In vivo degradation of nitric oxide synthase (NOS) and heat shock protein 90 (HSP90) by calpain is modulated by the formation of a NOS-HSP90 heterocomplex

We have shown previously that isolated heat shock protein 90 (HSP90) and nitric oxide synthase (NOS), once associated in a heterocomplex, become completely resistant to calpain digestion. In this study, it is shown that, in vivo, under conditions of calpain activation, the protection of NOS degradation occurs. In addition, the extent of NOS degradation is a function of the level of HSP90 expression. Thus, in rat brain, which contains a large excess of HSP90, almost all neuronal NOS is associated with the chaperone protein. In this condition, neuronal NOS retains its full catalytic activity, although limited proteolytic conversion to still active low-molecular-mass (130 kDa) products takes place. In contrast, in aorta, which contains much smaller amounts of HSP90, endothelial NOS is not completely associated with the chaperone, and undergoes extensive degradation with a loss of protein and catalytic activity. On the basis of these findings, we propose a novel role of the HSP90-NOS heterocomplex in protecting in vivo NOS from proteolytic degradation by calpain. The efficiency of this effect is directly related to the level of intracellular HSP90 expression, generating a high HSP90 to NOS ratio, which favours both the formation and stabilization of the HSP90-NOS heterocomplex. This condition seems to occur in rat brain, but not in aorta, thus explaining the higher vulnerability to proteolytic degradation of endothelial NOS relative to neuronal NOS.     

Proteolytic cleavage of inducible nitric oxide synthase (iNOS) by calpain I

Proteolytic degradation of inducible nitric oxide synthase (iNOS or NOS2; EC 1.14.13.39) is one of the key steps by which the synthetic glucocorticoid dexamethasone controls the amount of iNOS protein and thus the production of nitric oxide (NO) in interferon-γ-stimulated RAW 264.7 cells. In the present study we examined the role of the calmodulin (CaM)-binding site present within iNOS protein for the proteolytic degradation by the calcium-dependent neutral cysteine protease calpain I (EC 3.4.22.17). Using pulse chase experiments as well as cell-free degradation assays we show that the iNOS monomer is a direct substrate for cleavage by calpain I. Two structural determinants are involved in proteolytic cleavage, the canonical CaM-binding domain present at amino acids 501–532 and a conformational determinant located within iNOS. The access of the CaM-binding region appears to be critical for substrate cleavage as incubation of in vitro synthesized iNOS with purified CaM inhibits iNOS degradation by calpain I. Moreover, cytosolic CaM levels are decreased upon treatment of RAW 264.7 cells with dexamethasone as assessed by immunoprecipitation. The data shown herein provide novel insights into the underlying mechanisms involved in the anti-inflammatory actions of glucocorticoids.     

Proteolytic cleavage of inducible nitric oxide synthase (iNOS) by calpain I.

Proteolytic degradation of inducible nitric oxide synthase (iNOS or NOS2; EC 1.14.13.39) is one of the key steps by which the synthetic glucocorticoid dexamethasone controls the amount of iNOS protein and thus the production of nitric oxide (NO) in interferon-gamma-stimulated RAW 264.7 cells. In the present study we examined the role of the calmodulin (CaM)-binding site present within iNOS protein for the proteolytic degradation by the calcium-dependent neutral cysteine protease calpain I (EC 3.4.22.17). Using pulse chase experiments as well as cell-free degradation assays we show that the iNOS monomer is a direct substrate for cleavage by calpain I. Two structural determinants are involved in proteolytic cleavage, the canonical CaM-binding domain present at amino acids 501-532 and a conformational determinant located within iNOS. The access of the CaM-binding region appears to be critical for substrate cleavage as incubation of in vitro synthesized iNOS with purified CaM inhibits iNOS degradation by calpain I. Moreover, cytosolic CaM levels are decreased upon treatment of RAW 264.7 cells with dexamethasone as assessed by immunoprecipitation. The data shown herein provide novel insights into the underlying mechanisms involved in the anti-inflammatory actions of glucocorticoids.     

