S-nitroso-acetyl-penicillamine Research Articles

Role of glutathione in nitric oxide-mediated injury to rat gastric mucosal cells

Recent studies suggest that in some cell types, the activity of nitric oxide (NO) is influenced by the endogenous antioxidant, reduced glutathione (GSH). The present study has examined the role of GSH in NO-induced cytotoxicity in cells harvested from the rat gastric mucosa. Cell integrity was assessed by Trypan blue exclusion and alamar blue dye absorbance. Pretreatment of rats with bacterial endotoxin lipopolysaccharide increased Ca 2+-independent NO synthase (iNO synthase) activity (as detected by the radiolabeled conversion of [ 14C]arginine to [ 14C]citrulline, lowered GSH content and increased cell injury. Lipopolysaccharide treatment also resulted in a significant increase in the in vitro production of reactive oxygen metabolites as assessed by the fluorescent probe 2′,7′-dichlorofluorescein diacetate. Inhibition of iNO synthase activity by dexamethasone and N G-nitro- l-arginine methyl ester prevented these effects. Similarly, the NO donor, S-nitroso acetyl-penicillamine depleted GSH stores and damaged cells in a dose-dependent manner. The effects of S-nitroso acetyl-penicillamine were diminished by the NO scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide. In contrast, incubating cells with N-acetyl- l-cysteine to augment endogenous GSH synthesis, prevented the effects of S-nitroso acetyl-penicillamine. Reduction of GSH stores by pretreatment of rats with buthionine sulfoximine or incubating cells in vitro with diethyl maleate, increased oxidant production and exacerbated NO-induced cell injury. These results suggest that excessive levels of NO alter GSH homeostasis and increase the generation of oxidants leading to increased gastric cellular injury.

Role of nitric oxide and superoxide in Giardia lamblia killing.

Giardia lamblia trophozoites were incubated for 2 h with activated murine macrophages, nitric oxide (NO) donors or a superoxide anion generator (20 mU/ml xanthine oxidase plus 1 mM xanthine). Activated macrophages were cytotoxic to Giardia trophozoites (approximately 60% dead trophozoites). The effect was inhibited (> 90%) by an NO synthase inhibitor (200 microM) and unaffected by superoxide dismutase (SOD, 300 U/ml). Giardia trophozoites were killed by the NO donors, S-nitroso-acetyl-penicillamine (SNAP) and sodium nitroprusside (SNP) in a dose-dependent manner (LD50 300 and 50 microM, respectively). A dual NO-superoxide anion donor, 3-morpholino-sydnonimine hydrochloride (SIN-1), did not have a killing effect in concentrations up to 1 mM. However, when SOD (300 U/ml) was added simultaneously with SIN-1 to Giardia, a significant trophozoite-killing effect was observed (approximately 35% dead trophozoites at 1 mM). The mixtures of SNAP or SNP with superoxide anion, which yields peroxynitrite, abolished the trophozoite killing induced by NO donors. Authentic peroxynitrite only killed trophozoites at very high concentrations (3 mM). These results indicate that NO accounts for Giardia trophozoites killing and this effect is not mediated by peroxynitrite.

Effect of nitric oxide on heme metabolism in pulmonary artery endothelial cells.

Primary intracellular targets for nitric oxide (NO) include nonheme iron-containing enzymes and protein-bound iron. Because NO is an important effector molecule in lung inflammation and endothelial cell-associated iron is critical to numerous forms of oxidant-mediated lung injury, we studied the effects of the NO donor S-nitrosoacetylpenicillamine (SNAP) on heme and iron metabolism in cultured sheep pulmonary artery endothelial cells. SNAP (300 microM) caused a transient increase in heme oxygenase-1 (HO-1) mRNA associated with a fivefold increase in HO activity that was completely blocked by the competitive HO inhibitor, tin protoporphyrin IX (SnPP). SNAP-induced activation of HO caused SnPP-sensitive reduction of activity of the hemoprotein catalase and decrease in heme iron. SNAP caused increases in iron-responsive gene products, ferritin and mitochondrial aconitase, secondary to the release of iron from heme stores via HO induction, since these changes were also sensitive to SNPP. The NO-induced increase in nonheme iron was apparent via electron paramagnetic resonance, where an enhanced SNAP-induced (300 microM for 4 h) g = 2.04 signal (e.g., dinitrosyl-iron-sulfur complex) was noted after exposure to a dose of SNAP (200 microM for 14 h) that in itself did not produce a detectable signal. These data show that exposure of pulmonary endothelial cells to NO results in profound changes in intracellular heme- and nonheme-iron homeostasis and that HO plays a central role in affecting this balance.

