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PubMed2004

Angiogenesis and vasculogenesis are impaired in the precocious-aging klotho mouse.

BACKGROUND: The effects of aging on angiogenesis (vascular sprouting) and vasculogenesis (endothelial precursor cell [EPC] incorporation into vessels) are not well known. We examined whether ischemia-induced angiogenesis/vasculogenesis is altered in klotho (kl) mutant mice, an animal model of typical aging. METHODS AND RESULTS: After unilateral hindlimb ischemia, laser Doppler blood-flow (LDBF) analysis revealed a decreased ischemic-normal LDBF ratio in kl mice. Tissue capillary density was also suppressed in kl mice (+/+>+/kl>kl/kl). Aortic-ring culture assay showed impaired angiogenesis in kl/kl mice, accompanied by reduced endothelium-derived nitric oxide release. Moreover, the rate of transplanted homologous bone marrow cells incorporated into capillaries in ischemic tissues (vasculogenesis) was lower in kl/kl mice than in wild-type (+/+) mice, which was associated with a decrease in the number of c-Kit+CD31+ EPC-like mononuclear cells in bone marrow and in peripheral blood. Finally, the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor cerivastatin restored the impaired neovascularization in kl/kl mice, accompanied by an increase in c-Kit+CD31+ cells in bone marrow and peripheral blood, and enhanced angiogenesis in the aortic-ring culture. CONCLUSIONS: Angiogenesis and vasculogenesis are impaired in kl mutant mice, a model of typical aging. Moreover, the age-associated impairment of neovascularization might be a new target of statin therapy.

Aging, Premature↗
PubMed1993

A direct link between LY83583, a selective repressor of cyclic GMP formation, and glutathione metabolism.

LY83583 (6-anilino-5,8-quinolinedione), considered to be a relatively specific repressor of cyclic GMP formation, is shown in the present study to inhibit (K(i) = 3 microM) glutathione reductase from bovine intestinal mucosa. As glutathione disulphide has been reported to inhibit guanylate cyclase irreversibly [Braughler, Biochem Pharmacol 32: 811-818, 1983], the inhibition of glutathione reductase should affect the activity of guanylate cyclase and may thus have physiological implications in the action of endothelium-derived relaxation factor and the design of muscle relaxants. LY83583 is reduced by NADPH and glutathione reductase in aerobic media and this may offer a route to the metabolic activation of LY83583. These results may have significant implications for the design of heart-regulating drugs (e.g. those used in angina), such as glyceryl trinitrate, which act via guanylate cyclase.

Aminoquinolines↗
PubMed1997

Nitric oxide effects on cell growth: GMP-dependent stimulation of the AP-1 transcription complex and cyclic GMP-independent slowing of cell cycling.

1. The role of nitric oxide (NO) in the control of cell growth is controversial since both stimulation and (more often) inhibition have been demonstrated in various cell types. In order to reinvestigate the problem and identify the sites of NO action, we have employed murine NIH-3T3 fibroblasts overexpressing epidermal growth factor (EGF) receptors. 2. The effects of four structurally-unrelated NO donors: S-nitroso-N-acetyl penicillamine, S-nitroso-L-glutathione, 3-morpholinosydnonimine and isosorbide dinitrate (0.01-3 mM) on EGF (10 nM)-stimulated cell growth were estimated by both thymidine incorporation and the colorimetric MTT assay, while those of a messenger generated in response to NO, cyclic GMP, were revealed by the use of 8-Br cyclic GMP (0.01-3 mM) as well as of blockers of guanylyl cyclase and cyclic GMP-dependent kinase I. 3. Studies were focused on: (i) multiple signalling events, including receptor-induced tyrosine phosphorylations, phosphorylation of mitogen-activated protein kinase, activation of the AP-1 transcription complex and deoxyribonucleotide synthesis; (ii) the progression through the cell cycle, dissected out by the use of staurosporine (1 nM), lovastatin (10 microM), mimosine (200 microM), hydroxyurea (1 mM) and nocodazole (1.5 microM). 4. NO was found to have no effects on the phosphorylation events of the growth factor cascade. In contrast, later processes were modified by the messenger but with opposite effects. 5. A cyclic GMP-dependent stimulation of growth was shown to be sustained in part by the activation of the AP-1 transcription complex, while a predominant, cyclic GMP-independent inhibition was found to be mediated by both the negative regulation of ribonucleotide reductase and the marked slowing down of the cell cycle occurring at early and late G1 and during the S phase. 6. Although multiple and apparently conflicting, the effects of NO here described could work coordinately in a general programme of cell growth regulation. In particular, the cyclic GMP-dependent actions might function as rapid modulatory events, while the effects on cell cycle might operate collectively as a multi-switch process whenever growth inhibition is required.

