Effect of selective PKC isoform activation and inhibition on TNF-alpha-induced injury and apoptosis in human intestinal epithelial cells.

(1) We have investigated the effects of specific PKC isoforms in TNF-alpha mediated cellular damage using a human intestinal cell line (SCBN). (2) TNF-alpha treatment induced a decrease in the extent of intestinal cellular viability as determined by a formazan-based assay and an increase in the apoptotic index as assessed by immunohistology. These changes in cellular integrity were found to be related to the degradation of I-kappaBalpha, mobilization of NF-kappaB and release of mitochondrial cytochrome c. (3) TNF-alpha treatment also induced the activation of selective PKC isoforms which were associated with the decrease in cellular viability and an increase of cellular apoptosis. (4) Nonselective PKC antagonists, such as GF109203X and Gö6976 as well as isoform-selective PKC-inhibiting peptides would reverse the cellular injury as well as reduce the degradation of I-kappaBalpha and mitochondrial cytochrome c release. These effects were most highly correlated with changes in PKCdelta and epsilon primarily. (5) Intestinal cellular injury could be induced by treating cells with agonists selective for PKCdelta and epsilon mainly. (6) In conclusion, this study has shown that TNF-alpha treatment can induce the activation of PKCdelta and epsilon in the human intestinal cell line, SCBN, and this response is closely associated with an increase in cellular damage and apoptosis. PKCdelta and epsilon primarily mediate the release of mitochondrial cytochrome c and degradation of I-kappaBalpha and hence mobilization of NF-kappaB, which are responsible for the pathway leading to cell injury.

Gastroprotective effects of amtolmetin guacyl: a new non-steroidal anti-inflammatory drug that activates inducible gastric nitric oxide synthase.

BACKGROUND: The novel non-steroidal anti-inflammatory drug amtolmetin guacyl has been shown to possess markedly reduced ulcerogenic effects and nitric oxide-mediated gastroprotective activity against the damage induced by ethanol in the rat. AIMS: To investigate, in the rat, the role of nitric oxide and of inducible nitric oxide synthase isoform in the protective effect of amtolmetin guacyl against the gastric damage induced by ethanol. METHODS: The effects of amtolmetin guacyl on gastric transmucosal potential difference and on gastric mucosal blood flow were investigated in the anaesthetised rat; myeloperoxidase activity, inducible and endothelial nitric oxide synthase protein content were determined in rat gastric mucosal homogenates. The anti-inflammatory drug tolmetin and the bacterial lipopolysaccharide from Escherichia coli were studied for comparison. RESULTS: In the anaesthetised rat, amtolmetin guacyl, but not tolmetin, reduced by approximately 50% the fall in gastric potential difference and, to a lesser extent, the macroscopic damage induced by ethanol. The effect of amtolmetin guacyl on transmucosal potential difference was prevented by the selective inducible nitric oxide synthase inhibitor 1400W. In amtolmetin guacyl-treated rats, 1400W decreased gastric mucosal blood flow, whereas it was inactive in vehicle- and tolmetin-treated animals. In gastric mucosal homogenates, both amtolmetin guacyl and lipopolysaccharide, but not tolmetin, increased inducible, but not endothelial, nitric oxide synthase protein content, as revealed by Western immunoblotting. CONCLUSIONS: These data confirm that amtolmetin guacyl is a non-steroidal anti-inflammatory agent devoid of gastrolesive properties, that can actually reduce the damaging effects of ethanol through the increase in nitric oxide production, via the inducible isoform of nitric oxide synthase.

The role of protein kinase C isozymes in TNF-alpha-induced cytotoxicity to a rat intestinal epithelial cell line.

Tumor necrosis factor (TNF)-alpha can induce cytotoxicity and apoptosis in a number of cell types and has been implicated in the regulation of many inflammatory processes. It has been suggested that protein kinase C (PKC) is one of the intracellular mediators of the actions of TNF-alpha. In the present study, the role of PKC isoforms in TNF-alpha-mediated cytotoxicity and apoptosis in intestinal cells was investigated using the rat epithelial cell line, IEC-18. Cells were incubated with TNF-alpha in the presence or absence of the transcription inhibitor actinomycin D (AMD). The extent of cell damage was enhanced when AMD was added to incubation medium, suggesting that new protein synthesis plays a role in the cytotoxic action of TNF. TNF-alpha also induced the translocation of PKC-alpha, -delta, and -epsilon from cytosol to the membrane fraction of the intestinal cells. Furthermore, the cytotoxic and apoptotic effects of TNF were reduced by pretreating the cells with the PKC-epsilon translocation inhibitor, PKC-epsilonV1-2. In contrast, although cells incubated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) also displayed an increase in cell injury, the extent of cytotoxicity and apoptosis was not enhanced by AMD. Furthermore, PMA-induced cell damage was reduced by rottlerin, a PKC-delta inhibitor. Caspase-3, an enzyme implicated in the mediation of apoptosis, was activated in cells in response to either TNF-alpha or PMA stimulation, and its effects on this activity were reduced by selective inhibition of PKC-epsilon and -delta, respectively. Furthermore, inhibition of caspase-3 activity reduced apoptosis. These data suggest that activation of selective PKC isoforms mediate the effects of TNF-alpha on intestinal epithelial cell injury.

