Interaction between nitric oxide,reactive oxygen intermediates,and peroxynitrite in the regulation of 5-lipoxygenase metabolism

We have shown that overnight lipopolysaccharide (LPS) suppresses alveolar macrophage (AM) leukotriene (LT) synthesis mediated in part by induction of inducible nitric oxide synthase (iNOS) and NO production. Here we examined the possibility that reactive oxygen intermediates (ROI) generated by LPS pretreatment contribute to the suppression of 5-lipoxygenase (5-LO) metabolism. Pretreatment of AM with xanthine/xanthine oxidase, which generates high concentrations of ROI, resulted in suppression of LT synthetic capacity. Since NO and ROI reactive species are known to react and form peroxynitrite (ONOO(-)), we examined the effect of ONOO(-) on 5-LO metabolism. Exogenous ONOO(-) caused a dose-dependent suppression of recombinant 5-LO cell-free activity. ONOO(-) also suppressed LT synthesis in intact AM, which was reversed by the ONOO(-) scavenger tetrakis(4-benzoic acid)porphyrin. ONOO(-) treatment also resulted in dose-dependent nitrotyrosination and S-nitrosylation of the recombinant 5-LO enzyme. Since the direct 5-LO inhibitor zileuton prevents the LPS-induced suppression of LT synthesis, we examined if 5-LO itself was the source of ROI. Zileuton reduced ROI generation in LPS-treated cells. These studies identify an important role for ROI and ONOO(-) in the suppression of 5-LO metabolism by LPS.     

Prolonged exposure to lipopolysaccharide inhibits macrophage 5-lipoxygenase metabolism via induction of nitric oxide synthesis

LPS from bacteria can result in the development of sepsis syndrome and acute lung injury. Although acute exposure to endotoxin primes leukocytes for enhanced synthesis of leukotrienes (LT), little is known about the effect of chronic exposure. Therefore, we determined the effect of prolonged LPS treatment on 5-lipoxygenase (5-LO) metabolism of arachidonic acid in alveolar macrophages (AM) and in peripheral blood monocytes. Pretreatment of AM with LPS caused time- and dose-dependent suppression of LT synthetic capacity. LPS pretreatment failed to inhibit arachidonic acid (AA) release. The fact that LPS inhibited LT synthesis from endogenous AA more than from exogenous AA suggested an effect on 5-LO-activating protein (FLAP). In addition, an inhibitory effect of LPS treatment on AM 5-LO activity was suggested by cell-free 5-LO enzyme assay. No effect on the expression of either 5-LO or FLAP proteins was observed. New protein synthesis was necessary for LPS-induced reduction of 5-LO metabolism in AM, and immunoblotting demonstrated marked induction of NO synthase (NOS). Inhibition by LPS was reproduced by an NO donor and was abrogated by inhibitors of constitutive and inducible NOS. Compared with AM, peripheral blood monocytes exhibited no suppression by LPS of 5-LO metabolism and no induction of inducible NOS. We conclude that prolonged exposure to LPS impairs AM 5-LO metabolism by NO-mediated suppression of both 5-LO and FLAP function. Because LT contribute to antimicrobial defense, this down-regulation of 5-LO metabolism may contribute to the increased susceptibility to pneumonia in patients following sepsis.     

Role of conformational changes in the heme-dependent regulation of human soluble guanylate cyclase.

Soluble guanylate cyclase (sGC) is a receptor for endogenous and exogenous nitric oxide (NO) and is activated many fold upon its binding, making it a core enzyme in the nitric oxide signal transduction pathway. Much effort has been made to understand the link between binding of NO at the sGC heme and activation of the cyclase activity. We report here the first direct evidence for the role of conformational changes in transmitting the signal between the heme and cyclase domains. Using both circular dichroism (CD) and fluorescence spectroscopies, we have probed the effect that the sGC activators NO and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl-indazole (YC-1) and the inhibitor 1H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ) have on the structure of the protein. Surprisingly, binding of either ODQ or YC-1 to NO-bound sGC cause virtually identical changes in the far-UV CD spectra of sGC, reflecting a perturbation in the secondary structure of the enzyme. This change is absent upon binding of NO, YC-1 or ODQ alone. Using this and previous data, we propose a working model for the mechanism of activation of sGC by NO and YC-1 and inhibition by ODQ.     