Determination of the enhancing action of HSP90 on neuronal nitric oxide synthase by EPR spectroscopy

Recent studies showed that heatshock protein 90 (HSP90) enhances nitric oxide (NO) synthesis fromendothelial and neuronal NO synthase (eNOS and nNOS, respectively).However, these findings were based on indirect NO measurements.Moreover, although our previous studies showed that the action of HSP90involves increased Ca²⁺/calmodulin (Ca²⁺/CaM)binding, quantitative measurements of the effect of HSP90 on CaMbinding to nNOS have been lacking. With electron paramagnetic resonancespectroscopy, we directly measured NO signals from purified nNOS. HSP90augmented NO formation from nNOS in a dose-dependent manner. Tryptophanfluorescence-quenching measurements revealed that HSP90 markedlyreduced the K_d of CaM to nNOS (0.5 ± 0.1 nM vs. 9.4 ± 1.8 nM in the presence and absence of HSP90,P < 0.01). Ca²⁺ ionophore triggered strongNO production from nNOS-transfected cells, and this was significantlyreduced by the HSP90 inhibitor geldanamycin. Thus these studies providedirect evidence demonstrating that HSP90 enhances nNOS catalyticfunction in vitro and in intact cells. The effect of HSP90 is mediatedby the enhancement of CaM binding to nNOS.

     

Functional role of HSP90 complexes with endothelial nitric-oxide synthase (eNOS) and calpain on nitric oxide generation in endothelial cells

Although several reports have indicated that eNOS is a highly sensitive calpain substrate, the occurrence of a concomitant Ca(2+)-dependent activation of the synthase and of the protease has never been analyzed in specific direct experiments. In this study, we have explored in vivo how eNOS can undergo Ca(2+)-dependent translocation and activation, protected against degradation by activated calpain. Here we demonstrate that following a brief exposure to Ca(2+)-loading, the cytosolic eNOS-HSP90 complex recruits calpain in a form in which the chaperone and the synthase are almost completely resistant to digestion by the protease. Furthermore, in the presence of the HSP90 inhibitor geldanamycin, a significant decrease in NO production and an extensive degradation of eNOS protein occurs, indicating that dissociation from membranes and association with the chaperone is correlated to the protection of the synthase. Experiments with isolated membrane preparations confirm the primary role of HSP90 in dissociation of eNOS from caveolae. Prolonged exposure of cells to Ca(2+)-loading resulted in an extensive degradation of both eNOS and HSP90, accompanied by a large suppression of NO production. We propose that the protective effect exerted by HSP90 on eNOS degradation mediated by calpain represents a novel and critical mechanism that assures the reversibility of the intracellular trafficking and activation of the synthase.     

Inducible nitric oxide synthase expression is inhibited by myeloperoxidase.

Nitric oxide (NO) plays key roles in vasodilation and host defense, yet the overproduction of NO by inducible nitric oxide synthase (iNOS) at inflammatory sites can also be pathogenic. Here, we investigate the role of MPO in modulating the induction of iNOS by IFNgamma/LPS (IL). In monocyte-macrophages (Mvarphi) treated with IL, MPO gene expression was found to be downregulated as iNOS was upregulated. In Mvarphi from MPO-knockout (KO) mice, the induction of iNOS by IL was earlier and higher than in MPO-positive cells, suggesting MPO is inhibitory. Consistent with that interpretation, the addition of purified MPO enzyme to cultured macrophages inhibited iNOS induction by IL. In addition, an inhibitor of MPO enzyme, 4-aminobenzohydrazide, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. Similarly, taurine, a scavenger of MPO-generated HOCl, enhanced iNOS induction in MPO-positive cells, but not in MPO-KO cells. MPO affects an early event, suppressing iNOS induction when added within 2h of IL, but not when added several hours after IL. The suppression by MPO was alleviated by NO donor, sodium nitroprusside, suggesting the suppression results from scavenging of NO by MPO. This interpretation is consistent with earlier reports that MPO consumes NO, and that low levels of NO donor augment induction of iNOS by IFNgamma/LPS. The implication of these findings is that MPO acts as gatekeeper, suppressing the deleterious induction of iNOS at inflammatory sites by illegitimate signals. The combined signaling of IFNgamma/LPS overrides the gatekeeper function by suppressing MPO gene expression.     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