Colonic production and expression of IL-4, IL-6, and IL-10 in neonatal suckling rats after LPS challenge.

It has been demonstrated that the neonatal suckling rat is more susceptible to endotoxin [lipopolysaccharide (LPS)]-induced colonic damage compared with weaned littermates. There is evidence to suggest that differences in the production of certain cytokines, including interleukin (IL)-4, IL-6, and IL-10, are associated with intestinal inflammation in children. We have examined the production, localization, and mRNA detection of these cytokines in suckling and weaned rat colons after bacterial LPS challenge. Suckling (10 day old) and weaned (25 day old) rats were injected with LPS (3 mg/kg ip). Colon samples were taken up to 4 h after treatment, and cytokines were measured by ELISA. LPS-induced cytokine levels were significantly different in suckling rats compared with weaned rats. Cytokine localization to the colonic mucosa was evident in suckling rats up to 4 h after LPS administration but was not consistently seen in weaned rats. The mRNA for cytokines examined were detected by RT-PCR in suckling but not in weaned rat colons after LPS treatment. Induction of neutropenia via anti-neutrophil serum (ANS) administration did not affect cytokine mRNA detection in neonates after LPS treatment. Weaned animals displayed positive detection of all cytokines examined after ANS. Therefore, we have shown that the suckling rat displays a different production and expression of colonic IL-4, IL-6, and IL-10 compared with weaned littermates after LPS challenge. Furthermore, neutrophils may be implicated in colonic cytokine expression after LPS challenge in rats.

Protein kinase C mediates lipopolysaccharide- and phorbol-induced nitric-oxide synthase activity and cellular injury in the rat colon.

The role of protein kinase C (PKC) in lipopolysaccharide (LPS)- and phorbol ester-induced changes in rat colonic cellular integrity and Ca(2+)-independent inducible nitric-oxide synthase (iNOS) activity was investigated. LPS treatment (3 mg kg(-1) i.p.) increased colonic cellular PKC activity within 1 h after administration. The percentage of nonviable cells and iNOS activity in response to LPS were reduced by pretreatment with the selective PKC antagonist GF 109203X (25 ng kg(-1) i.v.). Pretreatment with the selective iNOS inhibitor 1400W (5 mg kg(-1) s.c.) reduced the extent of cellular injury and iNOS activity but did not affect the increase in LPS-mediated PKC activation. Reduction of circulating neutrophils with anti-neutrophil serum reduced cell damage as well as the increases in PKC and iNOS activities in response to LPS. Intracolonic administration of the phorbol ester phorbol-12-myristate-13-acetate (PMA; 3 mg kg(-1)) increased colonic cellular PKC activity within 2 h after instillation. Cellular iNOS activity did not increase until 6 h after PMA administration. The colonic responses to PMA were eliminated by GF 109203X. The selective iNOS inhibitor 1400W reduced the increase in cell injury but did not affect the PKC activation in response to PMA. LPS treatment also increased in the proteins for PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta. PMA treatment resulted in PKC-delta and PKC-epsilon translocation from cytosol to membrane. These data suggest that PKC mediates iNOS activation and subsequent colonic cell injury in response to LPS administration. The delta- and epsilon-isozymes appear to be most closely associated with these responses.

Alterations in protein kinase C isoforms in experimentally-induced colitis in the rat.