Nitric oxide decreases endothelin-1 secretion through the activation of soluble guanylate cyclase

The use of exogenous nitric oxide (NO) has been shown to alter the regulation of other endothelially derived mediators of vascular tone, such as endothelin-1 (ET-1). However, the interaction between NO and ET-1 appears to be complex and remains incompletely understood. One of the major actions of NO is the activation of soluble guanylate cyclase (sGC) with the subsequent generation of cGMP. Therefore, we undertook this study to test the hypothesis that NO regulates ET-1 production via the activation of the sGC/cGMP pathway. The results obtained indicated that the exposure of primary cultures of 4-wk-old ovine pulmonary arterial endothelial cells (4-wk PAECs) to the long-acting NO donor DETA NONOate induced both a dose- and time-dependent decrease in secreted ET-1. This decrease in ET-1 secretion occurred in the absence of changes in endothelin-converting enzyme-1 or sGC expression but in conjunction with a decrease in prepro-ET-1 mRNA. The changes in ET-1 release were inversely proportional to the cellular cGMP content. Furthermore, the NO-independent activator of sGC, YC-1, or treatment with a cGMP analog also produced significant decreases in ET-1 secretion. Conversely, pretreatment with the sGC inhibitor ODQ blocked the NO-induced decrease in ET-1. Therefore, we conclude that exposure of 4-wk PAECs to exogenous NO decreases secreted ET-1 resulting from the activation of sGC and increased cGMP generation.     

Generation and characterization of a stable soluble guanylate cyclase-overexpressing CHO cell line.

A stably transfected soluble guanylate cyclase (sGC, alpha1 and beta1 subunits of the rat lung enzyme)-overexpressing CHO cell line was generated for the characterization of different types of activators of the soluble guanylate cyclase. Polyclonal antibodies directed against both subunits of the rat enzyme were used to detect both subunits in the cytosol of the transfected CHO cells. We studied the effects of different nitric oxide (NO) donors like SNP and DEA/NO and, in particular, the direct, NO-independent stimulator of the soluble guanylate cyclase 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), on intracellular guanosine 3',5'-cyclic monophosphate (cGMP) production. DEA/NO (0.01-3 microM), SNP (1-10 microM), and YC-1 (1-10 microM) induced a concentration-dependent intracellular cGMP increase with maximal effects of 16-fold (3 microM DEA/NO), 8-fold (10 microM SNP), and 6-fold (10 microM YC-1) stimulation compared to controls, respectively. In addition, a synergistic effect of the combination of the NO donor and YC-1 could be observed with a maximal stimulation of 64-fold by SNP (10 microM) and YC-1 (10 microM). 1H-(1,2,4)-Oxadiazolo-(4,3-a)-6-bromo-quinoxazin-1-one (ODQ, 10 microM), a potent and selective inhibitor of sGC, inhibited both the single effects of NO donors [DEA/NO (3 microM), 77%; SNP (3 microM), 83%] and YC-1 [YC-1 (3 microM), 82%], but moreover the synergistic effects between NO donors and YC-1 [DEA/NO (3 microM) + YC-1 (3 microM), 81%; SNP (3 microM) + YC-1 (3 microM),89%] on intracellular cGMP production. In summary,we have generated a simple, sensitive, and useful bioassay method to characterize all types of sGC activators on the cellular level without the need of primary cell culture, several transfections, or purifying enzyme from biological materials.     

Functional characterization of nitric oxide and YC-1 activation of soluble guanylyl cyclase: structural implication for the YC-1 binding site?

