Nitric Oxide Donors GEA Research Articles

In-vitro bronchorelaxing effects of novel nitric oxide donors GEA 3268 and GEA 5145 in guinea-pigs and rats.

Endogenously released nitric oxide (NO) in airways might contribute to physiological bronchodilation; induced production of NO might play a role in the pathogenesis of asthma, although it could also be a compensatory mechanism to other factors that cause bronchoconstriction or inflammation. To investigate the efficacy of NO donors on bronchial tone, the bronchorelaxing efficacies of NO donors, new experimental GEA compounds 3268 and 5145 (oxatriazole sulphonylamides) were compared with those of sodium nitroprusside and SIN-1 (3-morpholinosydnonimine) and to the standard beta 2-adrenergic agonist, salbutamol, in bronchi of guinea-pigs and rats in-vitro. Their relaxing effects were also studied in rat mesentery arteries to compare the selectivity for airways. The capacity of the NO donors to produce nitrites and nitrates was assayed by the Griess reaction. The novel NO donors GEA 3268 and GEA 5145 were more potent bronchorelaxing agents than the old NO donors sodium nitroprusside and SIN-1. In guinea-pig bronchi, however, salbutamol was most potent. In rat bronchi the GEA compounds induced the strongest relaxation effect when compared with the old NO donors or with salbutamol. The airway selectivity of the drugs studied decreased in the order of salbutamol, SIN-1, GEA 5145, GEA 3268, sodium nitroprusside. The nitrites and nitrates produced spontaneously did not correlate with the efficacy of the relaxants. The results obtained suggest that NO is only partly responsible for the relaxation and the potency is dependent on the animal species and constricting agents used.

Ca 2+-dependent and -independent Cl − secretion stimulated by the nitric oxide donor, GEA 3162, in rat colonic epithelium

The lipophilic nitric oxide-liberating drug, 1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), concentration-dependently induced a Cl − secretion in rat colon. At a low concentration (5×10 −5 M), the action was Ca 2+-dependent, whereas at a high concentration (5×10 −4 M), the response was independent from extracellular Ca 2+. Fura-2 experiments at isolated colonic crypts revealed that GEA 3162 induced an increase of the cytoplasmic Ca 2+ concentration due to an influx of extracellular Ca 2+, probably mediated by an activation of a nonselective cation conductance as demonstrated by whole-cell patch-clamp studies. After depolarization of the basolateral membrane, GEA 3162 (5×10 −4 M) stimulated a current, which was suppressed by glibenclamide but was resistant against blockade of protein kinases by staurosporine, suggesting an activation of apical Cl − channels directly by the nitric oxide (NO) donor. After permeabilizing the apical membrane with the ionophore, nystatin, GEA 3162 (5×10 −4 M) activated basolateral K + conductances and the Na +–K +-ATPase. Thus, the lipophilic NO donor GEA 3162 stimulates a Cl − secretion in a Ca 2+-dependent and -independent manner.

Dissociation of DNA Fragmentation from Other Hallmarks of Apoptosis in Nitric Oxide-Treated Neutrophils: Differences between Individual Nitric Oxide Donor Drugs

The events of apoptotic cell death can be experimentally dissociated from each other in certain cell types. Here we demonstrate the ability of structurally diverse nitric oxide (NO) donating compounds to delay or enhance neutrophil apoptosis and to differentially influence distinct parameters of programmed cell death. We provide evidence that high concentrations of the NO donors GEA 3162, SPER/NO, and DEA/NO induce morphological and biochemical markers of neutrophil apoptosis, but that only DEA/NO causes a concomitant increase in DNA fragmentation as evidenced by nuclear propidium iodide intercalation and the classical laddering pattern of electrophoresed DNA. In contrast, both GEA 3162 and SPER/NO inhibit DNA cleavage in a time- and concentration-dependent manner. We are the first to show that DNA fragmentation can be dissociated from other changes of apoptosis in NO-treated neutrophils and that it may therefore be inappropriate to assess NO-induced apoptosis solely by measuring DNA fragmentation in this cell type.

Distinction between interleukin-1-induced necrosis and apoptosis of islet cells.

Interleukin (IL)-1beta is known to cause beta-cell death in isolated rat islets. This effect has been attributed to induction of nitric oxide (NO) synthase in beta-cells and subsequent generation of toxic NO levels; it was not observed, however, in dispersed rat beta-cells. The present study demonstrates that IL-1beta induces NO-dependent necrosis in rat beta-cells cultured for 3 days at high cell density or in cell aggregates but not as single cells. Its cytotoxic condition is not explained by higher NO production rates but might result from higher intercellular NO concentrations in statically cultured cell preparations with cell-to-cell contacts; nitrite levels in collected culture medium are not a reliable index for these intercellular concentrations. Absence of IL-1-induced necrosis in rat alpha-cells or in human beta-cells is attributed to the cytokine's failure to generate NO in these preparations, not to their reduced sensitivity to NO: the NO donor GEA 3162 (15 min, 50-100 micromol/l) exerts a comparable necrotic effect in rat and human alpha- or beta-cells. In preparations in which IL-1beta does not cause beta-cell necrosis, its combination with gamma-interferon (IFN-gamma) results in NO-independent apoptosis, starting after 3 days and increasing with the duration of exposure. Because IFN-gamma alone was apoptotic for rat alpha-cells, it is proposed that IL-1beta can make beta-cells susceptible to this effect, conceivably through altering their phenotype. It is concluded that IL-1beta can cause NO-dependent necrosis or NO-independent apoptosis of islet cells, depending on the species and on the environmental conditions. The experiments in isolated human beta-cell preparations suggest that these cells may preferentially undergo apoptosis when exposed to IL-1beta plus IFN-gamma unless neighboring non-beta-cells produce toxic NO levels.