Tolerance to nitroglycerin in rabbit aorta: Investigating the involvement of the mu isozyme of glutathione S-transferases

We have proposed that glutathione S-transferases (GSTs), especially the mu isozyme, play a critical role in the metabolism of nitroglycerin (glyceryl trinitrate, GTN), leading to pharmacologic effects. Here we study this enzyme(s) during tolerance development in male New Zealand white rabbits. Each aorta was divided into two segments designated as GTN pretreated and buffer control. Tolerance was induced in rabbit aortic strips so assigned by incubation with GTN (0.22 mM). The activity of the mu isozyme and of total GSTs was determined in portions of each segment. In each rabbit aorta, the response to GTN (0.5 μM) was determined in GTN-pretreated and buffer-pretreated strips by measuring cyclic GMP levels (N = 7 pairs) and percent relaxation (N = 4 pairs). In GTN-pretreated strips, a significant decrease was observed in the activity of the mu isozyme of GST, while the total GST activity was unchanged as compared with control strips. The decrease in isozyme activity correlated very well with the decrease in response to GTN. Two rabbit aortae did not become tolerant, and the activity of the mu isozyme was also not affected. The levels of thiols were not affected by GTN pretreatment and aortae tolerant to GTN did not develop tolerance to S-nitroso acetylpenicillamine (SNAP), indicating that thiol depletion and guanylate cyclase desensitization probably play a minor role in tolerance development to GTN in our model. These studies suggest that tolerance to GTN in rabbit aorta in vitro is associated with a decrease in GST mu activity, which correlates well with the decrease in GTN response.

Differential Effects of Nitric Oxide on Erythroid and Myeloid Colony Growth From CD34+ Human Bone Marrow Cells

Nitric oxide (NO) is a reactive molecule with numerous physiologic and pathophysiologic roles affecting the nervous, cardiovascular, and immune systems. In previous work, we have demonstrated that NO inhibits the growth and induces the monocytic differentiation of cells of the HL-60 cell line. We have also demonstrated that NO inhibits the growth of acute nonlymphocytic leukemia cells freshly isolated from untreated patients and increases monocytic differentiation antigens in some. In the present work, we studied the effect of NO on the growth and differentiation of normal human bone marrow cells in vitro. Mononuclear cells isolated from human bone marrow were cultured in semisolid media and treated with the NO-donating agents sodium nitroprusside (SNP) or S-nitroso-acetyl penicillamine (SNAP) (0.25 to 1 mmol/L). Both agents decreased colony-forming unit-erythroid (CFU-E) and colony-forming unit-granulocyte macrophage (CFU-GM) formation by 34% to 100%. When CD34+ cells were examined, we noted that these cells responded to SNP and SNAP differently than did the mononuclear cells. At a concentration range of 0.25 to 1 mmol/L, SNP inhibited the growth of CFU-E by 30% to 75%. However, at the same concentration range, SNP increased the number of CFU-GM by up to 94%. At concentrations of 0.25 to 1 mmol/L, SNAP inhibited the growth of CFU-E by 33% to 100%. At a concentration of 0.25 mmol/L, SNAP did not affect CFU-GM. At higher concentrations, SNAP inhibited the growth of CFU-GM. Although SNP increased intracellular levels of cGMP in bone marrow cells, increasing cGMP in cells by addition of 8-Br-cGMP (a membrane permeable cGMP analogue) did not reproduce the observed NO effects on bone marrow colonies. These results demonstrate that NO can influence the growth and differentiation of normal human bone marrow cells. NO (generated in the bone marrow microenvironment) may play an important role modulating the growth and differentiation of bone marrow cells in vivo.