3T3 Cells↗
PubMed1993

Aldose reductase inhibition restores endothelial cell function in diabetic rabbit aorta.

A possible relationship between increased aldose reductase activity and abnormal endothelium-dependent relaxation was examined in aorta from alloxan-induced diabetic rabbits. Isolated aorta of diabetic rabbits, contracted submaximally with phenylephrine, showed significantly decreased endothelium-dependent relaxations induced by acetylcholine or adenosine diphosphate compared to those from normal rabbits. Basal and acetylcholine-stimulated levels of cyclic GMP and the relaxations in response to an endothelium-independent vasodilator, sodium nitroprusside, were not significantly different between diabetic and normal rabbits, indicating that nitric oxide release and action on the vascular smooth muscle were unchanged. The release of thromboxane A2 from diabetic vessels was increased, as previously demonstrated. Treatment with an aldose reductase inhibitor, zopolrestat, normalized the elevated red blood cell sorbitol levels in diabetic rabbits. Zopolrestat also restored the abnormal acetylcholine- and adenosine diphosphate-induced relaxations of the aorta. The aldose reductase inhibitor had no effect on the levels of cyclic GMP or on the increased release of thromboxane A2 in diabetic aorta. These findings suggest that increased activity of the aldose reductase pathway in hyperglycemia is responsible for the abnormal endothelium-dependent relaxation in diabetic blood vessels. Significant alterations in endothelial production of neither nitric oxide nor vasoconstrictor prostanoids could be directly implicated in the improvement caused by the drug, suggesting another mechanism of action.

Acetylcholine↗
PubMed1997

Crystal structure of phosphoadenylyl sulphate (PAPS) reductase: a new family of adenine nucleotide alpha hydrolases.

BACKGROUND: Assimilatory sulphate reduction supplies prototrophic organisms with reduced sulphur for the biosynthesis of all sulphur-containing metabolites. This process is driven by a sequence of enzymatic steps involving phosphoadenylyl sulphate (PAPS) reductase. Thioredoxin is used as the electron donor for the reduction of PAPS to phospho-adenosine-phosphate (PAP) and sulphite. Unlike most electron-transfer reactions, there are no cofactors or prosthetic groups involved in this reduction and PAPS reductase is one of the rare examples of an enzyme that is able to store two electrons. Determination of the structure of PAPS reductase is the first step towards elucidating the biochemical details of the reduction of PAPS to sulphite. RESULTS: We have determined the crystal structure of PAPS reductase at 2.0 A resolution in the open, reduced form, in which a flexible loop covers the active site. The protein is active as a dimer, each monomer consisting of a central six-stranded beta sheet with alpha helices packing against each side. A highly modified version of the P loop, the fingerprint peptide of mononucleotide-binding proteins, is present in the active site of the protein, which appears to be a positively charged cleft containing a number of conserved arginine and lysine residues. Although PAPS reductase has no ATPase activity, it shows a striking similarity to the structure of the ATP pyrophosphatase (ATP PPase) domain of GMP synthetase, indicating that both enzyme families have evolved from a common ancestral nucleotide-binding fold. CONCLUSIONS: The sequence conservation between ATP sulphurylases, a subfamily of ATP PPases, and PAPS reductase and the similarities in both their mechanisms and folds, suggest an evolutionary link between the ATP PPases and PAPS reductases. Together with the N type ATP PPases, PAPS reductases and ATP sulphurylases are proposed to form a new family of homologous enzymes with adenine nucleotide alpha-hydrolase activity. The open, reduced form of PAPS reductase is able to bind PAPS, whereas the closed oxidized form cannot. A movement between the two monomers of the dimer may allow this switch in conformation to occur.