Expression of nitric oxide synthase isoforms is reduced in late-gestation ovine fetal brainstem

Fetal baroreflex responsiveness increases in late gestation. An important modulator of baroreflex activity is the generation of nitric oxide in the brainstem nuclei that integrate afferent and efferent reflex activity. The present study was designed to test the hypothesis that nitric oxide synthase (NOS) isoforms are expressed in the fetal brainstem and that the expression of one or more of these enzymes is reduced in late gestation. Brainstem tissue was rapidly collected from fetal sheep of known gestational ages (80, 100, 120, 130, 145 days gestation and 1 day and 1 wk postnatal). Neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) mRNA was measured using real-time PCR methodology specific for ovine NOS isoforms. The three enzymes were measured at the protein level using Western blot methodology. In tissue prepared for histology separately, the cellular pattern of immunostaining was identified in medullae from late-gestation fetal sheep. Fetal brainstem contained mRNA and protein of all three NOS isoforms, with nNOS the most abundant, followed by iNOS and eNOS, respectively. nNOS and iNOS mRNA abundances were highest at 80 days' gestation, with statistically significant decreases in abundance in more mature fetuses and postnatal animals. nNOS and eNOS protein abundance also decreased as a function of developmental age. nNOS and eNOS were expressed in neurons, iNOS was expressed in glia, and eNOS was expressed in vascular endothelial cells. We conclude that all three isoforms of NOS are constitutively expressed within the fetal brainstem, and the expression of all three forms is reduced with advancing gestation. We speculate that the reduced expression of NOS in this brain region plays a role in the increased fetal baroreflex activity in late gestation.     

Proteolytic cleavage confers nitric oxide synthase inducing activity upon prolactin

Prolactin (PRL), originally associated with milk secretion, is now known to possess a wide variety of biological actions and diverse sites of production beyond the pituitary. Proteolytic cleavage is a common post-translational modification that can either activate precursor proteins or confer upon the peptide fragment unique biological actions not exerted by the parent molecule. Recent studies have demonstrated that the 16-kDa N-terminal proteolytic cleavage product of PRL (16K-PRL) acts as a potent inhibitor of angiogenesis. Despite previous demonstrations of 16K-PRL production in vivo, biological functions beyond its antiangiogenic actions remain unknown. Here we show that 16K-PRL, but not full-length PRL, acts to promote the expression of the inducible isoform of nitric oxide synthase (iNOS) and nitric oxide (*NO) production by pulmonary fibroblasts and alveolar type II cells with potency comparable with the proinflammatory cytokines interleukin-1beta, interferon gamma, and tumor necrosis factor alpha. The differential effect of 16K-PRL versus PRL occurs through a receptor distinct from known PRL receptors. Additionally, pulmonary fibroblasts express the PRL gene and endogenously produce 16K-PRL, suggesting that this pathway may serve both autocrine and paracrine roles in the regulation of *NO production. These results reveal that proteolytic cleavage of PRL confers upon this classical hormone potent iNOS inducing activity, suggesting its role in inflammatory/immune processes.     

