Modulation of gastric mucosal inflammatory responses to <i>Helicobacter pylori</i> by ghrelin: Role of cNOS-dependent IKK-<i>β</i> S-nitrosylation in the regulation of COX-2 activation

Abstract

Disturbances in nitric oxide synthase (NOS) and cyclooxygenase (COX) isozyme systems, manifested by the excessive NO and prostaglandin (PGE 2 ) generation, are well-recognized features of gastric mucosal inflammatory responses to H. pylori infection. In this study, we report that H. pylori LPS-induced enhancement in gastric mucosal inducible (i) iNOS expression and COX-2 activation was accompanied by the impairment in constitutive (c) cNOS phosphorylation, up-regulation in the inhibitory κB kinase-β (IKKβ) activation and the increase in the transcriptional factor, NF-κB, nuclear translocation. Further, we show that abrogation of cNOS control over NF-κB activation has lead to induction of iNOS expression and COX-2 activation through S-nitrosylation. Moreover, we demonstrate that the modulatory effect of peptide hormone, ghrelin, on the LPS-induced changes was reflected in the increase in Src/Akt-dependent cNOS activation through phosphorylation and the suppression of IKK-β activity through cNOS-mediated IKK-β protein S-nitrosylation. As a result, ghrelin exerted the inhibitory effect on NF-κB nuclear translocation, thus causing the repression of iNOS gene induction and the inhibition in COX-2 activation through iNOSdependent S-nitrosylation. Our findings point to cNOS activation as a pivotal element in the signaling cascade by which ghrelin exerts modulatory control over proinflammatory events triggered in gastric mucosa by H. pylori infection.

Role of epidermal growth factor receptor transactivation in the amplification of Helicobacter pylori-elicited induction in gastric mucosal expression of cyclooxygenase-2 and inducible nitric oxide synthase

“…To measure the inhibitory κB kinase-β (IKK-β) activity, we utilized the ELI-SA-based detection kit, K-LISA (Calbiochem). The GST-IκB-α50-amino acid peptide that includes the Ser 32 and Ser 36 of IκB-α phosphorylation sites was used as a substrate 27,29 . The gastric mucosal cell cytosolic extracts were incubated with a glutathionecoated 96-well plate with GST-tagged IκB-α at room temperature for 30 min, and the phosphorylated GST-IκB-α substrate was detected using anti-phospho-IκB-α (Ser 32 /Ser 36 ) as first antibody, followed by horseradish peroxidase-conjugated secondary antibody.…”

“…The aliquots of the resulting supernatant were incubated for 30 min at 25°C in the presence of 50 µCi/mL of L-[ 3 H] arginine, 10 mM NAPDH, 5 µM tetrahydrobiopterin and 50 mM Tris-HCl buffer, pH 7.4, in a final volume of 250 µL. Following the addition of stop buffer and Dowex-50 W (Na + ) resin, the mixtures were transferred to spin cups, centrifuged and the formed L-[ 3 H] citrulline contained in the flowthrough was quantified by scintillation counting 27 .…”

“…For measurements of COX-2 activity, the gastric mucosal cells from the control and various experimental treatments were settled by centrifugation, rinsed with phosphate-buffered saline and homogenized in 0.3 mL of cold sample buffer containing 0.1 M Tris-HCl, pH 7.8, and 1 mM EDTA, centrifuged at 12,000×g for 10 min at 4°C and the supernatant collected 27 . The COX-2 activity in 40 µL sample aliquots of the resulting supernatant was measured using COX activity assay kit (Cayman) in the absence and presence of COX-1 inhibition (SC-560), by monitoring the appearance of oxidized TMPD at 590 nm 14 .…”

Helicobacter Pylori is associated with decrease serum level of the thyroid hormonal in healthy elderly population

“…1,7,8 Chronic inflammation that caused by H pylori can induces oxidative stress that has been shown to be associated with both hyperthyroidism and hypothyroidism. 8,9 however the mechanisms of oxidative stress generated in to hypothyroidism is low availability of antioxidants. 9',10 Therefore, we conducted a cross-sectional study to investigate whether correlation between H. pylori infection and T3 serum level also thyroid-stimulating hormone serum level.…”

<i>Helicobacter pylori</i>Induction in Gastric Mucosal Prostaglandin and Nitric Oxide Generation Is Dependent on MAPK/ERK-Mediated Activation of IKK-<i>β</i> and cPLA<sub>2</sub>: Modulatory Effect of Ghrelin

“…Indeed, Snitrosylation has been linked to the processes of COX-2 and cPLA 2 activation, as well as the regulation of inhibitory κB kinase-β (IKK-β) activity responsible for IκB-α degradation and NF-κB nuclear translocation [26][27][28][29]. Moreover, we have recently shown that S-nitrosylation of IKK-β plays a role in the modulatory influence of peptide hormone, ghrelin, on the gastric mucosal inflammatory responses to H. pylori [11,17,30].…”