Experiments were designed to explore the effects of nitric oxide (NO) donors on generation of superoxide (O2.−) and peroxynitrite (ONOO−) in rabbit aortic rings.
Following inhibition of endogenous ...superoxide dismutase (SOD), significant basal release of O2.− was revealed (0.9±0.01×10−12 mol min−1 mg−1 tissue). Generation of O2.− increased in a concentration‐dependent manner in response to NADH or NADPH (EC50=2.34±1.18×10−4 and 6.21±1.79×10−3 M respectively, n=4). NADH‐stimulated O2.− chemiluminescence was reduced by approximately 85% in the presence of exogenous SOD (15×103 U ml−1).
Incubation of aortic rings with S‐nitrosoglutathione (GSNO; 1×10−5–3×10−3 M) or sodium nitroprusside (SNP; 1×10−8–1×10−3 M), resulted in a concentration‐dependent quenching of O2.− chemiluminescence which was proportional to NO release.
ONOO− formation was assessed indirectly by determining protein tyrosine nitration in rabbit aorta using a specific antibody against nitrotyrosine. Basally and in the presence of NADH, a single band was detected. Incubation of aortic rings with either GSNO (1×10−3 M) alone or GSNO with NADH resulted in the appearance of additional nitrotyrosine bands. Incubation of serum albumin with GSNO alone did not cause nitrotyrosine formation. In contrast, incubation with 3‐morpholinosydonomine (SIN‐1; 1×10−3 M, 10 min), resulted in marked nitration of albumin which was reduced by oxyhaemoglobin or SOD. Incubation of albumin with GSNO and pyrogallol, a O2.− generator, also resulted in protein nitration.
Addition of exogenous NO results in nitrotyrosine formation in rabbit aortic rings. Nitrotyrosine formation is likely to result from the reaction of exogenous NO and basal endogenous O2.− resulting in the formation of ONOO−. Formation of ONOO− and nitration of tyrosine residues potentially could lead to vascular damage and might represent unexpected adverse effects of long‐term nitrate therapy.
British Journal of Pharmacology (1999) 127, 788–794; doi:10.1038/sj.bjp.0702590
The effects of inhibitors of nitric oxide (NO) synthase and other cationic amino acids on unidirectional l‐arginine transport were studied in porcine aortic endothelial cells cultured in microwell ...plates or perfused in microcarrier columns. l‐Homoarginine, l‐lysine and l‐ornithine inhibited transport of l‐arginine. The NO synthase inhibitors NG‐monomethyl‐l‐arginine and NG‐iminoethyl‐l‐ornithine also reduced l‐arginine uptake, whereas NG‐nitro‐l‐arginine and its methyl‐ester had no inhibitory effect. The ability to modulate selectively endothelial cell l‐arginine transport or NO synthase activity will allow further characterization of the arginine transporter and its role in regulating NO biosynthesis.
The effects of endotoxaemia on coronary vasodilator responses to bradykinin (BK), sodium nitroprusside (SNP) and nicardipine were investigated in the rat isolated heart perfused at constant flow ex ...vivo.
Dose‐dependent reductions in coronary perfusion pressure reaching a maximum of 56±3 and 57±5 mmHg were observed for BK and SNP respectively. The BK response was biphasic, consisting of a rapid dilator response that was insensitive to NGnitro‐L‐arginine methyl ester (L‐NAME, 0.1 mM) and a second slower component whose duration was attenuated by L‐NAME.
Hearts obtained from rats treated with endotoxin (2.5 mg kg−1, i.p.) for 2 or 6 h had increased basal coronary perfusion pressure and reduced vasodilator responses to BK or SNP. Dilator responses to nicardipine were not affected by endotoxin treatment. In vitro perfusion of hearts from endotoxin‐treated rats with L‐NAME (0.1 mM) restored SNP responses to control values.
Treatment with dexamethasone (1 mg kg−1), 1 h before endotoxin did not alter the endotoxin‐induced impairment of dilator responses to BK or SNP.
These results show that coronary microvascular responses are altered following endotoxin exposure. Endotoxin results in increased coronary microvascular tone despite induction of NO synthase and inhibits the dilator response to BK and SNP, vasodilators that act via the release of NO. Responses to SNP in endotoxin‐treated hearts were restored to control values in the presence of L‐NAME suggesting that enhanced endogenous NO synthesis might saturate guanylate cyclase resulting in reduced response to NO donors. The reduced response to vasodilators and increased coronary resistance might be important in determining the response of the coronary circulation to systemic inflammation and infection.
British Journal of Pharmacology (2000) 130, 118–124; doi:10.1038/sj.bjp.0703267
1
The kinetics, specificity, pH‐ and Na+‐dependency of l‐citrulline transport were examined in unstimulated and lipopolysaccharide (LPS)‐activated murine macrophage J774 cells. The dependency of ...nitric oxide production on extracellular arginine or citrulline was investigated in cells activated with LPS (1 μg ml−1) for 24 h.