Expression of nitric oxide synthase isoforms and detection of nitric oxide in rat placenta

Nitric oxide (NO) production in therat placenta was monitored and quantified by electron paramagneticresonance (EPR) spectroscopy with hemoglobin and anFe-N-(dithiocarboxy)sarcosine (DTCS) complex as NO-trappingreagents. Expression of nitric oxide synthase (NOS) isoformswas also examined by quantitative RT-PCR analysis. The EPR spectrum ofthe placenta with hemoglobin trapping showed a three-line hyperfinestructure (g = 2.008 and a = 1.66-mT). The EPR signal was diminished after the placenta was homogenized or the NOSinhibitor L-NAME was administered to pregnant rats.Therefore, the specific signal was definitely identified as beingderived from endogenous NO spin-trapped by hemoglobin, and the EPRspectrum showed that the NO adduct existed as a pentacoordinate -NOheme species. The EPR spectrum of the placenta with Fe-DTCS trapping showed a triplet signal (g = 2.038) derived from anNO-Fe-DTCS complex. The height of the triplet signal did not varysignificantly with gestational stage during the last few days ofgestation. At the gestational stages examined, the level of NOS II mRNAexpression was significantly higher than that of NOS III mRNA. NOS IIexpression in term (day 21.5) placenta was significantlyincreased compared with that in preterm (day 19.5) placenta(P < 0.01, n = 4 or 5). These resultssuggest that NOS II is the predominant producer of NO in the placentaand that NOS II-generated NO plays significant roles in the maintenanceof placental functions immediately before birth.

     

Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased nitric oxide (NO) release and impaired pulmonary vasodilation. We investigated the hypothesis that decreased association of heat shock protein 90 (HSP90) with endothelial NO synthase (eNOS) impairs NO release and vasodilation in PPHN. The responses to the NOS agonist ATP were investigated in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus, and in sham ligation controls. ATP caused dose-dependent vasodilation in control pulmonary resistance arteries, and this response was attenuated in PPHN vessels. The response of control pulmonary arteries to ATP was attenuated by NG-nitro-l-arginine methyl ester (l-NAME), a NOS antagonist, and geldanamycin, an inhibitor of HSP90-eNOS interaction. The attenuated response to ATP observed in PPHN was improved by pretreatment of vessels with l-NAME or 4,5-dihydroxy-1,3-benzene-disulfonate, a superoxide scavenger. Pulmonary arteries from PPHN lambs had decreased basal levels of HSP90 in association with eNOS. Association of HSP90 with eNOS and NO release increased in response to ATP in control pulmonary artery endothelial cells, but not in cells from PPHN lambs. Decreased HSP90-eNOS interactions may contribute to the impaired NO release and vasodilation observed in the ductal ligation model of PPHN.     

Role of nitric oxide synthase isoforms in glucose-stimulated insulin release

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.     

Inhibition of nitric oxide synthase isoforms by tris-malonyl-C(60)-fullerene adducts

C(3)-tris-malonyl-C(60)-fullerene and D(3)-tris-malonyl-C(60)-fullerene derivatives inhibit citrulline and NO formation by all three nitric oxide synthase isoforms in a manner fully reversible by dilution. The inhibition of citrulline formation by C(3)-tris-malonyl-C(60)-fullerene occurs with IC(50) values of 24, 17, and 123 microM for the neuronal, endothelial, and inducible nitric oxide synthase (NOS) isoforms, respectively. As measured at 100 microM l-arginine, neuronal NOS-catalyzed nitric oxide formation was inhibited 50% at a concentration of 25 microM C(3)-tris-malonyl-C(60)-fullerene. This inhibition was a multisite, positively cooperative inhibition with a Hill coefficient of 2.0. C(3)-tris-malonyl-C(60)-fullerene inhibited the arginine-independent NADPH-oxidase activity of nNOS with an IC(50) value of 22 microM but had no effects on its cytochrome c reductase activity at concentrations as high as 300 microM. The inhibition of nNOS activity by C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of product formation but did not alter the EC(50) value for activation by calmodulin. C(3)-tris-malonyl-C(60)-fullerene reduced the maximal velocity of citrulline formation by inducible NOS without altering the K(m) for l-arginine substrate or the EC(50) value for tetrahydrobiopterin cofactor. As measured by sucrose density gradient centrifugation, fully inhibitory concentrations of C(3)-tris-malonyl-C(60)-fullerene did not produce a dissociation of nNOS dimers into monomers. These observations are consistent with the proposal that C(3)-tris-malonyl-C(60)-fullerene inhibits the inter-subunit transfer of electrons, presumably by a reversible distortion of the dimer interface.     

