Nitric oxide stimulates prostaglandin synthesis in cultured rabbit gastric cells

Both prostaglandins (PGs) and nitric oxide (NO) have cytoprotective and hyperemic effects in the stomach. However, the effect of NO on PG synthesis in gastric mucosal cells is unclear. We examined whether sodium nitroprusside (SNP), a releaser of NO, stimulates PG synthesis in cultured rabbit gastric mucus-producing cells. These cells did not release NO themselves. Co-incubation with SNP (2 × 10⁻⁴, 5 × 10⁻⁴, 10⁻³ M) increased PGE₂ synthesis, and SNP (10⁻³ M) increased PGI₂ synthesis in these cells. Hemoglobin, a scavenger of NO, (10⁻⁵ M) eliminated the increase in PGE₂ synthesis by SNP, but methylene blue, an inhibitor of soluble guanylate cyclase, (5 × 10⁻⁵ M) did not affect the increase in PGE₂ synthesis by SNP. 8-bromo guanosine 3′ : 5′-cyclic monophosphate (8-bromo cGMP), a cGMP analogue, (10⁻⁶, 10⁻⁵, 10⁻⁴, 10⁻³ M) did not affect PGE₂ synthesis. These findings suggest that NO increased PGE₂ and PGI₂ synthesis via a cGMP-independent pathway in cultured rabbit gastric cells.     

ADAM-15 inhibits wound healing in human intestinal epithelial cell monolayers

The disintegrin metalloproteases (or ADAMs) are membrane-anchored glycoproteins that have been implicated in cell-cell or cell-matrix interactions and in proteolysis of molecules on the cell surface. The expression and/or the pathophysiological implications of ADAMs are not known in intestinal epithelial cells. Therefore, our aim was to investigate the expression and the role of ADAMs in intestinal epithelial cells. Expression of ADAMs was assessed by RT-PCR, Western blot analysis, and immunufluorescence experiments. Wound-healing experiments were performed by using the electric cell substrate impedence sensing technology. Our results showed that ADAMs-10, -12, and -15 mRNA are expressed in the colonic human cell lines Caco2-BBE and HT29-Cl.19A. An ADAM-15 complementary DNA cloned from Caco2-BBE poly(A)+ RNA, and encompassing the entire coding region, was found to be shorter and to present a different region encoding the cytoplasmic tail compared with ADAM-15 sequence deposited in the database. In Caco2-BBE cells and colonic epithelial cells, ADAM-15 protein was found in the apical, basolateral, and intracellular compartments. We also showed that the overexpression of ADAM-15 reduced cell migration in a wound-healing assay in Caco2-BBE monolayers. Our data show that 1) ADAM-15 is expressed in human intestinal epithelia, 2) a new variant of ADAM-15 is expressed in a human intestinal epithelial cell line, and 3) ADAM-15 is involved in intestinal epithelial cells wound-healing processes. Together, these results suggest that ADAM-15 may have important pathophysiological roles in intestinal cells.     

Specific signaling cascades involved in cell spreading during healing of micro-wounded gastric epithelial monolayers

Mechanisms that specifically modulate cell spreading and/or cell migration following epithelial wounding are poorly understood. Using micro-wounded human gastric epithelial monolayers, we show herein that EGF and TGFalpha maximally increase spreading of epithelial sheets under a cell proliferation-independent mechanism. Treatment of confluent HGE-17 cells with the phosphatidylinositol 3-kinase inhibitor, LY294002, and the epidermal growth factor receptor inhibitor, PD153035, strongly reduced basal and TGFalpha-stimulated cell spreading. While pharmacological inhibition of pp60src-kinase activity also attenuated basal epithelial spreading, addition of the mTOR/p70S6K inhibitor rapamycin or a specific siRNA targeting ILK sequence did not affect the kinetic rates of wound closure. Epithelial wound healing was initiated by actin purse-string contraction followed by lamellae formation. Conversely, disruption of actin and tubulin stability with cytochalasin D and nocodazole, respectively, inhibited epithelial sheet spreading. Finally, antibodies directed against the alpha3 integrin subunit, but not against the alpha6 or alpha2 subunits, attenuated epithelial sheet spreading as well as lamellae formation. In conclusion, the current investigation establishes that EGF/TGFalpha and the alpha3beta1 integrin, pp60c-src, EGFR and PI3K pathways are mainly associated with the cell spreading of the restitution process during healing of human gastric epithelial wounds.     

