We investigated the role of nitric oxide (NO) in the control of myocardial O2 consumption in Fischer 344 rats. In Fischer rats at 4, 14, and 23 mo of age, we examined cardiac function using echocardiography, the regulation of cardiac O2 consumption in vitro, endothelial NO synthase (eNOS) protein levels, and potential mechanisms that regulate superoxide. Aging was associated with a reduced ejection fraction [from 75 +/- 2% at 4 mo to 66 +/- 3% (P < 0.05) at 23 mo] and an increased cardiac diastolic volume [from 0.60 +/- 0.04 to 1.00 +/- 0.10 ml (P < 0.01)] and heart weight (from 0.70 +/- 0.02 to 0.90 +/- 0.02 g). The NO-mediated control of cardiac O2 consumption by bradykinin or enalaprilat was not different between 4 mo (36 +/- 2 or 34 +/- 3%) and 14 mo (29 +/- 1 or 25 +/- 3%) but markedly (P < 0.05) reduced in 23-mo-old Fischer rats (15 +/- 3 or 7 +/- 2%). The response to the NO donor S-nitroso-N-acetyl penicillamine was not different across groups (35%, 35%, and 44%). Interestingly, the eNOS protein level was not different at 4, 14, and 23 mo. The addition of tempol (1 mmol/l) to the tissue bath eliminated the depression in the control of cardiac O2 consumption by bradykinin (25 +/- 3%) or enalaprilat (28 +/- 3%) in 23-mo-old Fischer rats. We next examined the levels of enzymes involved in the production and breakdown of superoxide. The expression of Mn SOD, Cu/Zn SOD, extracellular SOD, and p67phox, however, did not differ between 4- and 23-mo-old rats. Importantly, there was a marked increase in gp91phox, and apocynin restored the defect in NO-dependent control of cardiac O2 consumption at 23 mo to that seen in 4-mo-old rats, identifying the role of NADPH oxidase. Thus increased biological activity of superoxide and not decreases in the enzyme that produces NO are responsible for the altered control of cardiac O2 consumption by NO in 23-mo-old Fischer rats. Increased oxidant stress in aging, by decreasing NO bioavailability, may contribute not only to changes in myocardial function but also to altered regulation of vascular tone and the progression of cardiac or vascular disease.

Background-Hyperhomocysteinemia (HHcy) has been considered a vascular disease associated with increased levels of oxidative stress that results in scavenging of NO. However, little is known of the impact of HHcy on cardiac function and especially myocardial metabolism. Methods and Results-L-Homocysteine was intravenously infused into conscious dogs, and the dogs were fed methionine to increase plasma homocysteine to 10 mol/L for acute and 24 mol/L for chronic HHcy. There was no significant change in hemodynamics with HHcy. Veratrine-induced, NO-dependent, coronary vasodilation (Bezold-Jarisch reflex) was reduced by 32% but was restored by simultaneous intravenous infusion of ascorbic acid or apocynin. Acute and chronic HHcy significantly increased uptake of glucose and lactate and decreased uptake of free fatty acid by the heart. HHcy significantly decreased bradykinin-or carbachol-induced reduction of myocardial oxygen consumption in vitro, and this effect was completely restored by coincubation with ascorbic acid, Tempol, or apocynin. Western blot analysis indicated an increase in Nox2 (82%) and a reduction in endothelial nitric oxide synthase (39%), phospho-endothelial nitric oxide synthase (39%), and superoxide dismutase-1 (45%). Microarray analysis of gene expression in heart tissue from chronic HHcy indicated a switch in cardiac phenotype to enzymes that metabolize glucose. Conclusions-HHcy directly modulates substrate use by the heart independent of changes in hemodynamics or ventricular function by reducing NO bioavailability through the generation of superoxide. The progression of cardiac or coronary heart disease associated with HHcy should be evaluated in light of the impact of alterations in the regulation of cardiac metabolism and substrate use.

X, Hintze TH. Role of nitric oxide in the coupling of myocardial oxygen consumption and coronary vascular dynamics during pregnancy in the dog. Am J Physiol Heart Circ Physiol 293: H2479-H2486, 2007. First published July 20, 2007; doi:10.1152/ajpheart.00036.2006.-We examined the ability of cardiac endothelial nitric oxide synthase (eNOS) to couple myocardial oxygen consumption (MV O2) and oxygen delivery during pregnancy. Awake dogs were studied using echocardiography before and at 40 days, 50 days, and 60 days (60D) of pregnancy and at ϳ14 days postpartum. Left ventricular eNOS, phosphorylated eNOS, and copper, zinc-superoxide dismutase (CuZnSOD or SOD-1) were determined by immunoblotting. MV O2 of left ventricular tissue samples was measured in vitro in response to increasing doses of bradykinin, enalapril maleate, and amlodipine. We examined the changes in passive diameter and flow-dependant arteriolar dilation of coronary arterioles. Echocardiography indicated increases in cardiac output (ϳ60%) during pregnancy. Myocardial eNOS (21 Ϯ 4%), phosphorylated eNOS (19 Ϯ 3%), and SOD-1 (61 Ϯ 2.7%) protein levels were significantly increased at 60D. Bradykinin, enalapril maleate, and amlodipine (10 Ϫ4 mol/l) decreased MV O2 in a nitric oxide-dependant manner (by 24 Ϯ 1.3% in controls and 34 Ϯ 2.2% at 60D; by 21 Ϯ 1.1% in controls and 29 Ϯ 1.1 at 60D; and by 22 Ϯ 2.5% in controls and 26 Ϯ 1.0% at 60D, respectively). Arterioles from pregnant dogs showed increased flow-dependant dilation in response to increased shear stress and larger passive diameter. Nitrite production was stimulated by bradykinin and carbachol in microvessels in vitro; pregnancy enhanced nitrite release. Myocardial eNOS, phosphorylated eNOS, and SOD-1 protein expression are increased during pregnancy, and this increase is associated with enhanced nitric oxidedependant control of MV O2. Thus increases in eNOS and SOD-1 promote the coupling of oxygen delivery and efficiency in the heart during pregnancy. gene expression PREGNANCY IN HUMANS IS ASSOCIATED with an early rise in plasma volume, which is increased further in the second trimester and then increases only slightly further until delivery. The average increase in plasma volume is 40% over normal values (12). Along with this volume overload state, there is an increase in cardiac output (20). In humans, cardiac output increases an average of 40% (from 5 to 7 l/min) in women at rest in the supine position. The increase in output is detectable starting at week 5 and then levels off at around week 20. A portion of the increase in cardiac output is attributable to an increase in stroke volume (3,6). By midpregnancy, the stroke volume has increased upward of 30%. The increase in cardiac output is at least partially achieved by an enhanced myocardial performance (9) and a relative tachycardia. The basal heart rates of women rise significantly by the 4th wk of pregnancy and reach a peak at the 36th wk (4).The increase in cardiac output is associated with a decrease in total peripheral resistance (23), ...

