Akin Yesilkaya

PEROXYNITRITE INACTIVATES PROSTACYCLIN SYNTHASE BY HEME–THIOLATE-CATALYZED TYROSINE NITRATION

Previous work has shown a sensitive inhibition of prostacyclin synthase activity by peroxynitrite as well as by superoxide in the presence of NO donors. Neither superoxide nor NO alone nor decomposed peroxynitrite is effective. The inhibition of activity was paralleled by a nitration of a tyrosine residue and both could be prevented by a stable substrate analog. The same IC50 value for peroxynitrite was also found for the cellular prostacyclin activity in endothelial and kidney mesangial cells, indicating that the antioxidant potential of the cell cannot prevent the inactivation. Aortic tissue shows a co-localization of prostacyclin synthase and nitrotyrosine staining after treatment of the tissue with 1 microM peroxynitrite. It can be speculated that this pathway of enzyme nitration is of pathophysiological significance.

Deformability and oxidant stress in red blood cells under the influence of halothane and isoflurane anesthesia

1. The effects of halothane and isoflurane anesthesia on red blood cell (RBC) deformability, lipid peroxidation and antioxidant enzymes were tested in rabbits. 2. RBC transit time was significantly increased to 2.12 +/- 0.07 msec after 1-hr halothane anesthesia preceded by 6 mg/kg pentobarbital injections from 1.98 +/- 0.07 msec preanesthesia value (p < 0.05). Thiobarbituric acid-reactive substances also were increased significantly, being 23.35 +/- 2.75 nmol/gHb and 33.11 +/- 5.34 nmol/gHb before and after anesthesia, respectively (p < 0.05). 3. Under halothane anesthesia without prior pentobarbital injection or under isoflurane anesthesia with or without pentobarbital injection, no significant alterations were observed in these parameters. 4. RBC superoxide dismutase activity was decreased in the group anesthetized with the pentobarbital-halothane combination. The impaired RBC deformability and increased oxidant damage might be related to the free radical formation during the metabolism of halothane. Pentobarbital can potentiate this effect either by inducing cytochrome P-450 or by altering antioxidant defense. 5. Alterations in RBC mechanical properties may contribute to the tissue perfusion problems that develop after surgery under general anesthesia.

The antioxidant effect of free bilirubin on cumene-hydroperoxide treated human leukocytes.

To examine the antioxidant effect of bilirubin (BR) on leukocyte, we treated leukocytes obtained from healthy subjects with an oxidant and various concentrations of BR. High concentrations of BR decreased thiobarbituric acid reactive substances (TBARS) and catalase activities, increased superoxide dismutase (SOD) activity, but had no effect on glutathione (GSH) concentration. Our results showed that under physiological conditions, BR has an antioxidant effect only in high concentrations.

Uric acid stimulates proliferative pathways in vascular smooth muscle cells through the activation of p38 MAPK, p44/42 MAPK and PDGFRβ

Abstract Hyperuricemia and angiotensin II (Ang II) may have a pathogenetic role in the development of hypertension and atherosclerosis as well as cardiovascular disease (CVD) and its prognosis. The purpose of this study was to investigate whether uric acid can induce proliferative pathways of vascular smooth muscle cell (VSMC) that are thought to be responsible for the development of CVD. The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), p44/42 mitogen-activated protein kinase (p44/42 MAPK) and platelet-derived growth factor receptor β (PDGFRβ) was measured by Elisa and Western blot techniques to determine the activation of proliferative pathways in primary cultured VSMCs from rat aorta. Results demonstrated that uric acid can stimulate p38 MAPK, p44/42 MAPK and PDGFRβ phosphorylation in a time- and concentration-dependent manner. Furthermore, treatment of VSMCs with the angiotensin II type I receptor (AT1R) inhibitor losartan suppressed p38 MAPK and p44/42 MAPK induction by uric acid. The stimulatory effect of uric acid on p38 MAPK was higher compared to that of Ang II. The results of this study show for the first time that uric acid-induced PDGFRβ phosphorylation plays a crucial role in the development of CVDs and that elevated uric acid levels could be a potential therapeutical target in CVD patients.

Inhibition of Human Erythrocyte (Na+-K+)ATPase by Organic Hydroperoxides and Protection by Ascorbic Acid and Butylated Hydroxytoluene

1. The in vitro effects of cumene hydroperoxide and t-butyl hydroperoxide on intact human erythrocyte membrane (Na(+)-K+)ATPase activities have been studied. 2. (Na(+)-K+)ATPase activities on erythrocyte membranes decreased in agreement with the results of chemiluminescence experiments. 3. Our results demonstrated that the organic hydroperoxides inhibit the activity of (Na(+)-K+) ATPase enzyme and that the antioxidants used prevent this inhibition.

