Association with nitric oxide synthase on insulin secretory granules regulates glucokinase protein levels

Glucokinase (GCK) association with insulin-secretory granules is controlled by interaction with nitric oxide synthase (NOS) and is reversed by GCK S-nitrosylation. Nonetheless, the function of GCK sequestration on secretory granules is unknown. Here we report that the S-nitrosylation blocking V367M mutation prevents GCK accumulation on secretory granules by inhibiting association with NOS. Expression of this mutant is reduced compared with a second S-nitrosylation blocking GCK mutant (C371S) that accumulates to secretory granules and is expressed at levels greater than wild type. Even so, the rate of degradation for wild type and mutant GCK proteins were not significantly different from one another, and neither mutation disrupted the ability of GCK to be ubiquitinated. Furthermore, gene silencing of NOS reduced endogenous GCK content but did not affect β-actin content. Treatment of GCK(C371S) expressing cells with short interfering RNA specific for NOS also blocked accumulation of this protein to secretory granules and reduced expression levels to that of GCK(V367M). Conversely, cotransfection of catalytically inactive NOS increased GCK-mCherry levels. Expression of GCK(C371S) in βTC3 cells enhanced glucose metabolism compared with untransfected cells and cells expressing wild type GCK, even though this mutant has slightly reduced enzymatic activity in vitro. Finally, molecular dynamics simulations revealed that V367M induces conformational changes in GCK that are similar to S-nitrosylated GCK, thereby suggesting a mechanism for V367M-inhibition of NOS association. Our findings suggest that sequestration of GCK on secretory granules regulates cellular GCK protein content, and thus cellular GCK activity, by acting as a storage pool for GCK proteins.     

A functional link between glucokinase binding to insulin granules and conformational alterations in response to glucose and insulin

Glucokinase (GK) activity is essential for the physiological regulation of insulin secretion by glucose. Because the enzyme exerts nearly total control over glucose metabolism in the beta-cell, even small changes in GK activity exert effects on glucose-stimulated insulin secretion and, consequently, the blood glucose concentration. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the association of GK with insulin granules is regulated by glucose in the beta-cell. Glucose stimulation increased the rate of fluorescence recovery after photobleaching of GK to insulin granules, indicating that GK is released into the cytoplasm after glucose stimulation. Changes in fluorescence resonance energy transfer between two different fluorescent protein variants inserted on opposing ends of GK were observed after glucose stimulation and correlated with increased enzyme activity. Furthermore, glucose-stimulated changes in GK regulation were blocked by two inhibitors of insulin secretion. Insulin treatment restored GK regulation in inhibited cells and stimulated GK translocation and activation by itself. Together, these data support a model for post-translational regulation of GK whereby insulin regulates both the association of GK with secretory granules and the activity of the enzyme within the pancreatic beta-cell.     

Intracellular degradation of insulin and crinophagy are maintained by nitric oxide and cyclo-oxygenase 2 activity in isolated pancreatic islets

BACKGROUND INFORMATION: Pancreatic beta-cells require an optimal insulin content to allow instantaneous secretion of insulin. This is maintained by insulin biosynthesis and intracellular degradation of insulin. Degradation may be effected by crinophagy, i.e. the fusion of secretory granules with lysosomes. IL-1beta (interleukin 1beta) induces distinct changes of beta-cell lysosomes. To study the mechanisms for intracellular insulin degradation and crinophagy, isolated mouse pancreatic islets were exposed to IL-1beta and known pathways for IL-1beta actions were blocked. Intracellular insulin degradation was determined by following the fate of radioactively labelled insulin. Crinophagy was studied by ultrastructural analysis. The effects of blocking pathways for IL-1beta were monitored by measurements of nitrite and PGE(2) (prostaglandin E(2)). RESULTS: IL-1beta caused an enhancement of islet intracellular insulin degradation and an increase in the lysosomal incorporation of beta-cell secretory granules. The effects of IL-1beta were abolished by aminoguanidine, a selective inhibitor of inducible NOS (nitric oxide synthase), or by rofecoxib, a specific inhibitor of COX-2 (cyclo-oxygenase 2). In the absence of IL-1beta, nitroarginine, which is a selective inhibitor of constitutive NOS, caused a decrease in intracellular degradation of insulin in parallel with a decreased production of NO and PGE(2) by the islets. CONCLUSIONS: The correlation between the enhanced intracellular insulin degradation and lysosomal changes caused by IL-1beta suggests that insulin degradation may be effected by crinophagy. Under physiological conditions, significant beta-cell degradation of insulin may depend on the activity of COX-2, possibly stimulated by endogenous NO.     

