that MLCK activation triggers caveolin-1–dependent endocytosis of occludin to effect structural and functional tight junction regulation.
During epithelialization, cell adhesions and polarity must be established to maintain tissue assemblies and separate the biological compartments in the body. However, the molecular basis of epithelial morphogenesis, in particular, a role of cell adhesion molecules in epithelial differentiation from stem cells, remains unclear. Here, we show that the stable and conditional expression of a tight-junction protein, claudin-6 (Cldn6), triggers epithelial morphogenesis in mouse F9 stem cells. We also demonstrate that Cldn6 induces the expression of other tight-junction and microvillus molecules including Cldn7, occludin, ZO-1α+, and ezrin/radixin/moesin-binding phosphoprotein50. These events were inhibited by attenuation of Cldn6 using RNA interference or the C-terminal half of Clostridium Perfringens enterotoxin. Furthermore, similar results were obtained in mouse embryonic stem cells. Thus, we have uncovered that the Cldn6 functions as a novel cue to induce epithelial differentiation.
Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal adenocarcinoma. The factors that result in IBD-associated carcinogenesis are not understood. We hypothesized that altered expression of intestinal epithelial tight junction proteins might contribute to neoplastic progression. Semi-quantitative immunohistochemical staining of human biopsies was used to assess expression of the tight junction proteins claudin-1, claudin-2, claudin-4, and occludin in IBD, IBD-associated dysplasia, acute, self-limited colitis (ASLC), and sporadic adenomas. Claudin-1 and claudin-2 expression was elevated in active IBD, adenomas, and IBD-associated dysplasia, but not ASLC. In contrast, claudin-4 expression was elevated in both active IBD and ASLC. Occludin expression was similar to control in all cases. Importantly, in IBD, claudin-1 and claudin-2 expression correlated positively with inflammatory activity. To investigate mechanisms underlying altered claudin expression, β-catenin activation was assessed as nuclear localization. Like claudin-1 and claudin-2, β-catenin was markedly activated in IBD, dysplasia, IBD-associated dysplasia, but only slightly activated in ASLC. Taken together, these data suggest that β-catenin transcriptional activity is elevated in chronic injury and that this may contribute to increased claudin-1 and claudin-2 expression. We speculate that increased claudin-1 and claudin-2 expression may be involved in early stages of transformation in IBD-associated neoplasia.
claudin-1; claudin-2; claudin-4; colon cancer; dysplasia; inflammatory bowel disease; occludin
Enteric bacteria and their products play an important role in intestinal inflammation; however, the complete mechanisms are not elucidated yet. Tumor suppressor Foxo3a regulates gene expression in the nucleus, and its translocation to the cytosol leads to inactivation. Proximally, Foxo3a is regulated by different pathways including the phosphoinositide 3-kinase (PI3K) pathway. The aim of this study was to determine the effect of bacterial infection on Foxo3a in intestinal epithelial cells and to examine the contribution of Foxo3a in intestinal inflammation. Bacterial lipopolysaccharide (LPS) and infection with mouse pathogen Citrobacter rodentium induce translocation of the nuclear Foxo3a into the cytosol, where it degrades in human HT-29 and mouse CMT-93 cells. In colonic epithelia of healthy mice, Foxo3a is localized in the epithelia at the bottom of the crypts in both the nucleus and the cytosol, while in C. rodentium-infected colon Foxo3a is expressed along the crypts and located mainly in the cytosol, suggesting its inactivation. LPS utilized the PI3K pathway to inhibit Foxo3a. Additionally, inhibition of PI3K attenuated LPS-induced proinflammatory interleukin-8 (IL-8). LPS-induced IL-8 is increased in HT-29 cells with silenced Foxo3a. Moreover, in HT-29 cells with silenced Foxo3a, the amount of IκBα, an NF-κB inhibitor, is decreased. In conclusion, LPS and bacterial infection inactivate Foxo3a in intestinal epithelia via the PI3K pathway and inactivated Foxo3a leads to the upregulation of IL-8 by suppressing inhibitory IκBα.