Effects of p38MAPK isoforms on renal mesangial cell inducible nitric oxide synthase expression

Several related isoforms of p38^MAPK have been identified and cloned in many species. Although they all contain the dual phosphorylation motif TGY, the expression of these isoforms is not ubiquitous. p38 and -2 are ubiquitously expressed, whereas p38 and - appear to have more restricted expression. Because there is evidence for selective activation by upstream kinases and selective preference for downstream substrates, the functions of these conserved proteins is still incompletely understood. We have demonstrated that the renal mesangial cell expresses the mRNA for all the isoforms of p38^MAPK, with p38 mRNA expressed at the highest level, followed by p38 and the lowest levels of expression by p382 and -. To determine the functional effects of these proteins on interleukin (IL)-1-induced inducible nitric oxide synthase (iNOS) expression, we transduced TAT-p38 chimeric proteins into renal mesangial cells and assessed the effects of wild-type and mutant p38 isoforms on ligand induced iNOS expression. We show that whereas p38 and - had minimal effects on iNOS expression, p38 and -2 significantly altered its expression. p38 mutant and p382 wild-type dose dependently inhibited IL-1-induced iNOS expression. These data suggest that p38 and 2 have reciprocal effects on iNOS expression in the mesangial cell, and these observations may have important consequences for the development of selective inhibitors targeting the p38^MAPK family of proteins. TAT proteins; p38 MAPK; inducible nitric oxide synthase; mesangial cell; interleukin-1     

Compromised proteasome degradation elevates neuronal nitric oxide synthase levels and induces apoptotic cell death

The significance of impairment of proteasome activity in PC12 cells was examined in connection with nitrative/nitrosative stress and apoptotic cell death. Treatment of differentiated PC12 cells with MG132, a proteasome inhibitor, elicited a dose- and time-dependent increase in neuronal nitric oxide synthase (nNOS) protein levels, decreased cell viability, and increased cytotoxicity. Viability and cytotoxicity were ameliorated by L-NAME (a broad NOS inhibitor). Nitric oxide/peroxynitrite formation was increased upon treatment of PC12 cells with MG132 and decreased upon treatment with the combination of MG132 and 7-NI (a specific inhibitor of nNOS). The decreases in cell viability appeared to be effected by an activation of JNK and its effect on mitochondrial Bcl-x_L phosphorylation. These effects are strengthened by the activation of caspase-9 along with increased caspase-3 activity upon treatment of PC12 cells with MG132. These results suggest that impairment of proteasome activity and consequent increases in nNOS levels lead to a nitrative stress that involves the coordinated response of JNK cytosolic signaling and mitochondrion-driven apoptotic pathways.     

Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation

Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 A resolution) and iNOS (2.25 A resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.     

Paradoxical roles of different nitric oxide synthase isoforms in colonic injury

Nitric oxide (NO) is a free radical that is largely produced by three isoforms of NO synthase (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). NO regulates numerous processes in the gastrointestinal tract; however, the overall role that NO plays in intestinal inflammation is unclear. NO is upregulated in both ulcerative colitis and Crohn's disease as well as in animal models of colitis. There have been conflicting reports on whether NO protects or exacerbates injury in colitis or is simply a marker of inflammation. To determine whether the site, timing, and level of NO production modulate the effect on the inflammatory responses, the dextran sodium sulfate model of colitis was assessed in murine lines rendered deficient in iNOS, nNOS, eNOS, or e/nNOS by targeted gene disruption. The loss of nNOS resulted in more severe disease and increased mortality, whereas the loss of eNOS or iNOS was protective. Furthermore, concomitant loss of eNOS reversed the susceptibility found in nNOS-/- mice. Deficiencies in specific NOS isoforms led to distinctive alterations of inflammatory responses, including changes in leukocyte recruitment and alterations in colonic lymphocyte populations. The present studies indicate that NO produced by individual NOS isoforms plays different roles in modulating an inflammatory process.