Nitric oxide donors

Nitric oxide (NO) donors are pharmacologically active substances that release NO in vivo or in vitro. NO has a variety of functions such as the release of prostanoids, inhibition of platelet aggregation, effect on angiogenesis, and production of oxygen free radicals. This report discusses the chemical and pharmacological characteristics of NO donors, their effect on platelet function and cyclooxygenase, their cardiac action including myocardial infarction, and release of superoxide anions. This review stresses NO tolerance and the effect of NO donors on angiogenesis in myocardial infarction and in solid tumors.     

Nitric oxide attenuates hydrogen peroxide-induced barrier disruption and protein tyrosine phosphorylation in monolayers of intestinal epithelial cell

The intestinal epithelium provides a barrier to the transport of harmful luminal molecules into the systemic circulation. A dysfunctional epithelial barrier is closely associated with the pathogenesis of a variety of intestinal and systemic disorders. We investigated here the effects of nitric oxide (NO) and hydrogen peroxide (H(2)O(2)) on the barrier function of a human intestinal epithelial cell line, Caco-2. When treated with H(2)O(2), Caco-2 cell monolayers grown on permeable supports exhibited several remarkable features of barrier dysfunction as follows: a decrease in transepithelial electrical resistance, an increase in paracellular permeability to dextran, and a disruption of the intercellular junctional localization of the scaffolding protein ZO-1. In addition, an induction of tyrosine phosphorylation of numerous cellular proteins including ZO-1, E-cadherin, and beta-catenin, components of tight and adherens junctions, was observed. On the other hand, combined treatment of Caco-2 monolayers with H(2)O(2) and an NO donor (NOC5 or NOC12) relieved the damage to the barrier function and suppressed the protein tyrosine phosphorylation induced by H(2)O(2) alone. These results suggest that NO protects the barrier function of intestinal epithelia from oxidative stress by modulating some intracellular signaling pathways of protein tyrosine phosphorylation in epithelial cells.     

Nitric oxide inhibits cell growth in cultured human thyrocytes

Effect of NO induced by interleukin-1 (IL-1) or IL-1/interferon-_γ (IL-1/IFN-_γ) was investigated on cell growth using primary cultures of human thyrocytes. Cytokine-induced NO production was associated not only with an increase in cyclic GMP (cGMP) formation but also with an inhibition of cell growth determined by bromo-deoxyuridine (Br-dU) incorporation into DNA. When NO synthesis was blocked by N^G-monomethyl-L-arginine (L-MMA), cGMP formation was prevented in parallel with NO production and inversely a restoration of cell growth was evident. S-nitroso-N-acetyl-penicillamine, a NO donor, but not a cell permeable cGMP analog, 8-bromo-cGMP, inhibited cell growth in a dose-dependent manner. The present findings strongly indicate that endogenous NO produced by the cytokine treatment as well as exogenous NO, has a cGMP-independent inhibitory action on human thyrocyte growth.     

Nitric oxide donor-induced hyperpermeability of cultured intestinal epithelial monolayers: role of superoxide radical,hydroxyl radical,and peroxynitrite

Many of the cytopathic effects of nitric oxide (NO^·) are mediated by peroxynitrite (PN), a product of the reaction between NO^· and superoxide radical (O^·?₂). In the present study, we investigated the role of PN, O^·?₂ and hydroxyl radical (OH^·) as mediators of epithelial hyperpermeability induced by the NO^· donor, S-nitroso-N-acetylpenicillamine (SNAP), and the PN generator, 3-morpholinosydnonimine (SIN-1). Caco-2_BBe enterocytic monolayers were grown on permeable supports in bicameral chambers. Epithelial permeability, measured as the apical-to-basolateral flux of fluorescein disulfonic acid, increased after 24 h of incubation with 5.0 mM SNAP or SIN-1. Addition of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO^· scavenger, or Tiron, an O^·?₂ scavenger, reduced the increase in permeability induced by both donor compounds. The SNAP-induced increase in permeability was prevented by allopurinol, an inhibitor of xanthine oxidase (a source of endogenous O^·?₂). Diethyldithiocarbamate, a superoxide dismutase inhibitor, and pyrogallol, an O^·?₂ generator, potentiated the increase in permeability induced by SNAP. Addition of the PN scavengers deferoxamine, urate, or glutathione, or the OH^· scavenger mannitol, attenuated the increase in permeability induced by both SNAP and SIN-1. Both donor compounds decreased intracellular levels of glutathione and protein-bound sulfhydryl groups, suggesting the generation of a potent oxidant. These results support a role for PN, and possibly OH^·, in the pathogenesis of NO^· donor-induced intestinal epithelial hyperpermeability.     

