Leptin suppresses Porphyromonas gingivalis lipopolysaccharide interference with salivary mucin synthesis

Leptin, a multifunctional hormone produced predominantly by adipocytes but also identified throughout the glandular tissue of alimentary tract, including salivary glands and oral mucosa, has emerged recently as an important regulator of mucosal inflammatory responses to bacterial infection. In this study, we report that leptin prevents (up to 88.4%) the reduction in mucin synthesis evoked in mucous cells of sublingual salivary gland by LPS of periodontopathic bacterium, Porphyromonas gingivalis. The effect of leptin, moreover, was reflected in a marked decrease in the LPS-induced apoptosis, expression of TNF-alpha, caspase-3 activity, and NO generation. The impedance by leptin of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of PI3K, which also obviated the inhibitory effect of leptin on the LPS-induced upregulation in apoptosis, caspase-3 activity, and NO generation. A potentiation in the impedance by leptin of the LPS-induced apoptosis, caspase-3 activity, and NO generation was, however, attained with NOS-2 inhibitor, 1400W, that also caused further enhancement in the impedance by leptin of the LPS detrimental effect on mucin synthesis. Taken together, our data are the first to demonstrate the nature of the involvement of leptin in countering the pathological consequences of P. gingivalis infection on the synthesis of salivary mucins.     

Src kinase-dependent epidermal growth factor receptor transactivation in PPARgamma ligand-induced suppression of Porphyromonas gingivalis interference with salivary mucin synthesis

Peroxisome proliferator-activated receptor gamma (PPARgamma) has emerged recently as an important participant in the resolution of inflammation by conveying signals that lead to mitogen-activated protein kinase (MAPK) cascade activation. In this study, we report that PPARgamma activation leading to the impedance of P. gingivalis lipopolysaccharide (LPS) inhibitory effect on salivary mucin synthesis requires epidermal growth factor receptor (EGFR) participation. We show that activation of PPARgamma with a specific agonist, ciglitazone, prevents the LPS-induced reduction in mucin synthesis, and the effect is reflected in a marked decrease in apoptosis, caspase-3 activity and NO generation. The impedance by ciglitazone of the LPS-induced reduction in mucin synthesis was countered (up to 68.9%) in a dose-dependent fashion by a specific inhibitor of EGFR kinase, PD153035, as well as wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K). Moreover, the inhibitory effect of ciglitazone on the LPS-induced reduction in mucin synthesis and upregulation in apoptosis, caspase-3 activity, and NO generation was blunted by a selective inhibitor of tyrosine kinase Src, PP2, responsible for ligand-independent EGFR transactivation. These findings indicate that PPARgamma activation leading to the suppression of P. gingivalis LPS inhibition of salivary mucin synthesis involves Src kinase-dependent EGFR transactivation.     

Disruption in gastric mucin synthesis by Helicobacter pylori lipopolysaccharide involves ERK and p38 mitogen-activated protein kinase participation

Helicobacter pylori is a primary factor in the etiology of gastric disease, and its early pathogenic effects are manifested by up-regulation of inflammatory processes and the loss of mucus coat continuity. We investigated the role of extracellular signal-regulated kinase (ERK) and p38 mitogen activated protein kinase (MAPK) in the disturbances in gastric mucin synthesis and apoptotic processes evoked by H. pylori lipopolysaccharide (LPS). Exposure of gastric mucosal cells to the LPS led to a dose-dependent decrease (up to 59.5%) in mucin synthesis, accompanied by a marked increase in caspase-3 activity and apoptosis. Inhibition of ERK with PD98059 accelerated (up to 36.1%) the LPS-induced decrease in mucin synthesis, and caused further enhancement in caspase-3 activity and apoptosis. Blockade of p38 kinase with SB203580 produced reversal in the LPS-induced reduction in mucin synthesis, and substantially countered the LPS-induced increases in caspas-3 activity and apoptosis. Moreover, inhibition of caspase-3 blocked the LPS-induced increase in caspse-3 activity and produced an increase in mucin synthesis. Thus the detrimental influence of H. pylori LPS on gastric mucin synthesis is closely linked to caspase-3 activation and apoptosis, and involves ERK and p38 kinase participation.     