Soluble guanylyl cyclase (sGC) is a heterodimeric enzyme formed by an alpha subunit and a beta subunit, the latter containing the heme where nitric oxide (NO) binds. When NO binds, the basal activity of sGC is increased several hundred fold. sGC activity is also increased by YC-1, a benzylindazole allosteric activator. In the presence of NO, YC-1 synergistically increases the catalytic activity of sGC by enhancing the affinity of NO for the heme. The site of interaction of YC-1 with sGC is unknown. We conducted a mutational analysis to identify the binding site and to determine what residues were involved in the propagation of NO and/or YC-1 activation. Because guanylyl cyclases (GCs) and adenylyl cyclases (ACs) are homologous, we used the three-dimensional structure of AC to guide the mutagenesis. Biochemical analysis of purified mutants revealed that YC-1 increases the catalytic activity not only by increasing the NO affinity but also by increasing the efficacy of NO. Effects of YC-1 on NO affinity and efficacy were dissociated by single-point mutations implying that YC-1 has, at least, two types of interaction with sGC. A structural model predicts that YC-1 may adopt two configurations in one site that is pseudosymmetric with the GTP binding site and equivalent to the forskolin site in AC.     

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells

5-Lipoxygenase (5-LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5-LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5-LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5-LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5-LO product synthesis.     

Modulation of neuronal nitric oxide release by soluble guanylyl cyclase in guinea pig colon

Peripheral autonomic neurones release nitric oxide (NO) upon nerve activation. However, the regulation of neuronal NO formation is poorly understood. We used the cyclic guanosine 3',5'-monophosphate (cGMP) analogue 8-Br-cGMP, the soluble guanylyl cyclase (sGC) stimulator YC-1, the phosphodiesterase inhibitor zaprinast and the sGC inhibitor ODQ to study whether the sGC/cGMP pathway is involved in regulation of neuronal NO release in nerve plexus-containing smooth muscle preparations from guinea pig colon. Electrical stimulation of the preparation evoked release of NO/NO(-)(2). In the presence of 8-Br-cGMP, YC-1 and zaprinast (all at 10(-4) M) the NO/NO(-)(2)-release increased to 152 +/- 16% (P < 0.05), 164 +/- 37% (P < 0.05) and 290 +/- 67% (P < 0.05) of controls, respectively. Conversely, ODQ (10(-5) M) decreased the evoked release of NO/NO(-)(2) to 49 +/- 7% (P < 0.05) of controls. Our data suggest that the sGC/cGMP pathway modulates NO release. Thus it is likely that NO exerts a positive feedback on its own release from peripheral autonomic neurones.     

Investigation of the vasorelaxant effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) and diethylamine/nitric oxide (DEA/NO) on the human radial artery used as coronary bypass graft