The tight junction defines epithelial organization. Structurally, the tight junction is comprised of transmembrane and membrane-associated proteins that are thought to assemble into stable complexes to determine function. In this study, we measure tight junction protein dynamics in live confluent Madin–Darby canine kidney monolayers using fluorescence recovery after photobleaching and related methods. Mathematical modeling shows that the majority of claudin-1 (76 ± 5%) is stably localized at the tight junction. In contrast, the majority of occludin (71 ± 3%) diffuses rapidly within the tight junction with a diffusion constant of 0.011 μm2s−1. Zonula occludens-1 molecules are also highly dynamic in this region, but, rather than diffusing within the plane of the membrane, 69 ± 5% exchange between membrane and intracellular pools in an energy-dependent manner. These data demonstrate that the tight junction undergoes constant remodeling and suggest that this dynamic behavior may contribute to tight junction assembly and regulation.
Glucose stimulates both insulin secretion and hydrolysis of arachidonic acid (AA) esterified in membrane phospholipids of pancreatic islet β-cells, and these processes are amplified by muscarinic agonists. Here we demonstrate that nonesterified AA regulates the biophysical activity of the pancreatic islet β-cell-delayed rectifier channel, Kv2.1. Recordings of Kv2.1 currents from INS-1 insulinoma cells incubated with AA (5 μM) and subjected to graded degrees of depolarization exhibit a significantly shorter time-to-peak current interval than do control cells. AA causes a rapid decay and reduced peak conductance of delayed rectifier currents from INS-1 cells and from primary β-cells isolated from mouse, rat, and human pancreatic islets. Stimulating mouse islets with AA results in a significant increase in the frequency of glucose-induced [Ca2+] oscillations, which is an expected effect of Kv2.1 channel blockade. Stimulation with concentrations of glucose and carbachol that accelerate hydrolysis of endogenous AA from islet phosphoplipids also results in accelerated Kv2.1 inactivation and a shorter time-to-peak current interval. Group VIA phospholipase A2 (iPLA2β) hydrolyzes β-cell membrane phospholipids to release nonesterified fatty acids, including AA, and inhibiting iPLA2β prevents the muscarinic agonist-induced accelerated Kv2.1 inactivation. Furthermore, glucose and carbachol do not significantly affect Kv2.1 inactivation in β-cells from iPLA2β−/− mice. Stably transfected INS-1 cells that overexpress iPLA2β hydrolyze phospholipids more rapidly than control INS-1 cells and also exhibit an increase in the inactivation rate of the delayed rectifier currents. These results suggest that Kv2.1 currents could be dynamically modulated in the pancreatic islet β-cell by phospholipase-catalyzed hydrolysis of membrane phospholipids to yield non-esterified fatty acids, such as AA, that facilitate Ca2+ entry and insulin secretion.
MMP25 (MT6-MMP) is one of the two glycosylphosphatidylinositol-anchored matrix metalloproteinases (MMPs) that have been suggested to play a role in pericellular proteolysis. However, its role in cancer is unknown, and its biochemical properties are not well established. Here we found a marked increase in MT6-MMP expression within in situ dysplasia and invasive cancer in 61 samples of human colon cancer. Expression of MT6-MMP in HCT-116 human colon cancer cells promoted tumorigenesis in nude mice. Histologically, the MT6-MMP-expressing tumors demonstrated an infiltrative leading edge in contrast to a rounded leading edge in vector control tumors. Biochemical and biosynthesis analyses revealed that MT6-MMP displayed on the cell surface exists as a major form of 120 kDa that likely represents enzyme homodimers linked by disulfide bonds. Upon reduction, a single 57-kDa active MT6-MMP was detected. Interestingly, neither membrane-anchored nor phosphatidylinositol-specific phospholipase C-released MT6-MMPs were found to be associated with tissue inhibitor of metalloproteinases (TIMPs) and did not activate pro-gelatinases (pro-MMP-2 and pro-MMP-9) even in the presence of exogenous TIMP-2 or TIMP-1. A catalytic domain of MT6-MMP was inhibited preferentially by TIMP-1 (Ki = 0.2 nm) over TIMP-2 (Ki = 2.0 nm), because of a slower association rate. These results show that MT6-MMP may play a role in colon cancer and exhibit unique biochemical and structural properties that may regulate proteolytic function at the cell surface.
