Particulate Nitric Oxide Synthase Research Articles

Superoxide activates synaptic particulate nitric oxide synthase: implications for superoxide signalling and neurotoxicity

Mounting evidence supports the view that nitric oxide may protect against superoxide‐induced oxidative damage by mechanisms involving nitrosylation, direct scavenging of oxygen radicals including superoxide, and up‐regulation of antioxidant enzymes. However, the ability of neurons to respond to superoxide by increasing nitric oxide synthesis is unknown. We report here that superoxide, added as potassium superoxide (KO2), produced a rapid activation (up to 12‐fold) of nitric oxide synthase (NOS), measured by the conversion of [3H]arginine to [3H]citrulline, and nitrite production in a synaptic particulate fraction from rat brain cerebral cortex. Stimulation of NOS by KO2 required the presence of ATP, which by itself inhibited basal NOS activity. Decayed KO2 or authentic hydrogen peroxide did not increase NOS activity and, importantly, activation of NOS by KO2 was blocked by addition of superoxide dismutase. In addition, stimulation of NOS activity by KO2 was abolished by the NOS inhibitor N‐monomethyl‐l‐arginine and EGTA but only partially reduced by the neuronal NOS inhibitor 7‐nitroindazole. These findings are the first to demonstrate directly that superoxide activates a synaptic NOS isoform. The nature of the particulate fraction, the experimental conditions, and the low sensitivity to 7‐nitroindazole strongly suggest that superoxide activated mitochondrial NOS (mtNOS). We propose that activation of the particulate NOS by superoxide reflects a novel mitochondrial response that may contribute to neuronal oxidant defenses although chronic activation of NOS under conditions of superoxide generation may produce toxic quantities of peroxynitrite and other reactive nitrogen oxides.

Effect of ovarian steroids on nitric oxide synthase in the rat uterus, cervix and vagina

The effects of estrogen (E 2), progesterone (P) and E 2 and P (E 2 + P) were examined on nitric oxide synthase (NOS) activity in both cytosolic and particulate fractions isolated from the rat uterus, vagina, cervix and cerebral cortex. Additionally plasma nitrate+nitrite (NO 3+ NO 2) levels were measured in control and hormone treated rats. Cytosolic NOS was the predominant form being approximately 80% of the total in all four tissues. NOS activity in both fractions from all tissues was highly Ca-dependent (> 90%). Among the reproductive tract tissues, the highest activity was found in the cervix, which was nearly 5- and 2-fold higher than the uterus and vagina, respectively. NOS activity in the cerebral cortex was by far the highest being 5-fold higher than in the cervix. In contrast to the cortex, E 2 treatment downregulated cytosolic NOS in all reproductive tract tissue, but this was statistically significant in only uterus. When compared with E 2 treated rats, P increased cytosolic NOS in uterus, vagina, and particulate NOS in the cervix. The data do not give any indication whatsoever of differential effects of P in the uterus and cervix.

Downregulation by estrogen of nitric oxide synthase activity in the female rabbit lower urinary tract

Objectives. Because female urinary tract tissues are considered to be targets for estrogen, and because nitric oxide (NO) is known to participate in the nerve-induced relaxation in the lower urinary tract, the effect of estrogen on nitric oxide synthase (NOS) in the upper and lower urinary tracts was examined. Methods. Ovariectomized rabbits were treated with polyestradiol phosphate, and NOS in both cytosolic and particulate fractions from kidney, urinary pelvis, ureter, urinary bladder, trigonum, and urethra was characterized. NOS activity was measured by the formation of [ 14C]- l-citrulline from [ 14C]- l-arginine. Results. NOS was considerably higher in cytosolic than in particulate fractions from all urinary tracts, and activity in both fractions was highly calcium dependent. NOS activity was much lower (fourfold to eightfold) in the kidney and pelvis than in the ureter. Estrogen treatment caused no change in NOS in either fraction from upper urinary tract tissues. In the lower urinary tract, NOS was slightly higher in the bladder and trigonum than in the urethra, and activities were comparable to NOS in the ureter. In contrast to the upper urinary tract, estrogen treatment led to a significant reduction of cytosolic NOS in the bladder, trigonum, and urethra. Estrogen, however, caused no significant change in the particulate NOS. Conclusions. Downregulation by estrogen in cytosolic NOS in the tissue of the lower urinary tract is consistent with the presence of estrogen receptors and suggests a physiologic significance.