BACKGROUND: Although protein kinase C (PKC) isoenzymes have been implicated as mediators for multiple physiological processes, PKC also mediates cellular and intestinal mucosal injury. We have investigated the expression of the isoenzymes, PKCalpha, PKCdelta, PKCepsilon and PKCzeta in colonic mucosal tissue from TNBS-treated and HLA-B27 spontaneous colitis animals. METHODS: Colonic mucosal samples were taken at various times (2 h-14 d) after instillation of TNBS (75 mg/kg in 50% ethanol) or from HLA-B27 rats at 16-18 weeks of age. Tissues were homogenized and separated into membrane and cytosolic fractions by centrifugation. PKC activity was measured radioenzymatically. PKC protein for isoforms alpha, delta, epsilon and delta was assessed by Western blot while corresponding mRNA was analyzed by RT-PCR. RESULTS: PKC activity increased in cytosolic and membrane fractions by 1d after TNBS and returned to normal by d3. PKCalpha protein was translocated from cytosol to membrane by 2 h after TNBS followed by down-regulation until d3. Increases in PKCdelta, PKCepsilon and PKCzeta protein occurred initially in membrane fractions as early as 2 h after TNBS. Increases in cytosolic protein occurred at later times after induction of colitis. Protein levels for all isoenzymes remained increased up to 7d after TNBS. RT-PCR revealed that mRNA for PKCalpha decreased while PKC mRNA increased correspondingly with their respective protein levels. In HLA-B27 rats, protein levels for all isoforms were less than was detected in normal colonic tissue. CONCLUSIONS: The early increase in gene expression and protein levels for PKCalpha and zeta suggest that these isozymes may play roles in an acute model of colitis induced by TNBS. In contrast, the increase in PKCdelta and epsilon protein was not associated with mRNA changes suggesting that these isozymes are not similarly regulated in the inflamed colonic mucosa. In a chronic model of experimental colonic inflammation (HLA-B27), all of these isoforms appeared to be down-regulated.

The effect of protein kinase C activation on colonic epithelial cellular integrity.

We have investigated whether activation of protein kinase C has a direct cytotoxic effect on colonic mucosal epithelial cells and whether oxidant-induced damage to colonocytes is mediated by activation of cellular protein kinase C. Incubation of freshly harvested cells from rat colon with the protein kinase C activator, phorbol 12-myristate, resulted in a concentration-dependent increase in the extent of cell injury. Phorbol 12-myristate acetate (0.1-10 microM) also increased cellular protein kinase C activity and this was reduced significantly by treating cells with the antagonists staurosporine or 2-[1-(3-dimethylaminopropyl)-indol-3-yl]3-(-indol-3-yl)maleimide (GF 109203X; 10 microM). Phorbol 12-myristate acetate treatment also resulted in increased translocation of proteins for protein kinase C isoforms alpha, delta and epsilon from cytosol to membrane particulate fractions. The antagonists reduced the extent of cell damage in response to phorbol 12-myristate acetate. Furthermore, cell injury in response to the phorbol acetate was also inhibited by the addition of the oxidant scavengers, superoxide dismutase or catalase to the cell suspension. Addition of H(2)O(2) to the incubation medium (0.1-100 microM) resulted in an increase in cellular protein kinase C activity, an increase in the expression of the alpha, beta and zeta isoforms and a reduction in cell integrity. The cellular damaging actions of H(2)O(2) were significantly reduced by the protein kinase C antagonists, staurosporine or 2-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(-indol-3-yl)maleimide (GF 109203X). These findings suggest that protein kinase C activation results in colonic cellular injury and this damage is mediated, at least in part, by release of reactive oxidants. Furthermore, oxidant-mediated damage to these cells also involves protein kinase C activation.

The involvement of protein kinase C in nitric oxide-induced damage to rat isolated colonic mucosal cells.

1 The role of protein kinase C (PKC) in colonic cellular injury in response to high concentrations of nitric oxide (NO) released from the donor, S-nitroso-N-acetyl-DL-penicillamine (SNAP) was investigated. 2 Addition of SNAP (0.1-1000 microM) to the cellular suspension resulted in a dose-dependent increase in the extent of damage to isolated colonic mucosal cells as assessed by Trypan blue dye uptake and release of the lysosmal enzyme, N-acetyl-beta-glucosaminidase. SNAP treatment also resulted in an increase in cellular total PKC activity. These increases were reduced or eliminated by pretreatment of the cells with the PKC antagonists staurosporine or GF 109203X or the NO scavenger, phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). 3 PKC-alpha, PKC-delta, PKC-epsilon and PKC-zeta were detected in colonic cellular lysates by immunoblotting. However, only PKC-epsilon protein was increased in response to SNAP treatment. Furthermore, SNAP treatment resulted in activation of PKC-epsilon by causing translocation of the enzyme from the cytosolic to membrane fraction of the cell. This effect was eliminated if cells were preincubated with the NO scavenger, PTIO. 4 The extent of cellular damage in response to addition of SNAP to the incubation medium was enhanced by coincubation with the PKC activator, phorbol 12-myristate 13-acetate (PMA; 1 and 10 microM). 5 PKC activity and the extent of cell damage in response to SNAP were reduced by preincubation of the cells with the peroxyl scavenger, ebselen (0.01-10 microM). 6 These data suggest that the PKC-epsilon isoform of the enzyme mediates NO-induced damage to colonic mucosal cells. This response may occur, at least in part, due to peroxynitrite formation.