The cardiac sarcolemmal Na-Ca exchanger (NCX) is allosterically regulated by [Ca]i such that when [Ca]i is low, NCX current (INCX) deactivates. In this study, we used membrane potential (Em) and INCX to control Ca entry into and Ca efflux from intact cardiac myocytes to investigate whether this allosteric regulation (Ca activation) occurs with [Ca]i in the physiological range. In the absence of Ca activation, the electrochemical effect of increasing [Ca]i would be to increase inward INCX (Ca efflux) and to decrease outward INCX. On the other hand, Ca activation would increase INCX in both directions. Thus, we attributed [Ca]i-dependent increases in outward INCX to allosteric regulation. Ca activation of INCX was observed in ferret myocytes but not in wild-type mouse myocytes, suggesting that Ca regulation of NCX may be species dependent. We also studied transgenic mouse myocytes overexpressing either normal canine NCX or this same canine NCX lacking Ca regulation (Δ680–685). Animals with the normal canine NCX transgene showed Ca activation, whereas animals with the mutant transgene did not, confirming the role of this region in the process. In native ferret cells and in mice with expressed canine NCX, allosteric regulation by Ca occurs under physiological conditions (KmCaAct = 125 ± 16 nM SEM ≈ resting [Ca]i). This, along with the observation that no delay was observed between measured [Ca]i and activation of INCX under our conditions, suggests that beat to beat changes in NCX function can occur in vivo. These changes in the INCX activation state may influence SR Ca load and resting [Ca]i, helping to fine tune Ca influx and efflux from cells under both normal and pathophysiological conditions. Our failure to observe Ca activation in mouse myocytes may be due to either the extent of Ca regulation or to a difference in KmCaAct from other species. Model predictions for Ca activation, on which our estimates of KmCaAct are based, confirm that Ca activation strongly influences outward INCX, explaining why it increases rather than declines with increasing [Ca]i.
Na/Ca exchanger; cardiac electrophysiology; ferret; mouse; dog
Quantifying changes in partial resistances of epithelial barriers in vitro is a challenging and time-consuming task in physiology and pathophysiology. Here, we demonstrate that electrical properties of epithelial barriers can be estimated reliably by combining impedance spectroscopy measurements, mathematical modeling and machine learning algorithms. Conventional impedance spectroscopy is often used to estimate epithelial capacitance as well as epithelial and subepithelial resistance. Based on this, the more refined two-path impedance spectroscopy makes it possible to further distinguish transcellular and paracellular resistances. In a next step, transcellular properties may be further divided into their apical and basolateral components. The accuracy of these derived values, however, strongly depends on the accuracy of the initial estimates. To obtain adequate accuracy in estimating subepithelial and epithelial resistance, artificial neural networks were trained to estimate these parameters from model impedance spectra. Spectra that reflect behavior of either HT-29/B6 or IPEC-J2 cells as well as the data scatter intrinsic to the used experimental setup were created computationally. To prove the proposed approach, reliability of the estimations was assessed with both modeled and measured impedance spectra. Transcellular and paracellular resistances obtained by such neural network-enhanced two-path impedance spectroscopy are shown to be sufficiently reliable to derive the underlying apical and basolateral resistances and capacitances. As an exemplary perturbation of pathophysiological importance, the effect of forskolin on the apical resistance of HT-29/B6 cells was quantified.
In steady state, the Ca content of the sarcoplasmic reticulum (SR) of cardiac myocytes is determined by a balance among influx and efflux pathways. The SR Ca content may be limited mainly by the ATP-supplied chemical potential that is inherent in the gradient between SR and cytosol. That is, forward Ca pumping from cytosol to SR may be opposed by energetically conservative reverse pumping dependent on intra-SR free [Ca]. On the other hand, SR Ca loading may be limited by dissipative pathways (pump slippage and/or pump-independent leak). To assess how SR Ca content is limited, we loaded voltage-clamped ferret ventricular myocytes cumulatively with known amounts of Ca via L-type Ca channels (ICa), using Na-free solutions to prevent Na/Ca exchange. We then measured the maximal resulting caffeine-released SR Ca content under control conditions, as well as when SR Ca pumping was accelerated by isoproterenol (1 μM) or slowed by thapsigargin (0.2–0.4 μM). Under control conditions, SR Ca content reached a limit of 137 μmol·liter cytosol−1 (nonmitochondrial volume) when measured by integrating caffeine-induced Na/Ca exchange currents (∫INaCaXdt) and of 119 μmol·liter cytosol−1 when measured using fluorescence signals dependent on changes in cytosolic free Ca ([Ca]i). When Ca-ATPase pumping rate was slowed 39% by thapsigargin, the maximal SR Ca content decreased by 5 (∫INaCaXdt method) or 23% (fluorescence method); when pumping rate was increased 74% by isoproterenol, SR Ca content increased by 10% (fluorescence method) or 20% (∫INaCaXdt method). The relative stability of the SR Ca load suggests that dissipative losses have only a minor influence in setting the SR Ca content. Indeed, it appears that the SR Ca pump in intact cells can generate a [Ca] gradient approaching the thermodynamic limit.