Fetal rabbit gastric epithelial cells cultured on floating collagen gels

Summary Cells were isolated from ∼ 30 d fetal rabbit stomachs and cultured on floating collagen gels. Electron microscopy showed monolayers in which only one cell type persisted. These columnar cells were joined at apical borders by tight junctions and contained an extensive endoplasmic reticular network with an occasional intracellular canaliculus. They also occasionally contained what appeared to be secretory granules (mucus?), and therefore had some characteristics of all the cell types of the intact fetal stomachs, which showed oxyntic, mucous, and undifferentiated cells. In Ussing chambers with Ringer's solution on both sides, cultures developed transepithelial potential (potential difference [PD], mV, mucosa ground)=13, resistance (resistance [R], Ω-cm²)=285, and short-circuit current (I _sc , μA/cm²)=45 (n=7), clearly indicating that cellular polarity and junctional integrity were maintained. These transport parameters were somewhat different for intact fetal stomachs (PD=20, R=70, and I _sc =220 [n=4]), which may be due to extensive folding of intact fetal stomachs or the presence of only one cell type in culture, or both. Although gastric stimulants histamine, dibutyryl cycle AMP (dbcAMP), and isobutyl-methylxanthine (IMX) (a phosphodiesterase inhibitor) did not elicit H⁺ secretion or electrophysiological changes in monolayers or intact stomachs, 10⁻⁴ M apical amiloride caused a decrease in I _sc in cultured monolayers (27%) and intact stomachs (50%). Thus, Na⁺ transport seems to be a significant fraction of ion transport in both preparations. This culture system may allow the study of oxyntic cell differentiation and the development of H⁺, Na⁺, and Cl⁻ transport in the gastric mucosa. This work was supported by NIH Grant AM 19520. The electron microscope was purchased in part by NSF Grant PM 76-80300. C. Bisbee was supported by National Cancer Institute Grants CA-05388 and CA-09041. C. Logsdon received support from the Systems and Integrative Biology Training Grant.     

The mathematical modelling of cell kinetics in corneal epithelial wound healing

This paper considers the comparison of experimental spatial and temporal data of mitotic rates measured during corneal epithelial wound healing (CEWH) of a rat model with the predictions of a computer modelling framework. We begin by briefly showing that previous models, used in the study of corneal epithelial wound healing speeds, are inadequate for the study of cell kinetics. We proceed to formulate a new modelling framework more suited to such a study. This framework is simulated in its simplest form, and the results from this motivate a new realisation of the modelling framework, including a caricature of age structuring. Finally, a model with a simple representation of juxtacrine signalling is considered. The final model captures many, though not all, of the trends of the experimental data. This paper thus lays a foundation for the modelling of the cell kinetics of corneal epithelial wound healing, and yields valuable insight regarding the important mechanisms a model should consider in order to reproduce the observed experimental trends.     

Characterization of bovine oviduct epithelial cell monolayers cultured under serum-free conditions

Summary We have developed a culture system for early bovine embryos in serum-free media conditioned by oviduct cell monolayers. A gentle mechanical procedure for oviduct cell isolation has been applied for this purpose avoiding the use of proteolytic enzymes. The aim of the present study was to identify the cell types present in the monolayers and to examine their fate in primary culture in serum-free or in serum-containing media by means of electronmicroscopical, immunocytochemical, and biochemical analyses. The cell dissociation procedure yielded two cell populations: ciliary cells and secretory cells that gradually dedifferentiate during culture. These cells formed a confluent monolayer after 6 d of culture in Tissue Culture Medium 199 medium supplemented with 10% fetal calf serum. Confluent cells displayed a typical epithelial cell morphology as assessed by phase contrast and electron microscopy and all the cells contained cytokeratin filaments as determined by immunocytochemistry. The overall histoarchitecture of the monolayer was preserved after washing and further culture for 7 d in serum-free medium. However, some degenerative signs indicate that the serum-free culture should not be extended for more than 7 d. Confluent oviduct cells also maintained their metabolic and protein secretory activity when deprived of serum. Total protein content in the culture supernatant linearly increased as a function of time and numerous peaks were detected after separation of proteins by high performance ion exchange chromatography. Protein elution patterns were reproducible and most of the proteins present in the culture medium were neosynthesized as determined by the incorporation of radiolabeled amino acids into nondialyzable proteins.     

