Research communications in molecular pathology and pharmacology, 2001
In the present study, we measured the concentrations of reduced glutathione (GSH) and malonyldial... more In the present study, we measured the concentrations of reduced glutathione (GSH) and malonyldialdehyde (MDA) and the activities of glutathione peroxidase (GSH-Px), glutathione S-transferase (GSH-S-T), superoxide dismutase (SOD), catalase (CAT) and glucose-6-phosphate dehydrogenase (G-6-PD) in erythrocytes obtained freshly from adult male donors which was preserved with CPDA-1 anticoagulant (citrate,phosphate, dextrose, adenine) on different days of storage. At the end of the study, storage-associated alterations in antioxidant activities were noted and discussed. GSH, GSH-Px, GSH-S-T, SOD, CAT and G-6-PD activities decreased, but erythrocyte MDA levels, as anindex of lipid peroxidation, increased during the storage period. According to our results, glutathione-dependent antioxidant systems in erythrocytes might be depleted during long storage in blood bags.
Nitric oxide (NO) plays a major role in vascular regulation. Modulation of NO synthesis is known ... more Nitric oxide (NO) plays a major role in vascular regulation. Modulation of NO synthesis is known to influence blood pressure. Inhibition of NO synthesis by NG-nitro-L-arginine methyl ester (L-NAME; 72 mg/kg/day, p.o., 21 days) resulted in 60% increase in blood pressure in rats. Red blood cell (RBC) transit time measured by the cell transit analyzer increased significantly in the L-NAME treated group, in comparison to normotensive rats. RBC aggregation measured in autologous plasma, by a photometric rheoscope also increased significantly in the hypertensive rats. RBC cytosolic free calcium concentration was also significantly higher in the hypertensive animals. Incubation of RBC from hypertensive and control animals with NO donor, sodium nitroprusside (SNP; 10-1000 microM) for 60 minutes resulted in a dose-dependent decrease in RBC aggregation, however aggregation index was significantly higher in hypertensive group at each SNP concentration. Incubation with SNP had no effect on RBC ...
1. The effects of halothane and isoflurane anesthesia on red blood cell (RBC) deformability, lipi... more 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.
ABSTRACT Aim: Ang II stimulates vascular smooth muscle cells (VSMCs) and activates mitogen protei... more ABSTRACT Aim: Ang II stimulates vascular smooth muscle cells (VSMCs) and activates mitogen protein kinases (MAPKs) in culture media. p38 MAPK, a member of MAPK family, is stimulated by both Ang II and reactive oxygen species. This study was carried out to show Ang II stimulated p38 MAPK activation through Ras and subsequent NADPH oxidase activation in VSMCs. Material and Method: In our study, primary VSMCs isolated from rat aorta were stimulated by Ang II and with different inhibitors, and western blot was used to measure p38 MAPK phosphorylation. Results: The optimum p38 MAPK phosphorylation was observed at 100 nM Ang II concentration for 5 minutes and the next experiments were carried out under this conditions. To analyze the effect of Ras, which is an upstream mediator of p38 MAPK phosphorylation, VSMCs were incubated with a Ras spesific inhibitor FTS (Farnesyl thiosalicylic acid). After the stimulation of VSMCs by Ang II, p38 MAPK phosphorylation was inhibited. p38 MAPK phosphorylation was also completely inhibited in the presence of DPI (Diphenyl Iodinoum), a spesific NAD(P)H oxidase inhibitor, after Ang II stimulation. Conclusion: These findings demonstrate that Ang II stimulated p38 MAPK phosphorylation is through AT1 receptor and Ras-NAD(P)H oxidase dependent pathway in VSMCs. Understanding this pathway may contribute to the cellular mechanisms underlying in cardiovascular diseases.
To examine the antioxidant effect of bilirubin (BR) on leukocyte, we treated leukocytes obtained ... more 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.
