Increased permeability of primary cultured brain microvessel endothelial cell monolayers following TNF-alpha exposure.

TNF-alpha is a cytokine that produces increased permeability in the peripheral vasculature; however, little is known about the effects of TNF-alpha on the blood-brain barrier (BBB). Using primary cultured bovine brain microvessel endothelial cells (BBMEC) as an in vitro model of the BBB, this study shows that TNF-alpha produces a reversible increase in the permeability of the brain microvessel endothelial cells. The BBMEC monolayers were pre-treated with 100 ng/ml of TNF-alpha for periods ranging from 2 to 12 hours. Permeability was assessed using three molecular weight markers, fluorescein (376 MW), fluorescein-dextran (FDX-4400; 4400 MW), and FDX-70000 (MW 70000). The permeability of BBMEC monolayers to all three fluorescent markers was increased two-fold or greater in the TNF-alpha treatment group compared to control monolayers receiving no TNF-alpha. Significant changes in permeability were also observed with TNF-alpha concentrations as low as 1 ng/ml. These results suggest that TNF-alpha acts directly on the brain microvessel endothelial cells in a dynamic manner to produce a reversible increase in permeability. Exposure of either the lumenal or ablumenal side of BBMEC monolayers to TNF-alpha resulted in similar increases in permeability to small macromolecules, e.g. fluorescein. However, when a higher molecular weight marker was used (e.g. FDX-3000), there was a greater response following lumenal exposure to TNF-alpha. Together, these studies demonstrate a reversible and time dependent increase in brain microvessel endothelial cell permeability following exposure to TNF-alpha. Such results appear to be due to TNF's direct interaction with the brain microvessel endothelial cell.     

Expanding the febrigenic role of cyclooxygenase-2 to the previously overlooked responses

Previous studies on the role of cyclooxygenase (COX)-1 and -2 in fever induced by intravenous LPS have failed to investigate the role of these isoenzymes in the earliest responses: monophasic fever (response to a low, near-threshold dose of LPS) and the first phase of polyphasic fever (response to higher doses). We studied these responses in 96 mice that were COX-1 or COX-2 deficient (-/-) or sufficient (+/+). Each mouse was implanted with a temperature telemetry probe into the peritoneal cavity and a jugular catheter. The study was conducted at a tightly controlled, neutral ambient temperature (31 degrees C). To avoid stress hyperthermia (which masks the onset of fever), all injections were performed through a catheter extension. The +/+ mice responded to intravenous saline with no change in deep body temperature. To a low dose of LPS (1 microg/kg iv), they responded with a monophasic fever. To a higher dose (56 microg/kg), they responded with a polyphasic fever. Neither monophasic fever nor the first phase of polyphasic fever was attenuated in the COX-1 -/- mice, but both responses were absent in the COX-2 -/- mice. The second and third phases of polyphasic fever were also missing in the COX-2 -/- mice. The present study identifies a new, critical role for COX-2 in the mediation of the earliest responses to intravenous LPS: monophasic fever and the first phase of polyphasic fever. It also suggests that no product of the COX-1 gene, including the splice variant COX-1b (COX-3), is essential for these responses.     

Induction of COX-2 enzyme and down-regulation of COX-1 expression by lipopolysaccharide (LPS) control prostaglandin E2 production in astrocytes

Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E(2) (PGE(2)) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE(2) and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE(2) production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE(2), and suggest that the coordinated down-regulation of COX-1 facilitates PGE(2) production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation.     

Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lungs exposed to endotoxin.