Argininosuccinate synthetase is reversibly inactivated by S-nitrosylation in vitro and in vivo

Prior studies have demonstrated that the substrate for NO synthesis, l-arginine, can be regenerated from the NOS co-product l-citrulline. This requires the sequential action of two enzymes, argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). AS activity has been shown to be rate-limiting for high output NO synthesis by immunostimulant-activated cells and represents a potential site for metabolic control of NO synthesis. We now demonstrate that NO mediates reversible S-nitrosylation and inactivation of AS in vitro and in lipopolysaccharide-treated cells and mice. Using a novel mass spectrometry-based method, we show that Cys-132 in human AS is the sole target for S-nitrosylation among five Cys residues. Mutagenesis studies confirm that S-nitrosylation of Cys-132 is both necessary and sufficient for the inhibition of AS by NO donors. S-nitroso-AS content is regulated by cellular glutathione levels and selectively influences NO production when citrulline is provided to cells as a protosubstrate of NOS but not when l-arginine is provided. A phylogenetic comparison of AS sequences suggests that Cys-132 evolved as a site for post-translational regulation of activity in the AS in NOS-expressing species, endowing NO with the capacity to limit its own synthesis by restricting arginine availability.     

Nitric Oxide Synthase Activity in Retinas from Non-Insulin-Dependent Diabetic Goto-Kakizaki Rats: Correlation with Blood–Retinal Barrier Permeability

The aim of this work was to examine whether the non-insulin-dependent diabetic Goto-Kakizaki (GK) rats develop retinal changes with similar characteristics to those observed in insulin-dependent diabetic rats in what concerns blood-retinal barrier (BRB) permeability, nitric oxide (NO) production, and retinal IL-1beta level. BRB permeability was evaluated by vitreous fluorophotometry. NO synthase (NOS) activity was assessed by the production of l-[(3)H]-citrulline and retinal IL-1beta level was determined by ELISA. The expression of the inducible isoform of NOS (iNOS) protein was evaluated by Western blot analysis and immunohistochemistry. The in vivo studies indicated that in GK rats the BRB permeability to fluorescein was increased (787.81 +/- 68 min(-1)) in comparison to that in normal Wistar rats (646.6 +/- 55 min(-1)). The ex vivo studies showed that in retinas from GK rats the NOS activity was higher (207 +/- 28.9 pmol l-[(3)H]-citrulline/mg protein/30 min) than that in normal Wistar rats (125 +/- 32.3 pmol l-[(3)H]-citrulline/mg protein/30 min). These results were correlated with an increase in the protein level of iNOS in the retinas of GK rats, which was confirmed not only by the study of the iNOS protein expression but also by the use of NOS activity inhibitors. Indeed, the data about the effect of specific inhibitors on the NOS activity revealed that in retinas from GK rats the most effective inhibitor was aminoguanidine, which predominantly inhibits the iNOS isoform whereas in retinas from normal Wistar rats it was N(G) nitro l-arginine that predominantly inhibits the constitutive isoforms of NOS. In summary, in retinas from GK rats there is an increased production of NO which may contribute to the BRB breakdown.     