Nitric oxide exposure and sulfhydryl modulation alter l-arginine transport in cultured pulmonary artery endothelial cells

The effect of nitric oxide (NO) exposure and sulfhydryl-reactive chemicals on L-arginine transport in pulmonary artery endothelial cells was evaluated. Exposure of pulmonary artery endothelial cells to 7.5 ppm (0.4μM) NO for 4 h resulted in a significant ( p < 0.05) reduction of Na +- dependent but not Na +- independent L- arginine transport. More prolonged exposure for 12–24 h reduced both Na +- dependent and Na +- independent transport of L- arginine with maximal loss of transport after 18 h of exposure ( p < 0.02 for both). Similarly, incubation of cells in the presence of 50–200 μM S- nitroso- acetyl- penicillamine ( SNAP) ( but not 500 AM each of nitrate or nitrite) for 2 h also reduced both the Na +- dependent and Na +- independent transport of L- arginine ( p < 0.05 for all concentrations). The SNAP-induced reduction of L-arginine transport was blocked by the NO scavenger oxyhemoglobin. When cell monolayers were exposed to varying concentrations of the sulfhydryl reactive chemicals n-ethylmaleimide (NEM) and acrolein, a dose-dependent reduction of L-arginine transport by both Na +-dependent and Na +-independent processes was observed. Na +-dependent L-arginine transport was more susceptible to inhibition by exposure to NO and to sulfhydryl reactive chemicals. Incubation of cells with 0.5 mM of the thiol-containing agent N-acetyl-L-cysteine prior to and during NEM or acrolein exposure blocked NEM and acrolein-induced reduction of L-arginine transport by both Na +-dependent and Na +-independent processes. Similarly, NO-induced reductions of Na +-dependent and Na +-independent L-arginine transport were reversed to control levels 24 h after termination of NO exposure. Treatment with the disulfide reducing agent dithiothreitol after exposure to NO resulted in partial reversal of the decreases in L-arginine transport. These results demonstrate that exposure to exogenous NO is responsible for reversible reductions of plasma membrane-dependent L-arginine transport mediated by both the Na +-dependent (system B 0,+) and the Na +-independent (system y +) transport processes. Modulation of the sulfhydryl status of plasma membrane proteins involved in L-arginine transport, such as L-arginine transporters and/or Na+/K+-ATPase, may be responsible, at least in part, for reductions in overall L-arginine transport in pulmonary artery endothelial cells.

Involvement of endogneous nitric oxide in the control of pituitary responsiveness to different elicitors of growth hormone release in prepubertal rats.

Recently, nitric oxide (NO) has emerged as an important modulator of several neuroendocrine events. Present studies were designed in order to evaluate the involvement of endogenous NO in the control of GH secretion. In a first set of experiments, in vivo pituitary responsiveness to the elicitors of GH release, GH-releasing hormone (GHRH), GH-releasing hexapeptide (GHRP-6) and the agonist of excitatory amino acid receptors, N-methyl-D-aspartate (NMDA) was tested in prepubertal male rats after blockade of endogenous NO by pretreatment (45 min before the tests) with the competitive NO synthase (NOS) inhibitors NW-nitro-L-arginine methyl ester (NAME) and NW-nitro-L-arginine (NA). In addition, simultaneous administration of NAME and GHRH to prepubertal males was performed and the effects of NO deprivation on GHRH- and NMDA-stimulated GH release in prepubertal female rats were studied. In a second set of experiments, the involvement of NO in the modulation of GH secretion was analyzed in vitro. GH release by dispersed adenohypophysial cells was evaluated after challenge with the NO donors, sodium nitroprusside (SNP) and S-nitrosoacetylpenicillamine (SNAP), and cGMP, the classical second messenger for NO actions. In addition, the ability of NAME, SNP and cGMP to affect the GHRH-induced GH release in vitro was also tested. Our results showed that endogenous NO deprivation significantly attenuated in vivo GHRH-, GHRP-6- and NMDA-induced GH secretion in prepubertal male rats. However, the releasing ability of GHRH was not abolished by simultaneous administration of NAME. In addition, pretreatment with NAME blunted GHRH- and NMDA-stimulated GH release in prepubertal females. In vitro, neither SNP, SNAP, NAME nor cGMP modified GH secretion by pituitary cells in culture but NO deprivation by NAME completely blocked GHRH-induced GH release. In addition, cGMP but not SNP slightly enhanced GHRH-stimulated GH secretion. Taken together, these data strongly suggest that endogenous NO plays a permissive role in the control of stimulated GH release and that endogenous NO is an important factor eliciting GH secretion in prepubertal rats.