Protein kinase C mediates experimental colitis in the rat.

Protein kinase C (PKC) plays an important role in the cell signal transduction of many physiological processes. In contrast to these physiological responses, increases in PKC activity have also been associated with inflammatory disease states, including ulcerative colitis. The objective of this study was to examine the role of PKC as a causative mediator in initiation of experimentally induced colitis in the rat. Colitis was induced in rats by intrarectal (0.6 ml) instillation of 2,4,6-trinitrobenzenesulfonic acid (TNBS; 75 mg/kg in 50% ethanol) or the PKC activator phorbol 12-myristate 13-acetate (PMA; 1.5-3.0 mg/kg in 20% ethanol). Gross and histological mucosal damage, mucosal neutrophil infiltration, mucosal PKC activity, and PKC protein content for PKC isoforms alpha, beta, delta, and epsilon were assessed 2 h to 14 days after an inflammatory challenge. Both PKC activity and mucosal injury increased significantly within 4 h of TNBS treatment. PKC activity was maximal at 7 days and declined at 14 days, whereas mucosal damage became maximal at 1 day and declined after 7 days. In contrast, neutrophil infiltration as assessed by myeloperoxidase activity only increased 12 h after TNBS treatment, became maximal 1 day after TNBS administration, and declined thereafter. PKCbeta, -delta, and -epsilon were increased in response to TNBS, whereas PKCalpha protein content was decreased. The PKC antagonists staurosporine and GF-109203X (25 ng/kg iv) reduced TNBS-induced changes in mucosal PKC activity and the degree of mucosal damage. In contrast, neutropenia induced by antineutrophil serum treatment did not significantly affect the degree of injury or mucosal PKC activity. Furthermore, activation of mucosal PKC activity with PMA also induced mucosal damage, which was also inhibited by pretreatment with a PKC antagonist. In conclusion, these results suggest that increases in PKC activity play a causative role in TNBS-induced colitis. The PKC-mediated response to TNBS does not appear to involve neutrophil infiltration.

The role of phospholipase A2 in calcium-ionophore-mediated injury to rat gastric mucosal cells.

Although transient increases in intracellular Ca2+ ([Ca2+]i) underlie a number of important physiological processes, sustained elevations in [Ca2+]i mediate damage to a number of tissues and cell types including gastric mucosal cells. Increases in [Ca2+]i can activate phospholipid hydrolysis via increases in phospholipase A2 (PLA2) activity and subsequent cell injury. In the present study we have examined whether [Ca2+]i-induced gastric cellular injury is mediated by PLA2 activation. Gastric mucosal cells were harvested from rat stomachs after pronase digestion. Cell integrity was assessed using trypan blue dye exclusion and release of lysozomal enzymes. PLA2 activity was estimated colorimetrically by determination of thiol release from the substrate, arachidonyl thio-PC. In these studies calcium ionophore A23187 (3-25 microM) resulted in an increase in cell injury. The damage produced by A23187 (12.5 microM) was inhibited by preincubation of cells with the PLA2 inhibitor, quinacrine (1-100 microM). Quinacrine did not reduce ethanol (10% w/v) mediated-cell damage. Similarly Ca2+ ionophore A23187 treatment resulted in a concentration-dependent increase in PLA2 activity in gastric cells. The increase in PLA2 activity was attenuated if cells were incubated in Ca2+-depleted medium containing EGTA (4 mM). Furthermore lysophospholipids generated by PLA2 (lysophosphatidylethanolamine and lysophosphatidylcholine; 100 microM) also increased the degree of cell injury. Pretreatment of cells with the PAF antagonist WEB 2086 (10(-6) and 10(-5) M), the leukotriene synthase inhibitor 5,6-dehydroarachidonic acid (10 microM), or the thromboxane synthase inhibitor furegrelate (1 microM) decrease A23187-mediated cell injury. These data suggest that Ca2+ ionophore-mediated increases in [Ca2+]i result in gastric cell injury and this effect is mediated in part by PLA2 activation and subsequent release of free fatty acids and lysophosphatides.

