cGMP-independent Manner Research Articles

Nitrergic modulation of neuronal excitability in the mouse hippocampus is mediated via regulation of Kv2 and voltage-gated sodium channels.

Regulation of neuronal activity is a necessity for communication and information transmission. Many regulatory processes which have been studied provide a complex picture of how neurons can respond to permanently changing functional requirements. One such activity-dependent mechanism involves signaling mediated by nitric oxide (NO). Within the brain, NO is generated in response to neuronal NO synthase (nNOS) activation but NO-dependent pathways regulating neuronal excitability in the hippocampus remain to be fully elucidated. This study was set out to systematically assess the effects of NO on ion channel activities and intrinsic excitabilities of pyramidal neurons within the CA1 region of the mouse hippocampus. We characterized whole-cell potassium and sodium currents, both involved in action potential (AP) shaping and propagation and determined NO-mediated changes in excitabilities and AP waveforms. Our data describe a novel signaling by which NO, in a cGMP-independent manner, suppresses voltage-gated Kv2 potassium and voltage-gated sodium channel activities, thereby widening AP waveforms and reducing depolarization-induced AP firing rates. Our data show that glutathione, which possesses denitrosylating activity, is sufficient to prevent the observed nitrergic effects on potassium and sodium channels, whereas inhibition of cGMP signaling is also sufficient to abolish NO modulation of sodium currents. We propose that NO suppresses both ion channel activities via redox signaling and that an additional cGMP-mediated component is required to exert effects on sodium currents. Both mechanisms result in a dampened excitability and firing ability providing new data on nitrergic activities in the context of activity-dependent regulation of neuronal function following nNOS activation.

Cardioprotective actions of the sodium-activated potassium channel Slack (aka Slo2.2, KNa1.1 or KCNT1)

Abstract Background Sodium-activated potassium channels (KNa) Slack (sequence like a calcium-activated potassium channel, aka Slo 2.2, KNa1.1 or KCNT1) are widely expressed throughout neuronal tissue, whereas their presence and function in the cardiovascular system is not well understood. Due to high intracellular sodium concentrations ([Na+]i) necessary to induce half-maximal channel activation, we hypothesized that Slack function is attributed to pathophysiological conditions such as myocardial ischemia. Purpose To elucidate the putative functions of Slack in the murine heart and in cardiomyocytes (CMs) and to explore whether the ischemia and reperfusion (I/R)-induced cardiac damage is affected by endogenous Slack channel activity. Methods I/R injury was evaluated in global and CM-specific Slack knockout mice (Slack gKO, CM Slack KO) and compared to litter-matched controls (Slack gWT, CM Slack CTR) by applying an in vivo model of acute myocardial infarction (MI). Infarct size (IS) was assessed at baseline, after ischemic pre- (iPre) and postconditioning (iPost) and in response to cinaciguat (CIN), a cGMP-elevating agent. Moreover, Slack expression and function in CMs was studied by biochemical and electrophysiological means and by utilizing the newly developed FRET-based K+ probe GEPII 1.0. Results IS in Slack gKO mice was increased in comparison to gWT littermates. In addition, the cardioprotection afforded by iPost was attenuated in the absence of Slack. To test if the increased vulnerability to I/R injury of the Slack gKO mouse model was originating from Slack activity in CMs, we subjected CM-specific Slack CTR and KO mutants to an identical MI procedure. IS measurements confirmed increased cardiac damage at baseline and reduced cardioprotective effects afforded by iPre and iPost in CM Slack KO mice. Interestingly, CIN (i.p., 30 min prior to I/R) reduced IS to a similar extent in both genotypes, suggesting that Slack functions in a cGMP-independent manner. Whole-cell patch clamp experiments on CMs demonstrated a reduction of the KNa-inhibitors clofilium- and chinidine-sensitive K+ outward currents in Slack gKO CM. Extracellular potassium ([K+]ex) accumulation measured with GEPII 1.0 was lower in Slack gKO versus gWT CM pools exposed to membrane permeabilizing agent digitonin. Accordingly, [K+]ex evoked by the Slack activators niclosamide and bithionol was lower in the absence of functional Slack in CMs. Conclusion The presented findings establish an important role of Slack channels for cardioprotective signalling mechanisms during I/R in vivo and for mediating beneficial effects of mechanical conditioning on IS. Corroborating in vitro studies on adult CMs exhibit an impaired [K+]ex dynamic in response to genetic or pharmacological modulation of Slack activity. Thus, we conclude that Slack-dependent K+ signalling pathways in CMs may represent a promising drug target that renders the heart muscle less vulnerable to the I/R-induced damage. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Work in the authors' laboratories is supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (to R.L.) and the DFG Research Unit 2060, “cGMP Signaling in Cell Growth and Survival” (to R.L. and P.R.).