Characterization of nitric oxide synthase activity in rabbit uterus and vagina: Downregulation by estrogen

Nitric oxide synthase (NOS) was determined in the soluble (cytosolic) and particulate fractions of rabbit uterus, vagina and cerebellum and the influence of estrogen treatment on NOS activity was studied. NOS in both the cytosolic and particulate fractions was highly calcium dependent. The activity in cytosolic fraction was nearly 4-fold higher than the particulate fractions from all three organs. The concentration of NOS was highest in cerebellum followed by vagina and uterus. Vaginal NOS activity was 3–4-fold higher than the uterine NOS. After a continuous treatment of rabbits for one week with estrogen, cytosolic NOS was reduced by nearly 7 and 4-fold in the uterus and vagina, respectively, whereas there was no significant change in the particulate NOS. Estrogen treatment caused no change in cutosolic or particulate NOS from the cerebellum. Downregulation of cytosolic NOS by estrogen in the estrogen target tissues like uterus and vagina and absence of effect in the cerebellum strongly suggests a physiological significance.

Monophosphoryl lipid A stimulated up-regulation of nitric oxide synthase and nitric oxide release by human monocytes in vitro

Monophosphoryl lipid A (MPL) is a derivative of lipopolysaccharide (LPS) with reduced toxicity which has been shown to modulate various immune functions in monocytes. We examined whether human monocytes can be stimulated to produce nitric oxide (NO) and its catalytic enzyme nitric oxide synthase (NOS). Monocytes were stimulated with LPS or MPL and both NOS and NO (as nitrite) production were measured. MPL at high doses (>100 μg/ml) stimulated monocytes to release NO that was significantly greater than both the control and LPS-treated monocytes (p<0.05). NO release by control cells and the LPS treated cells was not significantly different. Both arginase and N-monomethyl arginine (NMLA) inhibited the MPL stimulated release of NO (p<0.01). MPL significantly increased inducible NOS (iNOS) expression as measured by both fluorescent microscopy and flow cytometry (p<0.05). Similarly, both soluble NOS (sNOS) and particulate NOS (pNOS) activity were significantly up-regulated by MPL (p<0.05). Significant correlations were found between pNOS expression and sNOS release (r=0.72, p<0.0001) and between 12 h NO release and sNOS production (r=0.44, p<0.005). These experiments confirm that human monocytes can be stimulated with MPL to produce NO in vitro and suggest that up-regulation of pNOS does not preclude NO release.

Increase of particulate nitric oxide synthase activity and peroxynitrite synthesis in UVB-irradiated keratinocyte membranes.

Here we demonstrate that human keratinocytes possess a Ca2+/calmodulin-dependent particulate NO synthase that can be activated to release NO after exposure to UVB radiation. UVB irradiation (up to 20 mJ/cm2) of human keratinocyte plasma membranes resulted in a dose-dependent increase in NO and L-[3H]citrulline production that was inhibited by approx. 90% in the presence of N-monomethyl-L-arginine (L-NMMA). In time-course experiments with UVB-irradiated plasma membranes the changes in NO production were followed by analogous changes in soluble guanylate cyclase (sGC) activity. In reconstitution experiments, when particulate NO synthase was added to purified sGC isolated from keratinocyte cytosol, a 4-fold increase in cGMP was observed; the cGMP was increased by NO synthesized after UVB irradiation (up to 20 mJ/cm2) of particulate NO synthase. A 5-fold increase in superoxide (O2-) and a 7-fold increase in NO formation followed by an 8-fold increase in peroxynitrite (ONOO-) production by UVB (20 mJ/cm2)-irradiated keratinocyte microsomes was observed. UVB radiation (20 mJ/cm2) decreased plasma membrane lipid fluidity as indicated by steady-state fluorescence anisotropy. Membrane fluidity changes were prevented by L-NMMA. Changes in Arrhenius plots of particulate NO synthase in combination with changes in its allosteric properties induced by UVB radiation are consistent with a decreased fluidity of the lipid microenvironment of the enzyme. The present studies provide important new clues to the role of NO and ONOO- released by UVB-irradiated human keratinocytes in skin erythema and inflammation.