Role of mast cells, neutrophils and nitric oxide in endotoxin-induced damage to the neonatal rat colon.

1. The mechanisms involved in mediating bacterial endotoxin lipopolysaccharide (LPS)-induced injury in the colon of neonatal rat pups aged 10-12 days was examined. 2. Administration of LPS (3 mg kg(-1), i.p.) caused a time-related increase in the plasma concentration of rat mast cell protease-II (RMCP-II) which was attenuated dose-dependently, by the non-selective mast cell stabilizer doxantrazole (0.05-5 mg kg(-1), i.p.). The selective connective tissue mast cell stabilizer ketotifen (5-25 mg kg(-1), i.p.) was without effect at the lower dose and had only a limited inhibitory effect at the higher dose. 3. In addition, doxantrazole (5 mg kg(-1), i.p.) inhibited mast cell degranulation in response to LPS in sections of neonatal rat colon, but ketotifen (5 mg kg(-1), i.p.) was without effect. 4. The increase in plasma RMCP-II concentration in response to LPS treatment preceded increases in tissue myeloperoxidase (MPO) activity, inducible nitric oxide synthase (iNOS) activity and tissue lipid peroxidation. These events were all attenuated by pretreatment with doxantrazole (5 mg kg(-1), i.p.), antineutrophil serum (100 microl kg(-1), i.p.), dexamethasone (2 mg kg(-1), i.p.) and the selective iNOS inhibitor, aminoguanidine (25 mg kg(-1), i.p.). 5. In addition, lipid peroxidation was inhibited by pre-administration of the antioxidant enzymes superoxide dismutase (2000 u kg(-1), i.p.) and catalase (2000 u kg(-1), i.p.), the xanthine oxidase inhibitor allopurinol (100 mg kg(-1), i.p.) and the peroxyl scavenger deferoxamine (10 mg kg(-1), i.p.), suggesting the involvement of reactive oxygen metabolites in the colonic injury. 6. These findings suggest that the sequence of events resulting in colonic damage in the neonatal rat following administration of LPS include mast cell degranulation, neutrophil infiltration, elevation in iNOS activity and subsequent lipid peroxidation.

The role of cyclic guanylate monophosphate in nitric oxide-induced injury to rat small intestinal epithelial cells.

In our study we have examined the importance of cyclic guanylate monophosphate (cGMP) in NO-mediated intestinal cellular damage. Epithelial cells were harvested from a 20-cm segment of rat proximal small intestine by dispersion using citrate and ethylenediaminetetraacetic acid. Cell viability was assessed by trypan blue dye exclusion. Incubation of cells with the nitric oxide donors, S-nitroso-N-acetyl penicillamine (SNAP) or sodium nitroprusside (SNP) (10-1000 microM) produced a concentration-dependent increase in cell injury and an increase in cellular cGMP formation as determined by immunoassay. In addition, cell injury was also increased by treatment of cells with the cell permeable analogue, dibutryryl cGMP (db cGMP; 0.1-2.0 mM). Suppression of cellular cGMP production by incubating cells with the guanylate cyclase inhibitor LY83583 (5-20 microM) attenuated the damaging actions of SNAP or SNP. However, LY83583 treatment did not reduce ethanol-mediated (10% v/v) cell injury. Furthermore the cytotoxic actions of SNAP or SNP were enhanced by preincubation of cells with the selective cGMP phosphodiesterase inhibitor, zaprinast (10 mM). The damaging actions of SNAP, SNP and db cGMP were reduced by treating cells with superoxide dismutase (100 U/ml). Similarly SNAP, SNP and db cGMP treatments resulted in an increase in the in vitro production of reactive oxygen metabolites as assessed by the fluorescent probe 2'7' dichlorofluoresein diacetate. These findings indicate that cGMP mediates intestinal cell injury in response to high levels of nitric oxide as produced by the nitric oxide donors, SNAP and SNP. Furthermore these data suggest that the cGMP-induced damage to intestinal epithelial cells involves the generation of reactive oxidants.

Ontogeny of nitric oxide synthase activity and endotoxin-mediated damage in the neonatal rat colon.

