Michael J. Menconi

Ringer's ethyl pyruvate solution ameliorates ischemia/reperfusion-induced intestinal mucosal injury in rats.

ObjectivePyruvate has been shown to be protective in numerous in vitro and in vivo models of oxidant-mediated cellular or organ system injury. Unfortunately, the usefulness of pyruvate as a therapeutic agent is abrogated by its very poor stability in solution. In an effort to take advantage of the ability of pyruvate to scavenge reactive oxygen species while avoiding the problems associated with the instability of pyruvate in solution, we sought to determine whether a simple derivative, ethyl pyruvate, would be protective in an animal model of reactive oxygen species-mediated tissue injury, namely mesenteric ischemia and reperfusion in rats. DesignProspective, randomized trial. SettingAnimal research center. SubjectsMale Sprague-Dawley rats. InterventionsUnder general anesthesia, rats were subjected to 60 mins of mesenteric ischemia followed by 60 mins of reperfusion. Controls (n = 6) received intravenous lactated Ringer’s solution according this dosing schedule: 1.5 mL/kg bolus before ischemia, 3.0 mL/kg bolus before resuscitation, and 1.5 mL·kg−1·hr−1 by continuous infusion. Two experimental groups received similar volumes of either pyruvate (n = 6 each) or ethyl pyruvate (n = 9) solution made up exactly like lactated Ringer’s solution except for the substitution of eitherpyruvate or ethyl pyruvate for lactate, respectively. Measurements and Main Results To obtain tissues for assessing mucosal permeability and histology, five 10-cm long segments of small intestine were obtained at the following time points: baseline, after 30 and 60 mins of ischemia, and after 30 and 60 mins of reperfusion. Mucosal permeability to fluorescein isothiocyanate dextran (molecular weight 4000 Da) was assessed ex vivo by using an everted gut sac method. Compared with controls, treatment of rats with either pyruvate solution or ethyl pyruvate solution significantly ameliorated the development of intestinal mucosal hyperpermeability during the reperfusion. Treatment with ethyl pyruvate solution also significantly decreased the extent of histologic mucosal damage after mesenteric reperfusion. ConclusionsTreatment with Ringer’s ethyl pyruvate solution ameliorated structural and functional damage to the intestinal mucosa in a rat model of mesenteric ischemia/reperfusion. Ethyl pyruvate solution warrants further evaluation as a novel therapeutic agent for preventing oxidant-mediated injury in various disease states.

Cytokine-induced intestinal epithelial hyperpermeability: role of nitric oxide.

OBJECTIVE Incubation of enterocytic monolayers with interferon (IFN)-gamma increases nitric oxide (NO) production and permeability, but NO synthesis inhibitors ameliorate the development of IFN-gamma-induced hyperpermeability. Induction of inducible nitric oxide synthase (iNOS), an isoform of the enzyme responsible for NO biosynthesis, is often enhanced by the synergistic effects of multiple cytokines. Moreover, many of the cytopathic effects of NO are mediated by peroxynitrite, which is produced by the reaction of NO with superoxide radical anion. In the present study, we sought to determine whether combinations of several proinflammatory cytokines, including IFN-gamma, interleukin-1beta, and tumor necrosis factor-alpha, have synergistic effects on the induction of iNOS expression and/or hyperpermeability to hydrophilic solutes in cultured enterocytic monolayers. We also assessed the effects of aminoguanidine (a relatively selective iNOS inhibitor), L-N(G)-monomethyl arginine (an isoform-nonselective NO synthase inhibitor), and Tiron (a superoxide radical anion scavenger) on the development of cytokine-induced hyperpermeability. DESIGN Caco-2 monolayers were incubated under control conditions or with IFN-gamma, interleukin-1beta, or tumor necrosis factor-alpha alone, pairwise combinations of these cytokines, or all three cytokines together (cytomix; CM). iNOS messenger RNA (mRNA) expression was assessed using Northern blot analysis. The permeability of Caco-2 monolayers growing on permeable supports in bicameral chambers was assessed by measuring the apical-to-basolateral flux of fluorescein disulfonic acid. The concentration of nitrate plus nitrite in culture supernatants, an indirect measure of NO production, was determined using the Griess reaction. RESULTS After 24 hrs of incubation, up-regulation of iNOS mRNA expression was evident only in cells exposed to IFN-gamma-containing formulations. Expression of iNOS mRNA was far greater in cells incubated with CM than in cells treated with IFN-gamma alone or either of the two-component IFN-gamma-containing cytokine combinations. Compared with IFN-gamma, CM resulted in a higher rate of NO production over 48 hrs of incubation. The permeability of Caco-2 monolayers increased by approximately six-fold and approximately 20-fold after incubation for 48 hrs with IFN-gamma alone and CM, respectively. The increase in permeability induced by incubation with CM was significantly ameliorated by the addition of aminoguanidine, L-N(G)-monomethyl arginine, or Tiron. CONCLUSIONS IFN-gamma-containing combinations of cytokines are potent inducers of iNOS in cultured enterocytic monolayers. Peroxynitrite may be an important mediator of cytokine-induced gut epithelial hyperpermeability.