Activation of Large Conductance, Calcium-Activated Potassium Channels by Nitric Oxide Mediates Apelin-Induced Relaxation of Isolated Rat Coronary Arteries.

Apelin increases coronary blood flow, cardiac contractility, and cardiac output. Based on these favorable hemodynamic effects, apelin and apelin-like analogs are being developed for treating heart failure and related disorders; however, the molecular mechanisms underlying apelin-induced coronary vasodilation are unknown. This study aimed to elucidate the signaling pathways by which apelin causes smooth muscle relaxation in coronary arteries. Receptors for apelin (APJ receptors) were expressed in coronary arteries, as determined by Western blot and polymerase chain reaction analyses. Immunofluorescence imaging studies identified APJ receptors on endothelial and smooth muscle cells. In isolated endothelial cells, apelin caused an increase in 4,5-diaminofluorescein fluorescence that was abolished by nitro-l-arginine (NLA) and F13A (H-Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Ala-OH), an APJ receptor antagonist, consistent with increased nitric oxide (NO) production. In arterial rings, apelin caused endothelium-dependent relaxations that were abolished by NLA, F13A, and iberiotoxin. Neither oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) nor DT-2, a protein kinase G inhibitor, had any effect on apelin-induced relaxations, and apelin itself had no effect on intracellular cGMP accumulation in coronary arteries. Patch-clamp studies in isolated smooth muscle cells demonstrated that the NO donors, diethyl amine NONOate and sodium nitroprusside, caused increases in large conductance, calcium-activated potassium channel (BKCa) currents, which were inhibited by iberiotoxin but not ODQ. Thus, apelin causes endothelium-dependent relaxation of coronary arteries by stimulating endothelial APJ receptors and releasing NO, which acts in a cGMP-independent manner and increases BKCa activity in the underlying smooth muscle cells. These results provide a mechanistic basis for apelin-induced coronary vasodilation and may provide guidance for the future development of novel apelin-like therapeutic agents.

Nitric oxide-mediated posttranslational modifications control neurotransmitter release by modulating complexin farnesylation and enhancing its clamping ability.

Nitric oxide (NO) regulates neuronal function and thus is critical for tuning neuronal communication. Mechanisms by which NO modulates protein function and interaction include posttranslational modifications (PTMs) such as S-nitrosylation. Importantly, cross signaling between S-nitrosylation and prenylation can have major regulatory potential. However, the exact protein targets and resulting changes in function remain elusive. Here, we interrogated the role of NO-dependent PTMs and farnesylation in synaptic transmission. We found that NO compromises synaptic function at the Drosophila neuromuscular junction (NMJ) in a cGMP-independent manner. NO suppressed release and reduced the size of available vesicle pools, which was reversed by glutathione (GSH) and occluded by genetic up-regulation of GSH-generating and de-nitrosylating glutamate-cysteine-ligase and S-nitroso-glutathione reductase activities. Enhanced nitrergic activity led to S-nitrosylation of the fusion-clamp protein complexin (cpx) and altered its membrane association and interactions with active zone (AZ) and soluble N-ethyl-maleimide-sensitive fusion protein Attachment Protein Receptor (SNARE) proteins. Furthermore, genetic and pharmacological suppression of farnesylation and a nitrosylation mimetic mutant of cpx induced identical physiological and localization phenotypes as caused by NO. Together, our data provide evidence for a novel physiological nitrergic molecular switch involving S-nitrosylation, which reversibly suppresses farnesylation and thereby enhances the net-clamping function of cpx. These data illustrate a new mechanistic signaling pathway by which regulation of farnesylation can fine-tune synaptic release.