Neuronal nitric oxide synthase-induced S-nitrosylation of H-Ras inhibits calcium ionophore-mediated extracellular-signal-regulated kinase activity

nNOS (neuronal nitric oxide synthase) is a constitutively expressed enzyme responsible for the production of NO* from L-arginine and O2. NO* acts as both an intra- and an inter-cellular messenger that mediates a variety of signalling pathways. Previous studies from our laboratory have demonstrated that nNOS production of NO* blocks Ca2+-ionophore-induced activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) of the mitogen-activated protein kinases through a mechanism involving Ras G-proteins and Raf-1 kinase. Herein we describe a mechanism by which NO* blocks Ca2+-mediated ERK1/2 activity through direct modification of H-Ras. Ca2+-mediated ERK1/2 activation in NO*-producing cells could be restored by exogenous expression of constitutively active mitogen-activated protein kinase kinase 1. In contrast, exogenous expression of constitutively active mutants of Raf-1 and H-Ras only partially restored ERK1/2 activity, by 50% and 10% respectively. On the basis of these findings, we focused on NO*-mediated mechanisms of H-Ras inhibition. Assays for GTP loading and H-Ras interactions with the Ras-binding domain on Raf-1 demonstrated a decrease in H-Ras activity in the presence of NO*. We demonstrate that S-nitrosylation of H-Ras occurs in nNOS-expressing cells activated with Ca2+ ionophore. Mutation of a putative nitrosylation site at Cys118 inhibited S-nitrosylation and restored ERK1/2 activity by constitutively active H-Ras even in the presence of NO*. These findings indicate that intracellular generation of NO* by nNOS leads to S-nitrosylation of H-Ras, which interferes with Raf-1 activation and propagation of signalling through ERK1/2.     

Basal and stimulated protein S-nitrosylation in multiple cell types and tissues.

There is substantial evidence that protein S-nitrosylation provides a significant route through which nitric oxide (NO)-derived bioactivity is conveyed. However, most examples of S-nitrosylation have been characterized on the basis of analysis in vitro, and relatively little progress has been made in assessing the participant forms of nitric-oxide synthase (NOS) or the dynamics of protein S-nitrosylation in situ. Here we utilize antibodies specific for the nitrosothiol (SNO) moiety to provide an immunohistochemical demonstration that protein S-nitrosylation is coupled to the activity of each of the major forms of NOS. In cultured endothelial cells, SNO-protein immunoreactivity increases in response to Ca(2+)-stimulated endothelial NOS (eNOS) activity, and in aortic rings, endothelium-derived and eNOS-mediated relaxation (EDRF) is coupled to increased protein S-nitrosylation in both endothelial and associated smooth muscle cells. In cultured macrophages, SNO-protein levels increase upon cytokine induction of induced NOS (iNOS), and in PC12 cells, increased protein S-nitrosylation is linked to nerve growth factor induction of neuronal NOS (nNOS). In addition, we describe developmental and pathophysiological increases in SNO-protein immunoreactivity within human lung. These results, which demonstrate Ca(2+), neurohumoral, growth factor, cytokine, and developmental regulation of protein S-nitrosylation that is coupled to NOS expression and activity, provide unique evidence for the proposition that this ubiquitous NO-derived post-translational protein modification serves as a major effector of NO-related bioactivity.     

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.     

Excessive islet NO generation in type 2 diabetic GK rats coincides with abnormal hormone secretion and is counteracted by GLP-1

Background

A distinctive feature of type 2 diabetes is inability of insulin-secreting β-cells to properly respond to elevated glucose eventually leading to β-cell failure. We have hypothesized that an abnormally increased NO production in the pancreatic islets might be an important factor in the pathogenesis of β-cell dysfunction.