Knockdown of heme oxygenase-2 impairs corneal epithelial cell wound healing

Heme oxygenase (HO) represents an intrinsic cytoprotective system based on its anti‐oxidative and anti‐inflammatory properties mediated via its products biliverdin/bilirubin and carbon monoxide (CO). We showed that deletion of HO‐2 results in impaired corneal wound healing with associated chronic inflammatory complications. This study was undertaken to examine the role of HO activity and the contribution of HO‐1 and HO‐2 to corneal wound healing in an in vitro epithelial scratch injury model. A scratch wound model was established using human corneal epithelial (HCE) cells. These cells expressed both HO‐1 and HO‐2 proteins. Injury elicited a rapid and transient increase in HO‐1 and HO activity; HO‐2 expression was unchanged. Treatment with biliverdin or CORM‐A1, a CO donor, accelerated wound closure by 10% at 24 h. Inhibition of HO activity impaired wound closure by more than 50%. However, addition of biliverdin or CORM‐A1 reversed the effect of HO inhibition on wound healing. Moreover, knockdown of HO‐2 expression, but not HO‐1, significantly impaired wound healing. These results indicate that HO activity is required for corneal epithelial cell migration. Inhibition of HO activity impairs wound healing while amplification of its activity restores and accelerates healing. Importantly, HO‐2, which is highly expressed in the corneal epithelium, appears to be critical for the wound healing process in the cornea. The mechanisms by which it contributes to cell migration in response to injury may reside in the cytoprotective properties of CO and biliverdin. J. Cell. Physiol. 226: 1732–1740, 2011. © 2010 Wiley‐Liss, Inc.     

Nitric oxide inhibits wound collagen synthesis

Nitric oxide (NO) is a messenger molecule which regulates many physiological functions like immunity, vascular tone and serves as a neurotransmitter. Although it is known to participate in healing process, its role in collagen synthesis is not clear. Therefore, the present investigation was done to study the role of NO in wound collagen synthesis. Rats received full thickness, circular (8 mm), transdermal wounds which were treated with NO releaser, sodium nitroprusside (SNP, 0.001 100 M) topically for 5 days. Wound collagen content estimated in terms of hydroxyproline (HP) and confirmed histochemically was decreased significantly by all SNP doses. L-Arginine, a substrate for nitric oxide synthase (NOS) when applied topically decreased collagen content of the wounded tissues. N-Nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NOS, increased wound collagen content significantly as compared to untreated and SNP treated animal wounds when administered intraperitoneally at the doses 3, 10 and 30 mg/kg. Furthermore, histological findings also demonstrated laying down of thick collagen bundles and proliferation of fibroblasts together with prominent angiogenesis in L-NAME treated wound tissues as compared to untreated and SNP treated tissues. N-nitro-D-arginine methyl ester, an inactive isomer, was found to have no effect on wound collagen levels. When L-arginine was administered in L-NAME pretreated rats, it significantly elevated wound HP content. The results indicate that NO plays an important role in regulating the collagen biosynthesis in skin model of a healing wound.     

Double-mode impedance analysis of epithelial cell monolayers cultured on shear wave resonators

The viscoelastic behavior of epithelial cells (MDCK-I and MDCK-II) grown on AT-cut quartz crystals with a fundamental resonance at 5 MHz was investigated by impedance spectroscopy. Using the electromechanical model recently derived by Martin et al. [(1991) Anal Chem 63: 2272 – 2281] for Newtonian liquids in contact with shear wave resonators we quantified the viscous damping arising from the adherent cells by fitting the impedance data with a modified Butterworth-Van Dyke circuit in the region of the resonance frequency. Impedance spectroscopy was additionally performed in the frequency range from 1 Hz to 1 MHz to scrutinize the passive electrical properties of the epithelial cell layers using an additional platinum electrode. These data allow one to document the cell layers' integrity as well as the electrode coverage. We were able to confirm that the presence of a cell-layer mainly increases damping of the shear wave and does not exhibit a pure mass-load behavior. These findings were supported by the discovery that the inductance L in the electromechanical model was less influenced by the cell-layer than the resistance R. The apparent cell-viscosities determined by our method are 0.097 poise for MDCK-I and 0.142 poise for MDCK-II cell-layers. These low apparent viscosities may be explained in terms of a considerable spacing between the cells immobilized via their focal contacts and the quartz surface. Received: 5 June 1996 / Accepted: 6 August 1996     

Discrete limbal epithelial stem cell populations mediate corneal homeostasis and wound healing

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Nitric oxide in gastrointestinal epithelial cell carcinogenesis: linking inflammation to oncogenesis