Guanylyl cyclase sensitivity to nitric oxide is protected by a thiol oxidation-driven interaction with thioredoxin-1

Nitric oxide (NO) modulates many physiological events through production of cGMP from its receptor, the NO-sensitive guanylyl cyclase (GC1). NO also appears to function in a cGMP-independent manner, via S-nitrosation (SNO), a redox-based modification of cysteine thiols. Previously, we have shown that S-nitrosated GC1 (SNO-GC1) is desensitized to NO stimulation following prolonged NO exposure or under oxidative/nitrosative stress. In animal models of nitrate tolerance and angiotensin II-induced hypertension, decreased vasodilation in response to NO correlates with GC1 thiol oxidation, but the physiological mechanism that resensitizes GC1 to NO and restores basal activity is unknown. Because GC1 interacts with the oxidoreductase protein-disulfide isomerase, we hypothesized that thioredoxin-1 (Trx1), a cytosolic oxidoreductase, could be involved in restoring GC1 basal activity and NO sensitivity because the Trx/thioredoxin reductase (TrxR) system maintains thiol redox homeostasis. Here, by manipulating activity and levels of the Trx1/TrxR system and by using a Trx1-Trap assay, we demonstrate that Trx1 modulates cGMP synthesis through an association between Trx1 and GC1 via a mixed disulfide. A proximity ligation assay confirmed the endogenous Trx1–GC1 complex in cells. Mutational analysis suggested that Cys609 in GC1 is involved in the Trx1–GC1 association and modulation of GC1 activity. Functionally, we established that Trx1 protects GC1 from S-nitrosocysteine–induced desensitization. A computational model of Trx1–GC1 interaction illustrates a possible mechanism for Trx1 to maintain basal GC1 activity and prevent/rescue GC1 desensitization to NO. The etiology of some oxidative vascular diseases may very well be explained by the dysfunction of the Trx1–GC1 association.

AB281. SPR-08 Altered S-nitrosation of contractile proteins underlies dysfunctional quiescence in human preterm labor

ObjectivePreterm labor is defined as labor prior to 37 weeks of gestation. There are no FDA approved drugs available to treat spontaneous preterm labor. Uterine smooth muscle is unique in that it relaxes in a cGMP-independent manner in the presence of nitric oxide (NO). However, NO-mediated relaxation in tissues from mothers who deliver preterm is blunted. S-nitrosation of cysteines via NO acts as an important mediator in a number of disease states. Our analysis of the myometrial S-nitrosoproteome has revealed that several smooth muscle contractile proteins are differentially S-nitrosated based upon the state of labor in women. An important regulator of protein S-nitrosation is the availability of S-nitrosoglutathione (GSNO), an endogenously expressed NO donor. The enzyme GSNO reductase (GSNOR) regulates GSNO levels in smooth muscle. We performed experiments to test the hypotheses that GSNOR activity is altered in the myometrium from women in preterm labor and that inhibition of GSNOR attenuates myometrial contraction and restores quiescence in spontaneous preterm labor.MethodsExperiments were performed in the Myometrial Research Laboratory at UNR Med. Uterine biopsies were obtained with IRB approval and written informed consent from mothers undergoing Cesarean section and transported to the laboratory in physiological buffer for immediate processing. GSNOR expression was measured by western blot and normalized to GAPDH expression. GSNOR activity was measured by the decrease in absorbance at 340 nm via conversion of NADH to NAD+ by GSNOR in the presence of GSNO. Eight (8) nM N6022, a potent and specific inhibitor of GSNOR, was used to verify GSNOR specific activity. Human myometrial tissue was dissected and six uterine strips from each patient were mounted in tissue baths and tested for their ability to contract in response to KCl prior to addition of test agents.ResultsHuman preterm myometrium fails to relax to NO addition whether in the presence or absence of oxytocin stimulation. GSNOR expression is increased in myometrial tissue from women in spontaneous preterm labor compared to term laboring controls. GSNOR enzymatic activity was also increased in protein lysates from myometrial tissue from patients undergoing spontaneous preterm labor than those in labor at term. A potent and selective inhibitor of GSNOR (N6022) decreases the peak force of contraction in guinea pig myometrium and restores sensitivity to NO.ConclusionsGSNOR is more highly expressed in preterm laboring myometrium. Preterm laboring myometrium exhibits a blunted response to NO and the GSNOR inhibitor N6022 restores sensitivity to relaxation by NO. Our data suggests that increased GSNOR activity in spontaneous preterm labor contributes to a preterm contractile phenotype through the enzymatic degradation of endogenous GSNO.Funding Source(s)NIH 1U54GM 104944