Nitric oxide interferes with salivary mucin synthesis: involvement of ERK and p38 mitogen-activated protein kinase.

BACKGROUND: Nitric oxide (NO), a pluripotent molecule, is an important biological messenger that plays a role in the regulation of tissue homeostasis and pathophysiological processes. METHODS: Using sublingual salivary gland acinar cells in culture, we investigated the effect of NO on mucus glycoprotein synthesis, apoptotic processes, and the involvement of extracellular signal-regulated kinase (ERK) and p38 mitogen activated protein kinase (MAPK). RESULTS: Exposure of the acinar cells to NO donor led to a dose-dependent decrease (up to 42.8%) in mucus glycoprotein synthesis, and this effect of NO was accompanied by a marked increase in caspase-3 activity and apoptosis. Inhibition of ERK with PD98059 accelerated (up to 35.4%) the NO-induced decrease in the glycoprotein synthesis, and cause further enhancement in caspase-3 (up to 27.2%) activity and apoptosis (64.9%). On the other hand, blockade of p38 kinase with SB203580 produced a dose-dependent reversal (up to 42%) in the NO-induced reduction in the glycoprotein synthesis, and substantially countered the NO-induced increases in caspase-3 activity (by 62.8%) and apoptosis (by 57.6%). Moreover, caspase-3 inhibitor, Ac-DEVD-CHO, not only blocked the NO-induced increase in caspase-3 activity but also produced an increase in the glycoprotein synthesis. CONCLUSIONS: Together, our data indicate that the modulatory influence of NO on salivary mucin synthesis is closely linked to ERK and p38 protein kinase activation, in conjunction with caspase-3 activation and apoptosis.     

Porphyromonas gingivalis lipopolysaccharide-induced up-regulation in endothelin-1 interferes with salivary mucin synthesis via epidermal growth factor receptor transactivation

Endothelin-I (ET-1) is a 21 amino acid peptide produced from a biologically inactive big ET-1 by the action of endothelin-converting enzyme-1 (ECE-1) that acts through G protein-coupled ETA and ETB receptors. Using mucous cells of sublingual salivary gland, we show that P. gingivalis lipopolysaccharide (LPS) inhibitory effect on salivary mucin synthesis is accompanied by a marked increase in ET-I generation and the enhancement in ECE-1 activity. Inhibition of ECE-I with phosphoramidon led to the impedance of the LPS-induced ET-1 generation as well as countered the detrimental effect of the LPS on mucin synthesis. Moreover, the LPS inhibitory effect of on mucin synthesis was blocked by ETA receptor antagonist, BQ610, but not by ETB receptor antagonist, BQ788. The LPS-induced reduction in mucin synthesis, furthermore, was countered by PD153035 (76.8%), a specific inhibitor of EGFR kinase as well as PP2 (54.7%), a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR transactivation. Our findings are the first to demonstrate that P. gingivalis LPS detrimental effect on salivary mucin synthesis is intimately linked to the events controlled by EGFR transactivation, triggered by upregulation in ECE-1,enhancement in ET-1 production, and G protein-coupled ETA receptor activation.     

