Hyperinsulinaemia increases the gene expression of endothelial nitric oxide synthase and the phosphatidylinositol 3-kinase/Akt pathway in rat aorta.

1. Hyperinsulinaemia has been reported to be an independent risk factor for cardiovascular diseases. Insulin stimulates both the phosphatidylinositol 3-kinase (PI3-K)/Akt and mitogen-activated protein kinase (MAPK) pathways. To investigate the direct effects of insulin on vascular tissues, we examined the gene and protein expression of insulin signalling molecules, endothelial nitric oxide synthase (eNOS) and MAPK in aortas obtained from established hyperinsulinaemic rats under deep urethane anaesthesia (1.2 g/kg, i.p.). 2. High plasma insulin levels significantly enhanced the gene and protein expression of eNOS in aortas. This was accompanied not only by increased mRNA levels of insulin receptor substrate (IRS)-1, IRS-2, PI3-K and Akt, but also by a high protein content of Akt and phospho-Akt (Ser473). 3. In contrast, MAPK mRNA levels were decreased in hyperinsulinaemic rats compared with normoinsulinaemic rats. 4. Insulin receptor mRNA levels were also lower in insulin-treated rats rather than controls. The overexpression of mRNA for vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF)-I receptor was also observed in aortas from hyperinsulinaemic rats. 5. To our knowledge, these data provide the first direct measurements of the mRNA of insulin signalling molecules and the downstream eNOS and MAPK. We conclude that hyperinsulinaemia itself can lead to the upregulation of eNOS and the PI3-K/Akt pathway in the vasculature and may also induce the overexpression of VEGF and IGF-I receptor genes.

Calcium channel blockades exhibit anti-inflammatory and antioxidative effects by augmentation of endothelial nitric oxide synthase and the inhibition of angiotensin converting enzyme in the N(G)-nitro-L-arginine methyl ester-induced hypertensive rat aorta: vasoprotective effects beyond the blood pressure-lowering effects of amlodipine and manidipine.

Long-acting dihydropyridine calcium channel blockades have been shown to limit the progression of atherosclerosis and decrease the incidence of cardiovascular events in humans and animals. To investigate the vasoprotective effects beyond the blood pressure-lowering effects of these agents, amlodipine (20 mg/kg/ day) and manidipine (10 mg/kg/day) were administered by gavage to N(G)-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats for 2 weeks. L-NAME treatment (0.7 mg/ml in drinking water) significantly decreased the gene and protein expression of endothelial nitric oxide synthase (eNOS) and increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) mRNA levels in the aorta, as determined by Western blotting and reverse transcription (RT)-polymerase chain reaction (PCR). Amlodipine and manidipine normalized the decreased expression of eNOS gene and protein, and attenuated the overexpression of NADPH oxidase, VCAM-1, and MCP-1 mRNA. Furthermore, amlodipine and manidipine prevented the L-NAME-induced increase in the angiotensin converting enzyme (ACE) mRNA content, thereby restoring control levels in the aorta. On the other hand, hydralazine treatment had no such effect in L-NAME treated rats. Furthermore, the increased expression of manganese superoxide dismutase (Mn-SOD) by L-NAME treatment was not affected by amlodipine, manidipine, or hydralazine. We concluded that the direct anti-inflammatory and antioxidative effects of calcium channel blockades in the aorta of rats with L-NAME-induced hypertension were not likely to have been mediated by the blood pressure-lowering action of these agents, but instead these beneficial effects appear to have been mediated by an augmentation of eNOS expression and by the inhibition of the expression of ACE.

Cytokine-induced nitric oxide inhibits mitochondrial energy production and induces myocardial dysfunction in endotoxin-treated rat hearts.