Acid Anhydride Hydrolases↗
PubMed1990

Molybdopterin guanine dinucleotide: a modified form of molybdopterin identified in the molybdenum cofactor of dimethyl sulfoxide reductase from Rhodobacter sphaeroides forma specialis denitrificans.

The nature of molybdenum cofactor in the bacterial enzyme dimethyl sulfoxide reductase has been investigated by application of alkylation conditions that convert the molybdenum cofactor in chicken liver sulfite oxidase and milk xanthine oxidase to the stable, well-characterized derivative [di(carboxamidomethyl)]molybdopterin. The alkylated pterin obtained from dimethyl sulfoxide reductase was shown to be a modified form of alkylated molybdopterin with increased absorption in the 250-nm region of the spectrum and altered chromatographic behavior. The complex alkylated pterin was resolved into two components by treatment with nucleotide pyrophosphatase. These were identified as di(carboxamidomethyl)molybdopterin and GMP by their absorption spectra, coelution with standard compounds, and by further degradation by alkaline phosphatase to dephospho [di(carboxamidomethyl)]molybdopterin and guanosine. The GMP moiety was sensitive to periodate, identifying it as the 5' isomer. Chemical analysis of the intact alkylated pterin showed the presence of two phosphate residues per pterin. These results established that the pterin isolated from dimethyl sulfoxide reductase contains the phosphoric anhydride of molybdopterin and 5'-GMP, which is designated molybdopterin guanine dinucleotide.

Chromatography, High Pressure Liquid↗
PubMed1995

The napEDABC gene cluster encoding the periplasmic nitrate reductase system of Thiosphaera pantotropha.

The napEDABC locus coding for the periplasmic nitrate reductase of Thiosphaera pantotropha has been cloned and sequenced. The large and small subunits of the enzyme are coded by napA and napB. The sequence of NapA indicates that this protein binds the GMP-conjugated form of the molybdopterin cofactor. Cysteine-181 is proposed to ligate the molybdenum atom. It is inferred that the active site of the periplasmic nitrate reductase is structurally related to those of the molybdenum-dependent formate dehydrogenases and bacterial assimilatory nitrate reductases, but is distinct from that of the membrane-bound respiratory nitrate reductases. A four-cysteine motif at the N-terminus of NapA binds a [4Fe-4S] cluster. The DNA- and protein-derived primary sequence of NapB confirm that this protein is a dihaem c-type cytochrome and, together with spectroscopic data, indicate that both NapB haems have bis-histidine ligation. napC is predicted to code for a membrane-anchored tetrahaem c-type cytochrome that shows sequence similarity to the NirT cytochrome c family. NapC may be the direct electron donor to the NapAB complex. napD is predicted to encode a soluble cytoplasmic protein and napE a monotopic integral membrane protein, napDABC genes can be discerned at the aeg-46.5 locus of Escherichia coli K-12, suggesting that this operon encodes a periplasmic nitrate reductase system, while napD and napC are identified adjacent to the napAB genes of Alcaligenes eutrophus H16.

Amino Acid Sequence↗
PubMed1985

Inhibition of growth of Rhizobium japonicum by cyclic GMP.