Principal Findings

We show now that islets of type 2 spontaneous diabetes in GK rats display excessive NO generation associated with abnormal iNOS expression in insulin and glucagon cells, increased ncNOS activity, impaired glucose-stimulated insulin release, glucagon hypersecretion, and impaired glucose-induced glucagon suppression. Pharmacological blockade of islet NO production by the NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) greatly improved hormone secretion from GK islets suggesting islet NOS activity being an important target to inactivate for amelioration of islet cell function. The incretin hormone GLP-1, which is used in clinical practice suppressed iNOS and ncNOS expression and activity with almost full restoration of insulin release and partial restoration of glucagon release. GLP-1 suppression of iNOS expression was reversed by PKA inhibition but unaffected by the proteasome inhibitor MG132. Injection of glucose plus GLP-1 in the diabetic rats showed that GLP-1 amplified the insulin response but induced a transient increase and then a poor depression of glucagon.

Conclusion

The results suggest that abnormally increased NO production within islet cells is a significant player in the pathogenesis of type 2 diabetes being counteracted by GLP-1 through PKA-dependent, nonproteasomal mechanisms.     

Exophilin4/Slp2-a targets glucagon granules to the plasma membrane through unique Ca2+-inhibitory phospholipid-binding activity of the C2A domain

Rab27a and Rab27b have recently been recognized to play versatile roles in regulating the exocytosis of secretory granules and lysosome-related organelles by using multiple effector proteins. However, the precise roles of these effector proteins in particular cell types largely remain uncharacterized, except for those in pancreatic beta cells and in melanocytes. Here, we showed that one of the Rab27a/b effectors, exophilin4/Slp2-a, is specifically expressed in pancreatic alpha cells, in contrast to another effector, granuphilin, in beta cells. Like granuphilin toward insulin granules, exophilin4 promotes the targeting of glucagon granules to the plasma membrane. Although the interaction of granuphilin with syntaxin-1a is critical for the targeting activity, exophilin4 does this primarily through the affinity of its C2A domain toward the plasma membrane phospholipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Notably, the binding activity to phosphatidylserine is inhibited by a physiological range of the Ca(2+) concentration attained after secretagogue stimulation, which presents a striking contrast to the Ca(2+)-stimulatory activity of the C2A domain of synaptotagmin I. Analyses of the mutant suggested that this novel Ca(2+)-inhibitory phospholipid-binding activity not only mediates docking but also modulates the subsequent fusion of the secretory granules.     

Spontaneous and receptor-controlled soluble guanylyl cyclase activity in anterior pituitary cells

Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.     

Enhanced expression of neuronal nitric oxide synthase in islets of exercise-trained rats

It is well known that glucose-stimulated insulin secretion (GSIS) decreases after exercise training. In the present study, we investigated the effects of exercise training (9 weeks of running) on the activity of glucokinase (GK), the production of nitric oxide (NO), and the protein expressions of both glucose transporter-2 (GLUT-2) and NO synthase (NOS) in rat pancreatic islets. Exercise training significantly reduced GSIS, with decreases in GK activity and GLUT-2 protein expression. The NO releases and cGMP contents were higher in the islets of trained rats than in those of control rats. Exercise training enhanced cNOS activity, the protein expression of both neuronal nitric oxide synthase (nNOS) and calmodulin, and NADPH-cytochrome c reductase activity in the homogenates of islets. Thus, exercise training-induced reduction of GSIS would result from, at least in part, decreases in both glucose entry and the first step in glycolytic utilization of glucose. Moreover, exercise training could enhance the protein expression of nNOS, which in turn enhances two catalytic activities of nNOS, an NO production and a cytochrome c reductase activity.     

Differential expression of protein kinase C isoenzymes related to high nitric oxide synthase activity in a T lymphoma cell line

Protein kinase C (PKC) is critical for T lymphocyte activation and proliferation, while nitric oxide synthase (NOS) may function both as an activator or inhibitor of T cell apoptosis. Both enzymatic activities were studied in T lymphoma cells in comparison to normal and activated T lymphocytes. Here we show a higher translocation of PKC in BW5147 lymphoma cells than in mitogen-stimulated T lymphocytes. Tumor cells overexpressed PKC zeta isoform, while high levels of the PKC beta isotype were found in mitogen-stimulated T lymphocytes. Moreover, tumoral T cells showed high NOS activity, almost undetectable in normal or stimulated T lymphocytes. PKC and NOS inhibitors or the intracellular delivery of an anti-PKC zeta antibody diminished both NO production and proliferation in tumor cells.These results suggest that atypical PKC zeta isoform expression and its association with NOS activity regulation would participate in the multistep process leading to BW5147 cell malignant transformation.     