Impedance of Helicobacter pylori lipopolysaccharide interference with gastric mucin synthesis by peroxisome proliferator-activated receptor gamma activation involves phosphatidylinositol 3-kinase/ERK pathway

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a critical role in the regulation of the expression of genes associated with inflammation. In this study, we report that PPARgamma activation leading to the impedance of H. pylori lipopolysaccharide (LPS) inhibitory effect on gastric mucin synthesis occurs with the involvement of phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) pathways. Using gastric mucosal cells in culture, we show that activation of PPARgamma with a specific synthetic agonist, ciglitazone, prevents in a dose-dependent fashion (up to 90.2%) the LPS-induced reduction in mucin synthesis, and the effect is reflected in a marked decrease in the LPS-induced apoptosis (72.4%), NO generation (80.1%), and the expression of NOS-2 activity (90%). The impedance by ciglitazone of the LPS-induced reduction in mucin synthesis was blocked by wortmannin, a specific inhibitor of P13K and PD98059, an inhibitor of ERK. Both inhibitors, moreover, caused further enhancement in the LPS-induced NO generation and countered the inhibitory effect of ciglitazone on the LPS-induced upregulation in NOS-2. Our findings point to PI3K and ERK as mediators of PPARgamma agonist effect leading to the impedance of H. pylori LPS inhibition on gastric mucin synthesis.     

Nitric oxide as a modulator of gastric mucin synthesis: role of ERK and p38 mitogen-activated protein kinase activation

Nitric oxide (NO) is an important biological messenger in the regulation of tissue homeostasis and pathophysiological processes. Here, we investigated the effect of NO on gastric mucus glycoprotein (mucin) synthesis, apoptotic processes, and the involvement of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Exposure of gastric mucosal cells to NO donor led to a dose-dependent decrease (up to 48%) in mucin synthesis, accompanied by a marked increase in caspase-3 activity and apoptosis. Inhibition of ERK with PD98059 accelerated (up to 23.8%) the NO-induced decrease in mucin synthesis, and cause further enhancement in caspase-3 activity and apoptosis. Blockade of p38 kinase with SB203580 produced reversal in the NO-induced reduction in mucin synthesis, and substantially countered the induced increase in caspase-3 activity and apoptosis. Moreover, caspase-3 inhibitor not only blocked the NO-induced increase in caspase-3 activity but also produced an increase in mucin synthesis. Thus, the detrimental influence of NO on mucin synthesis is closely linked to caspase-3 activation and apoptosis, and involves ERK and p38 kinase participation. Activation of p38 kinase leads to the upregulation of proapoptotic signal, while ERK activation stimulates the anti-apoptotic pathway.     

Platelet-activating factor mediates Helicobacter pylori lipopolysaccharide interference with gastric mucin synthesis

Platelet-activating factor (PAF) is now recognized as the most proximal mediator of cellular events triggered by bacterial infection. In this study, we report that a specific PAF antagonist, BN52020, impedes the reduction in mucin synthesis evoked in gastric mucosal cells by H. pylori LPS. The impedance by BN52020 of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (P13K), which also obviated the inhibitory effect of BN52020 on the LPS-induced upregulation in apoptosis, TNF-alpha, and NO generation. A reduction in the impedance by BN52020 of the LPS detrimental effect on mucin synthesis was also attained with cNOS inhibitor, L-NNA, whereas NOS-2 inhibitor, 1400W caused a potentiation in the impedance effect of BN52020. However, while 1400W and BN52020 countered the potentiating effect of wortmannin on the LPS-induced decrease in mucin synthesis, a further exacerbation of the potentiating effect of wortmannin was attained in the presence of cNOS inhibitor, L-NNA. Our findings suggest that PAF, through the interference with PI3K-dependent cNOS activation, plays a critical role in influencing the extent of pathological consequences of H. pylori infection on the synthesis of gastric mucin.     

Role of leptin in modulation of Porphyromonas gingivalis lipopolysaccharide-induced up-regulation of endothelin-1 in salivary gland acinar cells