Exogenous cyclic guanosine-3',5'-monophosphate (cGMP) inhibited the growth of Rhizobium japonicum at less than 100 microM. Other nucleotides, including cyclic AMP, cyclic IMP, and cyclic CMP, had no inhibitory effect even at higher concentrations nor was the inhibition by cGMP reversed by cyclic AMP. The inhibitory effect was independent of the carbon and nitrogen source(s) used. cGMP did not inhibit the growth of any other species of bacterium tested, including several fast-growing Rhizobium species. The kinetics of growth inhibition are multiphasic, with no apparent effect for several hours after addition, followed by a period of total inhibition. Subsequently, growth resumed at a slower rate. Resumption of growth was not due to destruction of the nucleotide. Studies of the intracellular cGMP concentration did not reveal significant changes in cells grown under aerobic or microaerobic conditions. No effect of cGMP on the derepression of respiratory nitrate reductase was observed.

Bacterial Proteins↗
PubMedDate not supplied

[Lipid peroxidation, the enzyme antioxidative system and acid phosphatase content of the gastric mucosa in stomach ulcer].

Lipid peroxidation, a state of the antioxidative system and activity of acid phosphatase were studied in mucosal membrane of 53 patients with ulcerous disease of stomach. Increase of the acid phosphatase activity in cytoplasm, activation of lipid peroxidation and inhibition of the antioxidative system were detected in ulcer border and periulcerous region. These alterations in lipid peroxidation and the state of the antioxidative system were considered as distinct pathogenetic factors responsible for deterioration of lysosomal membranes and contributing to chronic and relapsing development of ulcerous disease.

Acid Phosphatase↗
PubMed2005

Hypoxia, red blood cells, and nitrite regulate NO-dependent hypoxic vasodilation.

Local vasodilation in response to hypoxia is a fundamental physiologic response ensuring oxygen delivery to tissues under metabolic stress. Recent studies identify a role for the red blood cell (RBC), with hemoglobin the hypoxic sensor. Herein, we investigate the mechanisms regulating this process and explore the relative roles of adenosine triphosphate, S-nitrosohemoglobin, and nitrite as effectors. We provide evidence that hypoxic RBCs mediate vasodilation by reducing nitrite to nitric oxide (NO) and ATP release. NO dependence for nitrite-mediated vasodilation was evidenced by NO gas formation, stimulation of cGMP production, and inhibition of mitochondrial respiration in a process sensitive to the NO scavenger C-PTIO. The nitrite reductase activity of hemoglobin is modulated by heme deoxygenation and heme redox potential, with maximal activity observed at 50% hemoglobin oxygenation (P(50)). Concomitantly, vasodilation is initiated at the P(50), suggesting that oxygen sensing by hemoglobin is mechanistically linked to nitrite reduction and stimulation of vasodilation. Mutation of the conserved beta93cys residue decreases the heme redox potential (ie, decreases E(1/2)), an effect that increases nitrite reductase activity and vasodilation at any given hemoglobin saturation. These data support a function for RBC hemoglobin as an allosterically and redox-regulated nitrite reductase whose "enzyme activity" couples hypoxia to increased NO-dependent blood flow.

Adenosine Triphosphate↗
PubMed1979

Effect of cyclic guanosine 3',5'-monophosphate on nitrogen fixation in Rhizobium japonicum.

The addition of exogenous cyclic guanosine 3',5'-monophosphate (cGMP) at a concentration of 0.1 mM to a free-living culture of Rhizobium japonicum 3I1b110 was found to completely inhibit the expression of nitrogenase activity and markedly inhibit the expression of hydrogenase and nitrate reductase activities. The effect was specific for cGMP. Experiments on the in vivo incorporation of radioactive methionine and subsequent analysis of the labeled proteins on polyacrylamide gels showed that the biosynthesis of nitrogenase polypeptides was inhibited. It appears that the time of addition of cGMP is important since the effect was only seen during the early stages of nif gene expression. The intracellular level of cGMP was found to respond to physiological changes in the cell, and there was a fall in cGMP concentrations when nitrogenase was induced. Microaerophilic-aerobic shift experiments showed that intracellular levels increased from 0.25 pmol/mg of cell protein under microaerophilic conditions to 2.6 pmol/mg of cell protein under aerobic conditions, suggesting that the cellular pool size of cGMP may be under redox control.

Cyclic GMP↗
PubMed1992

The role of cyclic adenosine 3',5'-monophosphate and polyol metabolism in diabetic neuropathy.