Nitric oxide show its survival role by NO-PKC pathway through cGMP-dependent or independent on the culture of cerebella granular neurons

The role of nitric oxide in the development of neurons is conflicting. In the present work, cerebellar granule neurons (CGNs) were used as a model to assess the survival role of nitric oxide and to find novel signal transduction pathways related to this role. It is reported that sustained inhibition of nitric oxide production induces apoptosis in differentiated cerebellar granule neurons. The antagonist (MK-801, or ODQ)-induced decrease of cell viability, caspase-3 activated, the expression of P-PKC decreased, which were normalized by the provision of the sodium nitroprusside, an NO donor. The data show that blockade of NO production induces apoptotic death in differentiating CGC through activation of caspase-3. This study provides direct evidence that NO plays an active role in sustaining the survival of developing CGNs and that NO-PKC pathway is important for the survival of CGNs in vitro. The endogenous NO exerts its effects in cGMP-dependent manner while the exogenous NO in cGMP-independent manner.

The Opposing Roles of Nitric Oxide and cGMP in the Age-Associated Decline in Rat Testicular Steroidogenesis

The molecular mechanism of the aging-associated dysfunction of Leydig cells (LCs) is complex and poorly understood. In this study, we analyzed the contribution of nitric oxide (NO) and cGMP signaling to the age-dependent decline in LC function. Significant (>50%) decreases in serum, intratesticular, and LC androgens in aging rats (15-24 months) were accompanied by a proportional increase in NO production, an up-regulation of cGMP levels, and the expression of soluble guanylyl cyclase-1B and protein kinase G1 in LCs. In contrast, LC cAMP levels decreased with age, most likely reflecting the up-regulation of cAMP-specific phosphodiesterase expression. Moreover, the expression of genes encoding enzymes responsible for cholesterol transport and its conversion to T were reduced. Exposing LCs from aged animals to NO further increased cGMP levels and decreased cAMP and androgen production, whereas the addition of cell-permeable 8-bromoguanosine-cGMP alone had the opposite effect. In vivo inhibition of cGMP-specific phosphodiesterase-5 for 3 and 6 months in aged rats led to a partial restoration of androgens, NO, and cyclic nucleotide levels, as well as the expression of steroidogenic and NO/cGMP signaling genes. These results indicate that a progressive increase in NO production contributes to the age-dependent decrease in steroidogenesis in a cGMP-independent manner, whereas the sustained elevation in cGMP levels significantly slows the decline in LC function.

H2O2 lowers the cytosolic Ca2+ concentration via activation of cGMP-dependent protein kinase Iα

The cGMP-dependent protein kinase I (cGKI) is a key mediator of cGMP signaling, but the specific functions of its two isoforms, cGKIα and cGKIβ, are poorly understood. Recent studies indicated a novel cGMP-independent role for cGKIα in redox sensing. To dissect the effects of oxidative stress on the cGKI isoforms, we used mouse embryonic fibroblasts and vascular smooth muscle cells (VSMCs) expressing both, one, or none of them. In cGKIα-expressing cells, but not in cells expressing only cGKIβ, incubation with H2O2 induced the formation of a disulfide bond between the two identical subunits of the dimeric enzyme. Oxidation of cGKIα was associated with increased phosphorylation of its substrate, vasodilator-stimulated phosphoprotein. H2O2 did not stimulate cGMP production, indicating that it activates cGKIα directly via oxidation. Interestingly, there was a mutual influence of H2O2 and cGMP on cGKI activity and disulfide bond formation, respectively; preoxidation of the kinase with H2O2 slightly impaired its activation by cGMP, whereas preactivation of the enzyme with cGMP attenuated its oxidation by H2O2. To evaluate the functional relevance of the noncanonical H2O2–cGKIα pathway, we studied the regulation of the cytosolic Ca2+ concentration ([Ca2+]i). H2O2 suppressed norepinephrine-induced Ca2+ transients in cGKIα-expressing VSMCs and, to a lower extent, in VSMCs expressing only cGKIβ or none of the isoforms. Thus, H2O2 lowers [Ca2+]i mainly via a cGKIα-dependent pathway. These results indicate that oxidative stress selectively targets the cGKIα isoform, which then modulates cellular processes in a cGMP-independent manner. A decrease in [Ca2+]i in VSMCs via activation of cGKIα might be a major mechanism of H2O2-induced vasodilation.