Nitric oxide modulation of the growth and differentiation of freshly isolated acute non-lymphocytic leukemia cells

Freshly isolated acute non-lymphocytic leukemia (ANLL) cells were treated with the nitric oxide (NO)-liberating compounds sodium nitroprusside or S-nitrosoacetyl penicillamine and analyzed for viability, growth, and differentiation at 3–5 days. NO decreased the viability and the growth of freshly isolated ANLL cells in vitro. NO treatment significantly increased expression of CD14 in blast cells from patients with M5 ANLL, and increased at least one differentiation parameter in M4 or M5 cells. It had little or no effect on parameters of differentiation in other ANLL cells. We conclude that in vitro culture with NO decreases the growth and viability of most freshly isolated ANLL cells. NO also induces the differentiation of ANLL cells with a monocytic phenotype.

Positive inotropic effect of nitric oxide in myocardium

Cardiac endothelium modulates underlying cardiac muscle performance probably by releasing certain regulatory factors. Nitric oxide (NO), which accounts for the biological activity of the vascular endothelium-derived relaxing factor and relaxes vascular smooth muscle by elevating intracellular cyclic GMP (cGMP), may be involved in this cardiac modulation. Many recent studies have examined inotropic effects of NO utilizing NO donors and NO-synthase inhibitors, both in vitro and in vivo, with apparently contradictory results. We examined the myocardial effects of NO-releasing nitrovasodilators (sodium nitroprusside (SNP), SIN-1 and S-nitrosoacetyl penicillamine (SNAP)), a cGMP analogue, 8-bromo-cGMP, and the cGMP phosphodiesterase inhibitor zaprinast, in isolated cat papillary muscle. A novel concentration-dependent positive inotropic effect of SNP and SIN-1 in muscles with damaged endocardial endothelium (EE) was observed which contrasted to their negative inotropic effect in muscles with intact EE. Both NO-induced positive and negative inotropic effects were attenuated by methylene blue, suggesting a role for cGMP. Concentration response curves with addition of SNAP and 8-bromo-cGMP resulted in a biphasic inotropic response. While administration of low concentrations of SNAP and 8-bromo cGMP induced a positive inotropic effect, higher concentrations induced a negative inotropic effect. Administration of zaprinast caused a monophasic concentration-dependent positive inotropic effect. We conclude that basal release of NO and consequent modest (physiological?) elevation in cGMP may preserve myocardial function, while large (pathological?) increases would depress myocardial function.

Nitric oxide: a cytokine-induced regulator of bone resorption.

Nitric oxide (NO) has been reported to inhibit osteoclastic bone resorption, yet potent stimulators of bone resorption, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF), are known to stimulate NO production. This paradox prompted us to reinvestigate the relationship between NO production and bone resorption in mouse calvarial organ cultures. Control cultures and those stimulated with calciotropic hormones and individual cytokines produced little NO, and under these conditions the NO synthase inhibitor, L-NG-monomethyl arginine (LMMA), had no significant effect on bone resorption. Cytokine combinations were much more potent stimulators of NO production than individual cytokines. Dramatic stimulation of NO production and inhibition of bone resorption resulted when gamma-interferon (IFN) was combined with IL-1 or TNF and these effects were reversed by LMMA. IFN had no effect on bone resorption and little effect on NO production when used alone or in combination with calciotropic hormones, however, suggesting that IFN selectively inhibits cytokine-induced bone resorption by generating large amounts of NO. IL-1 and TNF acted together to stimulate NO production but to a lesser degree than when combined with IFN. LMMA inhibited bone resorption induced by IL-1 and TNF, suggesting that lower concentrations of NO stimulate bone resorption. Experiments with the pharmacological NO donor S-nitroso-acetyl-penicillamine (SNAP) supported this view in showing generalized suppression of bone resorption at high SNAP concentrations, but potentiation of IL-1 induced bone resorption at lower SNAP concentrations. We conclude that cytokines are potent inducers of NO in bone and that cytokine-induced NO production has biphasic effects on bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