cardiac myocytes; sarcoplasmic reticulum; isoproterenol; thapsigargin; excitation–contraction coupling
Digestive proteases of the digestive tract of the apple snail Pomacea canaliculata were studied. Luminal protease activity was found in the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Several protease bands and their apparent molecular weights were identified in both tissue extracts and luminal contents by gel zymography: (1) a 125 kDa protease in salivary gland extracts and in the crop content; (2) a 30 kDa protease throughout all studied luminal contents and in extracts of the midgut gland and of the endosymbionts isolated from this gland; (3) two proteases of 145 and 198 kDa in the coiled gut content. All these proteases were inhibited by aprotinin, a serine-protease inhibitor, and showed maximum activity between 30°C and 35°C and pH between 8.5 and 9.5. Tissue L-alanine-N-aminopeptidase activity was determined in the wall of the crop, the style sac and the coiled gut and was significantly higher in the coiled gut. Our findings show that protein digestion in P. canaliculata is carried out through a battery of diverse proteases originated from the salivary glands and the endosymbionts lodged in the midgut gland and by proteases of uncertain origin that occur in the coiled gut lumen.
K-sensitive microelectrodes were used to measure K+ within the extracellular space (Ko) of pregnant rat myometrium. Contractile activity was monitored by measuring either force or bioelectrical signals. Single and double-barreled electrodes were used. Double-barreled electrodes allowed monitoring of electrical activity 15 microns from the site of Ko measurement. From double-barreled electrode experiments, the bioelectrical burst started first, and then Ko began to rise 0.6 ± 0.1 seconds later. This delay indicates that K+ leaves the cells in response to local electrical activity rather than vice versa. Four control experiments were performed to assess the influence of electrical artifacts caused by tissue motion on Ko values. When observed, artifacts were negative and transient, and hence would result in an underestimation of Ko rises. Artifacts were minimized when tissue motion was minimized by fixing the tissue at both ends. At 37°C, 7 single barreled experiments and 45 contractions were analyzed. Resting Ko was within 1 mM of bath K+ (5 mM) at the beginning and end of the experiments. Ko rose during the contraction, fell after the completion of the contraction, and normalized before the next contraction began. Peak Ko values observed during force production were 18.8 ± 5.9 mM, a value high enough to modulate tissue-level electrical activity. Ko required 15.7 ± 2.8 seconds to normalize halfway (t50). Six experiments expressing 38 contractions were performed at 24°C. The contraction period was longer at 24°C. Values for peak Ko (26.2 ± 9.9 mM) and t50 (29.8±16.2 sec) were both larger than at 37°C (p<0.0003 for both). The direct relationships between peak Ko, t50 and the contraction period, suggest elevations in Ko may modulate contraction frequency. The myometrial interstitial space appears to be functionally important, and Ko metabolism may participate in cell-cell interactions.