The uterine smooth muscle S‐nitrosylproteome in pregnancy and functional effects of S‐nitrosoglutathione

The molecular mechanisms involved in uterine quiescence during gestation and those responsible for induction of labor are not known. Nitric oxide relaxes uterine smooth muscle in a cGMP independent manner. S‐Nitrosylation, the covalent addition of an NO‐group to a cysteine thiol is a likely mechanism to explain the ability of NO to relax myometrium. For the first time we describe the myometrial S‐nitrosylproteome in pregnant and non‐pregnant guinea pig and the human labor, not in labor, and pre term laboring comprehensive proteome and S‐nitrosylproteome using multidimensional protein identification technology. We show that S‐nitrosylation of the intermediate filament protein desmin is significantly increased (5.7 fold, p < 0.005) in pregnancy and that this increase cannot be attributed solely to the increase in protein expression (1.8 fold, p < 0.005) that accompanies pregnancy. We also show that both myosin light regulatory polypeptide 9 and myosin light chain kinase are disparately S‐nitrosylated in the different states of pregnancy. We further describe both the S‐nitrosylation and activation of the potassium channel TREK‐1 by the NO donor S‐nitroso‐glutathione. Elucidation of the myometrial proteome and S‐nitrosylproteome provides a list of mechanistically important proteins that can constitute the basis of hypotheses formed to explain the regulation of uterine contraction‐relaxation.

Atypical PKCζ transduces electrophilic fatty acid signaling in pulmonary epithelial cells

Nitric oxide and secondary oxides of nitrogen react with unsaturated fatty acids such as linoleic acid to yield oxidized and nitrated products. Fatty acid nitroalkene derivatives, (e.g. nitrolinoleate [LNO2]) are produced by oxidative inflammatory reactions, detected clinically, display potent electrophilic reactivity and induce post-translational protein modifications that mediate adaptive inflammatory signaling responses. LNO2 signaling was examined in lung epithelial cells because the alveolar compartment is a rich site for the transduction of redox and inflammatory reactions. LNO2 did not directly induce Ca2+ influx in cultured lung epithelial cells, but inhibited bradykinin-induced Ca2+ influx in a cGMP-independent manner. In contrast, LNO2 activated MAP kinase (Erk1/2) by a mechanism independent of bradykinin. It was hypothesized that these unique responses were transduced by activation of different protein kinase C isotypes, supported by the observation that LNO2-mediated inhibition of Ca2+ influx was blocked by the non-selective PKC inhibitors chelerythine chloride and calphostin C, but not by the calcium dependent “classic” PKC inhibitor Gö6976. Western blot analysis showed that atypical PKCζ was activated by LNO2 stimulation, with PKCζ and Erk activation also demonstrated in primary culture of human lung type II cells. Addition of pseudotypical PKCζ substrate peptide reversed LNO2-mediated induction of Ca2+ influx and MAP kinase activation. Finally, the electrophilic nature of LNO2 resulted in a novel mode of PKCζ activation, covalent adduction of the enzyme. In summary, LNO2 mediated signaling in lung type II epithelial cells occurs via a unique pathway involving PKCζ.