The mechanism responsible for cardiac depression in septic shock remains unknown. The present study examined whether nitric oxide (NO) overproduced by inducible NO synthase (iNOS) can inhibit aerobic energy metabolism and impair the myocardial function in endotoxin-treated rat hearts. Lipopolysaccharide (LPS) significantly decreased systolic blood pressure (BP) to 44% of control during the 48 h treatment. Hearts from control and LPS-treated rats were perfused in a Langendorff apparatus. After LPS injection, left ventricular (LV) developed pressure (LVDP) was significantly depressed, plasma NO2-/NO3- (NO(x)) concentration was markedly increased, and myocardial adenosine 5'-triphosphate (ATP), creatine phosphate (CrP), and the ratio of ATP/adenosine 5'-diphosphate were progressively decreased with time. Immunological examination showed a significant expression of iNOS protein in the LPS-treated myocytes. Aminoguanidine, an inhibitor of iNOS, significantly attenuated these LPS-induced functional and metabolic changes. Myocardial cyclic guanosine 3',5'-monophosphate (cGMP) content was significantly increased after LPS injection. Methylene blue, an inhibitor of soluble guanylate cyclase, blunted this increase in cGMP and significantly restored the LPS-induced contractile dysfunction 6 h after LPS injection. In addition, there was a significant negative correlation between LVDP and myocardial cGMP levels as well as a significant negative correlation between LVDP and plasma NO(x) levels. In contrast, 48 h after LPS injection, methylene blue no longer affected cardiac performance, and there was a significant positive correlation between LVDP and myocardial ATP content. Furthermore, the normalized activities (as a ratio of the citrate synthase activity) of mitochondrial NADH-CoQ reductase, succinate-CoQ reductase, and ATPase, were significantly inhibited, and the swelling or disruption of mitochondria cristae was seen in the 48 h LPS treatment. These LPS-induced functional and morphological disorders in the mitochondria were significantly improved by aminoguanidine. The findings suggest that sustained production of NO by iNOS leads to contractile dysfunction via cGMP in the early stage, but that it can directly impair the mitochondrial function, lower myocardial energy production, and contribute significantly to the myocardial dysfunction in the later stage of septic shock.

Nitric oxide-cGMP pathway is involved in endotoxin-induced contractile dysfunction in rat hearts.

The mechanisms by which endotoxemia causes cardiac depression have not been fully elucidated. The present study examined the involvement of nitric oxide (NO) in this pathology. Rats were infused with lipopolysaccharide (LPS) or saline, and the plasma and myocardial NO(2)(-) and NO(3)(-) (NOx) concentrations were measured before or 3, 6, and 24 h after treatment. The hearts were then immediately isolated and mounted in a Langendorff apparatus, and left ventricular developed pressure (LVDP) was determined before biochemical analysis of the myocardium. LPS injection effected the expression of inducible NO synthase (iNOS) in the myocardium, a marked increase in plasma and myocardial NOx levels, and a significant decline in LVDP compared with saline controls. The LPS-induced NO production and concomitant cardiac depression were most pronounced 6 h after LPS injection and were accompanied by a significant increase in myocardial cGMP content. Myocardial ATP levels were not significantly altered after LPS injection. Significant negative correlation was observed between LVDP and myocardial cGMP content, as well as between LVDP and plasma NOx levels. Aminoguanidine, an inhibitor of iNOS, significantly attenuated the LPS-induced NOx production and contractile dysfunction. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, significantly decreased myocardial cGMP content and attenuated the contractile depression, although aminoguanidine or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was not able to completely reverse myocardial dysfunction. Our data suggest that endotoxin-induced contractile dysfunction in rat hearts is associated with NO production by myocardial iNOS and a concomitant increase in myocardial cGMP.

The dual effects of nitric oxide synthase inhibitors on ischemia-reperfusion injury in rat hearts.

OBJECTIVE: Nitric oxide (NO) is known to act as a mediator of tissue injury as well as being a potent endogenous vasodilator. The functional and metabolic effects of NO on ischemia-reperfusion injury are still controversial. The aim of this study was to clarify the relationship between the degree of NO synthase (NOS) inhibition and the effects on ischemia-reperfusion injury. METHODS AND RESULTS: Langendorff-perfused rat hearts were subjected to 30 minutes of global ischemia followed by 30 minutes of reperfusion. The recovery of left ventricular developed pressure (LVDP), creatine kinase (CK) release, and myocardial high energy phosphates were measured in hearts perfused with or without NOS inhibitors, L-N(G)-monomethyl arginine (L-NMMA) or N(G)nitro-L-arginine methylester (L-NAME). NOS inhibitors exerted different effects on the recovery of LVDP and CK release depending on the concentration. The low dose of L-NMMA improved the recovery of LVDP, decreased the CK release during reperfusion, and preserved the myocardial adenosine triphosphate content after reperfusion. In contrast, the high dose of L-NMMA had adverse effects. L-NMMA reduced NO release in coronary effluent in a dose-dependent fashion. Both effects of L-NMMA were abolished by excessive co-administration of L-arginine and the same doses of D-N(G)-monomethyl arginine (D-NMMA) showed no effect on ischemia-reperfusion injury. Therefore, both effects were due to NOS inhibition. In addition, L-NMMA suppressed the myocardial malondialdehyde accumulation, an indicator of oxidative stress, which might be attributed to the beneficial effects by partial NOS inhibition. On the other hand, the high dose L-NMMA significantly decreased coronary fl ow during aerobic perfusion and reperfusion. Therefore, it is conceivable that the vasoactive NOS inhibition contributes to the harmful effects, which might exceed the beneficial effects due to a decrease in oxidative stress. CONCLUSION: The present results showed that NO inhibitors had dual effects on mechanical function and energy metabolism depending on the concentration. Non-vasoactive inhibition of NOS had beneficial effects due to the suppression of oxidative injury. However, strong vasoactive inhibition of NOS exacerbated the ischemia-reperfusion injury.

