Carlos Hermenegildo

SUPEROXIDE PRODUCTION AND ANTIOXIDANT ENZYMES IN AMMONIA INTOXICATION IN RATS

Injection of large doses of ammonium salts lead to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We have tested the effect of injecting 7 mmol/kg of ammonium acetate on the production of superoxide and on the activities of some antioxidant enzymes in rat liver, brain, erythrocytes and plasma. Glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver and brain (both in cytosolic and mitochondrial fractions) and also in blood red cells, while glutathione reductase activity remained unchanged. Superoxide production in submitochondrial particles from liver and brain was increased by more than 100% in both tissues. Both diminished activity of antioxidant enzymes and increased superoxide radical production could lead to oxidative stress and cell damage, which could be involved in the mechanism of acute ammonia toxicity.

Acute ammonia toxicity is mediated by the NMDA type of glutamate receptors

Previous experiments in our laboratory suggested that ammonium toxicity could be mediated by the NMDA type of glutamate receptors. To assess this hypothesis we tested if MK‐801, a specific antagonist of the NMDA receptor, is able to prevent ammonium toxicity. Mice and rats were injected i.p. with 12 and 7 mmol/kg of ammonium acetate, respectively, 73% of the mice and 70% of the rats died. However, when the animals were injected i.p. with 2 mg/kg of MK‐801, 15 min before ammonium injection, only 5% of the mice and 15% of the rats died. The remarkable protection afforded by MK‐801 indicates that ammonia toxicity is mediated by the NMDA receptor.

NMDA Receptor Antagonists Prevent Acute Ammonia Toxicity in Mice

We proposed that acute ammonia toxicity is mediated by activation of NMDA receptors. To confirm this hypothesis we have tested whether different NMDA receptor antagonists, acting on different sites of NMDA receptors, prevent death of mice induced by injection of 14 mmol/Kg of ammonium acetate, a dose that induces death of 95% of mice. MK-801, phencyclidine and ketamine, which block the ion channel of NMDA receptors, prevent death of at least 75% of mice. CPP, AP-5, CGS 19755, and CGP 40116, competitive antagonists acting on the binding site for NMDA, also prevent death of at least 75% of mice. Butanol, ethanol and methanol which block NMDA receptors, also prevent death of mice. There is an excellent correlation between the EC50 for preventing ammonia-induced death and the IC50 for inhibiting NMDA-induced currents. Acute ammonia toxicity is not prevented by antagonists of kainate/AMPA receptors, of muscarinic or nicotinic acetylcholine receptors or of GABA receptors. Inhibitors of nitric oxide synthase afford partial protection against ammonia toxicity while inhibitors of calcineurin, of glutamine synthetase or antioxidants did not prevent ammonia-induced death of mice. These results strongly support the idea that acute ammonia toxicity is mediated by activation of NMDA receptors.

Menopause and Ovariectomy Cause a Low Grade of Systemic Inflammation that May Be Prevented by Chronic Treatment with Low Doses of Estrogen or Losartan

The incidence of cardiovascular diseases in premenopausal women is lower than in men or postmenopausal women. This study reports the discovery of a low grade of systemic inflammation, including monocyte adhesion to arterial endothelium, elicited by menopause or estrogen depletion. Chronic treatment with low dose of 17-β-estradiol or inhibition of the renin-angiotensin system reduced this inflammation. Using an in vitro flow chamber system with human arterial and venous endothelial cells, we found that leukocytes from healthy postmenopausal women were more adhesive to the arterial endothelium than those from premenopausal women regardless of the stimulus used on endothelial cells. Increased circulating levels of IL-8, MCP-1, RANTES, and MIP-1α and monocyte CD11b expression were also encountered in postmenopausal vs premenopausal subjects. This translational data led us to investigate the mechanisms in Sprague-Dawley rats. Using intravital microscopy, we imaged mesenteric arterioles and found significant increases in arteriolar leukocyte adhesion, cell adhesion molecule expression, and plasma levels of cytokine-induced neutrophil chemoattractant (CINC/KC), MCP-1, and MIP-1α in 1-mo ovariectomized rats. Chronic treatment of ovariectomized rats with low dose of 17-β-estradiol, losartan, both, or benazepril inhibited ovariectomy-induced arteriolar mononuclear leukocyte adhesion by 77%, 58%, 92%, and 65% respectively, partly by inhibition of cell adhesion molecule up-regulation and the increase in circulating chemokines. These results demonstrate that menopause and ovariectomy generate a low grade of systemic inflammation. Therefore, administration of low doses of estrogens or inhibition of the renin-angiotensin system, at early stages of estrogen deficiency, might prevent the systemic inflammation associated with menopause and decrease the risk of suffering further cardiovascular diseases.