2
In unstimulated J774 cells, transport of citrulline was saturable (Kt = 0.16 mm and Vmax = 32 pmol μg−1 protein min−1), pH‐insensitive and partially Na+‐dependent. In contrast to arginine, transport of citrulline was unchanged in LPS‐activated (1 μg ml−1, 24 h) cells.
3
Kinetic inhibition experiments revealed that arginine was a relatively poor inhibitor of citrulline transport, whilst citrulline was a more potent inhibitor (Ki = 3.4 mm) of arginine transport but only in the presence of extracellular Na+. Neutral amino acids inhibited citrulline transport (Ki = 0.2–0.3 mm), but were poor inhibitors of arginine transport.
4
Activated J774 cells did not release nitrite in the absence of exogenous arginine. Addition of citrulline (0.01 − 10 mm), in the absence of exogenous arginine, could only partially restore the ability of cells to synthesize nitrite, which was abolished by 100 μm NG‐nitro‐l‐arginine methyl ester or NG‐iminoethyl‐l‐ornithine.
5
Intracellular metabolism of l‐14C‐citrulline to l‐14C‐arginine was detected in unstimulated J774 cells and was increased further in cells activated with LPS and interferon‐γ.
6
We conclude that J774 macrophage cells transport citrulline via a saturable but nonselective neutral carrier which is insensitive to induction by LPS. In contrast, transport of arginine via the cationic amino acid system y+ is induced in J774 cells activated with LPS.
7
Our findings also confirm that citrulline can be recycled to arginine in activated J774 macrophage cells. Although this pathway provides a mechanism for enhanced arginine generation required for NO production under conditions of limited arginine availability, it cannot sustain maximal rates of NO synthesis.
A one hectare pond on the headwaters of a mercury-contaminated creek in Oak Ridge, Tennessee acted as a biochemical reactor for the production of methylmercury, increasing waterborne methylmercury ...concentrations in the stream below the pond discharge. The flow of the creek was diverted around the pond in order to eliminate this input. Waterborne total mercury, methylmercury, and mercury in fish, were monitored in the pond and stream before and after bypass. Waterborne methylmercury concentration in the creek downstream from the pond decreased over 80% following diversion of streamflow around the pond, but mercury in redbreast sunfish in the pond tailwater did not decline similarly. Within the pond, now isolated from fresh waterborne mercury inputs from the stream, methylmercury concentrations in the water column remained similar to levels present before bypass. However, mercury concentrations in sunfish in the pond decreased approximately 75% following bypass, despite the continued presence of highly contaminated sediments (approximately 50 mg Hg/kg dry weight). We concluded that a decrease in the fraction of `dissolved methylmercury' in the isolated pond relative to pre-bypass conditions explained the decrease in mercury in fish within the pond. That observation also indicates that mercury associated with pond sediments was relatively unavailable for eventual bioaccumulation when compared to `fresh' mercury contributed by upstream sources. The lack of a post-bypass decrease in mercury concentrations in tailwater fish was also likely to be associated with the particle-associated nature of waterborne methylmercury exported from the pond.
Mercury-bearing material enters municipal landfills from a wide array of sources, including fluorescent lights, batteries, electrical switches, thermometers, and general waste; however, the fate of ...mercury (Hg) in landfills has not been widely studied. Using automated flux chambers and downwind atmospheric sampling, we quantified the primary pathways of Hg vapor releases to the atmosphere at six municipal landfill operations in Florida. These pathways included landfill gas (LFG) releases from active vent systems, passive emissions from landfill surface covers, and emissions from daily activities at each working face (WF). We spiked the WF at two sites with known Hg sources; these were readily detected downwind, and were used to test our emission modeling approaches. Gaseous elemental mercury (Hg
0
) was released to the atmosphere at readily detectable rates from all sources measured; rates ranged from ∼1-10 ng m
−2
hr
−1
over aged landfill cover, from ∼8-20 mg/hr from LFG flares (LFG included Hg
0
at μg/m
3
concentrations), and from ∼200-400 mg/hr at the WF. These fluxes exceed our earlier published estimates. Attempts to identify specific Hg sources in excavated and sorted waste indicated few readily identifiable sources; because of effective mixing and diffusion of Hg
0
, the entire waste mass acts as a source. We estimate that atmospheric Hg releases from municipal landfill operations in the state of Florida are on the order of 10-50 kg/yr, substantially larger than our original estimates, but still a small fraction of current overall anthropogenic losses.
1
Effects of dexamethasone on induction of nitric oxide (NO) synathase and l‐arginine transport by lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line J774. Metabolism of ...l‐arginine to l‐citrulline and subsequent changes in intracellular amino acids pools were correlated with changes in nitrite production.
