Investigation of oxidant stress and vasodepression to glyceryl trinitrate in the obese Zucker rat in vivo.

1. We examined the relationship between oxidant stress and the vasodepressor activity of glyceryl trinitrate (GTN) in vivo, including rapid GTN tolerance development, in 13-week old obese and age-matched lean Zucker rats which had been maintained for 4 weeks on either control diet or diets enriched with the lipophilic, chain-breaking antioxidants vitamin E (0.5% w w(-1)) or probucol (0.5% w w(-1)) or the superoxide anion scavenger tiron (1% w v(-1) in drinking water). 2. The basal plasma level of the isoprostane 8-epi-PGF2alpha, an in vivo marker of lipid peroxidation, was elevated by approximately 5 fold in the obese Zucker rat and markedly reduced by dietary lipophilic antioxidants and depressed by dietary tiron. 3. Vasodepression to bolus does GTN (0.1-100 microg kg(-1) i.v.), but not endothelium-dependent vasodepression to bolus dose acetylcholine (ACh, 0.02-2.0 microg kg(-1) i.v.), was impaired in obese animals and completely restored by dietary antioxidants. 4. Nitrate tolerance developed in vivo during a I h infusion of GTN (40 microg kg(-1) min(-1) i.v.) appeared more severe in obese animals. However, rapid nitrate tolerance was not affected by dietary antioxidants in either the obese or lean Zucker rat. 5. We therefore provide evidence that elevated oxidant stress in the obese Zucker rat is associated with an impairment in nitrate vasodepressor activity. However, our data are not consistent with either a role for oxidant stress in rapid nitrate tolerance development in the anaesthetized Zucker rat or the aggravation of this tolerance by pre-existing oxidant stress.

Mechanism of gram-positive shock: identification of peptidoglycan and lipoteichoic acid moieties essential in the induction of nitric oxide synthase, shock, and multiple organ failure.

The incidence of septic shock caused by gram-positive bacteria has risen markedly in the last few years. It is largely unclear how gram-positive bacteria (which do not contain endotoxin) cause shock and multiple organ failure. We have discovered recently that two cell wall fragments of the pathogenic gram-positive bacterium Staphylococcus aureus, lipoteichoic acid (LTA) and peptidoglycan (PepG), synergize to cause the induction of nitric oxide (NO) formation, shock, and organ injury in the rat. We report here that a specific fragment of PepG, N-acetylglucosamine-beta-[1--> 4]-N-acetylmuramyl-L-alanine-D-isoglutamine, is the moiety within the PepG polymer responsible for the synergism with LTA (or the cytokine interferon gamma) to induce NO formation in the murine macrophage cell line J774.2. However, this moiety is also present in the PepG of the nonpathogenic bacterium Bacillus subtilis. We have discovered subsequently that S. aureus LTA synergizes with PepG from either bacterium to cause enhanced NO formation, shock, and organ injury in the rat, whereas the LTA from B. subtilis does not synergize with PepG of either bacterium. Thus, we propose that the structure of LTA determines the ability of a particular bacterium to cause shock and multiple organ failure (pathogenicity), while PepG acts to amplify any response induced by LTA.

Role of nitric oxide in the circulatory failure and organ injury in a rodent model of gram-positive shock.

1. The pathological features of Gram-positive shock can be mimicked by the co-administration of two cell wall components of Staphylococcus aureus, namely lipoteichoic acid (LTA) and peptidoglycan (PepG). This is associated with the expression of the inducible isoform of nitric oxide synthase (iNOS) in various organs. We have investigated the effects of dexamethasone (which prevents the expression of iNOS protein) or aminoguanidine (an inhibitor of iNOS activity) on haemodynamics, multiple organ dysfunction syndrome (MODS) as well as iNOS activity elicited by LTA + PepG in anaesthetized rats. 2. Co-administration of LTA (3 mg kg-1, i.v.) and PepG (10 mg kg-1, i.v.) resulted in a significant increase in the plasma levels of tumour necrosis factor-alpha (TNF alpha, maximum at 90 min) as well as a biphasic fall in mean arterial blood pressure (MAP) from 120 +/- 3 mmHg (time 0) to 77 +/- 5 mmHg (at 6 h, n = 8; P < 0.05). This hypotension was associated with a significant tachycardia (4-6 h, P < 0.05) and a reduction of the pressor response elicited by noradrenaline (NA, 1 microgram kg-1, i.v., at 1-6 h; n = 8, P < 0.05). Furthermore, LTA + PepG caused time-dependent increases in the serum levels of markers of hepatocellular injury, glutamate-pyruvate-transminase (GPT) and glutamate-oxalacetate-transaminase (GOT). In addition, urea and creatinine (indicators of renal dysfunction) were increased. There was also a fall in arterial oxygen tension (PaO2), indicating respiratory dysfunction, and metabolic acidosis as shown by the significant drop in pH, PaCO2 and HCO3-. These effects caused by LTA + PepG were associated with the induction of iNOS activity in aorta, liver, kidney and lungs as well as increases in serum levels of nitrite+nitrate (total nitrite). 3. Pretreatment of rats with dexamethasone (3 mg kg-1, i.p.) at 120 min before LTA + PepG administration significantly attenuated these adverse effects as well as the increases in the plasma levels of TNF alpha caused by LTA + PepG. The protective effects of dexamethasone were associated with a prevention of the increase in iNOS activity (in aorta, liver, lung, kidney), the expression of iNOS protein (in lungs), as well as in the increase in the plasma levels of total nitrite. 4. Treatment of rats with aminoguanidine (5 mg kg-1 + 10 mg kg-1 h-1) starting at 120 min after LTA + PepG attenuated most of the adverse effects and gave a significant inhibition of iNOS activity (in various organs) as well as an inhibition of the increase in total plasma nitrite. However, aminoguanidine did not improve renal function although this agent caused a substantial inhibition of NOS activity in the kidney. 5. Thus, an enhanced formation of NO by iNOS importantly contributes to the circulatory failure, hepatocellular injury, respiratory dysfunction and the metabolic acidosis, but not the renal failure, caused by LTA + PepG in the anaesthetized rat.