Nitroarginine, an Inhibitor of Nitric Oxide Synthase, Prevents Changes in Superoxide Radical and Antioxidant Enzymes Induced by Ammonia Intoxication

Injection of large doses of ammonium salts leads to the rapid death of animals. However, the molecular mechanisms involved in ammonia toxicity remain to be clarified. We reported that injecting ammonium acetate (7 mmol/kg) to rats increases the production of superoxide and reduces the activities of some antioxidant enzymes in rat liver and brain. We proposed that these effects induced by ammonia intoxication would be mediated by formation of nitric oxide. To test this possibility we tested whether injection of nitroarginine, an inhibitor of nitric oxide synthase, prevents the effects of ammonia intoxication on antioxidant enzymes and superoxide formation. Following injection of ammonia, glutathione peroxidase, superoxide dismutase and catalase activities were decreased in liver by 42%, 54% and 44%, respectively. In brain these activities were reduced by 35%, 46% and 65%, respectively. Glutathione reductase remained unchanged. Superoxide production in submitochondrial particles from liver and brain was increased by more than 100% in both tissues. Both reduction of activity of antioxidant enzymes and increased superoxide radical production were prevented by previous injection of 45 mg/kg of nitroarginine, indicating that ammonia induces increased formation of nitric oxide, which in turn reduces the activity of antioxidant enzymes, leading to increased formation of superoxide.

Trans- but Not Cis-Resveratrol Impairs Angiotensin-II–Mediated Vascular Inflammation through Inhibition of NF-κB Activation and Peroxisome Proliferator-Activated Receptor-γ Upregulation

Angiotensin II (Ang-II) displays inflammatory activity and is implicated in several cardiovascular disorders. This study evaluates the effect of cis- and trans (t)-resveratrol (RESV) in two in vivo models of vascular inflammation and identifies the cardioprotective mechanisms that underlie them. In vivo, Ang-II–induced arteriolar leukocyte adhesion was inhibited by 71% by t-RESV (2.1 mg/kg, i.v.), but was not affected by cis-RESV. Because estrogens influence the rennin-angiotensin system, chronic treatment with t-RESV (15 mg/kg/day, orally) inhibited ovariectomy-induced arteriolar leukocyte adhesion by 81%, partly through a reduction of cell adhesion molecule (CAM) expression and circulating levels of cytokine-induced neutrophil chemoattractant, MCP-1, and MIP-1α. In an in vitro flow chamber system, t-RESV (1–10 μM) undermined the adhesion of human leukocytes under physiological flow to Ang-II–activated human endothelial cells. These effects were accompanied by reductions in monocyte and endothelial CAM expression, chemokine release, phosphorylation of p38 MAPK, and phosphorylation of the p65 subunit of NF-κB. Interestingly, t-RESV increased the expression of peroxisome proliferator-activated receptor-γ in human endothelial and mononuclear cells. These results demonstrate for the first time that the in vivo anti-inflammatory activity of RESV is produced by its t-RESV, which possibly interferes with signaling pathways that cause the upregulation of CAMs and chemokine release. Upregulation of proliferator-activated receptor-γ also appears to be involved in the cardioprotective effects of t-RESV. In this way, chronic administration of t-RESV may reduce the systemic inflammatory response associated with the activation of the rennin-angiotensin system, thereby decreasing the risk of further cardiovascular disease.

Frailty and other geriatric conditions for risk stratification of older patients with acute coronary syndrome.

BACKGROUND Geriatric conditions may predict outcomes beyond age and standard risk factors. Our aim was to investigate a wide spectrum of geriatric conditions in survivors after an acute coronary syndrome. METHODS A total of 342 patients older than 65 years were included. At hospital discharge, 5 geriatric conditions were evaluated: frailty (Fried and Green scores), physical disability (Barthel index), instrumental disability (Lawton-Brody scale), cognitive impairment (Pfeiffer questionnaire), and comorbidity (Charlson and simple comorbidity indexes). The outcomes were postdischarge mortality and the composite of death/myocardial infarction during a 30-month median follow-up. RESULTS Seventy-four (22%) patients died and 105 (31%) suffered from the composite end point. Through univariable analysis, all individual geriatric indexes were associated with outcomes, mainly mortality. Of all of them, frailty using the Green score had the strongest discriminative accuracy (area under the receiver operating characteristic curve 0.76 for mortality). After full adjustment including clinical and geriatric data, the Green score was the only independent predictive geriatric condition (per point; mortality: hazard ratio 1.25, 95% CI 1.15-1.36, P = .0001; composite end point: hazard ratio 1.16, 95% CI 1.09-1.24, P = .0001). A Green score ≥ 5 points was the strongest mortality predictor. The addition of the Green score to the clinical model improved discrimination (area under the receiver operating characteristic curve 0.823 vs 0.846) and significantly reclassified mortality risk (net reclassification improvement 26.3, 95% CI 1.4-43.5; integrated discrimination improvement 4.0, 95% CI 0.8-9.0). The incremental predictive information was even greater over the GRACE score. CONCLUSIONS Frailty captures most of the prognostic information provided by geriatric conditions after acute coronary syndromes. The Green score performed better than the other geriatric indexes.