Angiotensin II (Ang II) induces a rapid increase in mitogen-activated protein kinase (MAPK) activ... more Angiotensin II (Ang II) induces a rapid increase in mitogen-activated protein kinase (MAPK) activity through the Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMCs). In the present study, we examined the effects of the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor GF109203X, and the Ras inhibitor farnesylthiosalicylic acid (FTS) on Ang II-induced activation of p42/p44 MAPKs in cultured VSMCs. Phosphorylation was shown using the Western blot technique with specific phospho-antibodies against MAPK proteins. The PLC inhibitor U73122 abolished the Ang II-induced MAPK activity, while the PKC inhibitor GF109203X only decreased it. There was also an inhibition observed with the Ras inhibitor, FTS on Ang II-induced MAPK activity. These data suggest that Ang II-induced MAPK phosphorylation through the Ang II type 1 receptor could be mediated by Ras and/or PLC-dependent phosphorylations but not by PKC phosphorylation.
Stimulation of the angiotensin II (Ang II) type 1 receptor (AT 1-R) causes phosphorylation of ext... more Stimulation of the angiotensin II (Ang II) type 1 receptor (AT 1-R) causes phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) via epidermal growth factor receptor (EGF-R) transactivation-dependent or-independent pathways in Ang II target cells. Here we examined the mechanisms involved in agonist-induced EGF-R transactivation and subsequent ERK1/2 phosphorylation in clone 9 (C9) hepatocytes, which express endogenous AT 1-R, and COS-7 and human embryonic kidney (HEK) 293 cells transfected with the AT 1-R. Ang II-induced ERK1/2 activation was attenuated by inhibition of Src kinase and of matrix metalloproteinases (MMPs) in C9 and COS-7 cells, but not in HEK 293 cells. Agonist-mediated MMP activation in C9 cells led to shedding of heparin-binding EGF (HB-EGF) and stimulation of ERK1/2 phosphorylation. Blockade of HB-EGF action by neutralizing antibody or its selective inhibitor, CRM197, attenuated ERK1/2 activation by Ang II. Consistent with its agonist action, HB-EGF stimulation of these cells caused marked phosphorylation of the EGF-R and its adapter molecule, Shc, as well as ERK1/2 and its dependent protein, p90 ribosomal S6 kinase, in a manner similar to that elicited by Ang II or EGF. Although the Tyr319 residue of the AT 1-R has been proposed to be an essential regulator of EGF-R transactivation, stimulation of wild-type and mutant (Y319F) AT 1-R expressed in COS-7 cells caused EGF-R transactivation and subsequent ERK1/2 phosphorylation through release of HB-EGF in a Src-dependent manner. In contrast, the noninvolvement of MMPs in HEK 293 cells, which may reflect the absence of Src activation by Ang II, was associated with lack of transactivation of the EGF-R. These data demonstrate that the individual actions of Ang II on EGF-R transactivation in specific cell types are related to differential involvement of MMP-dependent HB-EGF release. (Molecular Endocrinology 18: 2035-2048, 2004) G PROTEIN-COUPLED RECEPTORS (GPCRs) activate MAPK signaling cascades by a variety of biochemical pathways. These include the generation of second messengers [Ca 2ϩ , protein kinase C (PKC), and cAMP], G protein subunit coupling to novel effectors, and activation of receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor, and IGF receptor (1, 2). MAPKs are activated by a wide spectrum of stimuli, ranging from mitogens and growth factors to cellular stress and neurotoxic factors. ERK1/2 are closely related members of the MAPK family that are predominantly activated by growth factors and GPCRs, and are important factors in cell growth, survival, motility, and secretion (3). The type 1 angiotensin II (Ang II) receptor (AT 1-R) is a typical GPCR, and mediates a wide variety of physiological actions in the cardiovascular system, kidney, brain, adrenal glands, and liver (4). Ang II and the AT 1-R have critical roles in cardiovascular diseases including hypertension, atherosclerosis, and cardiac hypertrophy (5, 6). In most Ang II target cells, the
Intravascular hemolysis is one of the most emphasized mechanisms for destruction of erythrocytes ... more Intravascular hemolysis is one of the most emphasized mechanisms for destruction of erythrocytes during and after physical activity. Exercise-induced oxidative stress has been proposed among the different factors for explaining exercise-induced hemolysis. The validity of oxidative stress following exhaustive cycling exercise on erythrocyte damage was investigated in sedentary and trained subjects before and after antioxidant vitamin treatment (A, C, and E) for 2 mo. Exercise induced a significant increase in thiobarbituric acid-reactive substance and protein carbonyl content levels in sedentary subjects and resulted in an increase of osmotic fragility and decrease in deformability of erythrocytes, accompanied by signs for intravascular hemolysis (increase in plasma hemoglobin concentration and decrease in haptoglobulin levels). Administration of antioxidant vitamins for 2 mo prevented exercise-induced oxidative stress (thiobarbituric acid-reactive substance, protein carbonyl content...
