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The role of inducible nitric oxide synthase (iNOS) on cardiovascular function in rats with streptozotocin-induced diabetes Cheng, Xing


Cardiovascular complications are leading causes of death in Type 1 and Type 2 diabetes mellitus. Experimental studies and clinical trials have firmly established a link between hyperglycemia and cardiovascular complications. The precise mechanism involved, however, remains elusive. Recently, inducible nitric oxide synthase (iNOS) and oxidative stress have been implicated as factors that initiate cardiovascular complications in diabetes. However, there is a lack of direct evidence that link these biological changes with abnormalities in cardiovascular function. The three primary goals of the present investigation were: 1) to investigate if rats with streptozotocin (STZ, 60 mg/kg i.v.)-induced Type 1 diabetes had functional abnormalities of the cardiovascular system, and if these changes were associated with the activation of iNOS, formation of peroxynitrite, and reduced myocardium antioxidant capacity; 2) to examine if treatment with 1400W (inhibitor of iNOS) improved cardiovascular response to noradrenaline (NA) concurrently with inhibition of the activation of iNOS and formation of peroxynitrite in diabetes; 3) to examine if the chronic treatment of the antioxidant N-acetylcysteine (NAC, antioxidant and inhibitor of iNOS) reduced activation of iNOS, decreased formation of peroxynitrite, increased antioxidative capacity, and improved cardiac contractile function in diabetes. The results show that diabetic and control rats had similar mean arterial pressure (MAP) and total peripheral resistance (TPR) at three weeks after injection of STZ. NA increased in vivo MAP and TPR in both groups; however, the responses were markedly less in the diabetic than control rats. Rats with diabetes, relative to the controls, had reduced potency (increased ED50) for the pressor (2.5-fold of control) and mean circulatory filling pressure (MCFP, an index of venous tone, 4.3-fold of control) response to NA, as well as reduced maximum pressor response (efficacy) to NA. Diabetic rats also had reduced potency (ED50, 5-fold of control) of the pressor response to angiotensin II. Therefore, arterial and venous constrictions are impaired at an early phase of STZ-induced Type I diabetes. The diabetic rats in the present study also had reduced heart rate (HR) and maximal rate of increase as well as decrease of left ventricular pressure (+dP/dt of LV) relative to the controls at three weeks after injection of STZ. In addition, the diabetic rats had reduced inotropic and chronotropic responses to NA and dobutamine (β1-adrenoceptor agonist), exemplified by the decreases in the efficacy (reduced Emax ) and potency (increased EC50) of HR, and left ventricular developed tension as well as contractility (±dP/dt of LV) responses to dobutamine. The activation of iNOS and oxidative stress were confirmed in myocardium at three weeks after injection of STZ, evidenced by 1) detection of RT-PCR products (RNA) of iNOS in the hearts of the diabetic but not control rats; 2) the three-fold increase of activity of iNOS in the hearts of diabetic rats relative to the controls, and inhibition of this increase by 1400W; 3) clear identification of immunostaining (proteins) of iNOS and NT (in vivo marker of peroxynitrite, an oxidant and cytotoxic mediator) in the hearts of the diabetic rats, but not the controls; 4) similar baseline lipid peroxidation (formation of 15-F2t-isoprostane), but decreased tissue antioxidation capacity (increased thiobarbituric acid reactive substances, thiobarbituric acid reactive substance) upon peroxidation challenge. Acute treatment with 1400W (3 mg/kg followed by 3 mg/kg/h, i.v) did not alter baseline hemodynamic variables or responses to NA in the control rats but completely restored the efficacy (Emax) and insignificantly increased the potency (reduced ED50,) of MAP response to NA. This drug also completely restored the TPR response to NA, and increased MCFP response to a high dose of NA. Moreover, treatment with 1400W enhanced the influence of NA on cardiac contractility (LVP, LV ±dP/dt) in the diabetic rats and concurrently reduced the activity of iNOS in the diabetic myocardium. Therefore, an activation of iNOS contributes to depressed cardiovascular contractile response to NA at the acute phase of STZ-induced diabetes. Selective inhibition of iNOS partially restores cardiovascular responses to NA. Chronic treatment with the antioxidant NAC (1.2 g/day/kg) did not affect any measured variables in the control rats but improved cardiac contraction to dobutamine (1-57 μg/min/kg), exemplified by increased efficacy (Emax) and potency (EC50) of HR and left ventricular developed tension (LVP) as well as contractility (±dP/dt of LV) responses to dobutamine. NAC also concurrently reduced immunostaining of iNOS and NT, as well as decreased myocardial level of 15-F2t-isoprostane. Therefore, antioxidant supplementation is beneficial in the management of cardiac contractile dysfunction in diabetes mellitus. To our best knowledge, this is the first study that links cellular activation of iNOS in diabetes to abnormal in vivo adrenoceptor-mediated responses in the heart and blood vessels, and demonstrates the restoration of cardiovascular function after selective inhibition of the activity of iNOS. These results provide direct evidence that link depressed cardiac contractility with the activation of iNOS as well as the production of peroxynitrite and its associated nitration of tyrosine and reduction of antioxidative capacity. These results also provide the first evidence that NAC, an antioxidant/iNOS inhibitor, improved cardiac function concurrently with the reduction of myocardial level of 15-F2t-isoprostane at three week after the induction of diabetes. Our results suggest that antioxidant therapy or inhibition the activity of iNOS is beneficial in the management of cardiac contractile dysfunction at an early phase of diabetes, even when baseline levels of lipid peroxidation products are not yet elevated.

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