cNOS Phosphorylation Research Articles

Protein Kinase C<i>δ</i>-Mediated Posttranslational Phosphorylation of Constitutive Nitric Oxide Synthase Regulates Gastric Mucosal Inflammatory Responses to <i>Helicobacter pylori</i>: Effect of Ghrelin

Disturbances in constitutive nitric oxide synthase (cNOS) activation associated with H. pylori colonization of gastric mucosa are considered of major consequences in defining the extent of inflammatory involvement. As rapid changes in cNOS activation are linked to the enzyme phosphorylation at the specific Ser/Thr residues, we investigated the influence of H. pylori LPS and gastric hormone, ghrelin, on the processes of phosphorylation of these two critical sites in gastric mucosal cells. We show that the LPS-induced reduction in cNOS activity is reflected in the phosphorylation on Thr497, while the countering effect of ghrelin is associated with a rapid increase in cNOS phosphorylation on Ser1179. Further, we demonstrate that cNOS phosphorylation on Thr497 as well as Ser1179 displays dependence on PKCδ. However, while the LPS-induced suppression in cNOS activation shows reliance on the phosphorylation of PKCδ and PI3K on Ser, the effect of ghrelin is manifested by the increase in phosphorylation of PKCδ and PI3K on Tyr, as well as membrane translocation and phosphorylation of Akt on Ser493. Thus, our findings suggest that the LPS-induced suppression in cNOS activation is mediated by PKCδ-controlled phosphorylation of PI3K on Ser that interferes with the membrane recruitment of Akt and promotes cNOS phosphorylation on Thr497, and that ghrelin-elicited up-regulation in cNOS activation relies on the PKCδ-directed phosphorylation of PI3K on Tyr that stimulates the membrane localization of Akt and enhances cNOS phosphorylation on Ser1179.

Involvement of p38 MAPK-dependent activator protein (AP-1) activation in modulation of gastric mucosal inflammatory responses to Helicobacter pylori by ghrelin

A peptide hormone, ghrelin, plays an important role in modulation of gastric mucosal inflammatory responses to Helicobacter pylori infection by controlling the cross-talk between nitric oxide synthase (NOS) and cyclooxygenase (COX) enzyme systems. In this study, we report that H. pylori LPS-elicited induction in gastric mucosal COX-2 and inducible (i) iNOS protein expression, and the impairment in constitutive (c) cNOS phosphorylation, was associated with mitogen-activated protein kinase, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase and p38 activation, and occurred with the involvement of transcription factors, CCATT/enhancer-binding protein (C/EBP) δ, cAMP response element-binding protein, activator protein-1 (AP-1), and NF-κB. The modulatory effect of ghrelin on the LPS-induced changes was manifested in the inhibition of nuclear translocation of p65 NF-κB and C/EBPδ, and suppression in AP-1 activation, and the inhibition in phosphorylation of JNK and p38, as well as their respective downstream targets, c-Jun and ATF-2. However, only the inhibition of p38-mediated ATF-2 phosphorylation was reflected in the reduced expression of COX-2 protein. Further, the effect of ghrelin of the LPS-induced changes was reflected in the increase in Src/Akt-dependent cNOS activation through phosphorylation and the inhibition of cNOS-mediated IKK-β S-nitrosylation. Our findings indicate ghrelin counters the proinflammatory consequences of H. pylori by interfering with the p38/ATF-2-induced AP-1 activation in association with concurrent up-regulation in Src/Akt-dependent cNOS phosphorylation.