Previous work has shown a sensitive inhibition of prostacyclin synthase activity by peroxynitrite... more 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.
Observing better transfection efficiency in primary cultured VSMCs: Comparison and development of... more Observing better transfection efficiency in primary cultured VSMCs: Comparison and development of two different protocols [Primer VDKH kültüründe daha etkili bir transfeksiyon gözlenmesi: İki farklı protokolün karşılaştırılması ve geliştirilmesi]
Although effects of stress on the stomach have been extensively investigated in children and adul... more Although effects of stress on the stomach have been extensively investigated in children and adults, our knowledge about effects of fetal distress (FD) on the fetal stomach is quite limited. Therefore, an experimental study was planned to evaluate the effects of FD on fetal gastric physiology and histology. In this study, a model of FD was created by way of intermittent maternal aortic occlusion in pregnant rabbits. In total, 21 fetuses of 6 pregnant rabbits were available for surgical and laboratory procedures. Laboratory examinations showed that (1) fetal gastric acid secretion was 4.24 +/- 2.68 muEq/h in the control group and 18.08 +/- 6.34 muEq/h in the distress group (p < 0.01) and (2) fetal gastric PGE2 level was 16.59 +/- 6.15 mg/g wet weight in the control group and 9.86 +/- 3.46 mg/g wet weight in the distress group (p < 0.05). Histopathologically, there were mild hemorrhagic and errosive changes in the distressed fetuses, but not in control fetuses. These findings support that FD adversely affects fetal gastric physiology through two mechanisms consisting of increased gastric acid secretion and decreased fetal gastric protection in rabbits. Consequently, gastric injury should be noted as a potential problem among hypoxia-associated abnormalities encountered in the distressed fetus.
Fetal distress (FD) adversely affects fetal gastric physiology and histology, increasing gastric ... more Fetal distress (FD) adversely affects fetal gastric physiology and histology, increasing gastric acid secretion and disturbing gastric protective mechanism. Considering these findings, an experimental study was planned to test whether ranitidine prevents FD-related gastric physiological and histological changes during late gestational period. In this study, a rabbit model of FD was created by way of intermittent maternal aortic occlusion. In group 1 (SC), saline treated animals underwent control operation. In group 2 (SD), FD was created in saline treated animals. In group 3 (RC), ranitidine treated animals underwent control operation. In group 4 (RD), FD was created in ranitidine treated animals. Blood lactic acid levels of the fetuses were 2.3 +/- 1.0 mg/L in SC group and 4.7 +/- 1.8 mg/L in group SD (p < 0.01); 2.5 +/- 0.9 mg/L in group RC and 6.7 +/- 2.5 mg/L in group RD (p < 0.01). Fetal gastric acid secretion was 5.94 +/- 2.13 microEq/h in group SC and 8.26 +/- 2.24 microEq/h in group SD (p < 0.05); 6.63 +/- 2.3 microEq/h in group RC and 6.04 +/- 2.43 microEq/h in group RD (p < 0.05). Fetal gastric PGE2 level was 16.4 +/- 2.65 microg/g-wet weight in group SC and 7.62 +/- 1.86 microg/g-wet weight in group SD (p < 0.01); 15.6 +/- 2.61 microg/g-wet weight in group RC and 8.44 +/- 1.44 microg/g-wet weight in group RD (p < 0.01). In addition, histopathological examination showed normal gastric structure in groups SC and RC, but there were mild erosive and hemorrhagic changes in groups SD and RD. Because prophylactic ranitidine significantly decreased gastric acid secretion, but did not prevent harmful histopathologic effects in FD, it is suggested that gastric damage cannot be avoided by decreasing gastric acid secretion alone. However PGE2 analogs with or without H2 receptor blockers may have a potential role to prevent FD-related gastric damage.