Intestinal barrier dysfunction occurs in many intestinal diseases, in which proinflammatory cytokines play critical roles. However, researchers are still on the way to defining the underlying mechanisms and to evaluate therapeutic strategies for restoring intestinal barrier function. Berberine, a drug that has clinically been used to treat gastroenteritis and diarrhea for thousands of years, has been shown to protect barrier function in both endothelial and epithelial cells, but the mechanisms are completely unknown. In this study, we investigate the protective actions of berberine on barrier function and the underlying mechanisms in Caco-2 monolayers challenged with IFN-γ and TNF-α. Caco-2 monolayers were treated without or with simultaneous IFN-γ and TNF-α in the absence or presence of berberine. Both transepithelial electrical resistance (TER) and paracellular permeability were measured to evaluate barrier function. The expression and distribution of tight junction proteins ZO-1, occluding, and claudin-1 were respectively analyzed by immunoblot or immunofluorescence. The expressions of phosphorylated myosin light chain (pMLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were determined by immunoblot. The translocation of NF-κB p65 to nuclei was analyzed by immunofluorescence and immunoblot, respectively. The results showed that berberine significantly attenuated TER decrease and paracellular permeability increase in Caco-2 monolayers treated with IFN-γ and TNF-α. Berberine also dramatically alleviated IFN-γ and TNF-α-induced morphological alteration of tight junction proteins ZO-1, occluding, and claudin-1. The increase of both MLC phosphorylation and MLCK protein expression induced by IFN-γ and TNF-α was significantly inhibited by berberine treatment. Additionally, berberine suppressed the activation of HIF-1α, but not NF-κB. Taken together, it is suggested that berberine attenuates IFN-γ and TNF-α-induced intestinal epithelial barrier dysfunction by inhibiting the signaling pathway of MLCK-dependent MLC phosphorylation mediated by HIF-1α.
The nature of synaptic transmission at functionally distinct synapses in intestinal reflex pathways has not been fully identified. In this study, we investigated whether transmission between interneurons in the descending inhibitory pathway is mediated by a purine acting at P2Y receptors to produce slow excitatory synaptic potentials (EPSPs).
Myenteric neurons from guinea-pig ileum in vitro were impaled with intracellular microelectrodes. Responses to distension 15 mm oral to the recording site, in a separately perfused stimulation chamber and to electrical stimulation of local nerve trunks were recorded. A subset of neurons, previously identified as nitric oxide synthase immunoreactive descending interneurons, responded to both stimuli with slow EPSPs that were reversibly abolished by a high concentration of PPADS (30 μM, P2 receptor antagonist). When added to the central chamber of a three chambered organ bath, PPADS concentration-dependently depressed transmission through that chamber of descending inhibitory reflexes, measured as inhibitory junction potentials in the circular muscle of the anal chamber. Reflexes evoked by distension in the central chamber were unaffected. A similar depression of transmission was seen when the specific P2Y1 receptor antagonist MRS 2179 (10 μM) was in the central chamber. Blocking either nicotinic receptors (hexamethonium 200 μM) or 5-HT3 receptors (granisetron 1 μM) together with P2 receptors had no greater effect than blocking P2 receptors alone.
Slow EPSPs mediated by P2Y1 receptors, play a primary role in transmission between descending interneurons of the inhibitory reflexes in the guinea-pig ileum. This is the first demonstration for a primary role of excitatory metabotropic receptors in physiological transmission at a functionally identified synapse.
Despite two centuries of reports linking alcohol consumption with enhanced susceptibility to bacterial infections and in particular gut-derived bacteria, there have been no studies or model systems to assess the impact of long-term alcohol exposure on the ability of the epithelial barrier to withstand bacterial infection. It is well established that acute alcohol exposure leads to reduction in tight and adherens junctions, which in turn leads to increases in epithelial cellular permeability to bacterial products, leading to endotoxemia and a variety of deleterious effects in both rodents and human. We hypothesized that reduced fortification at junctional structures should also reduce the epithelial barrier’s capacity to maintain its integrity in the face of bacterial challenge thus rendering epithelial cells more vulnerable to infection. In this study, we established a cell-culture based model system for long-term alcohol exposure to assess the impact of chronic alcohol exposure on the ability of Caco-2 intestinal epithelial cells to withstand infection when facing pathogenic bacteria under the intact or wounded conditions. We report that daily treatment with 0.2% ethanol for two months rendered Caco-2 cells far more susceptible to wound damage and cytotoxicity caused by most but not all bacterial pathogens tested in our studies. Consistent with acute alcohol exposure, long-term ethanol exposure also adversely impacted tight junction structures, but in contrast, it did not affect the adherens junction. Finally, alcohol-treated cells partially regained their ability to withstand infection when ethanol treatment was ceased for two weeks, indicating that alcohol’s deleterious effects on cells may be reversible.