Leptin, a pleiotropic cytokine that regulates food intake and metabolic and endocrine responses, has emerged recently as an important regulator of mucosal inflammatory responses to bacterial infection. In this study, we report that in sublingual salivary gland acinar cells leptin plays a role in the suppression of up-regulation in endothelin-1 (ET-1), induced by the LPS of a periodontopathic bacterium P. gingivalis. We show that P. gingivalisLPS detrimental effect on salivary mucin synthesis, associated with up-regulation (3.9-fold) in ET-1 generation and the enhancement (3.2-fold) in endothelin-converting enzyme-1 (ECE-1) activity, was subject to a dose-dependent suppression by leptin. The impedance by leptin of the LPS inhibitory effect on mucin synthesis was blocked by wortmannin, an inhibitor of PI3K, as well as by ERK inhibitor, PD98059. However, while the blockade of ERK led also to amplification in the impedance by leptin of the LPS-induced expression of ECE-1 and ET-1, the effect was not observed in the presence of wortmannin. The findings are the first to demonstrate that leptin counters the pathological consequences of P. gingivalisinfection on the synthesis of salivary mucin through the involvement in signaling events of PI3K and ERK pathways. We also show that the ERK cascade represents a critical signaling target for leptin in the LPS-induced up-regulation in ET-1.     

Cytosolic phospholipase A2 activation in Helicobacter pylori lipopolysaccharide-induced interference with gastric mucin synthesis

Release of arachidonic acid from membrane glycerophospholipids by cytosolic phospholipase A2 (cPLA2) is a key step in the generation of platelet-activating factor (PAF), recognized as the most proximal mediator of inflammatory events triggered by bacterial infection. Here, we report on the role of cPLA2 in the disturbances in gastric mucin synthesis evoked by the LPS of H. pylori, a bacterium identified as a primary cause of gastric disease. Using rat gastric mucosal cells, we show that H. pylori LPS detrimental effect on gastric mucin synthesis, associated with up-regulation in PAF and endothelin-1 (ET-1) generation, was subject to suppression by a specific inhibitor of cPLA2, MAFP. Moreover, the LPS-induced changes in mucin synthesis and ET-1 generation were countered by PAF receptor antagonist, BN52020. The impedance by PAF antagonist of the LPS-induced reduction in mucin synthesis was countered by wortmannin, an inhibitor of PI3K, as well as by ERK inhibitor, PD98059. The blockade of ERK caused also inhibition of the LPS-induced cPLA2 activation and amplification in the impedance capacity of PAF antagonist on the LPS-induced ET-1 generation, while the inhibitor of PI3K had no effect. Our findings are the first to demonstrate that the detrimental consequences of H. pylori LPS on gastric mucin synthesis involve ERK-dependent cPLA2 activation that leads to up-regulation in PAF generation and ET-1 production.     

Saikosaponin C inhibits lipopolysaccharide-induced apoptosis by suppressing caspase-3 activation and subsequent degradation of focal adhesion kinase in human umbilical vein endothelial cells

Bacterial lipopolysaccharide (LPS) is an important mediator of inflammation and a potent inducer of endothelial cell damage and apoptosis. In this study, we investigated the protective effects of saikosaponin C (SSc), one of the active ingredients produced by the traditional Chinese herb, Radix Bupleuri, against LPS-induced apoptosis in human umbilical endothelial cells (HUVECs). LPS triggered caspase-3 activation, which was found to be important in LPS-induced HUVEC apoptosis. Inhibition of caspase-3 also inhibited LPS-induced degradation of focal adhesion kinase (FAK), indicating that caspase-3 is important in LPS-mediated FAK degradation as well as in apoptosis in HUVECs. SSc significantly inhibited LPS-induced apoptotic cell death in HUVECs through the selective suppression of caspase-3. SSc was also shown to rescue LPS-induced FAK degradation and other cell adhesion signals. Furthermore, the protective effects of SSc against LPS-induced apoptosis were abolished upon pretreatment with a FAK inhibitor, highlighting the importance of FAK in SSc activity. Taken together, these results show that SSc efficiently inhibited LPS-induced apoptotic cell death via inhibition of caspase-3 activation and caspase-3-mediated-FAK degradation. Therefore, SSc represents a promising therapeutic candidate for the treatment of vascular endothelial cell injury and cellular dysfunction.     