BACKGROUND: Lipopolysaccharides (LPS), widely used to study the mechanisms of gram-negative sepsis, increase airway resistance by constriction of terminal bronchioles. The role of the cyclooxygenase (COX) isoenzymes and their prostanoid metabolites in this process was studied. MATERIALS AND METHODS: Pulmonary resistance, the release of thromboxane (TX) and the expression of COX-2 mRNA were measured in isolated blood-free perfused rat lungs exposed to LPS. RESULTS: LPS induced the release of TX and caused increased airway resistance after about 30 min. Both TX formation and LPS-induced bronchoconstriction were prevented by treatment with the unspecific COX inhibitor acetyl salicylic acid, the specific COX-2 inhibitor CGP-28238, dexamethasone, actinomycin D, or cycloheximide. LPS-induced bronchoconstriction was also inhibited by the TX receptor antagonist BM-13177. The TX-mimetic compound, U-46619, increased airway resistance predominantly by constricting terminal bronchioles. COX-2-specific mRNA in lung tissue was elevated after LPS exposure, and this increase was attenuated by addition of dexamethasone or of actinomycin D. In contrast to LPS, platelet-activating factor (PAF) induced immediate TX release and bronchoconstriction that was prevented by acetyl salicylic acid, but not by CGP-28238. CONCLUSIONS: LPS elicits the following biochemical and functional changes in rat lungs: (i) induction of COX-2; (ii) formation of prostaglandins and TX; (iii) activation of the TX receptor on airway smooth muscle cells; (iv) constriction of terminal bronchioles; and (v) increased airway resistance. In contrast to LPS, the PAF-induced TX release is likely to depend on COX-1.     

COX-1 and COX-2 contribute differentially to the LPS-induced release of PGE2 and TxA2 in liver macrophages

LPS induces an immediate release of thromboxane TxA2 and a delayed release of PGE2. Dexamethasone suppresses the LPS-induced release of TxA2 and PGE2. In the first 8 h after LPS addition, the specific COX-2 inhibitor SC236 inhibits the PGE2 and TxA2 release by about 80% and 20%, whereas the release of PGE2 and TxA2 between 8 and 24 h is inhibited by about 40% and 35%, respectively. Resident liver macrophages express substantial amounts of COX-1, TxAS, cPGES and mPGES-2, small amounts of COX-2 but almost no detectable amounts of mPGES-1. LPS induces an increase of COX-2 and mPGES-1, but does not change COX-1, cPGES, mPGES-2 and TxAS at protein level. Dexamethasone suppresses almost completely the LPS-induced effects on COX-2 and mPGES-1. It is concluded that (1) COX-1 and COX-2 are involved in the LPS-induced synthesis of TxA2 and PGE2; (2) TxA2 release is catalyzed at early time-points by the combined action of COX-1 and TxAs, whereas at later time points the newly expressed COX-2 couples to TxAS and contributes to the TxA2 release; (3) PGE2 release within the first 8 h is predominantly catalyzed by COX-2, whereas at later time-points COX-1 couples to the newly expressed mPGES-1 and contributes to the PGE2 release.     

Role of reactive oxygen species in LPS-induced production of prostaglandin E2 in microglia

We determined the roles of reactive oxygen species (ROS) in the expression of cyclooxygenase-2 (COX-2) and the production of prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-activated microglia. LPS treatment increased intracellular ROS in rat microglia dose-dependently. Pre-treatment with superoxide dismutase (SOD)/catalase, or SOD/catalase mimetics that can scavenge intracellular ROS, significantly attenuated LPS-induced release in PGE2. Diphenylene iodonium (DPI), a non-specific NADPH oxidase inhibitor, decreased LPS-induced PGE2 production. In addition, microglia from NADPH oxidase-deficient mice produced less PGE2 than those from wild-type mice following LPS treatment. Furthermore, LPS-stimulated expression of COX-2 (determined by RT-PCR analysis of COX-2 mRNA and western blot for its protein) was significantly reduced by pre-treatment with SOD/catalase or SOD/catalase mimetics. SOD/catalase mimetics were more potent than SOD/catalase in reducing COX-2 expression and PGE2 production. As a comparison, scavenging ROS had no effect on LPS-induced nitric oxide production in microglia. These results suggest that ROS play a regulatory role in the expression of COX-2 and the subsequent production of PGE2 during the activation process of microglia. Thus, inhibiting NADPH oxidase activity and subsequent ROS generation in microglia can reduce COX-2 expression and PGE2 production. These findings suggest a potential therapeutic intervention strategy for the treatment of inflammation-mediated neurodegenerative diseases.     