1. The effects of halothane and isoflurane anesthesia on red blood cell (RBC) deformability, lipi... more 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 th...
Regular exercise has blood pressure-lowering effects, as shown in different types of experimental... more Regular exercise has blood pressure-lowering effects, as shown in different types of experimental hypertension models in rats, including the nitric oxide synthase (NOS) inhibition model. We aimed to investigate possible mechanisms implicated in the exercise effect by evaluating the vasoreactivity of resistance arteries. Exercise effects on agonist-induced vasodilatory responses and flow-mediated dilation were evaluated in vessel segments of the rat chronic NOS inhibition model. Normotensive and hypertensive rats were subjected to swimming exercise (1 h/day, 5 days/wk, 6 wk), while rats in other sedentary and hypertensive groups did not. Hypertension was induced by oral administration of the nonselective NOS inhibitor l-NAME (25 mg/kg day) for 6 wk. Systolic blood pressure, as measured by the tail-cuff method, was significantly decreased by the training protocol in exercising hypertensive rats. The vasoreactivity of resistance arteries was evaluated by both wire and pressure myography studies. An impaired nitric oxide-mediated relaxation pathway in untrained hypertensive rats led to decreased relaxation responses in vessels with intact endothelium. Exercise training significantly improved the responses to acetylcholine and flow-mediated dilation in exercise-trained hypertensive rats in parallel with a decrease in blood pressure. On the other hand contraction (norepinephrine and KCl) and relaxation (sodium nitroprusside) responses of vascular smooth muscle were not different between the groups. Vascular endothelial NOS protein expression was found to be increased in both exercising groups. In conclusion, these results revealed evidence of an increased role of the nitric oxide-dependent relaxation pathway in exercising hypertensive rats.
Fetal distress (FD) adversely affects fetal gastric physiology and histology, increasing gastric ... more Fetal distress (FD) adversely affects fetal gastric physiology and histology, increasing gastric acid secretion and disturbing gastric protective mechanism. Considering these findings, an experimental study was planned to test whether ranitidine prevents FD-related gastric physiological and histological changes during late gestational period. In this study, a rabbit model of FD was created by way of intermittent maternal aortic occlusion. In group 1 (SC), saline treated animals underwent control operation. In group 2 (SD), FD was created in saline treated animals. In group 3 (RC), ranitidine treated animals underwent control operation. In group 4 (RD), FD was created in ranitidine treated animals. Blood lactic acid levels of the fetuses were 2.3 +/- 1.0 mg/L in SC group and 4.7 +/- 1.8 mg/L in group SD (p &lt; 0.01); 2.5 +/- 0.9 mg/L in group RC and 6.7 +/- 2.5 mg/L in group RD (p &lt; 0.01). Fetal gastric acid secretion was 5.94 +/- 2.13 microEq/h in group SC and 8.26 +/- 2.24 microEq/h in group SD (p &lt; 0.05); 6.63 +/- 2.3 microEq/h in group RC and 6.04 +/- 2.43 microEq/h in group RD (p &lt; 0.05). Fetal gastric PGE2 level was 16.4 +/- 2.65 microg/g-wet weight in group SC and 7.62 +/- 1.86 microg/g-wet weight in group SD (p &lt; 0.01); 15.6 +/- 2.61 microg/g-wet weight in group RC and 8.44 +/- 1.44 microg/g-wet weight in group RD (p &lt; 0.01). In addition, histopathological examination showed normal gastric structure in groups SC and RC, but there were mild erosive and hemorrhagic changes in groups SD and RD. Because prophylactic ranitidine significantly decreased gastric acid secretion, but did not prevent harmful histopathologic effects in FD, it is suggested that gastric damage cannot be avoided by decreasing gastric acid secretion alone. However PGE2 analogs with or without H2 receptor blockers may have a potential role to prevent FD-related gastric damage.
