Role of constitutive nitric oxide synthase S-nitrosylation in Helicobacter pylori-induced gastric mucosal cell apoptosis: effect of ghrelin

Abstract

Infection with H. pylori is a primary factor in the etiology of gastric disease, and the excessive NO generation and a massive rise in apoptosis are well recognized features that characterize the mucosal inflammatory responses to the bacterium and its lipopolysaccharide (LPS). Here, we report that H. pylori LPS-induced enhancement in gastric mucosal cell apoptosis and NO generation was associated with the suppression in constitutive nitric oxide synthase (cNOS) activity and a marked up-regulation in the activity of inducible nitric oxide synthase (iNOS). Further, we demonstrate that the detrimental effect of the LPS on cNOS was manifested in the enzyme protein S-nitrosylation, that was susceptible to suppression by iNOS inhibitor, 1400W. Moreover, we show that the countering effect of peptide hormone, ghrelin, on the LPS-induced changes in apoptosis and cNOS activity was reflected in the loss in cNOS S-nitrosylation and the increase in the enzyme phosphorylation. These findings demonstrate that the disturbances in gastric mucosal NO generation system caused by H. pylori result from the iNOS-derived NO suppression of cNOS activation through S-nitrosylation. We also report that ghrelin protection against H. pylori-induced gastric mucosal proapoptotic events involves cNOS activation manifested by the increase in enzyme protein phosphorylation and a decrease in its S-nitrosylation.

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

“…Our results demonstrate that the LPS-induced abrogation of cNOS control over NF-κB activation results in the induction of iNOS expression and leads to COX-2 activation through S-nitrosylation. Moreover, our results show that peptide hormone, ghrelin, recognized for its modulatory control over NOS and COX enzyme systems [16,[22][23][24][25], suppresses these untoward consequences of the LPS through up-regulation in cNOS activation that interferes with NF-κB nuclear translocation, thus causing the repression of iNOS gene induction and the inhibition of COX-2 activation through iNOS-dependent S-nitrosylation.…”

“…The cross-talk between the products of NOS and COX pathways is supported by the studies indicating that stimulation of NO production through iNOS induction leads to COX enzymes activation and the increase in PGE 2 generation, whereas NOS gene deletion or inhibition of NOS enzymes with pharmacological agents results in a decrease in PGE 2 formation [9][10][11][12]. The role of cNOS in the iNOS-dependent COX-2 activation has also been suggested [10,13], and we have reported that the disturbances in NO and PGE 2 generation elicited by H. pylori LPS are reflected in the massive up-regulation of iNOS and COX-2 activity, and the suppression in Src/Aktdependent cNOS activation [14][15][16].…”

“…The cyclooxygenase activity of COX-1 and COX-2 isoforms in gastric mucosal cells was measured with the COX Activity Assay Kit (Cayman) by monitoring the appearance of oxidized TMPD at 590 nm [16]. The cells from the control and various experimental treatments were settled by centrifugation, rinsed with phosphatebuffered 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.…”

“…The mucosal cells from the control and experimental treatments were collected by centrifugation and resuspended for 30 min in ice-cold lysis buffer (20 mM TrisHCl, pH 7.4, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 2 mM EDTA, 1 mM sodium orthovanadate, 4 mM sodium pyrophosphate, 1 mM PMSF, and 1 mM NaF), containing 1 µg/ml leupeptin and 1 µg/ml pepstatin [16]. Following brief sonication, the lysates were centrifuged at 12,000 × g for 10 min, and the supernatants were collected and analyzed for protein concentration using BCA protein assay kit (Pierce).…”

“…Moreover, up-regulation in COX-2 activation has been linked to posttranslational modification of the enzyme protein through S-nitrosylation via LPS-elicited induction in iNOS expression [9,10]. Indeed, as demonstrated recently, the induction in iNOS expression by H. pylori LPS leads to COX-2 S-nitrosylation that results in an excessive PGE 2 generation [16].…”