Nitric oxide (NO) is synthesized by most regions of the gastrointestinal tract and is an important regulator of mucosal function and integrity. In this study we examined the ontogenic appearance of constitutively expressed Ca2+-dependent NO synthase (cNOS) and inducible Ca2+-independent NO synthase (iNOS) activity, in the colon of neonatal rats. Furthermore, the susceptibility of the colon to damage after induction of iNOS activity following bacterial endotoxin treatment was also examined. Segments of distal colon were removed from either control rat pups (aged between 10 and 25 d) or from animals pretreated with the following agents: 1) Escherichia coli lipopolysaccharide [LPS; 3 mg/kg, intraperitoneally (i.p.), 4 h before sacrifice], 2) dexamethasone (2 mg/kg, i.p., 1 h before administration of LPS) or aminoguanidine (25 mg/kg, i.p., at the same time as LPS). NOS activity was measured via the conversion of L-[14C]arginine to L-[14C]citrulline. Samples of colon were assessed for damage by either light microscopy or by measurement of the malondialdehyde content to estimate lipid peroxidation. In untreated animals cNOS activity increased during the first 20 postnatal days and fell postweaning at 25 d. LPS treatment resulted in a significant increase in iNOS activity in all age groups examined, with maximal activity occurring between 10 and 15 d of age. This coincided with the greatest histologic damage score and lipid peroxidation. Dexamethasone or aminoguanidine attenuated the effects of LPS suggesting the involvement of iNOS in these responses. These data suggest that colonic cNOS activity in the neonatal rat may be important during development and maturation of that tissue. Furthermore, the colon of the preweaned rat is more susceptible to the detrimental effects of LPS-induced NO production than the colon of postweaned animals.

Role of nitric oxide in hypoxia-induced colonic dysfunction in the neonatal rat.

In addition to being an important mediator in the regulation of intestinal integrity, nitric oxide (NO), when produced in large quantities by the inducible isoform of NO synthase, can also be cytotoxic. The aim of this study was to examine the role of NO in hypoxia-induced colonic injury in neonatal rats. Rats (10-12 days old) were exposed to a hypoxic environment of 14% O2-86% N2 for 30 min. NO synthase activity in colonic tissue was measured via the conversion of L-[14C]arginine to L-[14C]citrulline. Epithelial permeability was assessed by measuring the plasma-to-lumen flux of [3H]mannitol or the luminal protein content of colonic lavage. The role of neutrophils was assessed by pretreatment with antineutrophil serum (200 microl/kg ip) and measurement of tissue myeloperoxidase activity. Hypoxia resulted in an elevation in the activity of the inducible Ca2+-independent isoform of NO synthase in colonic tissue, which was maximal between 4 and 6 h posthypoxia and was associated with an increase in myeloperoxidase activity, [3H]mannitol flux, luminal protein content, and histological damage. These effects were attenuated by pretreatment with dexamethasone or the NO synthase inhibitors aminoguanidine and N(G)-nitro-L-arginine methyl ester, whereas the inactive stereoisomer N(G)-nitro-D-arginine methyl ester was without effect. Pretreatment with antineutrophil serum significantly reduced circulating neutrophils, myeloperoxidase activity, and Ca2+-independent NO synthase activity. These findings demonstrate that hypoxia-induced colonic injury in neonatal rats is associated with elevated NO synthase activity, which is related to an increase in neutrophil infiltration.

Role of glutathione in nitric oxide-mediated injury to rat gastric mucosal cells.

Recent studies suggest that in some cell types, the activity of nitric oxide (NO) is influenced by the endogenous antioxidant, reduced glutathione (GSH). The present study has examined the role of GSH in NO-induced cytotoxicity in cells harvested from the rat gastric mucosa. Cell integrity was assessed by Trypan blue exclusion and alamar blue dye absorbance. Pretreatment of rats with bacterial endotoxin lipopolysaccharide increased Ca(2+)-independent NO synthase (iNO synthase) activity (as detected by the radiolabeled conversion of [14C]arginine to [14C]citrulline, lowered GSH content and increased cell injury. Lipopolysaccharide treatment also resulted in a significant increase in the in vitro production of reactive oxygen metabolites as assessed by the fluorescent probe 2',7'-dichlorofluorescein diacetate. Inhibition of iNO synthase activity by dexamethasone and NG-nitro-L-arginine methyl ester prevented these effects. Similarly, the NO donor, S-nitroso acetyl-penicillamine depleted GSH stores and damaged cells in a dose-dependent manner. The effects of S-nitroso acetyl-penicillamine were diminished by the NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. In contrast, incubating cells with N-acetyl-L-cysteine to augment endogenous GSH synthesis, prevented the effects of S-nitroso acetyl-penicillamine. Reduction of GSH stores by pretreatment of rats with buthionine sulfoximine or incubating cells in vitro with diethyl maleate, increased oxidant production and exacerbated NO-induced cell injury. These results suggest that excessive levels of NO alter GSH homeostasis and increase the generation of oxidants leading to increased gastric cellular injury.