The effects of a stable prostacyclin analog, Iloprost, and aldose reductase inhibitors (ONO-2235 and isoliquiritigenin) were studied to elucidate the role of cAMP in diabetic neuropathy in relation to polyol metabolism. In in vivo experiments, the cAMP and myoinositol contents in sciatic nerves and motor nerve conduction velocity were significantly reduced in diabetic rats. Iloprost significantly restored the reduced cAMP content in sciatic nerves and improved motor nerve conduction velocity in diabetic rats. However, the contents of sorbitol or myoinositol in sciatic nerves were not affected by Iloprost in diabetic rats. On the other hand, aldose reductase inhibitors significantly reduced the sorbitol content and increased the cAMP and myoinositol contents in the sciatic nerves of diabetic rats. The motor nerve conduction velocity was also slightly but significantly improved by treatment with aldose reductase inhibitors. There was a negative correlation between cAMP and sorbitol in the sciatic nerves of diabetic rats treated with aldose reductase inhibitors and a positive correlation between cAMP and motor nerve conduction velocity. In in vitro experiments, Iloprost significantly increased cAMP, but did not affect the sorbitol content in sciatic nerves. Aldose reductase inhibitors inhibited sorbitol accumulation and increased cAMP in sciatic nerves. Our data suggest that polyol pathway activation somehow results in cAMP reduction in sciatic nerves and that the reduction of cAMP in peripheral nerves may be closely related to the pathogenesis of diabetic neuropathy.

Aldehyde Reductase↗
PubMed1997

Physiologic and molecular characterization of the role of nitric oxide in hemorrhagic shock: evidence that type II nitric oxide synthase does not regulate vascular decompensation.

To determine the role of nitric oxide (NO) in decompensated and irreversible hemorrhagic shock, rats were subjected to hemorrhagic shock (HS) for 3 or 5 h. Lung, liver, and plasma samples were studied for evidence of NO formation using Northern analysis and immunohistochemistry for Type II NOS, as well as measurement of plasma nitrite/nitrate, cyclic GMP, and nitrosylated hemoglobin levels. Comparisons were made with similarly instrumented time-matched sham rats. Type II NOS mRNA and protein were detectable in lung and liver only in the irreversible phase of HS (5 h). A large accumulation of nitrosylated hemoglobin and nitrite/nitrate appeared in the irreversible phase. Significant accumulation of cyclic GMP or nitrite/nitrate was not detectable in the decompensation phase. Despite the hemodynamic decompensation at 3 h of HS, Type II NOS mRNA and protein expression, as well as NO metabolites were not elevated. To assess whether NO plays a physiologically significant role in decompensation, rats in the decompensation phase and sham animals were subjected to nonspecific NOS inhibition. Both groups displayed a similar magnitude and duration of blood pressure elevation. Hemodynamic decompensation in HS is not mediated by Type II NOS induction. NO production increases only after prolonged HS; significant NO production is observed only in severe, irreversible HS.

Animals↗
PubMed2005

Rosuvastatin treatment protects against nitrate-induced oxidative stress.