Effects of ACE inhibition on myocardial apoptosis in an ischemia-reperfusion rat heart model.

Myocardial ischemia-reperfusion injury involves necrosis and apoptosis. The inhibition of angiotensin-converting enzyme (ACE) has been reported to suppress infarct size. In this study, it was investigated whether an ACE inhibitor affected myocardial apoptosis and apoptosis-related proteins in rats with experimental myocardial infarction. Anesthetized Sprague-Dawley rats were divided into four groups. Group I underwent 30 minutes of left coronary artery occlusion followed by 24 hours of reperfusion (control group); Group II underwent oral administration of the ACE inhibitor quinapril (10 mg/kg/day) before coronary occlusion (quinapril group); Group III underwent administration of the bradykinin B(2)-receptor antagonist Hoe 140 (250 microg/kg/day, subcutaneously) with quinapril (quinapril + Hoe 140 group); and Group IV underwent administration of Hoe 140 alone (Hoe 140 group). After reperfusion, myocardial infarct size was determined by triphenyltetrazolium chloride staining. Myocardial apoptosis was detected immunohistologically using terminal deoxynucleotidyl transferase-mediated nick end labeling staining and DNA electrophoresis. Myocardial caspase-3 activation was analyzed by Western blot and the expressions of Bcl-xL and Bax proteins were detected immunohistochemically. Quinapril significantly reduced the ratio of myocardial infarct size in the ischemic area at risk. In addition, quinapril significantly suppressed the incidence of apoptotic myocytes around the necrotic region (from 18.9 +/- 0.8% to 8.6 +/- 1.0%; P < 0.0001), the intensity of DNA ladder formation, and the activation of caspase-3. Hoe 140 attenuated these protective effects of quinapril. In the immunohistochemical study, Bax and Bcl-xL were expressed in myocytes, and ischemia-reperfusion abolished both proteins in the center region of ischemia. The Bax staining was equally observed among all groups. However, Bcl-xL staining remained in the ischemic area widely after quinapril treatment. In addition, Hoe 140 also depleted this effect of quinapril. These results suggest that inhibition of ACE reduces myocardial infarction and apoptosis via the bradykinin B(2) receptor in part. The antiapoptotic effect of the ACE inhibitor is attributed to the changing expression of Bcl-xL.

Amlodipine inhibits doxorubicin-induced apoptosis in neonatal rat cardiac myocytes.