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

Disturbances in nitric oxide synthase (NOS) and cyclooxygenase (COX) isozyme systems, manifested by the excessive NO and prostaglandin (PGE2) 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 iNOS-dependent 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 ghrelin-induced cSrc activation in modulation of gastric mucosal inflammatory responses to Helicobacter pylori

A peptide hormone, ghrelin, is recognized as an important modulator of gastric mucosal inflammatory responses to H. pylori through the regulation of nitric oxide synthase (NOS) system. As cSrc kinase plays a major role in transduction of signals that regulate the activity of NOS isozyme system, we investigated the influence of H. pylori LPS on the processes associated with Src activation in gastric mucosal cells. The LPS-induced drop in constitutive (c) cNOS activity and up-regulation in inducible (i) iNOS was associated with the suppression in cSrc kinase activity that was reflected in a decrease in its phosphorylation at Tyr⁴¹⁶. Further, the countering effect of ghrelin on the LPS-induced changes in cSrc activity and the extent of its phosphorylation was accompanied by a marked reduction in the activity of iNOS and an increase in cNOS activation through phosphorylation at Ser¹¹⁷⁹. Moreover, the effect of ghrelin on cSrc activation and its Tyr⁴¹⁶ phosphorylation was associated with the kinase S-nitrosylation that was susceptible to the blockage by cNOS inhibition. Our findings suggest that up-regulation in iNOS with H. pylori infection leads to disturbances in cNOS phosphorylation that exerts the detrimental effect on the processes of cSrc activation through cNOS-mediated S-nitrosylation. We also show that ghrelin attenuation of H. pylori-induced gastric mucosal inflammatory responses involves the enhancement in cSrc activation, elicited by the kinase S-nitrosylation and the increase in its phosphorylation at Tyr⁴¹⁶.

Cyclooxygenase-2 S-nitrosylation in salivary gland acinar cell inflammatory responses to <i>Porphyromonas gingivalis</i>: modulatory effect of ghrelin

Disturbances in nitric oxide synthase (NOS) system and the excessive prostaglandin (PGE2) generation are well-recognized features of oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis. Employing rat sublingual gland acinar cells, we show that P. gingivalis LPS-induced up-regulation in PGE2 generation and the enhancement in inducible (i) iNOS activity was associated with COX-2 activation through S-nitrosylation, and accompanied by the suppression in cSrc activity and the impairment in constitutive (c) cNOS phosphorylation. Further, we demonstrate that the countering effect of peptide hormone, ghrelin, on the LPS-induced changes was reflected in the increased cNOS activation through phosphorylation, repression in iNOS induction, and the reduction in PGE2 generation associated with the loss of COX-2 protein S-nitrosylation. Moreover, the effect of ghrelin on cNOS phosphorylation and the LPS-induced COX-2 S-nitrosylation was susceptible to the blockage by cSrc inhibition. Our findings suggest that P. gingivalis-induced up-regulation in iNOS leads to COX-2 S-nitrosylation and up-regulation in PGE2 generation, and that the countering effect of ghrelin is mediated through Src-dependent cNOS activation that is obligatory for the maintenance of iNOS gene suppression.

Ghrelin-induced cSrc activation through constitutive nitric oxide synthase-dependent S-nitrosylation in modulation of salivary gland acinar cell inflammatory responses to <i>Porphyromonas gingivalis</i>

A peptide hormone, ghrelin, recognized for its role in the regulation of nitric oxide production has emerged as an important modulator of oral mucosal inflammatory responses to periodontopathic bacterium, P. gingivalis. As cSrc kinase plays a major role in controlling the activity of nitric oxide synthase (NOS) system, in this study we investigated the influence of P. gingivalis LPS on the processes of Src activation in rat sublingual gland acinar cells. The LPS-induced enhancement in the activity of inducible (i) iNOS and the impairment in constitutive (c) cNOS were reflected in the suppression in cSrc activity and the extent of its phosphorylation at Tyr416. Further, we show that the countering effect of ghrelin on the LPS-induced changes in cSrc activity and the extent of its phosphorylation was accompanied by a marked reduction in iNOS and the increase in cNOS activation through phosphorylation at Ser1179. Moreover, the effect of ghrelin on cSrc activation was associated with the kinase S-nitrosylation that was susceptible to the blockage by cNOS inhibition. Our findings suggest that P. gingivalis-induced up-regulation in iNOS leads to disturbances in cNOS phosphorylation that exerts the detrimental effect on the processes of cSrc activation through cNOS mediated S-nitrosylation. We also show that the effect of ghrelin on P. gingivalis-induced inflammatory changes are manifested in the enhancement in cSrc activation through S-nitrosylation and the increase in its phosphorylation at Tyr416.