Nitrate tolerance is associated with an enhanced superoxide anion production and can be attenuated by statins, which interact with the 2 main [eNOS and NAD(P)H oxidase] pathways involved in producing this oxidative stress. Three groups of normocholesterolemic rats were treated: group 1 received rosuvastatin (10 mg/kg/d PO) for 5 weeks and in the last 3 days cotreatment with nitroglycerin (NTG 50 mg/kg/d, subcutaneous injections BID); group 2 received only NTG (50 mg/kg/d BID for the last 3 days); and group 3 served as control. Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM to 0.1 mM) were determined on phenylephrine-preconstricted rings and O2 production (RLU/10 s/mg dry weight) was assessed by lucigenin and the luminol analogue (L-012) chemiluminescence technique. In group 2 (NTG), the concentration-response curves to NTG were significantly shifted to the right: the pD2 (-log NTG concentration evoking a half-maximal relaxation) was 6.75+/-0.06 (n=7) versus 7.75+/-0.07 (n=7) in group 3 (not exposed to NTG, P<0.05); O2 production was enhanced (10,060+/-1,205, n=7 versus 5,235+/-1,052, n=7; P<0.05). In contrast, in group 1, the rightward shift was attenuated: pD2 value was 7.20+/-0.10 (n=8), P<0.05 versus group 2; O2 production was decreased (5911+/-663; n=9, P<0.05 versus group 2). In addition, before NTG exposure, rosuvastatin treatment decreased p22phox [the essential NAD(P)H oxidase subunit] abundance in the aortic wall and decreased NAD(P)H oxidase activity. In contrast, this treatment did not alter either eNOS abundance or the basal release of endothelium-derived NO. Interestingly, in vivo treatment with apocynin, an NAD(P)H oxidase inhibitor, produced a protection similar to that with rosuvastatin. Long-term rosuvastatin treatment protects against nitrate tolerance in the rat aorta by counteracting NTG-induced increase in O2 production. This protection seems to involve a direct interaction with the NAD(P)H oxidase pathway rather than an up-regulation of the eNOS pathway.

Acetylcholine↗
PubMed1994

Mo(V) electron paramagnetic resonance signals from the periplasmic nitrate reductase of Thiosphaera pantotropha.

A Mo(V) electron paramagnetic resonance (EPR) study of the periplasmic respiratory nitrate reductase of the denitrifying bacterium Thiosphaera pantotropha has revealed that the molybdenum centre of this enzyme is very similar to that in the assimilatory nitrate reductase of Azotobacter vinelandii but is somewhat different from that of the membrane-bound bacterial respiratory nitrate reductases such as those of Escherichia coli and Paracoccus denitrificans. We have identified the Mo(V) species most likely to be the catalytically relevant one and characterised two other sets of Mo(V) EPR signals. As well as exhibiting EPR signals with g values typical of bacterial molybdenum-containing reductases, molybdenum-hydroxylase-like EPR signals can be elicited in the nitrate reductase of T. pantotropha upon treatment with excess dithionite. The only other enzyme known to display this phenomenon is the periplasmic dimethylsulphoxide reductase of Rhodobacter capsulatus. A mechanism for the generation of these signals is proposed which invokes reduction of the pterin ring of the molybdenum cofactor linked to GMP from the dihydro to the tetrahydro state. The possibilities and implications of there being cysteine ligands to the molybdenum centres of these two enzymes are discussed.

Azotobacter↗
PubMed2001

Cerivastatin, a hydroxymethylglutaryl coenzyme a reductase inhibitor, improves endothelial function in elderly diabetic patients within 3 days.

BACKGROUND: The short-term effects of hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) on endothelial function at doses that do not affect plasma lipid levels are not known. METHODS AND RESULTS: We investigated the short-term effects of cerivastatin, a hydroxymethylglutaryl coenzyme A reductase inhibitor, on endothelial function and endothelium-related products in elderly diabetic patients. Twenty-seven elderly diabetic patients (aged 69.3+/-3.4 years), with or without mild hypercholesterolemia, were enrolled in this study, which tested cerivastatin treatment (0.15 mg/d) for 3 days. Endothelium-dependent flow-mediated dilatation, endothelium-independent dilatation by nitroglycerin in the brachial artery, nitric oxide-related products (nitrite/nitrate and cGMP), endothelium-related products (von Willebrand Factor, soluble vascular cell adhesion molecule-1, and soluble intercellular adhesion molecule-1), and a marker of oxidant stress (8-isoprostane) were assessed. Levels of plasma lipids were not changed before and after treatment with cerivastatin. Flow-mediated dilatation was significantly increased by cerivastatin treatment, as were plasma nitrite/nitrate levels (from 16.9+/-3.4 to 22.0+/-3.7 micromol/L, P<0.05) and cGMP values. The percent of nitroglycerin-induced dilatation was not changed. Plasma concentrations of 8-isoprostane decreased, and levels of soluble vascular cell adhesion molecule also tended to decrease with cerivastatin. CONCLUSIONS: Improvement of endothelial function was in line with antiatherosclerotic effects. Cerivastatin improved impaired endothelial function in the short-term without affecting lipid profiles in elderly diabetic patients. This effect may be partly due to upregulation of endothelial nitric oxide synthase.