The radial artery (RA) is used as a spastic coronary bypass graft. This study was designed to investigate the mechanism of vasorelaxant effects of YC-1 (3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole), a nitric oxide (NO)-independent soluble guanylate cyclase (sGC) activator, and DEA/NO (diethylamine/nitric oxide), a NO-nucleophile adduct, on the human RA. RA segments (n = 25) were obtained from coronary artery bypass grafting patients and were divided into 3-4 mm vascular rings.Using the isolated tissue bath technique, the endothelium-independent vasodilatation function was tested in vitro by the addition of cumulative concentrations of YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) following vasocontraction by phenylephrine in the presence or absence of 10-5 mol/L ODQ (1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one), the selective sGC inhibitor, 10-7 mol/L iberiotoxin, a blocker of Ca2+-activated K+ channels, or 10-5 mol/L ODQ plus 10-7 mol/L iberiotoxin. We also evaluated the effect of YC-1 and DEA/NO on the cGMP levels in vascular rings obtained from human radial artery (n = 6 for each drug). YC-1 (10-10 to 3 x 10-7 mol/L) and DEA/NO (10-8 to 3 x 10-5 mol/L) caused the concentration-dependent vasorelaxation in RA rings precontracted with phenylephrine (10-5 mol/L) (n = 20 for each drug). Pre-incubation of RA rings with ODQ, iberiotoxin, or ODQ plus iberiotoxin significantly inhibited the vasorelaxant effect of YC-1, but the inhibitor effect of ODQ plus iberiotoxin was significantly more than that of ODQ and iberiotoxin alone (p < 0.05). The vasorelaxant effect of DEA/NO almost completely abolished in the presence of ODQ and iberiotoxin plus ODQ, but did not significantly change in the presence of iberiotoxin alone (p > 0.05). The pEC50 value of DEA/NO was significantly lower than those for YC-1 (p < 0.01), with no change Emax values in RA rings. In addition, YC-1-stimulated RA rings showed more elevation in cGMP than that of DEA/NO (p < 0.05). These findings indicate that YC-1 is a more potent relaxant than DEA/NO in the human RA. The relaxant effects of YC-1 could be due to the stimulation of the sGC and Ca2+-sensitive K+channels, whereas the relaxant effects of DEA/NO could be completely due to the stimulation of the sGC. YC-1 and DEA/NO may be effective as vasodilator for the short-term treatment of perioperative spasm of coronary bypass grafts.     

Butyl isocyanide as a probe of the activation mechanism of soluble guanylate cyclase. Investigating the role of non-heme nitric oxide

Nitric oxide (NO) is a physiologically relevant activator of the hemoprotein soluble guanylate cyclase (sGC). In the presence of NO, sGC is activated several hundredfold above the basal level by a mechanism that remains to be elucidated. The heme ligand n-butyl isocyanide (BIC) was used to probe the mechanism of NO activation of sGC. Electronic absorption spectroscopy was used to show that BIC binds to the sGC heme, forming a 6-coordinate complex with an absorbance maximum at 429 nm. BIC activates sGC 2-5-fold, and synergizes with the allosteric activator YC-1, to activate the enzyme 15-25-fold. YC-1 activates the sGC-BIC complex, and leads to an increase in both the V(max) and K(m). BIC was also used to probe the mechanism of NO activation. The activity of the sGC-BIC complex increases 15-fold in the presence of NO, without displacing BIC at the heme, which is consistent with previous reports that proposed the involvement of a non-heme NO binding site in the activation process.     

Potentiation of carbon monoxide-induced relaxation of rat aorta by YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole

The hypothesis that endogenous carbon monoxide (CO), produced during the oxidation of heme catalyzed by heme oxygenase (HO), plays a role similar to that of nitric oxide (NO) in the regulation of cardiovascular tone has been criticized because of the low potency of CO compared with NO in relaxing blood vessels and stimulating soluble guanylyl cyclase (sGC). This criticism has been muted by the demonstration that, in the presence of YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole], CO has similar potency to NO in stimulating sGC activity. In this study, we determined that YC-1 potentiated CO-induced relaxation of rat aortic strips (RtAS) by approximately ten-fold. Furthermore, CO-induced relaxation of RtAS was shown to be mediated through stimulation of sGC because vasorelaxation was inhibited by ODQ (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one), a selective sGC inhibitor, in the absence and presence of YC-1. A gas chromatographic-headspace method was used to measure CO concentration in Krebs' solution following the addition of CO-saturated saline solution to the tissue bath, in order to provide an accurate determination of RtAS exposure to CO. The tissue bath concentration of CO was shown to be approximately one-half of that calculated to be present. We conclude that should an endogenous compound exist with properties similar to that of YC-1, then the potency of CO as a vasorelaxant in the presence of this factor would be increased. As a consequence, CO could play a role in the regulation of cardiovascular tone, comparable to that of NO.     