Helicobacter pylori Induces Disturbances in Gastric Mucosal Akt Activation through Inducible Nitric Oxide Synthase-Dependent S-Nitrosylation: Effect of Ghrelin

Gastric mucosal inflammatory response to H. pylori and its key virulence factor, lipopolysaccharide (LPS), are characterized by a massive rise in apoptosis and the disturbances in NO signaling pathways. Here, we report that H. pylori LPS-induced enhancement in the mucosal inducible nitric oxide synthase (iNOS) was associated with the suppression in Akt kinase activity and the impairment in constitutive nitric oxide synthase (cNOS) phosphorylation. Further, we demonstrate that the LPS effect on Akt inactivation, manifested in the kinase protein S-nitrosylation and a decrease in its phosphorylation at Ser473, was susceptible to suppression by iNOS inhibition. Moreover, the countering effect of hormone, ghrelin, on the LPS-induced changes in Akt activity was reflected in the loss in Akt S-nitrosylation and the increase in its phosphorylation at Ser473, as well as cNOS activation through phosphorylation. Our findings demonstrate that up-regulation in iNOS with H. pylori infection leads to Akt inactivation through S-nitrosylation that exerts the detrimental effect on the processes of cNOS activation through phosphorylation. We also report that ghrelin protection against H. pylori-induced disturbances is manifested in a marked increase in Akt activity and evoked by a decrease in the kinase S-nitrosylation and the increase in its phosphorylation at Ser473.

Constitutive nitric oxide synthase-mediated caspase-3 S-nitrosylation in ghrelin protection against Porphyromonas gingivalis-induced salivary gland acinar cell apoptosis

Recent advances in identifying the salivary constituents capable of influencing the oral mucosal inflammatory responses have brought to focus the importance of a peptide hormone, ghrelin. Here, we report on the involvement of ghrelin in controlling the apoptotic processes induced in sublingual salivary gland acinar cells by the lipopolysaccharide (LPS) of a periodontopathic bacterium, Porphyromonas gingivalis. We show that the countering effect of ghrelin on the LPS-induced acinar cell apoptosis was associated with the increase in constitutive nitric oxide synthase (cNOS) activity, and the reduction in caspase-3 and inducible nitric oxide synthase (iNOS). The loss in countering effect of ghrelin on the LPS-induced changes in apoptosis and caspase-3 activity was attained with Src kinase inhibitor, PP2, as well as Akt inhibitor, SH-5, and cNOS inhibitor, L-NAME, but not the iNOS inhibitor, 1400W. The effect of ghrelin on the LPS-induced changes in cNOS activity, moreover, was reflected in the increased cNOS phosphorylation that was sensitive to PP2 as well as SH-5. Furthermore, the ghrelin-induced up-regulation in cNOS activity was associated with the increase in caspase-3 S-nitrosylation that was susceptible to the blockage by SH-5 and L-NAME. The findings point to the involvement of ghrelin in Src/Akt kinase-mediated cNOS activation and the apoptogenic signal inhibition through the NO-induced caspase-3 S-nitrosylation.