OBJECTIVES: We examined whether amlodipine, a calcium channel antagonist with potent antioxidant activity, inhibits doxorubicin-induced apoptosis in cultured neonatal rat cardiac myocytes. BACKGROUND: Recent studies have shown that doxorubicin induces apoptosis as well as necrosis in myocytes through generation of reactive oxygen species. METHODS: The effects of amlodipine and several other antioxidants on doxorubicin-induced oxidative stress and mitochondria-mediated apoptosis were examined. RESULTS: Treatment of myocytes with doxorubicin (10(-6) mol/l) for 14 h increased the number of cells with elevated peroxides, as histochemically estimated by 2',7'-dichlorofluorescin (DCF) diacetate, and the percentage of apoptotic myocytes, as estimated by Hoechst 33258 nuclear staining, compared with control myocytes (25.0 +/- 1.6% vs. 5.2 +/- 1.2%). Moreover, doxorubicin-induced myocyte apoptosis was also confirmed by annexin V-fluorescein isothiocyanate binding assay. Doxorubicin induced a reduction in myocyte adenosine 5'-triphosphate content, a loss of mitochondrial membrane potential, cytochrome c release from the mitochondria into the cytosol, and caspase-3 activation to 1.9-fold of control. Amlodipine significantly attenuated increased DCF fluorescence, inhibited the mitochondria-mediated apoptotic responses described earlier, and decreased apoptosis in the doxorubicin-treated myocytes in a dose-dependent fashion. Amlodipine at 10(-6) mol/l significantly decreased apoptosis to 15.4 +/- 0.7%, and this antiapoptotic action was more effective than that seen with other antioxidants, including probucol, ascorbic acid, and alpha-tocopherol. In contrast, the calcium channel antagonist nifedipine (10(-6) mol/l) did not inhibit apoptosis. Catalase, glutathione, and N-acetylcysteine, but not mannitol or superoxide dismutase, significantly decreased DCF fluorescence and attenuated myocyte apoptosis induced by doxorubicin to 18.7 +/- 1.2%, 19.1 +/- 1.7%, and 18.7 +/- 0.6%, respectively. CONCLUSIONS: Amlodipine significantly inhibits doxorubicin-induced myocyte apoptosis by suppressing the mitochondrial apoptotic pathway. This effect is attributed to the antioxidant properties of amlodipine, affecting mainly hydrogen peroxide.

Lethal effect of cytokine-induced nitric oxide and peroxynitrite on cultured rat cardiac myocytes.

We examined the cytotoxic effect of iNOS-generated NO in cultured cardiac myocytes treated with IL-1 beta, IFN- gamma and LPS. Treatment of the myocytes with cytokines for 48 h resulted in a marked NO production, a significant decline in cellular ATP content, and a significant increase in myocyte death with morphological characteristics of necrosis. Moreover, immunohistochemical examination showed that the cytokines caused nitrotyrosine formation in the injured myocytes. Uric acid and L-cysteine which have the ability to quench peroxynitrite significantly attenuated these cytokine-induced effects, although they did not alter NO production or the decline in cellular ATP. These data suggest that NO production induced by cytokines can not only cause deleterious effects in the myocardial energy balance but also induce myocytes necrosis, through the formation of peroxynitrite.

Effects of pravastatin on exercise electrocardiography test performance and cardiovascular mortality and morbidity in patients with hypercholesterolemia: Lipid Intervention Study in Kyoto.

The long-term effects of the 3-hydoxy-3-methyl-glutaryl coenzyme A reductase inhibitor, pravastatin, on exercise electrocardiography (ECG) test performance and cardiovascular mortality and morbidity were compared with those of conventional lipid-lowering drugs in hypercholesterolemic patients with no history of myocardial infarction or stroke. One thousand two hundred and seventeen patients were randomly assigned with mean serum cholesterol, triglyceride, high-density lipoprotein (HDL) cholesterol, and low-density lipoprotein (LDL) cholesterol levels of 6.98 +/- 0.91mmol/L, 2.08 +/- 1.87mmol/L, 1.38 +/- 0.44mmol/L, and 5.07 +/- 1.14 mmol/L, respectively, and received either pravastatin at a dose of 10-20mg/day (group P) or one of the conventional lipid-lowering drugs such as fibrates, nicotinic acid, and probucol (group C). The numbers of patients available for analysis in groups P and C were 305 and 278 at year 1, 261 and 216 at year 2, 206 and 184 at year 3, 159 and 122 at year 4, and 103 and 81 at year 5. Over the 3.2 year mean follow-up period, the reduction in serum LDL cholesterol levels was significantly greater (p<0.01) in group P (-24.3%) than in group C (-16.0%). Serum HDL cholesterol levels increased in group P (+11.6%), but decreased in group C (-0.3%) (p<0.01). There were no significant differences in the rate of patients who exhibited ischemic changes to exercise ECG test (ischemic responders) between the 2 groups. Coronary heart diseases (CHD) occurred in 6 patients in group P and 13 in group C; pravastatin significantly reduced CHD risk (reduction rate 0.369; 95% confidence interval 0.140-0.970; p<0.05). No significant differences existed between the treatment groups in terms of the number of strokes (group P, 6; group C, 7) or deaths unrelated to CHD (group P, 3; group C, 2). Although pravastatin did not improve the proportion of ischemic responders on exercise testing, it reduced CHD risk and serum LDL cholesterol levels more significantly than conventional lipid-lowering drugs without adversely affecting the risk of stroke and non-CHD death in hypercholesterolemic patients.