Nitric Oxide Stimulates NCX1 and NCX2 but Inhibits NCX3 Isoform by Three Distinct Molecular Determinants

In this study, the role of nitric oxide (NO) in the modulation of the activity of NCX1, NCX2, and NCX3 exchangers was investigated in baby hamster kidney cells singly transfected with each of these isoforms by single-cell Fura-2-microfluorometry and patch clamp. Furthermore, the molecular determinants of NO on each isoform were identified by deletion, site-directed mutagenesis, and chimera strategies. Our data revealed four main findings. First, the NO-donor S-nitroso-N-acetylpenicillamine (SNAP; 10 nM) and the NO-precursor L-arginine (10 mM) were both able to increase NCX1 activity in a cGMP-independent way. Moreover, within the amino acid sequence 723 to 734 of the f-loop, Cys730 resulted as the target of NO on NCX1. Second, SNAP and L-arginine were able to increase NCX2 activity, but this effect was prevented by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). In addition, the membrane-permeable 8-bromoguanosine-cGMP alone was able to mimic the stimulatory effect of the gaseous mediator, suggesting the involvement of a cGMP-dependent mechanism. Within the amino acid sequence 699 to 744 of the f-loop, Ser713 was the NO molecular determinant on the NCX2 protein; Third, NCX3 activity was instead down-regulated by NO in a cGMP-independent manner. This NO-inhibitory action was exerted at the level of Cys156 in the α1-region outside the f-loop. Finally, the activity of the two NCX3 chimeras-obtained by the replacement of the NO-insensitive NCX3 region with the homologous NO-sensitive segments of NCX1 or NCX2-was potentiated by SNAP. Together, the present data demonstrate that NO differently regulates the activity of the three gene products NCX1, NCX2, and NCX3 by modulating specific molecular determinants.

Agonist-Specific Compartmentation of cGMP Action in Myometrium

Nitric oxide relaxes myometrium in a cGMP-independent manner. Although cGMP activates its cognate kinase, this is not required for the inhibitory effect of nitric oxide. Thus, nitric oxide-mediated cGMP elevation does not enjoy the same set of substrates as it does in other smooth muscles. To further understand the regulation of relaxation of uterine muscle by cGMP, we have studied the actions of peptide-mediated cGMP action in guinea pig myometrium. We used both functional and biochemical studies of the action of the particulate guanylyl cyclase activator uroguanylin and its receptor, particulate guanylyl cyclase type C, to address the relationship between cGMP elevation acting in the membrane signaling domain to that of the nonmembrane region of the cell. Uroguanylin relaxed oxytocin-induced contractions in a dose-dependent fashion only in pregnant myometrium. Both relaxation and cGMP accumulation after uroguanylin stimulation were blocked by the putative particulate guanylyl cyclase type C inhibitors 2-chloro-ATP and isatin (1H-indole-2,3-dione), but not by the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-A]quinoxalin-1-one (ODQ). Uroguanylin stimulated cGMP accumulation only in the pregnant myometrium. Caveolin-1 expression increased in pregnancy toward term. In the caveolin-1-containing membrane domain, uroguanylin, but not the nitric-oxide donor, led to the elevation of cGMP that was insensitive to ODQ. Particulate guanylyl cyclase C was expressed and prouroguanylin was detected in pregnant myometrium. We conclude that a uroguanylin-particulate cyclase-cGMP relaxation pathway is present and cGMP is compartmented in myometrium. The agonist-mediated selectivity of relaxation to cGMP is of fundamental pharmacological interest in understanding signal transduction in smooth muscle.

Inducible nitric oxide synthase gene deletion exaggerates MAPK-mediated cyclooxygenase-2 induction by inflammatory stimuli