Research communications in molecular pathology and pharmacology, 2001
In the present study, we measured the concentrations of reduced glutathione (GSH) and malonyldial... more In the present study, we measured the concentrations of reduced glutathione (GSH) and malonyldialdehyde (MDA) and the activities of glutathione peroxidase (GSH-Px), glutathione S-transferase (GSH-S-T), superoxide dismutase (SOD), catalase (CAT) and glucose-6-phosphate dehydrogenase (G-6-PD) in erythrocytes obtained freshly from adult male donors which was preserved with CPDA-1 anticoagulant (citrate,phosphate, dextrose, adenine) on different days of storage. At the end of the study, storage-associated alterations in antioxidant activities were noted and discussed. GSH, GSH-Px, GSH-S-T, SOD, CAT and G-6-PD activities decreased, but erythrocyte MDA levels, as anindex of lipid peroxidation, increased during the storage period. According to our results, glutathione-dependent antioxidant systems in erythrocytes might be depleted during long storage in blood bags.
Nitric oxide (NO) plays a major role in vascular regulation. Modulation of NO synthesis is known ... more Nitric oxide (NO) plays a major role in vascular regulation. Modulation of NO synthesis is known to influence blood pressure. Inhibition of NO synthesis by NG-nitro-L-arginine methyl ester (L-NAME; 72 mg/kg/day, p.o., 21 days) resulted in 60% increase in blood pressure in rats. Red blood cell (RBC) transit time measured by the cell transit analyzer increased significantly in the L-NAME treated group, in comparison to normotensive rats. RBC aggregation measured in autologous plasma, by a photometric rheoscope also increased significantly in the hypertensive rats. RBC cytosolic free calcium concentration was also significantly higher in the hypertensive animals. Incubation of RBC from hypertensive and control animals with NO donor, sodium nitroprusside (SNP; 10-1000 microM) for 60 minutes resulted in a dose-dependent decrease in RBC aggregation, however aggregation index was significantly higher in hypertensive group at each SNP concentration. Incubation with SNP had no effect on RBC ...
1. The effects of halothane and isoflurane anesthesia on red blood cell (RBC) deformability, lipi... more 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 &lt; 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 &lt; 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.
ABSTRACT Aim: Ang II stimulates vascular smooth muscle cells (VSMCs) and activates mitogen protei... more ABSTRACT Aim: Ang II stimulates vascular smooth muscle cells (VSMCs) and activates mitogen protein kinases (MAPKs) in culture media. p38 MAPK, a member of MAPK family, is stimulated by both Ang II and reactive oxygen species. This study was carried out to show Ang II stimulated p38 MAPK activation through Ras and subsequent NADPH oxidase activation in VSMCs. Material and Method: In our study, primary VSMCs isolated from rat aorta were stimulated by Ang II and with different inhibitors, and western blot was used to measure p38 MAPK phosphorylation. Results: The optimum p38 MAPK phosphorylation was observed at 100 nM Ang II concentration for 5 minutes and the next experiments were carried out under this conditions. To analyze the effect of Ras, which is an upstream mediator of p38 MAPK phosphorylation, VSMCs were incubated with a Ras spesific inhibitor FTS (Farnesyl thiosalicylic acid). After the stimulation of VSMCs by Ang II, p38 MAPK phosphorylation was inhibited. p38 MAPK phosphorylation was also completely inhibited in the presence of DPI (Diphenyl Iodinoum), a spesific NAD(P)H oxidase inhibitor, after Ang II stimulation. Conclusion: These findings demonstrate that Ang II stimulated p38 MAPK phosphorylation is through AT1 receptor and Ras-NAD(P)H oxidase dependent pathway in VSMCs. Understanding this pathway may contribute to the cellular mechanisms underlying in cardiovascular diseases.
To examine the antioxidant effect of bilirubin (BR) on leukocyte, we treated leukocytes obtained ... more 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.