Modulation of particulate nitric oxide synthase activity and peroxynitrite synthesis in cholesterol enriched endothelial cell membranes

Endothelium-derived relaxing factor/nitric oxide (EDRF/NO) is produced by the vascular wall and is a key modulator of vascular tone and blood pressure. Since reduced EDRF/NO release from the endothelium is a major key event in the development of atherosclerosis, we investigated the effect of cholesterol on endothelial cell particulate (membrane-bound) NO synthase activity. Low concentrations (up to 0.2 mM) of liposomal cholesterol progressively activated plasma membrane-bound NO synthase. Increasing cholesterol concentration above that which maximally stimulated enzyme activity produced a progressive inhibition with respect to the control value. In time course experiments using endothelial cell plasma membranes enriched with cholesterol, changes in NO production were followed by analogous changes in soluble guanylate cyclase activity (sGC). N-Monomethyl-L-arginine (L-NMMA) (1 mM) inhibited particulate NO synthase activity at all cholesterol concentrations used with subsequent decreases in cGMP production. Egg lecithin liposomes (free of cholesterol) had no effect on NO synthase activity. A three-fold increase in Superoxide (O 2 −) and a 2.5 fold increase in NO formation followed by an eight-fold increase in peroxynitrite (ONOO −) production by cholesterol-treated microsomes isolated from endothelial cells was observed, one which rose further up to eight-fold in the presence of Superoxide dismutase (SOD) (10 U/mL). Cholesterol had no effect on Lubrol-PX solubilized membrane-bound NO synthase or on cytosolic (soluble) NO synthase activities of endothelial cells. Cholesterol modulated lipid fluidity of plasma membranes labelled with 1,6-diphenyl-1, 3,5-hexatriene (DPH) as indicated by the steady state fluorescence anisotropy [( r o r ) − 1] − 1 . Arrhenius plots of [( r o r ) − 1] −1 indicated that the lipid phase separation of the membranes at 26.2 ± 1.5 ° was elevated to 34.4 ± 1.9 ° in cholesterol-enriched membranes, consistent with a general decrease in membrane fluidity. Cholesterol-enriched plasma membranes treated with egg lecithin liposomes showed a lipid phase separation at 27.5 ± 1.6 °, indicating the reversible effect of cholesterol on membrane lipid fluidity. Arrhenius plots of NO synthase activity exhibited break point at 26.9 ± 1.8 ° which rose to 35.6 ± 2.1 ° in 0.5 mM cholesterol-treated plasma membranes and decreased to 21.5 ± 1.4 ° in plasma membranes treated with 0.2 mM cholesterol. The allosteric properties of plasma membrane-bound NO synthase inhibited by Mn 2+ (as reflected by changes in the Hill coefficient) were changed by cholesterol, consistent with modulations of the fluidity of the lipid microenvironment of the enzyme. Our findings suggest that incorporation of high concentrations of cholesterol into endothelial cell membranes causes down-regulation of NO synthase by producing an increased packing of bulk lipids. In contrast, cholesterol incorporation at low concentrations up-regulates NO synthase by increasing the fluidity of the lipid microenvironment of the enzyme. The present studies concerning the behaviour of particulate NO synthase and rate of NO release with respect to the structure and function of the biomembranes provide important new clues as to the role of this fascinating molecule in atherosclerosis.