Roles of iNOS and nNOS in sepsis-induced pulmonary apoptosis

Apoptosis(programmed cell death) is induced in pulmonary cells and contributes to the pathogenesis of acute lung injury in septic humans. Previous studies have shown that nitric oxide (NO) is an important modulator of apoptosis; however, the functional role of NO derived from inducible NO synthase (iNOS) in sepsis-induced pulmonary apoptosis remains unknown. We measured pulmonary apoptosis in a rat model of Escherichia coli lipopolysaccharide (LPS)-induced sepsis in the absence and presence of the selective iNOS inhibitor 1400W. Four groups were studied 24 h after saline (control) or LPS injection in the absence and presence of 1400W pretreatment. Apoptosis was evaluated using DNA fragmentation, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and caspase activation. LPS administration significantly augmented pulmonary cell apoptosis and caspase-3 activity in airway and alveolar epithelial cells. Pretreatment with 1400W significantly enhanced LPS-induced pulmonary apoptosis and increased caspase-3 and -7 activation. The antiapoptotic effect of iNOS was confirmed in iNOS-/- mice, which developed a greater degree of pulmonary apoptosis both under control conditions and in response to LPS compared with wild-type mice. By comparison, genetic deletion of the neuronal NOS had no effect on LPS-induced pulmonary apoptosis. We conclude that NO derived from iNOS plays an important protective role against sepsis-induced pulmonary apoptosis.     

Partial deletion of argininosuccinate synthase protects from pyrazole plus lipopolysaccharide-induced liver injury by decreasing nitrosative stress

Argininosuccinate synthase (ASS) is the rate-limiting enzyme in the urea cycle. Along with nitric oxide synthase (NOS)-2, ASS endows cells with the L-citrulline/nitric oxide (NO·) salvage pathway to continually supply L-arginine from L-citrulline for sustained NO· generation. Because of the relevant role of NOS in liver injury, we hypothesized that downregulation of ASS could decrease the availability of intracellular substrate for NO· synthesis by NOS-2 and, hence, decrease liver damage. Previous work demonstrated that pyrazole plus LPS caused significant liver injury involving NO· generation and formation of 3-nitrotyrosine protein adducts; thus, wild-type (WT) and Ass+/- mice (Ass+/+ mice are lethal) were treated with pyrazole plus LPS, and markers of nitrosative stress, as well as liver injury, were analyzed. Partial ablation of Ass protected from pyrazole plus LPS-induced liver injury by decreasing nitrosative stress and hepatic and circulating TNFα. Moreover, apoptosis was prevented, since pyrazole plus LPS-treated Ass+/- mice showed decreased phosphorylation of JNK; increased MAPK phosphatase-1, which is known to deactivate JNK signaling; and lower cleaved caspase-3 than treated WT mice, and this was accompanied by less TdT-mediated dUTP nick end labeling-positive staining. Lastly, hepatic neutrophil accumulation was almost absent in pyrazole plus LPS-treated Ass+/- compared with WT mice. Partial Ass ablation prevents pyrazole plus LPS-mediated liver injury by reducing nitrosative stress, TNFα, apoptosis, and neutrophil infiltration.     

Activation of PKC is sufficient to induce an apoptotic program in salivary gland acinar cells

Accumulating evidence suggests that specific isoforms of PKC may function to promote apoptosis. We show here that activation of the conventional and novel isoforms of PKC with 12-O-tetradecanoyl phorbol-13- ester (TPA) induces apoptosis in salivary acinar cells as indicated by DNA fragmentation and activation of caspase-3. TPA-induced DNA fragmentation, caspase-3 activation, and morphologic indicators of apoptosis, can be enhanced by pretreatment of cells with the calpain inhibitor, calpeptin, prior to the addition of TPA. Analysis of PKC isoform expression by immunoblot shows that TPA-induced downregulation of PKC alpha and PKC delta is delayed in cells pre-treated with calpeptin, and that this correlates with an increase of these isoforms in the membrane fraction of cells. TPA-induced apoptosis is accompanied by biphasic activation of the c-jun-N-terminal kinase (JNK) pathway and inactivation of the extracellular regulated kinase (ERK) pathway. Expression of constitutively activated PKC alpha or PKC delta, but not kinase negative mutants of these isoforms, or constitutively activated PKC epsilon, induces apoptosis in salivary acinar cells, suggesting a role for these isoforms in TPA-induced apoptosis. These studies demonstrate that activation of PKC is sufficient for initiation of an apoptotic program in salivary acinar cells. Cell Death and Differentiation (2000) 7, 1200 - 1209.     