Background-Hyperhomocysteinemia (HHcy) is a reliable indicator of cardiovascular disease, in part because of the production of superoxide and scavenging of nitric oxide (NO). The present study assessed the impact of HHcy on the NO-dependent control of cardiac O 2 consumption and examined enzymatic sources of superoxide. Methods and Results-Rats and mice were fed methionine in drinking water for 5 to 9 weeks to increase plasma homocysteine, a process that did not cause significant changes in hemodynamic function. The ability of the NO agonists bradykinin and carbachol to reduce myocardial O 2 consumption in vitro was impaired by Ϸ40% in methionine-fed rats, and this impairment was proportional to their individual plasma homocysteine concentration. However, responses were restored in the presence of ascorbic acid, tempol, and apocynin, which inhibits NADPH oxidase assembly. Western blots showed no difference in Cu/Zn or Mn superoxide dismutase, endothelial NO synthase, or inducible NO synthase protein, but HHcy caused a 100% increase in the p22 phox subunit of NADPH oxidase. Western blots with plasma membrane-enriched fractions of cell lysate detected elevated levels of p22 phox , p67 phox , and rac-1, which indicates increased oxidase assembly. Finally, mice lacking a functional gp91phox subunit of NADPH oxidase demonstrated normal NO-dependent regulation of myocardial O 2 consumption after methionine feeding. Conclusions-In

.7 pg/mL). Veratrine-induced (5 g/kg) NO-mediated vasodilation was inhibited by 44% in LS; however, the simultaneous intravenous infusion of ascorbic acid or apocynin acutely and completely reversed this inhibition. In LS heart tissues, lucigenin chemiluminescence was increased 2.3-fold to angiotensin II (10 ؊8 mol/L), and bradykinin (10 ؊4 mol/L) induced reduction of myocardial oxygen consumption in vitro was decreased (40؎1.3% to 16؎6.3%) and completely restored by coincubation with tiron, tempol or apocynin. Switching of substrate uptake from free fatty acid to glucose by the heart was observed (free fatty acid: 8.97؎1.39 to 4.53؎1.12 mol/min; glucose: 1.31؎0.52 to 6.86؎1.78 mol/min). Western blotting indicated an increase in both p47 phox (121%) and gp91 phox (44%) as did RNA microarray analysis (433 genes changed) showed an increase in p47 phox (1.6-fold) and gp91 phox (2.0 fold) in the LS heart tissue. Conclusions: LS diet induces the activation of the renin-angiotensin system, which increases oxidative stress via the NADPH oxidase and attenuates NO bioavailability in the heart. (Circ Res. 2010;106:593-600.)Key Words: low salt diet Ⅲ nitric oxide Ⅲ free radicals Ⅲ angiotensin II Ⅲ heart diseases T he role of angiotensin in the control of plasma volume and especially sodium homeostasis is well known. 1,2 Historically, restriction of sodium intake has resulted in an increase in plasma renin and angiotensin I and II, an increase in plasma aldosterone, 3 and enhanced sodium reabsorption. In lower species, including fishes and amphibians, it is the action of angiotensin II that conserves sodium. 4 In addition, it has recently become clear that angiotensin II through its interaction with the angiotensin II type 1 (AT 1 ) receptor activates the NADPH oxidase to increase superoxide production. 5 In fact, a portion, perhaps 50%, of the increase in arterial pressure during chronic angiotensin infusion in the rat, is dependent on increased superoxide production, 6 as evidenced by the fact that the hypertension is reduced by scavenging superoxide. In tissues from dogs, angiotensin II increases superoxide as measured by lucigenin chemiluminescence in vitro and reduces NO bioactivity both of which are restored to control by addition of agents that scavenge superoxide or by apocynin. 7 In hearts from human, primate, dog, hamster, mouse, rat, and frog, in vitro, bradykinin (BK) reduces cardiac oxygen consumption and this is blocked by an NO synthase inhibitor. [7][8][9] In hearts from endothelial NO synthase (eNOS) knockout mice, NOdependent (BK) signaling to mitochondria does not occur, suggesting the importance of eNOS. 8 Low salt (LS)/angiotensin II may regulate the development of atherosclerosis and renal hormone production. 10 Thus, there is an NO-dependent regulation of mitochondrial function that is important in the control of tissue oxygen metabolism.Most importantly, the ability of NO to reduce oxygen consumption in vitro in the normal mouse heart is almost abolished during incubation with angiotensin II via a...