Nitric oxide synthase activity in portal-hypertensive and cirrhotic rats.

BACKGROUND/AIMS: The hyperdynamic circulation of cirrhosis and portal hypertension has been postulated to be due to the vasodilatory effects of nitric oxide. However, studies using pharmacological inhibitors of nitric oxide synthase have yielded conflicting results. We aimed to measure nitric oxide synthase activity in tissues from two different rat models of cirrhosis and portal hypertension. METHODS: Cirrhosis was induced by chronic bile duct ligation, and prehepatic portal hypertension by portal vein stenosis. Controls were sham-operated. A fourth group was treated with lipopolysaccharide endotoxin. Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthase activity was assayed by measuring the conversion rate of 14C-arginine to 14C-citrulline in homogenates of stomach, jejunum, liver, kidney and aorta. RESULTS: Jejunal homogenates from the portal vein-stenosed rats showed a significant 10-fold elevation of Ca(2+)-dependent nitric oxide synthase activity. Cirrhotic rat kidney showed significantly decreased Ca(2+)-dependent and Ca(2+)-independent nitric oxide synthase activity. Endotoxin treatment increased Ca(2+)-independent nitric oxide synthase activity in jejunum and liver. There was no increase in Ca(2+)-independent nitric oxide synthase activity in any tissues from cirrhotic or portal hypertensive rats. CONCLUSIONS: We conclude that the lack of increase in Ca(2+)-independent nitric oxide synthase activity does not support the hypothesis that nitric oxide is the major cause of hyperdynamic circulation in cirrhosis.

Role of calcium in nitric oxide-mediated injury to rat gastric mucosal cells.

BACKGROUND & AIMS: Perturbations in Ca2+ homeostasis as well as high levels of nitric oxide have been associated with gastric cellular injury. The purpose of this study was to examine whether high levels of endogenous or exogenous NO damage gastric cells by altering intracellular Ca2+. METHODS: Epithelial cells were isolated from the rat stomach, and cell integrity was estimated by trypan blue exclusion and alamar blue dye absorption. Cytosolic intracellular Ca2+ concentrations ([Ca2+]i) were determined by indo-1 dye fluorescence. NO synthase activity was assessed radioenzymatically. RESULTS: Induction of Ca2+-independent NO synthase in response to endotoxin challenge resulted in decreased viability and an increase in [Ca2+]i in gastric mucosal cells. These responses were ameliorated by pretreatment with NG-nitro-L-arginine methyl ester or dexamethasone. Treatment of cells with the NO donor S-nitrosoacetyl-penicillamine also decreased cell integrity and increased [Ca2+]i. The actions of S-nitroso-acetyl-penicillamine could be reduced by decreasing intracellular or extracellular Ca2+, by chelating Ca2+ with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid or 1,2,-bis(2-aminophenoxy)ethane-N,N,N,N'-tetraacetic acid acetoxymethyl ester, by Ca2+ channel antagonism (nifedipine), or by displacing surface-bound Ca2+ (lanthanum). Furthermore, cell damage was reduced by inhibiting protein kinase C activity with either H-7 or staurosporine. CONCLUSIONS: Excessive levels of NO from either endogenous or exogenous sources results in a reduction in gastric cellular viability. This response seems to be related causally to an increase in [Ca2+]i and protein kinase C activation.

Role of endonuclease activity and DNA fragmentation in Ca2+ ionophore A23187-mediated injury to rabbit isolated gastric mucosal cells.

In the current study, the role of endonuclease activity in calcium ionophore A23187-induced gastric mucosal cellular disruption was examined using rabbit gastric mucosal cells. Cell integrity was assessed using trypan blue dye exclusion and Alamar blue dye absorbance. Ionophore A23187 (1.6-25 microM) induced a concentration-dependent decrease in dye exclusion and cell metabolism in cells suspended in a medium containing Ca2+ (2 mM), while no such effect was observed in cells incubated in the absence of extracellular Ca2+. Cells that were pretreated with the endonuclease inhibitors aurintricarboxylic acid (ATCA; 0.2 or 0.5 mM or Zn2+; 0.01 and 0.1 mM) exhibited significant reduction in the total extent of cell injury when incubated with A23187 in the presence of Ca2+. DNA fragmentation as assessed by measurement of [3H]thymidine liberation or gel electrophoresis was increased in response to ionophore A23187 (12.5 or 25 microM) treatment. A minimal degree of fragmentation was observed when cells were suspended in a Ca(2+)-free medium or incubated in the presence of ATCA or Zn2+. Addition of ethanol (8% w/v) induced a significant increase in cell injury, which was not affected by either removal of extracellular Ca2+ or ATCA pretreatment. Furthermore, treatment with the antioxidants catalase (50 micrograms/ml) or 2',2'-dipyridyl (2 mM) reduced ionophore-induced cell injury but did not reduce the extent of DNA fragmentation. These data suggest that sustained increases in intracellular Ca2+ result in increased endonuclease activity in gastric mucosal cells, leading to extensive DNA lysis and cell damage. Ethanol-induced cell damage does not involve Ca2+ influx and therefore is not mediated by endonuclease activation. Furthermore, sustained increases in cellular Ca2+ may also mediate their effects via formation of reactive oxygen metabolites, but this mechanism of cell damage does not appear to involve DNA fragmentation.