Distribution of NOS in normoxic vs. hypoxic rat lung: upregulation of NOS by chronic hypoxia.

Expression and localization of nitric oxide synthase (NOS) in the lungs of chronically hypoxic and normoxic rats were studied using both immunohistochemistry and NADPH diaphorase (NADPH-d) staining techniques. In the normoxic and in the hypoxic rat, NOS was detected by both methods in the endothelium of large pulmonary vessels and in the epithelium of bronchi and bronchioli. NOS expression was not detected in the endothelium of normoxic pulmonary resistance vessels but was prominently expressed in the endothelium of these vessels after 2-4 wk of chronic hypoxia. In contrast to small pulmonary vessels, the endothelium of small bronchial vessels exhibited NOS immunostaining in both normoxic and hypoxic lungs. Hypoxia was also found to induce de novo NOS expression in the smooth muscle of large and small pulmonary vessels and in bronchial smooth muscle. NOS enzyme activity in lung homogenates was assessed by [3H]arginine to [3H]citrulline conversion. The activity of soluble NOS, but not particulate NOS, was increased in the hypoxic lungs. These results demonstrate chronic hypoxia-induced upregulation of NOS protein expression and activity in the rat lung, suggesting a potentially important role of nitric oxide in adaptation of the pulmonary circulation to chronic hypoxia. The lack of immunostaining in small pulmonary resistance vessels is also consistent with physiological studies suggesting that NO may not be involved in the mechanism for maintaining the normally low pulmonary vascular resistance.

Involvement of nitric oxide and cyclic GMP in rabbit urethral relaxation

Soluble and particulate fractions from rabbit urethra converted [ 14C]arginine to [ 14C]citrulline, indicating the presence of nitric oxide synthase activity in these fractions. Both soluble and particulate nitric oxide synthase activities were NADPH dependent, and the soluble activity was Ca 2+ dependent. Three nitric oxide synthase (NOS) inhibitors affected transmural nerve stimulation induced relaxation responses in the rabbit urethra and the activity of soluble nitric oxide synthase with the same rank order of potency, i.e., N G - nitro- l-arginine (NNA) > N G - methyl- l-arginine (NMA) > canavanine (CAN) . The rank order of potency with respect to particulate NOS activity was CAN > NMA = NNA. The relaxation responses to electrical stimulation were accompanied by increases in cyclic GMP. These results suggest that NOS activity found in the soluble fraction of urethral homogenates produces nitric oxide that in turn increases cyclic GMP levels which mediates the relaxation responses induced by transmural nerve stimulation in the rabbit urethra.

Identification of an endothelial-like type III NO synthase in LLC-PK1 kidney epithelial cells.

Porcine kidney tubular epithelial cells (LLC-PK1) produce nitric oxide or a related compound (e.g., a nitrosothiol) after stimulation with various agonists. We now report the identification and characterization of a constitutive, particulate nitric oxide (NO) synthase from LLC-PK1 cells. After partial purification on adenosine 2',5'-bisphosphate-Sepharose, the particulate NO synthase activity eluted anomalously from Superose 6 gel permeation columns near the total included volume, similar to that observed for the endothelial (type III) NO synthase. Substrate/cofactor requirements of the epithelial and endothelial NO synthases were identical, i.e., dependency on L-arginine, (6R)-5,6,7,8-tetrahydrobiopterin, FAD, calcium and calmodulin. The epithelial enzyme activity was inhibited by the arginine analogues, NG-methyl-L-arginine (100 microM) and NG-nitro-L-arginine (100 microM), as well as the calmodulin antagonists, trifluoperazine (100 microM) and calmidazolium (30 microM). Anti-type III (H32), but not anti-type I (brain, 6763-5) or anti-type II (macrophage, 8196) NO synthase antibodies, detected a single immunoreactive band in the LLC-PK1 particulate fraction of approximately 140 kDa by Western blot analysis. Finally, the presence of type III NO synthase mRNA in LLC-PK1 cells was demonstrated using the polymerase chain reaction. These data indicate that LLC-PK1 kidney epithelial cells contain type III NO synthase, which has been classically associated with the vascular endothelium.