Angiotensin II (Ang II) induces a rapid increase in mitogen-activated protein kinase (MAPK) activ... more Angiotensin II (Ang II) induces a rapid increase in mitogen-activated protein kinase (MAPK) activity through the Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMCs). In the present study, we examined the effects of the phospholipase C (PLC) inhibitor U73122, the protein kinase C (PKC) inhibitor GF109203X, and the Ras inhibitor farnesylthiosalicylic acid (FTS) on Ang II-induced activation of p42/p44 MAPKs in cultured VSMCs. Phosphorylation was shown using the Western blot technique with specific phospho-antibodies against MAPK proteins. The PLC inhibitor U73122 abolished the Ang II-induced MAPK activity, while the PKC inhibitor GF109203X only decreased it. There was also an inhibition observed with the Ras inhibitor, FTS on Ang II-induced MAPK activity. These data suggest that Ang II-induced MAPK phosphorylation through the Ang II type 1 receptor could be mediated by Ras and/or PLC-dependent phosphorylations but not by PKC phosphorylation.
Stimulation of the angiotensin II (Ang II) type 1 receptor (AT 1-R) causes phosphorylation of ext... more Stimulation of the angiotensin II (Ang II) type 1 receptor (AT 1-R) causes phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) via epidermal growth factor receptor (EGF-R) transactivation-dependent or-independent pathways in Ang II target cells. Here we examined the mechanisms involved in agonist-induced EGF-R transactivation and subsequent ERK1/2 phosphorylation in clone 9 (C9) hepatocytes, which express endogenous AT 1-R, and COS-7 and human embryonic kidney (HEK) 293 cells transfected with the AT 1-R. Ang II-induced ERK1/2 activation was attenuated by inhibition of Src kinase and of matrix metalloproteinases (MMPs) in C9 and COS-7 cells, but not in HEK 293 cells. Agonist-mediated MMP activation in C9 cells led to shedding of heparin-binding EGF (HB-EGF) and stimulation of ERK1/2 phosphorylation. Blockade of HB-EGF action by neutralizing antibody or its selective inhibitor, CRM197, attenuated ERK1/2 activation by Ang II. Consistent with its agonist action, HB-EGF stimulation of these cells caused marked phosphorylation of the EGF-R and its adapter molecule, Shc, as well as ERK1/2 and its dependent protein, p90 ribosomal S6 kinase, in a manner similar to that elicited by Ang II or EGF. Although the Tyr319 residue of the AT 1-R has been proposed to be an essential regulator of EGF-R transactivation, stimulation of wild-type and mutant (Y319F) AT 1-R expressed in COS-7 cells caused EGF-R transactivation and subsequent ERK1/2 phosphorylation through release of HB-EGF in a Src-dependent manner. In contrast, the noninvolvement of MMPs in HEK 293 cells, which may reflect the absence of Src activation by Ang II, was associated with lack of transactivation of the EGF-R. These data demonstrate that the individual actions of Ang II on EGF-R transactivation in specific cell types are related to differential involvement of MMP-dependent HB-EGF release. (Molecular Endocrinology 18: 2035-2048, 2004) G PROTEIN-COUPLED RECEPTORS (GPCRs) activate MAPK signaling cascades by a variety of biochemical pathways. These include the generation of second messengers [Ca 2ϩ , protein kinase C (PKC), and cAMP], G protein subunit coupling to novel effectors, and activation of receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGF-R), platelet-derived growth factor receptor, and IGF receptor (1, 2). MAPKs are activated by a wide spectrum of stimuli, ranging from mitogens and growth factors to cellular stress and neurotoxic factors. ERK1/2 are closely related members of the MAPK family that are predominantly activated by growth factors and GPCRs, and are important factors in cell growth, survival, motility, and secretion (3). The type 1 angiotensin II (Ang II) receptor (AT 1-R) is a typical GPCR, and mediates a wide variety of physiological actions in the cardiovascular system, kidney, brain, adrenal glands, and liver (4). Ang II and the AT 1-R have critical roles in cardiovascular diseases including hypertension, atherosclerosis, and cardiac hypertrophy (5, 6). In most Ang II target cells, the
Intravascular hemolysis is one of the most emphasized mechanisms for destruction of erythrocytes ... more Intravascular hemolysis is one of the most emphasized mechanisms for destruction of erythrocytes during and after physical activity. Exercise-induced oxidative stress has been proposed among the different factors for explaining exercise-induced hemolysis. The validity of oxidative stress following exhaustive cycling exercise on erythrocyte damage was investigated in sedentary and trained subjects before and after antioxidant vitamin treatment (A, C, and E) for 2 mo. Exercise induced a significant increase in thiobarbituric acid-reactive substance and protein carbonyl content levels in sedentary subjects and resulted in an increase of osmotic fragility and decrease in deformability of erythrocytes, accompanied by signs for intravascular hemolysis (increase in plasma hemoglobin concentration and decrease in haptoglobulin levels). Administration of antioxidant vitamins for 2 mo prevented exercise-induced oxidative stress (thiobarbituric acid-reactive substance, protein carbonyl content...