Ultrastructural localization of factor VIII-related antigen in cultured human brain microvessel endothelial cells.

Immunogold staining of primary cultures of human brain microvessel endothelial cells demonstrated the presence of Factor VIII-related antigen within cytoplasmic vesicles in close association with the rough endoplasmic reticulum and Golgi apparatus. Immunoperoxidase staining, at the light microscopic level, revealed a similar granular, perinuclear staining. The morphology and location of these vesicular profiles indicate that they are part of the trans-Golgi region where terminal processing and short-term storage of Factor VIII-related antigen takes place. Weibel-Palade bodies, specific storage organelles for von Willebrand factor in large vessel endothelium, were not observed in cerebral microvessel endothelium. The release of Factor VIII-related antigen from the cytoplasmic vesicles was influenced by some of the factors known to stimulate or inhibit the regulated pathway of secretion from Weibel-Palade bodies. Thus, stimulation of endothelial cells with calcium ionophore A23187 resulted in almost complete loss of staining, while addition of EGTA to the culture medium led to slight increase of intracellular pools of Factor VIII-related antigen. Pre-incubation of monolayers with interferon-gamma was associated with significant increase in the number of labeled vesicles, suggesting an additional role of this cytokine in the localized immune reaction within the central nervous system.     

Localized vs. systemic inflammation in guinea pigs: a role for prostaglandins at distinct points of the fever induction pathways?

In guinea pigs, dose-dependent febrile responses were induced by injection of a high (100 microg/kg) or a low (10 microg/kg) dose of bacterial lipopolysaccharide (LPS) into artificial subcutaneously implanted Teflon chambers. Both LPS doses further induced a pronounced formation of prostaglandin E(2) (PGE(2)) at the site of localized subcutaneous inflammation. Administration of diclofenac, a nonselective cyclooxygenase (COX) inhibitor, at different doses (5, 50, 500, or 5,000 microg/kg) attenuated or abrogated LPS-induced fever and inhibited LPS-induced local PGE(2) formation (5 or 500 microg/kg diclofenac). Even the lowest dose of diclofenac (5 microg/kg) attenuated fever in response to 10 microg/kg LPS, but only when administered directly into the subcutaneous chamber, and not into the site contralateral to the chamber. This observation indicated that a localized formation of PGE(2) at the site of inflammation mediated a portion of the febrile response, which was induced by injection of 10 microg/kg LPS into the subcutaneous chamber. Further support for this hypothesis derived from the observation that we failed to detect elevated amounts of COX-2 mRNA in the brain of guinea pigs injected subcutaneously with 10 microg/kg LPS, whereas subcutaneous injections of 100 microg/kg LPS, as well as systemic injections of LPS (intra-arterial or intraperitoneal routes), readily caused expression of the COX-2 gene in the guinea pig brain, as demonstrated by in situ hybridization. Therefore, fever in response to subcutaneous injection of 10 microg/kg LPS may, in part, have been evoked by a neural, rather than a humoral, pathway from the local site of inflammation to the brain.     

Lipopolysaccharide reduces electrical coupling in microvascular endothelial cells by targeting connexin40 in a tyrosine-, ERK1/2-, PKA-, and PKC-dependent manner