Activation of Nitric Oxide Synthase from Cultured Aortic Endothelial Cells by Phospholipids

Nitric oxide is a recently discovered biomolecule with a broad range of actions and synthesized by nitric oxide synthases. We investigated effects of lipids on particulate nitric oxide synthase purified from cultured bovine aortic endothelial cells by monitoring the conversion of L-[3H]arginine to L-[3H]citrulline. Phosphatidylcholine, lysophosphatidylcholine, and phosphatidylethanolamine dose-dependently enhanced the enzyme activity up to 3 fold in the presence of Ca2+, calmodulin, NADPH, FAD, and (6R)-5,6,7,8-tetrahydrobiopterin. These phospholipids increased the Vmax of nitric oxide synthase without altering the Km for L-arginine and the affinities for Ca2+ and calmodulin. These findings suggest that phospholipids play an important role in modulating endothelial nitric oxide synthase activity.

Developmental changes of cytosolic and particulate nitric oxide synthase in rat brain

In the presence of added flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), both cytosolic and particulate nitric oxide synthase (NOS) activities can be detected in rat brain. Developmental changes of the cytosolic and particulate NOS in rat cerebellum and cerebrum were determined biochemically and immunochemically. Particulate NOS activity in the cerebrum increased during the first week of development, but then decreased and became almost undetectable in adult rats. In contrast, the cytosolic NOS in cerebellum showed low activity in newborns, but then constantly increased reaching an 8-fold higher level in adult rats. The activities of cerebellar particulate and cerebral cytosolic NOS also increased slightly during maturation. Western blot analysis using a polyclonal antibody raised against rat cerebellar cytosolic NOS revealed that the particulate and cytosolic fractions of 1-week-old and adult rat brains contained the same 160 kDa NOS protein. The relative content of the NOS protein correlated well with the relative amount of NOS activity in all brain fractions. These results indicate that distribution of NOS in the rat brain changes during maturation, but the same NOS protein is likely to be responsible for activities in immature and mature brains, cytosolic and particulate fractions. We suggest that nitric oxide might play a role in functional differentiation of the brain.

Induced RAW 264.7 macrophages express soluble and particulate nitric oxide synthase: inhibition by transforming growth factor-ß

RAW 264.7 macrophages induced with lipopolysaccharide and interferon-γ expressed nitric oxide (NO) synthase. Approximately two-thirds of the total induced NO synthase activity was found in the cytosolic fraction, whereas one-third was associated with the particulate fraction. Both enzymes formed L-citrulline in addition to NO-like material. NO and L-citrulline formation by both enzymes were calcium-independent and inhibited by N G-nitro-L-arginine and N G-methyl-L-arginine. Transforming growth factor-ß1 prevented the induction of both enzymes.

Characterization of nitric oxide synthases in non‐adrenergic non‐cholinergic nerve containing tissue from the rat anococcygeus muscle

Tissue homogenates prepared from rat anococcygeus muscle converted L-arginine to L-citrulline indicating the presence of nitric oxide (NO) synthase. NO synthase activity was also found in crude and partially-purified soluble and particulate fractions prepared from the homogenates. Both soluble and particulate NO synthase were dependent on NADPH, 5,6,7,8-tetrahydrobiopterin and calcium, and inhibited by NG-nitro-L-arginine. Tissue homogenates or crude cytosolic and membrane fractions from rat vas deferens, which does not contain NO releasing non-adrenergic non-cholinergic neurones, had no NO synthase activity.