Estradiol selectively stimulates endothelial prostacyclin production through estrogen receptor-α

Estradiol (E(2)) acts on the endothelium to promote vasodilatation through the release of several compounds, including prostanoids, which are products of arachidonic acid metabolism. Among these, prostacyclin (PGI2) and thromboxane A2 (TXA2) exert opposite effects on vascular tone. The role of different estrogen receptors (ERs) in the PGI2/TXA2 balance, however, has not been fully elucidated. Our study sought to uncover whether E(2) enhances basal production of PGI2 or TXA2 in cultured human umbilical vein endothelial cells (HUVECs), to analyze the enzymatic mechanisms involved, and to evaluate the different roles of both types of ERs (ERalpha and ERbeta). HUVECs were exposed to E(2), selective ERalpha (1,3,5-tris(4-hydroxyphenyl)-4-propyl-1h-pyrazole, PPT) or ERbeta (diarylpropionitrile, DPN) agonists and antagonists (unspecific: ICI 182 780; specific for ERalpha: methyl-piperidino-pyrazole, MPP). PGI2 and TXA2 production was measured by ELISA. Expression of phospholipases, cyclooxygenases (COX-1 and COX-2), PGI2 synthase (PGIS), and thromboxane synthase (TXAS) was analyzed by western blot and quantitative RT-PCR. E(2) (1-100 nM) dose dependently increased PGI2 production (up to 50%), without affecting TXA2 production. COX-1 and PGIS protein and gene expressions were increased, whereas COX-2, phospholipases, and TXAS expression remained unaltered. All these effects were mediated through ERalpha, since they were produced not only in the presence of E(2), but also in that of PPT, while they were abolished in the presence of MPP. In conclusion, E(2), acting through ERalpha, up-regulates COX-1 and PGIS expression, thus directing prostanoid balance toward increased PGI2 production.

Estrogens Inhibit Angiotensin II–Induced Leukocyte–Endothelial Cell Interactions In Vivo via Rapid Endothelial Nitric Oxide Synthase and Cyclooxygenase Activation

Abstract— Angiotensin II (Ang II) may be a key molecule in the development of atherosclerosis. Because the incidence of coronary atherosclerosis in premenopausal women is lower than that observed in men or postmenopausal women, we have investigated the effect of estrogens on Ang II–induced leukocyte recruitment in vivo using intravital microscopy in the rat mesenteric microcirculation. Superfusion for 60 minutes with Ang II induced a significant increase in leukocyte rolling flux, adhesion, and emigration. Administration of 17-&bgr;-estradiol (17-&bgr;-E) after 30 minutes of Ang II superfusion produced a reduction of these leukocyte responses by 55.1%, 72.7%, and 70.9%, respectively, an additional 30 minutes later. The effect observed with 17-&bgr;-E was receptor-mediated and specific. 17-&bgr;-E superfusion did not modify either L-NAME or indomethacin-induced leukocyte responses. Inhibitory responses caused by 17-&bgr;-E were not altered by either 7-nitroindazole or actinomycin D cosuperfusion. Stimulation of endothelial cells with 17-&bgr;-E caused a rapid and dose-dependent release of prostacyclin. Finally, tamoxifen or ICI 182,780 administration provoked a significant increase in leukocyte–endothelial cell interactions 90 minutes later, which were significantly attenuated by systemic preadministration with an Ang II AT1 receptor antagonist. Tamoxifen-induced leukocyte responses were also reduced by systemic pretreatment with an anti–P-selectin mAb and an anti–CD18 mAb. Hence, the antiatherogenic effects of estrogens may be mediated by inhibition of Ang II–induced leukocyte recruitment through endothelial NO and prostacyclin release. Furthermore, scarcity of estrogens resulted in decreased levels of vasodilators and the exposure of the endothelium to the deleterious action of Ang II, which may explain the higher incidence of coronary atherosclerosis in men and postmenopausal women.

Oxygen Toxicity in the Nervous Tissue: Comparison of the Antioxidant Defense of Rat Brain and Sciatic Nerve

Nervous tissue, central and peripheral, is, as any other, subject to variations in oxygen tension, and to the attack of different xenobiotics; these situations may promote the generation of activated oxygen species of free radical character. Results are presented showing that the content of total glutathione (GSH) in brain is 10-fold that found in the sciatic nerve of the rat (2620 vs. 261 nmol/g wet weight, respectively). The existence of a relatively high superoxide dismutase activity in peripheral nervous tissue, when compared with brain or liver, in combination with the DT-diaphorase activity detected in the sciatic nerve might represent an effective defense mechanism against quinone toxicity, as is also discussed. Nervous tissue, both central and peripheral lack Se-independent GSH peroxidase activity. Finally, the activities of other glutathione-related enzymes studied in the sciatic nerve are very low, when compared with the central nervous tissue, thus suggesting a higher susceptibility of peripheral tissue to oxidative stress damage, since GSH concentration and/or any GSH-related enzymatic activities, e.g. GSH peroxidase or glutathione disulfide reductase, might become limiting.