Ca(2+)-mediated damage to rabbit gastric mucosal cells: modulation by nitric oxide.

Pertubations in cellular Ca2+ homeostasis can lead to oxidative stress whereas nitric oxide has been shown to inactivate oxygen radicals. Therefore the effects of inhibition of nitric oxide (NO) synthase activity on Ca2+-mediated disruption to rabbit dispersed gastric mucosal cells have been examined. Addition of the Ca2+ ionophore A23187 (3-25 microM) to the incubation medium induced a concentration-dependent increase in cell damage is assessed by trypan blue dye uptake and decreased cellular metabolic activity as estimated by alamar blue absorbance. These responses were exacerbated by inhibition of NO synthase activity with NG-monomethyl-L-arginine (300 microM). The deleterious effects of ionophore A23187 and NG-monomethyl-L-arginine were ameliorated by addition of the NO donor S-nitroso-acetyl-penicillamine to the cell suspension. An increase in cellular Ca2+ in response to ionophore A23187 (12.5 microM) resulted in enhanced 2'7'-dichlorofluorescein fluorescence suggesting an elevation in oxidative stress. Ca2+-mediated cell injury was abolished by the oxygen radical scavengers, catalase and 2',2'-dipyridyl. However, the cytotoxic effect of combined treatment with A23187 and NG-monomethyl-L-arginine was not reduced by administration of oxygen radical scavengers. NG-monomethyl-L-arginine treatment exacerbated the increase in cytosolic Ca2+ in response to ionophore A23187 as assessed by indo-1 fluorescence. Furthermore this increase in cytosolic Ca2+ was reduced by addition of S-nitroso-acetyl-penicillamine to the incubation medium. These data suggest that NO synthase inhibition in gastric mucosal cells exacerbates the damaging actions of the Ca2+ ionophore A23187. The increase in cell damage in response to the NO synthase inhibitor NG-monomethyl-L-arginine does not appear to be mediated by an increase in oxidative stress and may be associated in part with changes in cellular Ca2+ flux.

Role of calcium in thromboxane B2-mediated injury to rabbit gastric mucosal cells.

A sustained increase in cytosolic Ca2+ can damage gastric mucosal cells. The present study has examined the role of Ca2+ in thromboxane B2 (TXB2)-mediated damage of rabbit isolated gastric mucosal cells. Cells were isolated from rabbit oxyntic mucosa by collagenase-EDTA digestion. Cell metabolic activity and cell damage were estimated by alamar blue dye absorbance and trypan blue uptake, respectively. Cellular Ca2+ was monitored by indo-1 dye fluorescence. Addition of TXB2 (10(-6) and 10(-8) M) to the cell suspension resulted in a decrease in metabolic activity, and this effect was reduced when Ca2+ was removed from the incubation medium. TXB2 addition to the incubation medium resulted in an increase in cytosolic Ca2+ and incubation of cells with the intracellular Ca2+ chelator, BAPTA-AM (20 microM), reduced cell injury in response to TXB2. Incubation of cells with the Ca2+ ionophore A23187 (1-25 microM) resulted in a dose-dependent increase in trypan blue uptake and a reduction in cell metabolism. Cell injury in response to A23187 were exacerbated by addition of TXB2 (10(-8) M) to the cell suspension. TXB2 treatment reduced cellular content of reduced glutathione (GSH), while exogenous GSH addition (10 mM) reduced TXB2-mediated cell injury. These data demonstrate that TXB2 can directly injure gastric mucosal cells. Gastric mucosal cellular damage in response to TXB2 is mediated in part by a disruption of Ca2+ homeostasis as well as a reduction in cellular GSH content.