Plasma levels of nitrites, PGF1α and nitrotyrosine in LPS-treated rats : functional and histochemical implications in aorta

We investigated the effects of lipopolysaccharide (LPS) administration on plasma nitrite, nitrotyrosine and 6-keto prostaglandin F1α (PGF1α) levels and the related resultant changes in function and histochemistry of aorta in rats. Plasma nitrite and PGF1α, nitrotyrosine levels were analysed after 5 mg/kg intravenous LPS was administered to rats compared with those in non-treated rats. The distribution of nitrotyrotic rings to phenylephrine (PE) from both the LPS-treated and control rats were studied in the organ baths. There were increases in plasma nitrite, PGF1α, and nitrotyrosine concentrations of LPS-treated rats compared to non-treated rats. Immunoreactivity of nitrotyrosine residues were detected in the endothelial and smooth muscle cells in LPS-treated but not in control rat aorta. The contractile responses to PE of the LPS-treated rat aortic rings were significantly reduced as compared with those of control rat’s. Incubation of the aortic rings from LPS-treated rats with cyclooxygenase inhibitor indomethacine or with a combination of indomethacine and nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) increased the contractile responses to the levels observed in control rats suggesting that both prostanoids and particularly nitric oxide (NO) are involved in the reduced contractile responses in LPS-treated rats. These results supported the view that LPS might cause an increment in both NO and PGI2 levels. This increase in the NO and PGI2 levels may be responsible from the reduction in responses of aorta to contractile agents in LPS-treated rats. Increased peroxynitrite formation in LPS-treated rats may lead to nitration of the tyrosil residues of the proteins in the aorta.ResumenSe investigan los efectos de la administración de lipopolisacárido (LPS) sobre los niveles plasmáticos de nitritos, nitrotirosina y 6-ceto-prostaglandina Fα (PGF1α) y los cambios histoquímicos y funcionales en aorta de rata. Los niveles plasmáticos de los citados compuestos se analizaron tras administración intravenosa de 5 mg/Kg de LPS en comparación con animales control. La distribución de nitrotirosina en la aorta se estudió mediante inmunohistoquímica y las respuestas de anillos aórticos a la fenilefrina (FE) de ratas tratadas y control se midió en baño de órganos. Los niveles plasmáticos de nitritos, nitrotirosina y PGF1α resultaron mayores en ratas tratadas que en las controles. Se detectó inmunorreactividad a residuos de nitrotirosina en el endotelio y en músculo liso de aorta de ratas tratadas, no de las control. La respuesta contráctil a la fenilefrina de los anillos aórticos de ratas tratadas con LPS fue significativamente menor que la correspondiente a ratas control. La preincubación de los anillos aórticos de ratas tratadas con indometacina (inhibidor de la ciclooxigenasa) o con una combinación de indometacina y L-NAME (inhibidor de la nitrato sintasa) aumenta las respuestas contráctiles hasta alcanzar los niveles de las ratas control, lo que sugiere que los prostanoides y el NO están implicados en la reducción de la respuesta contráctil en ratas tratadas. Estos resultados parecen indicar que el LPS podría causar aumento de los niveles de NO y PGI2 que explicaría la reducción en las respuestas contráctiles en la aorta de las ratas tratadas con LPS.

Effect of uric acid on inflammatory COX-2 and ROS pathways in vascular smooth muscle cells

Abstract Hyperuricemia is thought to play a role in cardiovascular diseases (CVD), including hypertension, coronary artery disease and atherosclerosis. However, exactly how uric acid contributes to these pathologies is unknown. An underlying mechanism of inflammatory diseases, such as atherosclerosis, includes enhanced production of cyclooxygenase-2 (COX-2) and superoxide anion. Here, we aimed to examine the effect of uric acid on inflammatory COX-2 and superoxide anion production and to determine the role of losartan. Primarily cultured vascular smooth muscle cells (VSMCs) were time and dose-dependently induced by uric acid and COX-2 and superoxide anion levels were measured. COX-2 levels were determined by ELISA, and superoxide anion was measured by the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c method. Uric acid elevated COX-2 levels in a time-dependent manner. Angiotensin-II receptor blocker, losartan, diminished uric-acid-induced COX-2 elevation. Uric acid also increased superoxide anion level in VSMCs. Uric acid plays an important role in CVD pathogenesis by inducing inflammatory COX-2 and ROS pathways. This is the first study demonstrating losartan’s ability to reduce uric-acid-induced COX-2 elevation.