Effect of mesenteric ischemia and reperfusion or hemorrhagic shock on intestinal mucosal permeability and ATP content in rats.

In a reductionist in vitro system, intestinal epithelial permeability appears to be dependent on cellular ATP content. In order to extend these prior observations, we used rat models of mesenteric ischemia/reperfusion (I/R) and pressure-controlled hemorrhagic shock to test the hypothesis that intestinal barrier function is directly proportional to tissue ATP content. I/R was induced by clamping the mesenteric vessels for 60 min followed by 60 min of reperfusion. Normal, ischemic, and reperfused ileal segments were prepared from each rat (n = 12). Hemorrhagic shock was induced by bleeding rats (n = 9) to a mean arterial pressure of 30 mmHg and maintaining this pressure for 4 h by infusing Ringers lactate solution as necessary. Ileal segments were harvested before induction of hemorrhage and at 1 h intervals thereafter. Everted gut sacs were prepared to measure the mucosal-to-serosal passage of fluorescein-conjugated dextran (FD4; M.W. = 4 kDa). Tissue ATP levels were determined using a luciferase assay. FD4 clearance rates were plotted as a function of tissue ATP content. Linear regression analyses showed a strong inverse relationship between intestinal permeability and tissue ATP level in rats subjected to I/R (r2 = 0.78; P < 0.001) or hemorrhage (r2 = 0.82; P < 0.001). These data support the idea that ATP content is a determinant of intestinal epithelial barrier function in vivo.

Nitric oxide mediates interferon-gamma-induced hyperpermeability in cultured human intestinal epithelial monolayers.

OBJECTIVE Incubation with interferon-gamma has been shown to increase the permeability of cultured monolayers of intestinal epithelial cells. We sought to determine whether this phenomenon is mediated, at least in part, by increased production of nitric oxide. DESIGN Prospective, controlled, laboratory study. Human intestinal epithelial (Caco-2BBe) cells were grown as monolayers on permeable supports mounted in bicameral chambers. Permeability was assessed by adding fluorescein sulfonic acid (molecular weight = 478 daltons) to the apical compartment and determining the apical-to-basolateral clearance of the probe over a 24-hr period of incubation. SETTING Basic science laboratory. MEASUREMENTS AND MAIN RESULTS The permeability of monolayers to fluorescein sulfonic acid was significantly increased after incubation in the presence of interferon-gamma (250 to 1000 U/mL). The effect of interferon-gamma on permeability was dependent on both the concentration of the cytokine and the duration of exposure to it. Concentrations of nitric oxide oxidation products, nitrite and nitrate, in incubation media were increased after exposure of cells to interferon-gamma. When intestinal epithelial (Caco-2BBe) monolayers were incubated with interferon-gamma in the presence of inhibitors of nitric oxide synthase (NG-monomethyl-L-arginine NG-nitro-L-arginine-methyl ester, or NG-nitro-L-arginine), both of the effects of the cytokine (i.e., increased epithelial permeability and increased production of nitrite/nitrate) were attenuated. CONCLUSIONS These results suggest that upregulation of nitric oxide biosynthesis plays a pivotal role in the increase in permeability of intestinal epithelial (Caco-2BBe) monolayers induced by interferon-gamma. Increased production of nitric oxide induced by proinflammatory cytokines, such as interferon-gamma, may be an important factor contributing to gut mucosal hyperpermeability in sepsis.

Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes

Dexamethasone‐treated L6 (a rat cell line) and C2C12 (a mouse cell line) myotubes are frequently used as in vitro models of muscle wasting. We compared the effects of different concentrations of dexamethasone and corticosterone (the naturally occurring glucocorticoid in rodents) on protein breakdown rates, myotube size, and atrogin‐1 and MuRF1 mRNA levels in the two cell lines. In addition, the expression of the glucocorticoid receptor (GR) and its role in glucocorticoid‐induced metabolic changes were determined. Treatment with dexamethasone or corticosterone resulted in dose‐dependent increases in protein degradation rates in both L6 and C2C12 myotubes accompanied by 25–30% reduction of myotube diameter. The same treatments increased atrogin‐1 mRNA levels in L6 and C2C12 myotubes but, surprisingly, upregulated the expression of MuRF1 in L6 myotubes only. Both cell types expressed the GR and treatment with dexamethasone or corticosterone downregulated total cellular GR levels while increasing nuclear translocation of the GR in both L6 and C2C12 myotubes. The GR antagonist RU38486 inhibited the dexamethasone‐ and corticosterone‐induced increases in atrogin‐1 and MuRF1 expression in L6 myotubes but not in C2C12 myotubes. Interestingly, RU38486 exerted agonist effects in the C2C12, but not in the L6 myotubes. The present results suggest that muscle wasting‐related responses to dexamethasone and corticosterone are similar, but not identical, in L6 and C2C12 myotubes. Most notably, the regulation by glucocorticoids of MuRF1 and the role of the GR may be different in the two cell lines. These differences need to be taken into account when cultured myotubes are used in future studies to further explore mechanisms of muscle wasting. J. Cell. Biochem. 105: 353–364, 2008.