Cyclooxygenase (COX)-2 and inducible nitric oxide (NO) synthase (iNOS) are responsive to a wide array of inflammatory stimuli, have been localized to vascular smooth muscle cells (SMCs), and are intimately linked to the progression of vascular disease, including atherosclerotic lesion formation. We and others have shown that the production and subsequent impact of COX products appear to be correlative with the status of NO synthesis. This study examined the impact of inflammation-driven NO production on COX-2 expression in SMCs. Concurrent stimulation of quiescent rat aortic SMCs with lipopolysaccharide (LPS) and interferon (IFN)-gamma increased COX-2, iNOS, and nitrite production. Pharmacological inhibition of NO synthase (N(G)-monomethyl-l-arginine) concentration- and time-dependently magnified LPS + IFN-gamma-mediated COX-2 mRNA and protein induction in a cGMP-independent manner. COX-2 induction was associated with activation of the ERK, p38, and JNK mitogen-activated protein kinase (MAPK) pathways. Interestingly, NO synthase inhibition enhanced ERK, p38, and to a lesser extent JNK phosphorylation but suppressed MAPK phosphatase (MKP)-1 induction in response to LPS + IFN-gamma. Similarly, the exposure of SMCs from iNOS(-/-) mice to LPS + IFN-gamma produced an enhancement of COX-2 induction, p38, and JNK phosphorylation and an attenuated upregulation of MKP-1 versus their wild-type counterparts. Taken together, our data indicate that NO, in part derived from iNOS, negatively regulates the immediate early induction of COX-2 in response to inflammatory stimuli.

S -Nitrosylation in Cardiovascular Signaling

Well over 2 decades have passed since the endothelium-derived relaxation factor was reported to be the gaseous molecule nitric oxide (NO). Although soluble guanylyl cyclase (which generates cyclic guanosine monophosphate, cGMP) was the first identified receptor for NO, it has become increasingly clear that NO exerts a ubiquitous influence in a cGMP-independent manner. In particular, many, if not most, effects of NO are mediated by S-nitrosylation, the covalent modification of a protein cysteine thiol by an NO group to generate an S-nitrosothiol (SNO). Moreover, within the current framework of NO biology, endothelium-derived relaxation factor activity (ie, G protein-coupled receptor-mediated, or shear-induced endothelium-derived NO bioactivity) is understood to involve a central role for SNOs, acting both as second messengers and signal effectors. Furthermore, essential roles for S-nitrosylation have been implicated in virtually all major functions of NO in the cardiovascular system. Here, we review the basic biochemistry of S-nitrosylation (and denitrosylation), discuss the role of S-nitrosylation in the vascular and cardiac functions of NO, and identify current and potential clinical applications.

Endothelin-1 Impairs Alveolar Epithelial Function via Endothelial ETB Receptor

Endothelin-1 (ET-1) is increased in patients with high-altitude pulmonary edema and acute respiratory distress syndrome, and these patients have decreased alveolar fluid reabsorption (AFR). To determine whether ET-1 impairs AFR via activation of endothelial cells and nitric oxide (NO) generation. Isolated perfused rat lung, transgenic rats deficient in ETB receptors, coincubation of lung human microvascular endothelial cells (HMVEC-L) with rat alveolar epithelial type II cells or A549 cells, ouabain-sensitive 86Rb+ uptake. The ET-1-induced decrease in AFR was prevented by blocking the endothelin receptor ETB, but not ETA. Endothelial-epithelial cell interaction is required, as direct exposure of alveolar epithelial cells (AECs) to ET-1 did not affect Na,K-ATPase function or protein abundance at the plasma membrane, whereas coincubation of HMVEC-L and AECs with ET-1 decreased Na,K-ATPase activity and protein abundance at the plasma membrane. Exposing transgenic rats deficient in ETB receptors in the pulmonary vasculature (ET-B(-/-)) to ET-1 did not decrease AFR or Na,K-ATPase protein abundance at the plasma membrane of AECs. Exposing HMVEC-L to ET-1 led to increased NO, and the ET-1-induced down-regulation of Na,K-ATPase was prevented by the NO synthase inhibitor l-NAME, but not by a guanylate cyclase inhibitor. We provide the first evidence that ET-1, via an endothelial-epithelial interaction, leads to decreased AFR by a mechanism involving activation of endothelial ETB receptors and NO generation leading to alveolar epithelial Na,K-ATPase down-regulation in a cGMP-independent manner.