Aldose reductase mediates endotoxin-induced production of nitric oxide and cytotoxicity in murine macrophages

Aldose reductase (AR) is a ubiquitously expressed protein with pleiotrophic roles as an efficient catalyst for the reduction of toxic lipid aldehydes and mediator of hyperglycemia, cytokine, and growth factor-induced redox-sensitive signals that cause secondary diabetic complications. Although AR inhibition has been shown to be protective against oxidative stress signals, the role of AR in regulating nitric oxide (NO) synthesis and NO-mediated apoptosis has not been elucidated to date. We therefore investigated the role of AR in regulating lipopolysaccharide (LPS)-induced NO synthesis and apoptosis in RAW 264.7 macrophages. Inhibition or RNA interference ablation of AR suppressed LPS-stimulated production of NO and overexpression of iNOS mRNA. Inhibition or ablation of AR also prevented the LPS-induced apoptosis, cell cycle arrest, activation of caspase-3, p38-MAPK, JNK, NF-kappaB, and AP1. In addition, AR inhibition prevented the LPS-induced down-regulation of Bcl-xl and up-regulation of Bax and Bak in macrophages. L-Arginine increased and L-NAME decreased the severity of cell death caused by LPS and AR inhibitors prevented it. Furthermore, inhibition of AR prevents cell death caused by HNE and GS-HNE, but not GS-DHN. Our findings for the first time suggest that AR-catalyzed lipid aldehyde-glutathione conjugates regulate the LPS-induced production of inflammatory marker NO and cytotoxicity in RAW 264.7 cells. Inhibition or ablation of AR activity may be a potential therapeutic target in endotoximia and other inflammatory diseases.     

An inhibitor of c-Jun NH2-terminal kinase, SP600125, protects mice from D-galactosamine/lipopolysaccharide-induced hepatic failure by modulating BH3-only proteins

Fulminant hepatic failure (FHF) is a dramatic clinical syndrome characterized by massive hepatocyte apoptosis and very high mortality. The c-Jun-N-terminal kinase (JNK) pathway is an important stress-responsive kinase activated by several forms of liver injury. The aim of this study is to assess the role of JNK during D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver injury, an experimental model of FHF, using SP600125, a small molecule JNK-specific inhibitor. Mice were given an intraperitoneal dose of GalN (800 microg/g body weight)/LPS (100 ng/g body weight) with and without subcutaneous SP600125 (50 mg/kg body weight) treatment (at 6 and 2 h before and 2 h after GalN/LPS administration). GalN/LPS treatment induced sustained JNK activation. Administration of SP600125 diminished JNK activity, suppressed lethality and the elevation of both serum alanine aminotransferase and aspartate aminotransferase, but had no effect on serum tumor necrosis factor-alpha, and reduced hepatocyte apoptosis after GalN/LPS administration. In support of the role of JNK in promoting the mitochondria-mediated apoptosis pathway, SP600125 prevented cytochrome c release, caspase-9 and caspase-3 activity. Moreover, SP600125 downregulated the mRNA and protein expression of Bad in the early periods following GalN/LPS injection and prevented Bid cleavage in the late periods. These results confirm the role of JNK as a critical apoptotic mediator in GalN/LPS-induced FHF. SP600125 has the potential to protect FHF by downregulating Bad and inhibiting Bid cleavage.     