Previous work has shown a sensitive inhibition of prostacyclin synthase activity by peroxynitrite... more 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.
Observing better transfection efficiency in primary cultured VSMCs: Comparison and development of... more Observing better transfection efficiency in primary cultured VSMCs: Comparison and development of two different protocols [Primer VDKH kültüründe daha etkili bir transfeksiyon gözlenmesi: İki farklı protokolün karşılaştırılması ve geliştirilmesi]
Although effects of stress on the stomach have been extensively investigated in children and adul... more Although effects of stress on the stomach have been extensively investigated in children and adults, our knowledge about effects of fetal distress (FD) on the fetal stomach is quite limited. Therefore, an experimental study was planned to evaluate the effects of FD on fetal gastric physiology and histology. In this study, a model of FD was created by way of intermittent maternal aortic occlusion in pregnant rabbits. In total, 21 fetuses of 6 pregnant rabbits were available for surgical and laboratory procedures. Laboratory examinations showed that (1) fetal gastric acid secretion was 4.24 +/- 2.68 muEq/h in the control group and 18.08 +/- 6.34 muEq/h in the distress group (p &lt; 0.01) and (2) fetal gastric PGE2 level was 16.59 +/- 6.15 mg/g wet weight in the control group and 9.86 +/- 3.46 mg/g wet weight in the distress group (p &lt; 0.05). Histopathologically, there were mild hemorrhagic and errosive changes in the distressed fetuses, but not in control fetuses. These findings support that FD adversely affects fetal gastric physiology through two mechanisms consisting of increased gastric acid secretion and decreased fetal gastric protection in rabbits. Consequently, gastric injury should be noted as a potential problem among hypoxia-associated abnormalities encountered in the distressed fetus.
Fetal distress (FD) adversely affects fetal gastric physiology and histology, increasing gastric ... more Fetal distress (FD) adversely affects fetal gastric physiology and histology, increasing gastric acid secretion and disturbing gastric protective mechanism. Considering these findings, an experimental study was planned to test whether ranitidine prevents FD-related gastric physiological and histological changes during late gestational period. In this study, a rabbit model of FD was created by way of intermittent maternal aortic occlusion. In group 1 (SC), saline treated animals underwent control operation. In group 2 (SD), FD was created in saline treated animals. In group 3 (RC), ranitidine treated animals underwent control operation. In group 4 (RD), FD was created in ranitidine treated animals. Blood lactic acid levels of the fetuses were 2.3 +/- 1.0 mg/L in SC group and 4.7 +/- 1.8 mg/L in group SD (p &lt; 0.01); 2.5 +/- 0.9 mg/L in group RC and 6.7 +/- 2.5 mg/L in group RD (p &lt; 0.01). Fetal gastric acid secretion was 5.94 +/- 2.13 microEq/h in group SC and 8.26 +/- 2.24 microEq/h in group SD (p &lt; 0.05); 6.63 +/- 2.3 microEq/h in group RC and 6.04 +/- 2.43 microEq/h in group RD (p &lt; 0.05). Fetal gastric PGE2 level was 16.4 +/- 2.65 microg/g-wet weight in group SC and 7.62 +/- 1.86 microg/g-wet weight in group SD (p &lt; 0.01); 15.6 +/- 2.61 microg/g-wet weight in group RC and 8.44 +/- 1.44 microg/g-wet weight in group RD (p &lt; 0.01). In addition, histopathological examination showed normal gastric structure in groups SC and RC, but there were mild erosive and hemorrhagic changes in groups SD and RD. Because prophylactic ranitidine significantly decreased gastric acid secretion, but did not prevent harmful histopathologic effects in FD, it is suggested that gastric damage cannot be avoided by decreasing gastric acid secretion alone. However PGE2 analogs with or without H2 receptor blockers may have a potential role to prevent FD-related gastric damage.