Chronic inflammation of gastrointestinal tissues is a well-recognized risk factor for the development of epithelial cell-derived malignancies. Although the inflammatory mediators linking chronic inflammation to carcinogenesis are numerous, current information suggests that nitric oxide (NO) contributes to carcinogenesis during chronic inflammation. Inducible nitric oxide synthase (iNOS), expressed by both macrophages and epithelial cells during inflammation, generates the bioreactive molecule NO. In addition to causing DNA lesions, NO can directly interact with proteins by nitrosylation and nitosation reactions. The consequences of protein damage by NO appear to be procarcinogenic. For example, NO inhibits DNA repair enzymes such as human 8-oxodeoxyguanosine DNA glycosylase 1 and blocks apoptosis via nitrosylation of caspases. These cellular events permit DNA damage to accumulate, which is required for the numerous mutations necessary for development of invasive cancer. NO also promotes cancer progression by functioning as an angiogenesis factor. Strategies to inhibit NO generation during chronic inflammation or to scavenge reactive nitrogen species may prove useful in decreasing the risk of cancer development in chronic inflammatory gastrointestinal diseases.     

FAK regulates intestinal epithelial cell survival and proliferation during mucosal wound healing

Background

Following damage to the intestinal epithelium, restoration of epithelial barrier integrity is triggered by a robust proliferative response. In other tissues, focal adhesion kinase (FAK) regulates many of the cellular processes that are critical for epithelial homeostasis and restitution, including cell migration, proliferation and survival. However, few studies to date have determined how FAK contributes to mucosal wound healing in vivo.

Methodology and Principal Findings

To examine the role of FAK in intestinal epithelial homeostasis and during injury, we generated intestinal epithelium (IE)-specific conditional FAK knockout mice. Colitis was induced with dextran-sulfate-sodium (DSS) and intestinal tissues were analyzed by immunohistochemistry and immunoblotting. While intestinal development occurred normally in mice lacking FAK, FAK-deficient animals were profoundly susceptible to colitis. The loss of epithelial FAK resulted in elevated p53 expression and an increased sensitivity to apoptosis, coincident with a failure to upregulate epithelial cell proliferation. FAK has been reported to function as a mechanosensor, inducing cyclin D1 expression and promoting cell cycle progression under conditions in which tissue/matrix stiffness is increased. Collagen deposition, a hallmark of inflammatory injury resulting in increased tissue rigidity, was observed in control and FAK knockout mice during colitis. Despite this fibrotic response, the colonic epithelium in FAK-deficient mice exhibited significantly reduced cyclin D1 expression, suggesting that proliferation is uncoupled from fibrosis in the absence of FAK. In support of this hypothesis, proliferation of Caco-2 cells increased proportionally with matrix stiffness in vitro only under conditions of normal FAK expression; FAK depleted cells exhibited reduced proliferation concomitant with attenuated cyclin D1 expression.

Conclusions

In the colon, FAK functions as a regulator of epithelial cell survival and proliferation under conditions of mucosal injury and a mechanosensor of tissue compliance, inducing repair-driven proliferation in the colonic epithelium through upregulation of cyclin D1.     

Rho kinases regulate corneal epithelial wound healing

We have previously shown that Rho small GTPase is required for modulating both cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding. In the present study, we investigated the role of Rho kinases (ROCKs), major effectors of Rho GTPase, in mediating corneal epithelial wound healing. Both ROCK 1 and 2 were expressed and activated in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, in response to wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) stimulations. The ROCK inhibitor Y-27632 efficiently antagonized ROCK activities without affecting Rho activation in wounded HCECs. Y-27632 promoted basal and HB-EGF-enhanced scratch wound healing and enhanced cell migration and adhesion to matrices, while retarded HB-EGF induced cell proliferation. E-cadherin- and beta-catenin-mediated cell-cell junction and actin cytoskeleton organization were disrupted by Y-27632. Y-27632 impaired the formation and maintenance of tight junction barriers indicated by decreased trans-epithelial resistance and disrupted occludin staining. We conclude that ROCK activities enhance cell proliferation, promote epithelial differentiation, but negatively modulate cell migration and cell adhesion and therefore play a role in regulating corneal epithelial wound healing.     

Effects of valerate on intestinal barrier function in cultured Caco-2 epithelial cell monolayers

Molecular Biology Reports - Short-chain fatty acids (SCFAs) are a group of microbial metabolites of undigested dietary fiber, protein and unabsorbed amino acids in the colon, well-known for their...