Allostery in recombinant soluble guanylyl cyclase from Manduca sexta

Soluble guanylyl/guanylate cyclase (sGC), the primary biological receptor for nitric oxide, is required for proper development and health in all animals. We have expressed heterodimeric full-length and N-terminal fragments of Manduca sexta sGC in Escherichia coli, the first time this has been accomplished for any sGC, and have performed the first functional analyses of an insect sGC. Manduca sGC behaves much like its mammalian counterparts, displaying a 170-fold stimulation by NO and sensitivity to compound YC-1. YC-1 reduces the NO and CO off-rates for the approximately 100-kDa N-terminal heterodimeric fragment and increases the CO affinity by approximately 50-fold to 1.7 microm. Binding of NO leads to a transient six-coordinate intermediate, followed by release of the proximal histidine to yield a five-coordinate nitrosyl complex (k(6-5) = 12.8 s(-1)). The conversion rate is insensitive to nucleotides, YC-1, and changes in NO concentration up to approximately 30 microm. NO release is biphasic in the absence of YC-1 (k(off1) = 0.10 s(-1) and k(off2) = 0.0015 s(-1)); binding of YC-1 eliminates the fast phase but has little effect on the slower phase. Our data are consistent with a model for allosteric activation in which sGC undergoes a simple switch between two conformations, with an open or a closed heme pocket, integrating the influence of numerous effectors to give the final catalytic rate. Importantly, YC-1 binding occurs in the N-terminal two-thirds of the protein. Homology modeling and mutagenesis experiments suggest the presence of an H-NOX domain in the alpha subunit with importance for heme binding.     

Dissociation of nitric oxide from soluble guanylate cyclase and heme-nitric oxide/oxygen binding domain constructs

Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length alpha1beta1 sGC, and the constructs beta1(1-194), beta1(1-385), and beta2(1-217), at 37 and 10 degrees C with and without the substrate analogue guanosine-5'-[(alpha,beta-methylene]triphosphate (GMPCPP) or the activator 3-(5'-hydroxymethyl-3'-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and beta2(1-217) at 37 degrees C and dramatically accelerated NO dissociation from sGC at 10 degrees C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 degrees C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity "closed" form, which releases NO slowly, and a higher activity "open" form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states.     

Alterations in the pattern of arachidonate metabolism accompany rat macrophage differentiation in the lung

The present study was undertaken to investigate the changes in arachidonic acid (AA) metabolism which accompany rat macrophage (m phi) differentiation in the lung in order to determine whether these changes occur in the alveolar space or in the pulmonary interstitium, as well as the mechanisms responsible for such changes. Metabolism of endogenous and exogenous AA by cultured m phi obtained from the peritoneum (PM), the pulmonary interstitium (IM), and the alveolar spaces (AM) was examined by using HPLC and RIA. Although PM and AM released similar amounts of endogenous AA in response to both ionophore A23187 and the particulate zymosan, PM metabolized AA predominantly to cyclooxygenase (CO) products, whereas AM produced predominantly 5-lypoxygenase (5-LO) metabolites. IM synthesized a profile of eicosanoids which more closely resembled that of PM. Studies of the metabolism of exogenously supplied AA demonstrated that AM indeed had less CO activity than did PM. PM, but not AM, CO activity decreased during prolonged culture in air, suggesting the possibility that oxidative inactivation of CO plays a role in the decline in CO capacity which accompanies m phi differentiation in the lung. In contrast, the greater expression of 5-LO metabolism in AM than PM did not reflect mere differences in enzyme capacity, since upon activation of protein kinase C with PMA or oleoylacetylglycerol, ionophore-stimulated PM produced amounts of 5-LO products which were comparable to the amounts produced by AM stimulated with A23187 alone. These results indicate that increases in 5-LO metabolism and decreases in CO metabolism accompany rat m phi differentiation in the lung, that these changes occur largely in the alveolar space, and that the increased 5-LO capacity and decreased CO capacity are independently regulated by different mechanisms.     