1. The effects of halothane and isoflurane anesthesia on red blood cell (RBC) deformability, lipi... more 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 th...
Regular exercise has blood pressure-lowering effects, as shown in different types of experimental... more Regular exercise has blood pressure-lowering effects, as shown in different types of experimental hypertension models in rats, including the nitric oxide synthase (NOS) inhibition model. We aimed to investigate possible mechanisms implicated in the exercise effect by evaluating the vasoreactivity of resistance arteries. Exercise effects on agonist-induced vasodilatory responses and flow-mediated dilation were evaluated in vessel segments of the rat chronic NOS inhibition model. Normotensive and hypertensive rats were subjected to swimming exercise (1 h/day, 5 days/wk, 6 wk), while rats in other sedentary and hypertensive groups did not. Hypertension was induced by oral administration of the nonselective NOS inhibitor l-NAME (25 mg/kg day) for 6 wk. Systolic blood pressure, as measured by the tail-cuff method, was significantly decreased by the training protocol in exercising hypertensive rats. The vasoreactivity of resistance arteries was evaluated by both wire and pressure myography studies. An impaired nitric oxide-mediated relaxation pathway in untrained hypertensive rats led to decreased relaxation responses in vessels with intact endothelium. Exercise training significantly improved the responses to acetylcholine and flow-mediated dilation in exercise-trained hypertensive rats in parallel with a decrease in blood pressure. On the other hand contraction (norepinephrine and KCl) and relaxation (sodium nitroprusside) responses of vascular smooth muscle were not different between the groups. Vascular endothelial NOS protein expression was found to be increased in both exercising groups. In conclusion, these results revealed evidence of an increased role of the nitric oxide-dependent relaxation pathway in exercising hypertensive rats.
Fetal distress (FD) adversely affects fetal gastric physiology and histology, increasing gastric ... more Fetal distress (FD) adversely affects fetal gastric physiology and histology, increasing gastric acid secretion and disturbing gastric protective mechanism. Considering these findings, an experimental study was planned to test whether ranitidine prevents FD-related gastric physiological and histological changes during late gestational period. In this study, a rabbit model of FD was created by way of intermittent maternal aortic occlusion. In group 1 (SC), saline treated animals underwent control operation. In group 2 (SD), FD was created in saline treated animals. In group 3 (RC), ranitidine treated animals underwent control operation. In group 4 (RD), FD was created in ranitidine treated animals. Blood lactic acid levels of the fetuses were 2.3 +/- 1.0 mg/L in SC group and 4.7 +/- 1.8 mg/L in group SD (p &lt; 0.01); 2.5 +/- 0.9 mg/L in group RC and 6.7 +/- 2.5 mg/L in group RD (p &lt; 0.01). Fetal gastric acid secretion was 5.94 +/- 2.13 microEq/h in group SC and 8.26 +/- 2.24 microEq/h in group SD (p &lt; 0.05); 6.63 +/- 2.3 microEq/h in group RC and 6.04 +/- 2.43 microEq/h in group RD (p &lt; 0.05). Fetal gastric PGE2 level was 16.4 +/- 2.65 microg/g-wet weight in group SC and 7.62 +/- 1.86 microg/g-wet weight in group SD (p &lt; 0.01); 15.6 +/- 2.61 microg/g-wet weight in group RC and 8.44 +/- 1.44 microg/g-wet weight in group RD (p &lt; 0.01). In addition, histopathological examination showed normal gastric structure in groups SC and RC, but there were mild erosive and hemorrhagic changes in groups SD and RD. Because prophylactic ranitidine significantly decreased gastric acid secretion, but did not prevent harmful histopathologic effects in FD, it is suggested that gastric damage cannot be avoided by decreasing gastric acid secretion alone. However PGE2 analogs with or without H2 receptor blockers may have a potential role to prevent FD-related gastric damage.
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