An increase in nitric oxide produced by rat peritoneal neutrophils is not involved in cell apoptosis

Polymorphonuclear neutrophils (PMN) obtained from carrageenin-stimulated peritoneal cavities of rats, but not blood PMN, spontaneously produced nitric oxide (NO) when incubated in vitro. Incubation of the cells with the NO synthase inhibitors, L-imino-ethyl-L-ornithine (L-NIO) or NG-monomethyl-L-arginine (L-NMMA), inhibited NO production. This inhibition could be reversed by L-arginine. Incubation of PMN with lipopolysaccharide (LPS) failed to enhance NO production. Pretreatment of the rats with dexamethasone (DEXA) prior to carrageenin injection or incubation of PMN with the glucocorticoid in vitro partially inhibited the spontaneous release of NO. On the other hand, when PMN obtained from DEXA pretreated rats were incubated in vitro with DEXA, NO synthase activity and hence NO generation were almost abolished. A similar inhibition was also observed following the addition of L-NIO or cycloheximide to cultures of carrageenin-elicited PMN. The NO production by PMN did not appear to be related to cell viability or apoptosis. Indeed, neither the blockade of NO generation by L-NIO nor the incubation of the neutrophils with a NO donor, S-nitroso-acetylpenicillamine (SNAP) modified the pattern of LDH release or DNA fragmentation. In summary, it appears that PMN migration triggers a continuous NO synthesis, and that NO produced by these cells is not related to their apoptosis.

Role of nitric oxide and endothelium in rat pial vessel dilation response to isoflurane.

Isoflurane induces cerebral hyperemia. We sought to assess whether isoflurane induces cerebral microvessel dilation in vivo, and if so, to determine whether nitric oxide (NO) and endothelium are involved. By using a rat closed cranial window model, pial arterioles and venules of 30-70 microns in diameter were measured using intravital microscopy. The cerebral microvascular dilatory response was recorded as percent change of diameter from baseline. The pial vessels were suffused with sodium nitroprusside (SNP) or S-nitroso-acetyl-penicillamine (SNAP) to verify intact vascular smooth muscle relaxation function, and with adenosine diphosphate (ADP) and/or acetylcholine (ACh) to verify endothelial NO-generating capability. To isolate NO's role in the cerebral microvascular effects of isoflurane (Protocol I), microvessels were studied with and without nitric oxide synthase (NOS) inhibition by topically applied nitro-L-arginine methyl ester (L-NAME). In controls, L-NAME was replaced by its inactive enantiomer, nitro-D-arginine methyl ester (D-NAME). Mercury light plus fluorescein dye (LD) endothelial injury (Protocol II) was used to delineate an endothelium-mediated mechanism. Subsequently, vasodilator applications were repeated to verify the desired effects of the interventions and followed by suffusion of isoflurane 1%, 2%, and 3% (Protocol I) or isoflurane 3% (Protocol II). Suffusions of SNP, ADP, and ACh induced diameter increases of 15%-30%. NOS inhibition with L-NAME greatly attenuated ADP and ACh responses, but did not alter the SNP response, confirming that NO generation was blocked, but not NO action. These responses were unaffected in D-NAME-suffused rats. Isoflurane dilated arterioles 17% and venules 6% in the presence of D-NAME suffusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Feedback inhibition of nitric oxide synthase activity by nitric oxide.

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.