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Cardiovascular depression in streptozotocin-diabetic rats : role of inducible nitric oxide synthase and effects of antioxidant therapy Nagareddy, Prabhakara Reddy


Diabetes mellitus is associated with an increased incidence of cardiovascular complications. Among these, the development of cardiovascular depression is quite common, particularly in animal models of Type 1 diabetes such as streptozotocin (STZ) diabetic rats. These rats exhibit depressed mean arterial blood pressure (MABP) and heart rate (HR). In addition, they also demonstrate attenuated pressor responses in vivo yet enhanced contractile responses to various vasoconstrictor agents in vitro, despite exhibiting impaired endothelial function. Since endothelium derived nitric oxide (NO) is the major vasodilator involved in, it is unclear as to how endothelial dysfunction and subsequent changes in NO bioavailability contribute to the development of cardiovascular depression. Chronic hyperglycemia is associated with increased oxidative stress and expression of inducible nitric oxide synthase (iNOS) in various cardiovascular tissues. It is possible that the interaction of reactive oxygen species with NO result in the formation of reactive nitrogen species (nitrosative stress) such as peroxynitrite that are detrimental to cardiovascular tissue. Thus, we hypothesized that diabetes causes the induction of iNOS resulting in the elevation of nitrosative stress that may cause cardiovascular depression. Treatment with an antioxidant and/or iNOS inhibitor may therefore improve the cardiovascular depression. Using the STZ diabetic rat model, we measured the MABP and HR in freely moving conscious rats at different time points in the progression of diabetes (1, 3, 9 and 12 week duration). Using 1400W, a specific inhibitor of iNOS, we investigated the effects of angiotensin-2 (AT II) and methoxamine on changes in MABP and HR. In addition, using immunohistochemistry, we investigated the localization of iNOS, endothelial nitric oxide synthase (eNOS) and nitrotyrosine (NT) proteins in different cardiovascular tissues. We also studied the acute and chronic effects of antioxidant therapy using N-acetylcysteine (NAC), a strong antioxidant on iNOS mediated cardiovascular abnormalities in STZ diabetic rats. Further, we investigated the effects of NAC treatment on cardiovascular depression as measured by the MABP and HR in 3 and 9 week diabetic rats. In addition, using immunohistochemical techniques, the expression of eNOS, iNOS and NT were examined in different cardiovascular tissues. Also, the effect of NAC treatment on plasma nitrite and nitrate levels (NOx) and antioxidant defense mechanisms were studied. There are several observations from our studies. STZ diabetic rats had depressed MABP and HR by three weeks of diabetes. Treatment with N A C significantly improved the MABP and HR. In addition, STZ diabetic rats demonstrated impaired endothelial function as shown by attenuated pressor response to a non-selective nitric oxide synthase inhibitor, Nanitro- L-arginine methyl ester hydrochloride (L-NAME). Pressor responses to bolus doses of methoxamine and AT II were attenuated by 3 weeks of diabetes. Methoxamine produced attenuated pressor responses in 12-week diabetic rats compared to 1-week diabetic rats. Similarly, the pressor responses to AT II were also significantly reduced in 9 and 12-week diabetic rats compared to 1-week diabetic rats. These results suggest that the duration of diabetes significantly affects the pressor responses of both methoxamine and AT II. Pretreatment with 1400W did not affect the MABP and HR responses of either methoxamine or AT II in control rats. However, selective inhibition of iNOS by 1400W completely restored the pressor responses to both AT II and methoxamine in all diabetic groups except the 12-week diabetic group, where methoxamine increased the pressor response but failed to normalize it. These data suggest that iNOS plays a significant role in modulating the pressor responses to vasoactive agents in diabetes. Diabetic rats exhibited a differential regulation of iNOS and eNOS wherein prolonged diabetic state led to a downregulation of eNOS with a concomitant upregulation of iNOS protein expression. iNOS protein was first detected in the myocardial tissue of 3-week diabetic rats and the intensity increased with the duration of diabetes. This was associated inversely with the expression of eNOS, wherein prolonged diabetes led to decreased expression of eNOS in diabetic rat heart, aorta and superior mesenteric arteries. Increased expression of iNOS in concert with increased oxidative stress results in the formation of reactive nitrogen species such as peroxynitrite. Immunohistochemical localization of NT, an indirect marker of peroxynitrite reveals an increased formation of reactive nitrogen species in the heart and arteries of diabetic rats. A triphasic response of unchanged, decreased and elevated levels of plasma nitrite/nitrate (NOx) concentrations was observed in 3, 9 and 12-week diabetic rats respectively. This observation is difficult to explain with the data available but it may be possible that a simultaneous increase of reactive oxygen species (ROS) and NO in the beginning (unchanged), followed by increased scavenging of NO by ROS (decreased) by 9 weeks and finally leading to an uncontrollable production of NO from iNOS (increased) in 12 weeks may affect the plasma NOx levels. However, further studies are required to validate the conclusions. NAC treatment normalized the expression of iNOS, eNOS and NT in these tissues. Our results indicate that chronic hyperglycemia may facilitate the downregulation of eNOS by producing large quantities of NO and /or reactive nitrogen species from iNOS. This was supported by the reduced levels of nitrosative stress (NT) observed in diabetic rats treated with NAC. In addition, a significant reduction in plasma 15-F2t isoprostanes, increased plasma NOx concentration, enhanced myocardial antioxidant capacity and total antioxidant concentrations were observed in NAC treated diabetic rats. Results presented in this thesis therefore suggest that depressed MABP and. HR in STZ diabetic rats may be due to increased levels of nitrosative stress. The activation of iNOS, which depends on the duration of diabetes, may assume a critical role under oxidative conditions in modulating pressor responses to vasoactive agents. The interactions between iNOS and oxidative stress in diabetes could thus influence the subsequent hemodynamic outcomes.

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