Electrical coupling along the endothelium is central in the arteriolar conducted response and in control of vascular resistance. It has been shown that exposure of endothelium to lipopolysaccharide (LPS, an initiating factor in sepsis) reduces intercellular communication in vitro and in vivo. The molecular basis for this reduction is not known. We examined the effect of LPS on electrical coupling in monolayers of cultured mouse microvascular endothelial cells (MMEC) derived from the mouse hindlimb skeletal muscle. To assess coupling, we measured the spread of electrical current injected into the monolayer and computed the monolayer intercellular resistance (inverse measure of coupling). LPS (10 microg/ml, 1 h) reduced coupling (i.e., increased resistance) in MMEC isolated from wild-type, connexin37 (Cx37) null and Cx43(G60S) (nonfunctional mutant) mice, but not in MMEC derived from Cx40 null mice. LPS also activated JNK1/2, p38 and ERK1/2 MAP kinases. Pretreatment of WT monolayers with ERK1/2 inhibitor U0126 (20 microM, 1 h) prevented the LPS-induced decrease in coupling, while inhibition of JNK1/2 with SP600125 (20 microM, 1 h) and p38 with a p38 inhibitor (10 nM, 1 h) had no effect. Furthermore, inhibition of tyrosine kinases with PP-2 (10 nM, 1 h), activation of PKA by 8-bromo-cAMP (1 mM, 5 min), and activation of PKC by bryostatin-2 (10 nM, 1 h) also prevented the reduction in coupling. We propose that LPS reduces inter-endothelial electrical coupling via tyrosine-, ERK1/2-, PKA-, and PKC-dependent signaling that targets Cx40. We suggest that this mechanism contributes to compromised arteriolar function following LPS exposure.     

Nitric oxide mediates hypoxia-induced changes in paracellular permeability of cerebral microvasculature

Ischemic stroke from a reduction in blood flow to the brain microvasculature results in a subsequent decreased delivery of oxygen (i.e., hypoxia) and vital nutrients to endothelial, neuronal, and glial cells. Hypoxia associated with stroke has been shown to increase paracellular permeability of the blood-brain barrier, leading to the release of cellular mediators and brain tissue injury. Whereas reperfusion does not occur in all ischemic strokes, increased permeability has been seen in posthypoxic reoxygenation. Currently, it is unknown whether these deleterious effects result from cellular mechanisms stimulated by decreased oxygen during stroke or posthypoxic reoxygenation stress. This study used primary bovine brain microvessel endothelial cells (BBMECs) to examine the involvement of nitric oxide (NO) as a mediator in hypoxia-induced permeability changes. Hypoxia-induced increased transport of [14C]sucrose across BBMEC monolayers compared with normoxia was attenuated by either posthypoxic reoxygenation or inhibition of NO synthase (NOS). The hypoxia-induced permeability effect was further reduced when NOS inhibition was combined with posthypoxic reoxygenation. Additionally, a significant increase in total NO was seen in BBMECs after hypoxic exposure. This correlation was supported by the increased [14C]sucrose permeability observed when BBMECs were exposed to the NO donor diethylenetriaamine NONOate. Western blot analyses of NOS isoforms showed a significant increase in the inducible isoform after hypoxic exposure with a subsequent reduction in expression on reoxygenation. Results from this study suggest that hypoxia-induced blood-brain barrier breakdown can be diminished by inhibition of NO synthesis, decreased concentration of NO metabolites, and/or reoxygenation.     

Lipopolysaccharide enhances the production of nicotine-induced prostaglandin E2 by an increase in cyclooxygenase-2 expression in osteoblasts

Previous studies have indicated that lipopolysaccharide(LPS)from Gram-negative bacteria inplaque induces the release of prostaglandin E_2(PGE_2),which promotes alveolar bone resorption in periodontitis,and that tobacco smoking might be an important risk factor for the development and severity of periodontitis.We determined the effect of nicotine and LPS on alkaline phosphatase(ALPase)activity,PGE_2 production,and the expression of cyclooxygenase(COX-1,COX-2),PGE_2 receptors Ep1-4,and macrophage colonystimulating factor(M-CSF)in human osteoblastic Saos-2 cells.The cells were cultured with 10~(-3)M nicotinein the presence of 0,1,or 10μg/ml LPS,or with LPS alone.ALPase activity decreased in cells cultured withnicotine or LPS alone,and decreased further in those cultured with both nicotine and LPS,whereas PGE_2production significantly increased in the former and increased further in the latter.By itself,nicotine did notaffect expression of COX-1,COX-2,any of the PGE_2 receptors,or M-CSF,but when both nicotine and LPSwere present,expression of COX-2,Ep3,Ep4,and M-CSF increased significantly.Simultaneous addition of10~(-4)M indomethacin eliminated the effects of nicotine and LPS on ALPase activity,PGE_2 production,and M-CSF expression.Phosphorylation of protein kinase A was high in cells cultured with nicotine and LPS.Theseresults suggest that LPS enhances the production of nicotine-induced PGE_2 by an increase in COX-2 expres-sion in osteoblasts,that nicotine-LPS-induced PGE_2 interacts with the osteoblast Ep4 receptor primarily inautocrine or paracrine mode,and that the nicotine-LPS-induced PGE_2 then decreases ALPase activity andincreases M-CSF expression.     