Induction of human neutrophil apoptosis by nitric oxide donors: evidence for a caspase-dependent, cyclic-GMP-independent, mechanism

This study investigated the regulatory effects of the major inflammatory mediator, nitric oxide (NO), on human neutrophil apoptosis in vitro. Co-culture of human neutrophils with the NO donors GEA 3162 (1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride) (10–100 μM) and 3-morpholino-sydnonimine (SIN-1) (0.3–3 mM) caused a dramatic and concentration-dependent induction of apoptosis. However, N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced neutrophil activation (actin reorganization and chemotaxis) was inhibited by GEA 3162 treatment. The pro-apoptotic effects of the NO donors were (i) unaffected by the soluble guanylate cyclase inhibitor LY-83583 (6-anilino-5,8-quinolinedione; 100 μM), (ii) antagonized by superoxide dismutase (6 μg/mL), (iii) mimicked by exogenous peroxynitrite (at concentrations >100 μM), and (iv) inhibited by the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (100 μM). The pro-apoptotic effect of the NO donors was not mimicked by the cell-permeable cyclic nucleotide analogue, N 6,2- O-dibutyrylguanosine-3′,5′-cyclic monophosphate (dibutyryl-cGMP) at concentrations ≤0.2 mM. Indeed, at high concentrations (≥2 mM), dibutyryl-cGMP caused an inhibition of apoptosis. These results suggest that NO-mediated apoptosis, although caspase-dependent, is mediated by a cGMP-independent mechanism and involves the concurrent generation of oxygen free radicals and, potentially, peroxynitrite. Our data reveal a unique role for NO in inflammatory responses with differential effects upon neutrophil activation and survival, with important implications for the successful resolution of inflammation.

Effects of nitric oxide donors GEA 3162 and SIN-1 on ethanol-induced gastric ulceration in rats

Low doses of the intragastrically (i.g.) administered nitric oxide (NO) donors, 1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162; 0.3 mg/kg) and 3-morpholino-sydnonimine (SIN-1; 1 mg/kg), inhibited gastric ulceration induced by ethanol (94%) in anesthetized rats. In contrast, higher doses of these NO donors administered i.g. exacerbated the damage. When administered intravenously, the NO donors had no effect on ethanol-induced gastric lesions although a clear blood pressure-lowering effect was seen. Neither the inhibition nor the exacerbation of ulceration was correlated with changes in blood pressure or prostaglandin E 2 release from the mucosal tissue. The relatively small difference between the gastroprotective and damaging doses suggests that orally administered NO donors, especially in the case of GEA 3162, may have a narrow gastric safety margin.

Adenosine 5’-Diphosphate-Induced Platelet Aggregation in Uremia Shows Resistance to Inhibition by the Novel Nitric Oxide Donor GEA 3175 but Not by S-Nitroso-N-Acetylpenicillamine

Both bleeding and thrombosis are complications of uremia in patients on regular hemodialysis. An excessive endogenous formation of the vasodilator and platelet inhibitor nitric oxide (NO) has been proposed to contribute to the bleeding defect. Since exposure to pharmacological donors of NO, nitrovasodilators, can cause tolerance to NO, we investigated whether platelets from uremic patients on regular hemodialysis are influenced differently by NO donors than platelets from healthy subjects. A frequently used S-nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), was compared to a recently synthezised mesoionic oxatriazole derivate, GEA 3175, regarding its capacity to inhibit adenosine 5′-diphosphate (ADP)-induced platelet aggregation in vitro. The final products of NO production, nitrite + nitrate, were found to be significantly increased in uremic patients. The capacity to inhibit platelet aggregation by SNAP was only slightly different between the groups. However, GEA 3175 showed a significantly marked and reduced capacity to inhibit aggregation of uremic platelets compared to controls. Interactions of erythropoietin (EPO) with NO have earlier been reported. Addition of EPO to platelets from healthy donors in vitro did not significantly influence the NO donor capacity to inhibit platelet aggregation, but showed a tendency to enhance the effect of SNAP while the effect of GEA 3175 was inhibited. These results suggest compound-specific resistance to NO donors in uremic platelet activation.

Nitric oxide donor GEA 3162 inhibits endothelial cell-mediated oxidation of LDL

Nitric oxide donor GEA 3162 inhibits endothelial cell-mediated oxidation of LDL

Nitric oxide donor GEA 3162 inhibits endothelial cell-mediated oxidation of low density lipoprotein

The effect of a nitric oxide (NO) donor GEA 3162 on the endothelial cell (EC)-mediated oxidation of low density lipoprotein (LDL) was studied. In comparison to LDL incubated with EC without GEA 3162, the presence of GEA 3162 inhibited LDL oxidation by EC, as indicated by the following findings. (a) The degradation rate of LDL in macrophages was reduced to control levels. (b) The electrophoretic mobility of LDL decreased in a dose-dependent manner, (c) The concentrations of thiobarbituric acid-reactive substances and hydroperoxide-derived hydroxy fatty acids were lower. (d) The breakdown of apolipoprotein B was reduced. The results indicate that GEA 3162 prevents EC-mediated oxidation of LDL.