Interference by leptin with Helicobacter pylori lipopolysaccharide-induced cytosolic phospholipase A2 activation in gastric mucosal cells.

Activation of cytosolic phospholipase A(2) (cPLA(2)) by bacterial LPS for the rapid release of arachidonic acid from membrane phospholipids is considered a key step in the generation of platelet-activating factor (PAF), recognized as the most proximal mediator of inflammatory events triggered by bacterial infection. In this study, we report on the role of leptin in modulation of the detrimental consequences of H. pylori LPS-induced cPLA(2) activation that result in the disturbances in gastric mucin synthesis. Employing gastric mucosal cells labeled with [(3)H] arachidonic acid, we show that H. pylori LPS-induced cPLA(2) activation, associated with up-regulation in apoptosis and PAF generation, and the impairment in gastric mucin synthesis, was subject to a dose-dependent suppression by leptin, as well as the inhibition by MAFP, a specific inhibitor of cPLA(2). A potentiation in the countering capacity of leptin on the LPS-induced up-regulation in apoptosis, arachidonic acid release and PAF generation was attained in the presence of ERK inhibitor, PD98059, while PI3K inhibitor, wortmannin had no effect. On the other hand, the prevention by leptin of the LPS detrimental effect on mucin synthesis was subject to suppression by wortmannin, an inhibitor of PI3K as well as the inhibitor of ERK, PD98059. Moreover, potentiation in the effect of leptin on the LPS-induced decrease in mucin synthesis was attained with cPLA(2) inhibitor, MAFP as well as PAF receptor antagonist, BN52020. The results of our findings point to H. pylori LPS-induced ERK-dependent cPLA(2) activation as a critical factor influencing the level of PAF generation, and hence the extent of pathological consequences of H. pylori infection on the synthesis of gastric mucin. Furthermore, we show that leptin counters the pathological consequences of H. pylori-induced cPLA(2) activation on gastric mucin synthesis through the involvement in signaling events controlled by MAPK/ERK and PI3K pathways.     

Endotoxaemia in rats: role of leukocyte sequestration in rapid pulmonary nitric oxide synthase-2 expression.

Nitric oxide (NO), depending on the amount, time and source of generation may exert both, protective and deleterious actions during endotoxic acute lung injury (ALI). Evaluation of the expression and localization of NOS isoforms in the lung of lipopolysaccharide (LPS)-treated rats may contribute to understanding the role of NO in pathogenesis of ALI. Tissue samples (lung, heart, liver, kidney and spleen) as well as peripheral blood polymorphonuclear cells (PMNs) were collected from control male Wistar rats and LPS - treated animals, 15, 30, 60, 120 and 180 min after LPS injection (2 mg kg(-1) min(-1) for 10 minutes, i.v.). Levels of NOS-2 and NOS-3 mRNA and protein in tissues and PMNs were estimated by RT-PCR, Northern blotting and Western blotting. Additionally, myeloperoxidase (MPO) activity in tissue samples was assayed. NOS-3 mRNA as well as protein were detected in lungs of control animals; pulmonary NOS-3 expression was not influenced by LPS. The induction of NOS-2 mRNA in rat lungs and in PMNs isolated from peripheral blood was observed 15 minutes after LPS challenge. In contrast, increase of NOS-2 mRNA in the heart, kidneys, liver and spleen was observed 2-3 hours after LPS injection. In all tissues rise in NOS-2 mRNA was followed after 1-2 hours by increase of NOS-2 protein. Importantly, progressive leukocyte sequestration in the lung parenchyma that started as early as 15 min after LPS injection was revealed only in the lungs; in other organs no significant changes in MPO activity were detected up to 180 min after LPS injection. In conclusion, infusion of LPS caused much more rapid expression of NOS-2 in lungs as compared to the heart, kidneys, liver and spleen. Early induction of NOS-2 may depend on the LPS-stimulated rapid neutrophil sequestration within lung vasculature and fast induction of NOS-2 in sequestrated neutrophils.