2
Despite a high intracellular concentration of arginine in activated J774 cells, LPS (1 μg ml−1, 8 h) induced a 2.4 fold increase in arginine transport. Treatment of cells with cycloheximide (1 μg ml−1) inhibited the time‐dependent (1–8 h) induction of NO synthase and arginine transport mediated by LPS.
3
Induction of NO synthase by LPS (1 μg ml−1, 24 h) alone was accompanied by a marked increase in arginine utilisation leading to decreased intracellular arginine levels and elevated intracellular and extracellular l‐citrulline levels. These changes were further enhanced in the presence of interferon‐γ (IFN‐γ, 100 units ml−1, 24 h).
4
Dexamethasone (1 μm) abolished the increases in both nitrite and citrulline production induced by LPS alone but only partially reversed the combined effects of LPS and IFN‐γ. In contrast, treatment of cells with dexamethasone (10 μm) had no effect on the LPS‐mediated induction of arginine transport or the decrease in intracellular arginine concentration.
5
We conclude that induction of arginine transporter activity in LPS‐stimulated J774 cells involves de novo synthesis of carrier proteins, which increases transport of exogenous arginine during enhanced NO production. Moreover, the intracellular signalling pathways mediating induction of arginine transport and of NO synthase by LPS in activated macrophages diverge, since only the latter is sensitive to dexamethasone.
Summary
Aims: This short report was designed to provide 2‐year follow‐up data from a previous study carried out in a primary care practice in the UK to assess the clinical and practical implications ...of switching to generic drugs.
Methods: All patients previously switched from atorvastatin to simvastatin or losartan to candesartan were reviewed retrospectively 2 years after the switch. Total serum cholesterol and clinic blood pressure readings were used along with records of cardiovascular events occuring during the 2 year period to assess the clinical impact of the switch.
Results: Of the 69 patients switched from atorvastatin to simvastatin between March and September 2005, 65 are still registered at the practice. Of these, 61 (94%) are still on simvastatin and 58 (89%) on the same dose. There was no significant change in mean total cholesterol over this 2 year period between 4.04 ± 0.52 mmol/l prior to the switch and 3.90 ± 0.63 mmol/l 2 years after the switch (p = 0.06). Of the 108 patients switched from losartan to candesartan, 94 are still registered at the practice and taking an angiotensin receptor blocker (ARB), 92 of these (98%) are still on candesartan and there was a significant reduction in blood pressure 2 years post‐switch between 138/79 ± 12.9/6.6 prior to the switch and 131/77 ± 13.1/7.6 mmHg 2 years after the switch (p<<0.05). No adverse events attributable to the switch were reported in either group.
Conclusion: This small study provides evidence that switching drugs in primary care can be cost effective and safe in the medium term, if care is taken with selection of patients and there is structured follow‐up in place.
Approximately 250 000 kg of mercury was lost towater and soils at the U.S. Dept. of Energy Y-12 Plantin Oak Ridge, Tennessee in the 1950s and early 1960s. A creek originating within the plant ...receivedcontinuous inputs of waterborne mercury, predominantlyas dissolved inorganic mercury, from groundwater,streambed contamination, and sump and process waterdischarges to the contaminated storm sewer network.These produce aqueous total mercury concentrations of1-2 μg L^sup -1^ in the upper reaches of the stream,decreasing to about 0.1-0.2 μg L^sup -1^ in its lowerreaches. A program to reduce mercury concentrationsin the creek identified specific sources (buildingsumps, contaminated springwater seeps, foundationdrains, and contaminated piping) and rerouted wateraround contaminated portions of the drain system orcollected and treated mercury-contaminated waterbefore discharging it. As a result, waterbornemercury concentrations in the creek and total mercuryloading were reduced from 1.8 μg L^sup -1^ to0.6 μg L^sup -1^ and 100 to 20 g d^sup -1^, respectively, in the last 5 yr.Mean mercury concentrations in fish nearest sourceareas in the creek headwaters decreased at roughly thesame rate as waterborne total mercury concentrationsover the past five years, but at the facility boundarydownstream the decline in mercury bioaccumulation wasmuch less. At sites 5-15 km farther downstream, nodecrease was evident. Dissolved methylmercury tendedto increase with distance downstream in a patterninverse to that noted for its dissolved inorganicmercury precursor.Improvements in water quality and modification ofweirs to allow the passage of fish have resulted inthe establishment of large populations of fish inmercury-contaminated headwater areas previously devoidof fish. It may be that the accumulation, retention,and eventual downstream transport of this reservoir ofbiologically incorporated methylmercury has acted tobuffer against expected reductions in mercury in fishat downstream sites.PUBLICATION ABSTRACT
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