Arginase inhibition restores in vivo coronary microvascular function in type 2 diabetic rats

Nitric oxide (NO) is crucial for maintaining normal endothelial function and vascular integrity. Increased arginase activity in diabetes might compete with NO synthase (NOS) for their common substrate arginine, resulting in diminished production of NO. The aim of this study was to evaluate coronary microvascular function in type 2 diabetic Goto-Kakizaki (GK) rats using in vivo coronary flow velocity reserve (CFVR) and the effect of arginase inhibition to restore vascular function. Different groups of GK and Wistar rats were given vehicle, the arginase inhibitor N(ω)-hydroxy-nor-l-arginine (nor-NOHA), l-arginine, and the NOS inhibitor N(G)-monomethyl -l-arginine (l-NMMA). GK rats had impaired CFVR compared with Wistar rats (1.31 ± 0.09 vs. 1.87 ± 0.05, P < 0.001). CFVR was restored by nor-NOHA treatment compared with vehicle in GK rats (1.71 ± 0.13 vs. 1.23 ± 0.12, P < 0.05) but remained unchanged in Wistar rats (1.88 ± 0.10 vs. 1.79 ± 0.16). The beneficial effect of nor-NOHA in GK rats was abolished after NOS inhibition. CFVR was not affected by arginine compared with vehicle. Arginase II expression was increased in the aorta and myocardium from GK rats compared with Wistar rats. Citrulline-to-ornithine and citrulline-to-arginine ratios measured in plasma increased significantly more in GK rats than in Wistar rats after nor-NOHA treatment, suggesting a shift of arginine utilization from arginase to NOS. In conclusion, coronary artery microvascular function is impaired in the type 2 diabetic GK rat. Treatment with nor-NOHA restores the microvascular function by a mechanism related to increased utilization of arginine by NOS and increased NO availability.     

Identification of a chromogranin A domain that mediates binding to secretogranin III and targeting to secretory granules in pituitary cells and pancreatic beta-cells

Chromogranin A (CgA) is transported restrictedly to secretory granules in neuroendocrine cells. In addition to pH- and Ca(2+)-dependent aggregation, CgA is known to bind to a number of vesicle matrix proteins. Because the binding-prone property of CgA with secretory proteins may be essential for its targeting to secretory granules, we screened its binding partner proteins using a yeast two-hybrid system. We found that CgA bound to secretogranin III (SgIII) by specific interaction both in vitro and in endocrine cells. Localization analysis showed that CgA and SgIII were coexpressed in pituitary and pancreatic endocrine cell lines, whereas SgIII was not expressed in the adrenal glands and PC12 cells. Immunoelectron microscopy demonstrated that CgA and SgIII were specifically colocalized in large secretory granules in male rat gonadotropes, which possess large-type and small-type granules. An immunocytochemical analysis revealed that deletion of the binding domain (CgA 48-111) for SgIII missorted CgA to the constitutive pathway, whereas deletion of the binding domain (SgIII 214-373) for CgA did not affect the sorting of SgIII to the secretory granules in AtT-20 cells. These findings suggest that CgA localizes with SgIII by specific binding in secretory granules in SgIII-expressing pituitary and pancreatic endocrine cells, whereas other mechanisms are likely to be responsible for CgA localization in secretory granules of SgIII-lacking adrenal chromaffin cells and PC12 cells.     