Inhibition of NO-dependent soluble human platelet guanylate cyclase by isatin

Isatin (indole-dione-2,3) is an endogenous indole that exhibits a wide spectrum of biological and pharmacological activities. Physiologically relevant concentrations of isatin (ranged from 1 nM to 10 μM) did not influence basal activity of soluble human platelet guanylate cyclase (sGC), but caused a bell-shaped inhibition of the NO-activated enzyme. Inhibition of the NO-dependent activation by isatin did not depend on a chemical nature of the NO donors. The inhibitory effects of ODC (a heme-dependent inhibitor of sGC) and isatin were non-additive suggesting that the inhibitory effect of isatin may involve the heme binding domain (possibly heme iron) and experiments with hemin revealed some isatin-dependent changes in its spectrum. Isatin also inhibited sGC activation by the allosteric activator YC-1. It is suggested that the bell shaped inhibition of the NO-dependent activation of sGC by isatin may be attributed to complex interaction of isatin with the heme binding domain and the allosteric YC-1-binding site of sGC.     

PI3K-dependent lysosome exocytosis in nitric oxide-preconditioned hepatocytes

We investigated the signal mediators and the cellular events involved in the nitric oxide (NO)-induced hepatocyte resistance to oxygen deprivation in isolated hepatocytes treated with the NO donor (Z)-1-(N-methyl-N-[6-(N-methylammoniohexyl)amino])diazen-1-ium-1,2-diolate (NOC-9). NOC-9 greatly induced PI3K activation, as tested by phosphorylation of PKB/Akt. This effect was prevented by either 1H-(1,2,4)-oxadiazolo-(4,3)-quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (sGC), or KT5823, an inhibitor of cGMP-dependent kinase (cGK), as well as by farnesyl protein transferase inhibitor, which blocks the function of Ras GTPase. Bafilomycin A, an inhibitor of the lysosome-type vacuolar H+-ATPase, cytochalasin D, which disrupts the cytoskeleton-dependent organelle traffic, and wortmannin, which inhibits the PI3K-dependent traffic of lysosomes, all abolished the NOC-9-induced hepatocyte protection. The treatment with NOC-9 was associated with the PI3K-dependent peripheral translocation and fusion with the plasma membrane of lysosomes and the appearance at the cell surface of the vacuolar H+-ATPase. Inhibition of sGC, cGK, and Ras, as well as the inhibition of PI3K by wortmannin, prevented the exocytosis of lysosomes and concomitantly abolished the protective effect of NOC-9 on hypoxia-induced pHi and [Na+]i alterations and cell death. These data indicate that NO increases hepatocyte resistance to hypoxic injury by activating a pathway involving Ras, sGC, and cGK that determines PI3K-dependent exocytosis of lysosomes.     

The design and synthesis of YC-1 analogues as probes for soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is highly activated in the presence of both YC-1 (1-benzyl-3-(5'-hydroxymethyl-2'-furyl)-indazole) and CO. In this report, the design, synthesis, and activity (i.e., sGC activation) of photolabile analogues of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) are presented. Initial results with 6-azido-3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole led to the synthesis of a tritium-labeled analogue. When photoactivated, this analogue labeled the alpha-subunit of sGC.     

4-Hydroxynonenal enhances CD36 expression on murine macrophages via p38 MAPK-mediated activation of 5-lipoxygenase