CGMP-independent anti-apoptotic effect of nitric oxide on thapsigargin-induced apoptosis in the pancreatic β-cell line INS-1

Aims Low concentrations of nitric oxide (NO) produced by constitutive NO synthase (cNOS) in pancreatic β-cells have been suggested to be a physiological regulator of insulin secretion. In contrast, excessive NO produced by inducible NO synthase is known to mediate β-cell apoptosis. The aim of the present study was to investigate the effect of low concentrations of NO on β-cell apoptosis. Main methods Apoptosis of the pancreatic β-cell line INS-1 was quantitatively determined by Annexin V flow cytometry. Key findings The 24-h incubation with 1 mM DETA/NO, a long half-life NO donor, induced β-cell apoptosis, which was insensitive to the soluble guanylate cyclase (sGC) inhibitor ODQ. In contrast, DETA/NO at lower concentrations until 300 μM concentration-dependently decreased the apoptosis induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+-ATPase. ODQ did not affect the anti-apoptotic effect of DETA/NO. Moreover, neither the cGMP analogue 8-Br-cGMP nor the sGC activator YC-1 mimicked the anti-apoptotic effect of DETA/NO. Significance These results suggest that low levels of NO protect β-cells from thapsigargin-induced apoptosis in a cGMP-independent manner.

Highlights From The Literature

Highlights From The Literature

Signal transduction pathways affected by nitric oxide donors during neutrophil functional response in vitro

We investigated the intracellular signalling pathways by which nitric oxide (NO) donors: diethylamine/NO (DEA/NO) and 3-morpholinosydnonimine (SIN-1) regulate the functional response of human neutrophils to activating stimuli. The phosphorylation and nitration of signalling proteins, cyclic GMP level, neutrophil respiratory burst and adhesive activities and CD11b/CD18 molecule expression on neutrophils in the presence and absence of soluble guanylate cyclase inhibitors were determined. NO donors showed strong inhibitory effect on activated neutrophils. NO donors nitrated the tyrosine residues in signalling proteins causing a decrease in tyrosine phosphorylation and neutrophils response to activation. Diethylamine/NO employed cyclic GMP as a signalling molecule in its action on neutrophils, whereas peroxynitrite anion donor affected neutrophil functions in a cGMP-independent manner. Moreover, we observed that peroxynitrite anion can overcome the nitric oxide molecule action. We conclude that each NO donor depending on its concentration and chemical nature may act on different elements of neutrophil signalling pathways capable of inducing distinct neutrophil functions.

Modulation of human monocyte-derived dendritic cells maturation by a soluble guanylate cyclase activator, YC-1, in a cyclic nucleotide independent manner

This study evaluated how YC-1, a guanylate cyclase activator, affects the maturation of human monocyte-derived dendritic cells. Maturation markers and intracellular signaling pathways were evaluated. YC-1 inhibited the lipopolysaccharide up-regulation of mature markers, including CD40, CD80 or CD86 in a concentration-dependent manner with IC 50 values of 4.6 ± 0.4, 4.9 ± 0.6 or 4.5 ± 0.5 μM, respectively. YC-1, at a higher concentration, inhibited lipopolysaccharide-induced HLADR expression. These effects of YC-1 were not reversed by ODQ (10 μM), which is a soluble guanylate cyclase inhibitor, nor by KT5823 (1 μM), which is a PKG inhibitor. Additionally, YC-1 did not increase levels of cyclic nucleotides in dendritic cells, supporting the claim that YC-1 affects dendritic cells maturation in a cGMP-independent manner. YC-1, in a cGMP-independent manner, inhibited lipopolysaccharide-induced Akt activation, IκBα degradation and NF-κB translocation, all of which are associated with co-stimulatory molecules expression. YC-1 inhibited the capacity of dendritic cell to activate allogenic T cells with an IC 50 value of 1.2 ± 0.3 μM. YC-1-treated dendritic cells have mature phenotypes that exhibit up-regulated CCR7, enhanced IL-10 release and low phagocytosis activity in the presence of lipopolysaccharide. In conclusion, YC-1 inhibited the lipopolysaccharide-induced co-stimulatory molecular expression of dendritic cells by inhibiting Akt activation, IκBα degradation and NF-κB translocation. These inhibitory effects on co-stimulatory molecules suppressed the capacity of dendritic cells to activate allogenic T cells. Additionally, YC-1 treated dendritic cells exhibit the up-regulation of CCR7, enhanced IL-10 release and the down-regulation of phagocytosis in the presence of lipopolysaccharide. Accordingly, YC-1 might be a useful tool for evaluation of dendritic cells on autoimmune or allergic disease.