Dephosphorylation of beta2-syntrophin and Ca2+/mu-calpain-mediated cleavage of ICA512 upon stimulation of insulin secretion

Islet cell autoantigen (ICA) 512 is a receptor-tyrosine phosphatase-like protein associated with the secretory granules of neuroendocrine cells, including pancreatic beta-cells. Binding of its cytoplasmic tail to beta2-syntrophin suggests that ICA512 connects secretory granules to the utrophin complex and the actin cytoskeleton. Here we show that stimulation of insulin secretion from INS-1 cells triggers the biosynthesis of pro-ICA512 and the degradation of its mature form. Inhibition of calpain, which is activated upon stimulation of insulin secretion, prevents the Ca2+-dependent proteolysis of ICA512. In vitro mu-calpain cleaves ICA512 between a putative PEST domain and the beta2-syntrophin binding site, whereas binding of ICA512 to beta2-syntrophin protects the former from cleavage. beta2-syntrophin and its F-actin-binding protein utrophin are enriched in subcellular fractions containing secretory granules. ICA512 preferentially binds phospho-beta2-syntrophin and stimulation of insulin secretion induces the Ca2+-dependent, okadaic acid-sensitive dephosphorylation of beta2-syntrophin. Similarly to calpeptin, okadaic acid inhibits ICA512 proteolysis and insulin secretion. Thus, stimulation of insulin secretion might promote the mobilization of secretory granules by inducing the dissociation of ICA512 from beta2-syntrophin-utrophin complexes and the cleavage of the ICA512 cytoplasmic tail by mu-calpain.     

Activation of cytosolic phospholipase A2alpha through nitric oxide-induced S-nitrosylation. Involvement of inducible nitric-oxide synthase and cyclooxygenase-2

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha) is the rate-limiting key enzyme that cleaves arachidonic acid (AA) from membrane phospholipids for the biosynthesis of eicosanoids, including prostaglandin E(2) (PGE(2)), a key lipid mediator involved in inflammation and carcinogenesis. Here we show that cPLA(2)alpha protein is S-nitrosylated, and its activity is enhanced by nitric oxide (NO). Forced expression of inducible nitric-oxide synthase (iNOS) in human epithelial cells induced cPLA(2)alpha S-nitrosylation, enhanced its catalytic activity, and increased AA release. The iNOS-induced cPLA(2)alpha activation is blocked by the specific iNOS inhibitor, 1400W. The addition of the NO donor, S-nitrosoglutathione, to isolated cell lysates or purified recombinant human cPLA(2)alpha protein induced S-nitrosylation of cPLA(2)alpha in vitro. Incubation of cultured cells with the iNOS substrate L-arginine and NO donor significantly increased cPLA(2)alpha activity and AA release. These findings demonstrate that iNOS-derived NO S-nitrosylates and activates cPLA(2)alpha in human cells. Site-directed mutagenesis revealed that Cys-152 of cPLA(2)alpha is critical for S-nitrosylation. Furthermore, COX-2 induction or expression markedly enhanced iNOS-induced cPLA(2)alpha S-nitrosylation and activation, leading to 9-, 23-, and 20-fold increase of AA release and 100-, 38-, and 88-fold of PGE(2) production in A549, SG231, and HEK293 cells, respectively, whereas COX-2 alone leads to less than 2-fold change. These results indicate that COX-2 has the ability to enhance iNOS-induced cPLA(2)alpha S-nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA(2)alpha, and COX-2. Since COX-2 enhances the formation of cPLA(2)alpha-iNOS binding complex, it appears that COX-2-induced augmentation of cPLA(2)alpha S-nitrosylation is mediated at least in part through increased association between iNOS and cPLA(2)alpha. These findings disclose a novel link among cPLA(2)alpha, iNOS, and COX-2, which form a multiprotein complex leading to cPLA(2)alpha S-nitrosylation and activation. Therefore, therapy aimed at disrupting this interplay may represent a promising strategy to effectively inhibit PGE(2) production and prevent inflammation and carcinogenesis.