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 (O2*-). In the present study, we investigated the role of PN, O2*- 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-2BBe 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 O2*- 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 O2*-). Diethyldithiocarbamate, a superoxide dismutase inhibitor, and pyrogallol, an O2* 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.

Role of glucocorticoids in the molecular regulation of muscle wasting.

Objective:To review glucocorticoid-regulated molecular mechanisms of muscle wasting. Design:Review of recent literature describing the role of glucocorticoids in the regulation of proteolytic mechanisms, transcription factors, and nuclear cofactors in skeletal muscle during various catabolic conditions. Main Results:Catabolic doses of glucocorticoids induce muscle atrophy both in vivo and in vitro by stimulating protein breakdown and inhibiting protein synthesis. Signaling pathways that regulate muscle protein synthesis at the translational level are inhibited by glucocorticoids. Glucocorticoids increase the expression and activity of the ubiquitin-proteasome pathway, a major proteolytic mechanism of muscle atrophy. The expression and activity of muscle wasting-related transcription factors, including C/EBP&bgr; and &dgr; and Forkhead box O 1, 3, and 4, as well as the nuclear cofactor p300, are up-regulated by glucocorticoid excess. Conclusions:Muscle wasting in various catabolic conditions is, at least in part, regulated by glucocorticoids. The role of glucocorticoids in muscle wasting is complex and reflects regulation at the molecular level of multiple mechanisms influencing both synthesis and degradation of muscle proteins.

Role Of Oxidant Stress In The Adult Respiratory Distress Syndrome: Evaluation Of A Novel Antioxidant Strategy In A Porcine Model Of Endotoxin-induced Acute Lung Injury

Reactive oxygen metabolites (ROMs) are thought to play a key role in the pathogenesis of the adult respiratory distress syndrome (ARDS). Accordingly, the use of ROM scavengers, such as N-acetyl-cysteine or dimethylthiourea, as therapeutic adjuncts to prevent oxidant-mediated damage to the lung have been evaluated extensively in animal models of ARDS. Results with this approach have been quite variable among studies. Another strategy that has been examined in animal models of ARDS is the administration of various enzymes, particularly superoxide dismutase (SOD) or catalase (CAT), in an effort to promote the conversion of ROMs to inactive metabolites. In theory, this strategy should be more effective than the use of ROM scavengers since a single molecule of a catalytically active molecule can neutralize a large number of molecules of a reactive species, whereas most scavengers act in a stoichiometric fashion to neutralize radicals on a mole-for-mole basis. This notion is supported by studies showing that prophylactic treatment with CAT provides impressive protection against acute lung injury induced in experimental animals by the administration of lipopolysaccharide (LPS). Results with SOD have been more variable. Recently, we have utilized a porcine model of LPS-induced ARDS to investigate the therapeutic potential of EUK-8, a novel, synthetic, low molecular salen-manganese complex that exhibits both SOD-like and CAT-like activities in vitro. Using both pre- and post-treatment designs, we have documented that treatment with EUK-8 significantly attenuates many of the features of LPS-induced acute lung injury, including arterial hypoxemia, pulmonary hypertension, decreased dynamic pulmonary compliance, and pulmonary edema. These findings support the view that salen-manganese complexes warrant further evaluation as therapeutic agents for treatment or prevention of sepsis-related ARDS in humans.

Expression and activity of C/EBPβ and δ are upregulated by dexamethasone in skeletal muscle

The influence of glucocorticoids on the expression and activity of the transcription factors CCAAT/enhancer binding protein (C/EBP)β and δ in skeletal muscle was examined by treating rats or cultured L6 myotubes with dexamethasone. Treatment of rats with 10 mg/kg of dexamethasone resulted in increased C/EBPβ and δ DNA binding activity in the extensor digitorum longus muscle as determined by electrophoretic mobility shift assay (EMSA) and supershift analysis. A similar response was noticed in dexamethasone‐treated myotubes. In other experiments, myocytes were transfected with a plasmid containing a promoter construct consisting of multiple C/EBP binding elements upstream of a luciferase reporter gene. Treatment of these cells with dexamethasone resulted in a fourfold increase in luciferase activity, suggesting that glucocorticoids increase C/EBP‐dependent gene activation in muscle cells. In addition, dexamethasone upregulated the protein and gene expression of C/EBPβ and δ in the myotubes in a time‐ and dose‐dependent fashion as determined by Western blotting and real‐time PCR, respectively. The results suggest that glucocorticoids increase C/EBPβ and δ activity and expression through a direct effect in skeletal muscle.

P‐Glycoprotein‐170 inhibition significantly reduces cortisol and ciclosporin efflux from human intestinal epithelial cells and T lymphocytes

To assess the role of P‐glycoprotein‐170 (P‐gp) in transporting cortisol and ciclosporin from human intestinal epithelium and T lymphocytes.