Role of ghrelin in modulation of s-nitrosylation-Dependent akt inactivation induced in salivary gland acinar cells by porphyromonas gingivalis

Ghrelin, a peptide hormone, newly identified in oral mucosal tissue, has emerged re-cently as a principal modulator of the in-flammatory responses to bacterial infection through the regulation of nitric oxide syn-thase system. In this study, using rat sub-lingual salivary gland acinar cells, we report that lipopolysaccharide (LPS) of periodon-topathic bacterium, P. gingivalis- induced enhancement in the activity of inducible ni-tric oxide synthase (iNOS) was associated with the suppression in Akt kinase activity and the impairment in constitutive (c) cNOS phosphorylation. Further, we show that the detrimental effect of the LPS on Akt activa-tion, manifested in the kinase protein S-nitrosylation and a decrease in its phos-phorylation at Ser473, was susceptible to suppression by iNOS inhibitor, 1400W. Moreover, we demonstrate that a peptide hormone, ghrelin, countered the LPS- induced changes in Akt activity and NOS system. This effect of ghrelin was reflected in the decreased in Akt S-nitrosylation and the increase in its phosphorylation at Ser473, as well as cNOS activation through phos-phorylation. Our findings suggest that P. gingivalis-induced up-regulation in iNOS leads to Akt kinase inactivation through S-nitrosylation that impacts cNOS activation through phosphorylation. We also show that the countering effect of ghrelin on P. gingivalis-induced disturbances in Akt ac-tivation are manifested in a decrease in the kinase S-nitrosylation and the increase in its phosphorylation.

Ghrelin Protection against Lipopolysaccharide-Induced Gastric Mucosal Cell Apoptosis Involves Constitutive Nitric Oxide Synthase-Mediated Caspase-3 S-Nitrosylation

Ghrelin, a peptide hormone produced mainly in the stomach, has emerged as an important modulator of the inflammatory responses that are of significance to the maintenance of gastric mucosal integrity. Here, we report on the role of ghrelin in controlling the apoptotic processes induced in gastric mucosal cells by H. pylori lipopolysaccharide (LPS). The countering effect of ghrelin on the LPS-induced mucosal cell apoptosis was associated with the increase in constitutive nitric oxide synthase (cNOS) activity, and the reduction in caspase-3 and inducible nitric oxide synthase (NOS-2). The loss in countering effect of ghrelin on the LPS-induced changes in apoptosis and caspase-3 activity was attained with Src kinase inhibitor, PP2, as well as Akt inhibitor, SH-5, and cNOS inhibitor, L-NAME. Moreover, the effect of ghrelin on the LPS-induced changes in cNOS activity was reflected in the increased cNOS phosphorylation that was sensitive to SH-5. Furthermore, the ghrelin-induced up-regulation in cNOS activity was associated with the increase in caspase-3 S-nitrosylation that was susceptible to the blockage by L-NAME. Therefore, ghrelin protection of gastric mucosal cells against H. pylori LPS-induced apoptosis involves Src/Akt-mediated up-regulation in cNOS activation that leads to the apoptotic signal inhibition through the NO-induced caspase-3 S-nitrosylation.

Leptin protection of salivary gland acinar cells against ethanol cytotoxicity involves Src kinase-mediated parallel activation of prostaglandin and constitutive nitric oxide synthase pathways

Leptin, a pleiotropic cytokine secreted by adipocytes but also identified in salivary glands and saliva, is recognized as an important element of oral mucosal defense. Here, we report that in sublingual salivary glands leptin protects the acinar cells of against ethanol cytotoxicity. We show that ethanol- induced cytotoxicity, characterized by a marked drop in the acinar cell capacity for NO production, arachidonic acid release and prostaglandin generation, was subject to suppression by leptin. The loss in countering capacity of leptin on the ethanol-induced cytotoxicity was attained with cyclooxygenase inhibitor, indomethacin and nitric oxide synthase (cNOS) inhibitor, L-NAME, as well as PP2, an inhibitor of Src kinase. Indomethacin, while not affecting leptin-induced arachidonic acid release, caused the inhibition in PGE2 generation, pretreatment with L-NAME led to the inhibition in NO production, whereas PP2 exerted the inhibitory effect on leptin-induced changes in NO, arachidonic acid, and PGE2. The leptin-induced changes in arachidonic acid release and PGE2 generation were blocked by ERK inhibitor, PD98059, but not by PI3K inhibitor, wortmannin. Further, leptin suppression of ethanol cytotoxicity was reflected in the increased Akt and cNOS phosphorylation that was sensitive to PP2. Moreover, the stimulatory effect of leptin on the acinar cell cNOS activity was inhibited not only by PP2, but also by Akt inhibitor, SH-5, while wortmannin had no effect. Our findings demonstrate that leptin protection of salivary gland acinar cells against ethanol cytotoxicity involves Src kinase-mediated parallel activation of MAPK/ERK and Akt that result in up-regulation of the respective prostaglandin and nitric oxide synthase pathways.