Aged↗
PubMed1999

Morphological and biochemical investigation of nitric oxide synthase and related enzymes in the rat and pig urothelium.

We investigated the enzymes involved in the NADPH-diaphorase (d) reaction in the rat and pig bladder urothelium. The urothelial cell layer displayed intense and uniform NADPH-d activity. Preincubation with the flavoprotein inhibitor diphenyleneiodionium chloride (DPI) and the alkaline phosphatase inhibitor levamisole concentration-dependently decreased the urothelial NADPH-d activity. Immunoreactivities to neuronal (n), endothelial (e), or inducible (i) nitric oxide synthase (NOS) were not detected in rat or pig urothelial cells. In rats, the urothelium was uniformly immunoreactive for NADPH cytochrome P450 reductase, whereas the pig urothelium displayed inconsistent labeling. In lipopolysaccharide (LPS)-treated rats, the bladder urothelium showed positive iNOS immunoreactivity. The iNOS labeling was found predominantly in cells located in the basal layer of the urothelium. In the pig bladder mucosa, a Ca2+-dependent NOS activity was evident in cytosolic and particulate fractions that was quantitatively comparable to the NOS activity found in the smooth muscle. In ultrastructural studies of urothelial cells, NADPH-d reaction products were found predominantly on membranes of the nuclear envelope, endoplasmatic reticulum and mitochondria. In conclusion, NADPH-d staining of the urothelium cannot be taken as an indicator for the presence of constitutively expressed NOS. Activity of alkaline phosphatase and cytochrome P450 reductase may account for part of the NADPH-d reaction in urothelial cells. However, LPS treatment of rats caused expression of iNOS in urothelial cells.

Animals↗
PubMed2004

Nitric oxide (NO)-releasing statin derivatives, a class of drugs showing enhanced antiproliferative and antiinflammatory properties.

Inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, namely statins, exert pleiotropic actions beyond lipid-lowering effects. Their pharmacological activity on atherosclerotic plaque stability and vascular inflammation appears to be mediated, at least in part, by nitric oxide (NO). With the aim of enhancing the nonlipid-lowering properties of selected statins, we introduced a NO-releasing moiety into the structure of pravastatin (NCX 6550) and fluvastatin (NCX 6553). NO release was evaluated as nitrosylhemoglobin adduct formation by using EPR spectroscopy in rat blood. Both compounds produced a linear time-dependent increase in nitrosylhemoglobin formation, which is consistent with slow NO release kinetics. In PC12 cells, unlike their native statins, both compounds stimulated cGMP formation (NCX 6550, EC(50) = 2.3 +/- 0.2 microM; NCX 6553, EC(50) = 2.7 +/- 0.2 microM). Moreover, NCX 6550 potently inhibited cell proliferation in rat aortic smooth muscle cells (IC(50) = 2.2 +/- 0.3 microM) with a mechanism that involved both the polyamine and HMG-CoA reductase signaling pathways. Hence, mevalonate or putrescine partially reverted the effects of NCX 6550 and their combination was fully effective. In RAW 264.7 murine macrophage cells stimulated with lipopolysaccharide (1 microg/ml), NCX 6550, but not pravastatin, significantly decreased inducible NO synthase and cyclooxygenase-2 protein expression as well as nitrite accumulation. All together, the data show that the previously undescribed NO-releasing statins retain HMG-CoA reductase inhibitory activity and release bioactive NO slowly. Among the additional properties, compared with native statins, the NO-releasing statins show enhanced antiinflammatory effects. Thus, NO-releasing statins represent an interesting class of drugs having potential in the therapy of disorders associated with endothelial dysfunction and vascular inflammation.

Animals↗