Metabolism of Catecholamine Esters by Cultured Bovine Brain Microvessel Endothelial Cells

The metabolism of epinine (N-methyldopamine) and epinine diesters (acetyl, benzoyl, pivaloyl, and isobutyryl) by brain endothelium was investigated using primary cultures of bovine brain microvessel endothelial cells. 3,4-Dihydroxyphenylacetic acid (DOPAC), the product of monoamine oxidase (MAO)-mediated degradation of epinine, was the only metabolite detected by HPLC with electrochemical detection following incubation of the cell monolayers with epinine or its esters. This metabolism could be inhibited by the MAO inhibitors pargyline, clorgyline, and deprenyl, with the system being most sensitive to inhibition by clorgyline. Compared with epinine, incubation of cell monolayers with the diester prodrugs led to increased drug (epinine plus epinine diesters) tissue levels. With the exception of the diacetyl ester, lower levels of DOPAC were observed with the diester prodrugs than with the parent compound. Hydrolysis by serine-dependent esterases appears to be necessary for the subsequent oxidation by MAO. The permeabilities of epinine and the diester prodrugs through endothelial cell monolayers grown on porous supports were related to their lipophilicity and molecular weight.     

Disruption of COX-2 and eNOS does not confer protection from cardiovascular failure in lipopolysaccharide-treated conscious mice and isolated vascular rings

It was hypothesized that a serial stimulation of vascular cyclooxygenase-2 (COX-2) with subsequent activation of endothelial nitric oxide synthase (eNOS) is responsible for decrease in blood pressure, cardiac performance, and vascular reactivity in endotoxemia caused by LPS. The hypothesis was tested in catheterized, conscious, freely moving, wild-type mice and mice (C57BL/6J background) with targeted deletion of COX-2 and eNOS that were given an intravenous LPS bolus (2 mg/kg, 055:B5). In vitro studies were performed on murine aorta rings. LPS caused a concomitant decrease in mean arterial blood pressure (MAP) and heart rate (HR) that was significant after 3 h and was sustained throughout the experiment (8 h). The LPS-induced changes in MAP and HR were not different from control in COX-2(-/-) and eNOS(-/-) mice. A prostacyclin receptor antagonist (BR5064) blocked the hypotensive effect of a prostacyclin agonist (beraprost), but did not attenuate the LPS-induced decrease in MAP and HR. LPS decreased eNOS and neuronal NOS mRNA abundances in several organs, while inducible NOS mRNA was enhanced. In aortic rings, LPS suppressed α(1)-adrenoceptor-mediated vascular tone. Inhibition of COX-2 activity (NS 398), disruption of COX-2, endothelium removal, or eNOS deletion (eNOS(-/-)) did not improve vascular reactivity after LPS, while the NO synthase blockers 1400W and N(G)-nitro-l-arginine methyl ester prevented loss of tone. COX-2 and eNOS activities are not necessary for LPS-induced decreases in blood pressure, heart rate, and vascular reactivity. Inducible NOS activity appears crucial. COX-2 and eNOS are not obvious therapeutic targets for cardiovascular rescue during gram-negative endotoxemic shock.     