Increased levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) coexist in atherosclerotic lesions but their relationship in atherogenesis is unclear. This study investigated the role of 5-LO in HNE-induced CD36 expression and macrophage foam cell formation, and the link between HNE and 5-LO. In J774A.1 murine macrophages, HNE (10 μM) enhanced CD36 expression in association with an increased uptake of oxLDL, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor, or with 5-LO siRNA. In peritoneal macrophages from 5-LO-deficient mice, HNE-induced CD36 expression was markedly attenuated, confirming a pivotal role of 5-LO in HNE-induced CD36 expression. In an assay for 5-LO activity, stimulation of macrophages with HNE led to increased leukotriene B₄ production in the presence of exogenous arachidonic acid in association with an increased association of 5-LO to the nuclear membrane. Among the mitogen-activated protein kinase (MAPK) pathways involved in 5-LO phosphorylation, HNE predominantly activated p38 MAPK in macrophages, and the p38 MAPK inhibitor SB203580, but not an extracellular signal-regulated kinase inhibitor, suppressed HNE-induced LTB₄ production. Collectively, these data suggest that p38 MAPK-mediated activation of 5-LO by HNE might enhance CD36 expression, consequently leading to the formation of macrophage foam cells.     

Interaction of soluble guanylate cyclase with YC-1: kinetic and resonance Raman studies

The enzyme-soluble guanylate cyclase (sGC), which converts GTP to cGMP, is a receptor for the signaling agent nitric oxide (NO). YC-1, a synthetic benzylindazole derivative, has been shown to activate sGC in an NO-independent fashion. In the presence of carbon monoxide (CO), which by itself activates sGC approximately 5-fold, YC-1 activates sGC to a level comparable to stimulation by NO alone. We have used kinetic analyses and resonance Raman spectroscopy (RR) to investigate the interaction of YC-1 and CO with guanylate cyclase. In the presence of CO and 200 microM YC-1, the V(max)/K(m GTP) increases 226-fold. While YC-1 does not perturb the RR spectrum of the ferrous form of baculovirus/Sf9 cell expressed sGC, it induces a shift in the Fe-CO stretching frequency for the CO-bound form from 474 to 492 cm(-1). Similarly, YC-1 has no effect on the RR spectrum of ferrous beta1(1-385), the isolated sGC heme-binding domain, but shifts the nu(Fe-CO) of CO-beta1(1-385) from 478 to 491 cm(-1), indicating that YC-1 binds in heme-binding region of sGC. In addition, the CO-bound forms of sGC and beta1(1-385) in the presence of YC-1 lie on the nu(Fe-CO) vs nu(C-O) correlation curve for proximal ligands with imidazole character, which suggests that histidine remains the heme proximal ligand in the presence of YC-1. Interestingly, YC-1 does not shift nu(Fe-CO) for the CO-bound form of H105G(Im), the imidazole-rescued heme ligand mutant of beta1(1-385). The data are consistent with binding of CO and YC-1 to the sGC heme-binding domain leading to conformational changes that give rise to an increase in catalytic turnover and a change in the electrostatic environment of the heme pocket.     

Macrophage endothelial nitric-oxide synthase autoregulates cellular activation and pro-inflammatory protein expression

Expression of inducible nitric-oxide (NO) synthase (iNOS) and "high-output" production of NO by macrophages mediates many cytotoxic actions of these immune cells. However, macrophages have also been shown to express a constitutive NOS isoform, the function of which remains obscure. Herein, bone marrow-derived macrophages (BMDM?s) from wild-type and endothelial NOS (eNOS) knock-out (KO) mice have been used to assess the role of this constitutive NOS isoform in the regulation of macrophage activation. BMDM?s from eNOS KO animals exhibited reduced nuclear factor-kappaB activity, iNOS expression, and NO production after exposure to lipopolysaccharide (LPS) as compared with cells derived from wild-type mice. Soluble guanylate cyclase (sGC) was identified in BMDM?s at a mRNA and protein level, and activation of cells with LPS resulted in accumulation of cyclic GMP. Moreover, the novel non-NO-based sGC activator, BAY 41-2272, enhanced BMDM? activation in response to LPS, and the sGC inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one attenuated activation. These observations provide the first demonstration of a pathophysiological role for macrophage eNOS in regulating cellular activation and suggest that NO derived from this constitutive NOS isoform, in part via activation of sGC, is likely to play a pivotal role in the initiation of an inflammatory response.