Constitutive NO synthase regulates the Na+/Ca2+ exchanger in human T cells: role of [Ca2+]i and tyrosine phosphorylation.

For many types of cells, heat stress leads to an increase in intracellular free calcium concentration ([Ca2+](i)) that has been shown to trigger a wide variety of cellular responses. In T lymphocytes, for example, heat stress stimulates pathways that make them more susceptible to Fas/CD95-mediated apoptosis. Because of our interest in understanding more about the response of lymphocytes to various stressors, we used human peripheral and Jurkat T lymphocytes to investigate the effect of heat stress on calcium homeostasis. We found that peripheral and Jurkat T cells both exhibit cNOs activity but not iNOs activity. Heat stress increased NO production, which was inhibited by LNNA (a cNOs inhibitor) but not L-NIL (an iNOs inhibitor). Heat stress increased [Ca2+](i) in Jurkat T cells by decreasing the K(m) of the cell surface membrane Na+/Ca2+ exchanger for extracellular Ca2+. Heating also increased cNOs phosphorylation at tyrosine residues. In cells incubated with LNNA, heat stress promoted an increase in [Ca2+](i) and a decrease in [Na+](i) greater than in cells heated without LNNA, a larger decrease in K(m) of the Na+/Ca2+ exchanger for extracellular Ca2+, and decreased phosphorylation of cNOs. Our results suggest that cNOs plays an important regulatory role after heat stress. Heating appears to increase the phosphorylation of cNOs that is complexed with the Na+/Ca2+ exchanger to decrease its activity. This process is related to increased expression of Fas/CD95 on the cell surface, which might explain the apoptotic diathesis of lymphocytes after heat stress.

Different isoforms of tocopherols enhance nitric oxide synthase phosphorylation and inhibit human platelet aggregation and lipid peroxidation: implications in therapy with vitamin E.

alpha-Tocopherol has received much attention in the primary and secondary prevention of coronary artery disease. Absence of other isoforms, such as gamma- and delta-tocopherol, in commercial preparations of vitamin E may account for the inconsistent results of clinical trials. Since platelet aggregation is intimately involved in thrombogenesis, the relative effects of alpha-, gamma-, and delta-tocopherol and their combination were examined on human platelet aggregation, lipid peroxidation, and constitutive nitric oxide synthase (cNOS) activity. Human platelets were incubated with the three different isoforms of tocopherol and their combination for 30 minutes, and then ADP-induced platelet aggregation measured. All three isoforms of tocopherol markedly and similarly decreased platelet aggregation in a concentration (120--480 microM)-dependent manner. All three tocopherols also decreased the level of the lipid peroxidation product, malondialdehyde (MDA), and increased NO release (P < 0.05 vs control). These isoforms of tocopherol did not affect cNOS protein expression, but enhanced cNOS phosphorylation in platelets. The combination of three tocopherols in a concentration found in nature was more potent than alpha-, gamma-, or delta-tocopherol alone in this regard. These observations suggest that all three major isoforms of tocopherol have a similar effect on human platelet aggregation. The three isoforms appear to attenuate platelet aggregation at least in part via a decrease in free radical generation and an increase in platelet cNOS activity. The combination of tocopherols has a synergistic platelet inhibitory effect. Future clinical trials should concentrate on the combination of these three isoforms of tocopherols.