Cytosolic phospholipase A2alpha regulates induction of brain cyclooxygenase-2 in a mouse model of inflammation

The products of arachidonic acid metabolism are key mediators of inflammatory responses in the central nervous system, and yet we do not know the mechanisms of their regulation. The phospholipase A(2) enzymes are sources of cellular arachidonic acid, and the enzymes cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) are essential for the synthesis of inflammatory PGE(2) in the brain. These studies seek to determine the function of cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in inflammatory PGE(2) production in the brain. We wondered whether cPLA(2)alpha functions in inflammation to produce arachidonic acid or to modulate levels of COX-2 or mPGES-1. We investigated these questions in the brains of wild-type mice and mice deficient in cPLA(2)alpha (cPLA(2)alpha(-/-)) after systemic administration of LPS. cPLA(2)alpha(-/-) mice had significantly less brain COX-2 mRNA and protein expression in response to LPS than wild-type mice. The reduction in COX-2 was most apparent in the cells of the cerebral blood vessels and the leptomeninges. The brain PGE(2) concentration of untreated cPLA(2)alpha(-/-) mice was equal to their wild-type littermates. After LPS treatment, however, the brain concentration of PGE(2) was significantly less in cPLA(2)alpha(-/-) than in cPLA(2)alpha(+/+) mice (24.4 +/- 3.8 vs. 49.3 +/- 11.6 ng/g). In contrast to COX-2, mPGES-1 RNA levels increased equally in both mouse genotypes, and mPGES-1 protein was unaltered 6 h after LPS. We conclude that cPLA(2)alpha regulates COX-2 levels and modulates inflammatory PGE(2) levels. These results indicate that cPLA(2)alpha inhibition is a novel anti-inflammatory strategy that modulates, but does not completely prevent, eicosanoid responses.     

Cerebral microvascular changes in permeability and tight junctions induced by hypoxia-reoxygenation

Cerebral microvessel endothelial cells that form the blood-brain barrier (BBB) have tight junctions (TJ) that are critical for maintaining brain homeostasis and low permeability. Both integral (claudin-1 and occludin) and membrane-associated zonula occluden-1 and -2 (ZO-1 and ZO-2) proteins combine to form these TJ complexes that are anchored to the cytoskeletal architecture (actin). Disruptions of the BBB have been attributed to hypoxic conditions that occur with ischemic stroke, pathologies of decreased perfusion, and high-altitude exposure. The effects of hypoxia and posthypoxic reoxygenation in cerebral microvasculature and corresponding cellular mechanisms involved in disrupting the BBB remain unclear. This study examined hypoxia and posthypoxic reoxygenation effects on paracellular permeability and changes in actin and TJ proteins using primary bovine brain microvessel endothelial cells (BBMEC). Hypoxia induced a 2.6-fold increase in [(14)C]sucrose, a marker of paracellular permeability. This effect was significantly reduced (~58%) with posthypoxic reoxygenation. After hypoxia and posthypoxic reoxygenation, actin expression was increased (1.4- and 2.3-fold, respectively). Whereas little change was observed in TJ protein expression immediately after hypoxia, a twofold increase in expression was seen with posthypoxic reoxygenation. Furthermore, immunofluorescence studies showed alterations in occludin, ZO-1, and ZO-2 protein localization during hypoxia and posthypoxic reoxygenation that correlate with the observed changes in BBMEC permeability. The results of this study show hypoxia-induced changes in paracellular permeability may be due to perturbation of TJ complexes and that posthypoxic reoxygenation reverses these effects.     

Effects of pro-inflammatory cytokines,lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE₂, PGF_2α) or their corresponding ethanolamides (PGE₂-EA or PGF_2α-EA) over 48 h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20 h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10⁻⁵ M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10⁻⁴ M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE₂ transiently increased Caco-2 monolayer permeability at 24 h, while LPS (10 μg/ml) increased permeability over 24–48 h.These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE₂ transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.