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.

Oxidant stress is an important contributor to renal dysfunction and hypertension. We have previously demonstrated that regulation of renal oxygen consumption by nitric oxide (NO) is impaired in the kidney of spontaneously hypertensive rats (SHR) due to increased superoxide production. We further explored the mechanisms of enhanced oxidant stress in the kidney of SHR. Suppression of cortical oxygen consumption by bradykinin (BK) or enalaprilat (Enal), which act through stimulation of endogenous NO, was impaired in SHR (BK: -14.1 +/- 1.2%; Enal: -15.5 +/- 1.2%) and was restored by addition of apocynin, an inhibitor of assembly of the NAD(P)H oxidase complex (BK: -21.0 +/- 0.6%; Enal: -25.3 +/- 1.4%), suggesting this as the source of enhanced superoxide production. Addition of an angiotensin type 1 receptor blocker, losartan, also restored responsiveness to control levels (BK: -22.0 +/- 1.1%; Enal: -23.6 +/- 1.3%), suggesting that ANG II is responsible for enhanced oxidase activity. A similar defect in responsiveness to BK and Enal could be induced in Wistar-Kyoto kidneys by ANG II and was reversed by a superoxide scavenger (tempol), apocynin or losartan. Immunoblotting of cortical samples demonstrated enhanced expression of endothelial NO synthase (eNOS 1.9x) and NAD(P)H oxidase components (gp91(phox) 1.6x and Rac-1 4.5x). Expression of SOD-1 and -2 were unchanged, but SOD-3 was significantly decreased in SHR (0.5x). Thus NO bioavailability is impaired in SHR owing to an ANG II-mediated increase in superoxide production in association with enhanced expression of NAD(P)H oxidase components, despite increased expression of eNOS. Loss of SOD-3, an important superoxide scavenger, may also contribute to enhanced oxidant stress.

To determine the effects of acute myocardial infarction on the regulation of angiotensin II (Ang II) receptors and contractile performance of left and right ventricular myocytes, coronary artery ligation was surgically induced in rats, and Ang II receptor density and affinity and the mechanical properties of surviving muscle cells were examined 1 week later. Physiological determinations of cardiac pump function revealed the presence of ventricular failure, which was associated at the cellular level with a depression in the velocity of myocyte shortening and relengthening, a prolongation of time to peak shortening, and a reduction in the extent of cell shortening. These abnormalities in single-cell function were more prominent in left than in right ventricular myocytes. Cellular hypertrophy was documented by increases in cell length and width, which were also greater in the spared myocytes of the infarcted left ventricle. Reactive hypertrophy was accompanied by a 1.84- and 1.85-fold increase in the density of Ang II receptors on left and right myocytes, respectively. On the other hand, the affinity of Ang II receptors for the radiolabeled antagonist was not altered. However, Ang II-stimulated phosphoinositol turnover was enhanced by 3.7- and 2.5-fold in left and right myocytes, respectively, after infarction. Ventricular myocytes were found to possess the AT1 receptor subtype exclusively. In conclusion, myocardial infarction leads to impairment in the contractile behavior of the remaining cells and to the activation of Ang II receptors and effector pathway associated with these receptors, which may be involved in the reactive growth adaptation of the viable myocytes.

Abstract-Endothelial nitric oxide synthase (eNOS) plays an important role in the control of myocardial oxygen consumption (MVO 2 ) by nitric oxide (NO). A NOS isoform is present in cardiac mitochondria and it is derived from neuronal NOS (nNOS). However, the role of nNOS in the control of MVO 2 remains unknown. MVO 2 in left ventricular tissues from nNOS Ϫ/Ϫ mice was measured in vitro. Stimulation of NO production by bradykinin or carbachol induced a significant reduction in MVO 2 in wild-type (WT) mice. In contrast to WT, bradykinin-or carbachol-induced reduction in MVO 2 was attenuated in nNOS Ϫ/Ϫ . S-methyl-L-thiocitrulline, a potent isoform selective inhibitor of nNOS, had no effect on bradykinin-induced reduction in MVO 2 in WT. Bradykinin-induced reduction in MVO 2 in eNOS Ϫ/Ϫ mice, in which nNOS still exists, was also attenuated. The attenuated bradykinin-induced reduction in MVO 2 in nNOS Ϫ/Ϫ was restored by preincubation with Tiron, ascorbic acid, Tempol, oxypurinol, or SB203850, an inhibitor of p38 kinase, but not apocynin. There was an increase in lucigenin-detectable superoxide anion (O 2 Ϫ ) in cardiac tissues from nNOScompared with WT. Tempol, oxypurinol, or SB203850 decreased O 2 Ϫ in all groups to levels that were not different from each other. There was an increase in phosphorylated p38 kinase normalized by total p38 kinase protein level in nNOS Key Words: endothelial nitric oxide synthase Ⅲ neuronal nitric oxide synthase Ⅲ superoxide anion Ⅲ p38 Ⅲ oxygen consumption N itric oxide (NO) attenuates mitochondrial respiration by nitrosylating the iron-sulfur centers of aconitase, complexes I and II of the electron transport chain, and through a very potent reversible alteration in the activity of cytochrome c oxidase. [1][2][3] We and others have shown that NO can modulate mitochondrial respiration and tissue oxygen consumption in whole body, 4 heart, skeletal muscle, and kidney both in vivo 5-8 and in vitro. 8 -10 Furthermore, we have shown that NO derived from endothelial NO synthase (eNOS) plays an important role in these processes. 11 Immunohistochemical studies have shown that a NOS is present in the mitochondria. 12-14 Giulivi et al have provided evidence for the production of NO by intact, purified mitochondria using two spectroscopic techniques. [15][16][17][18] In other laboratories, the production of NO by mitochondria has been shown by formation of L-citrulline from radiolabeled L-arginine. 12,19,20 Furthermore, Giulivi et al 17 have reported that mitochondrial NOS was identified as neuronal NOS (nNOS) with two posttranslational modifications in isolated mitochondria from rat liver. Kanai et al 21 identified mitochondrial NOS as nNOS in the isolated cardiac mitochondria from nNOS wild-type (WT) and knockout (nNOS Ϫ/Ϫ ) mice. Thus, nNOS may provide a local source of NO, which can modulate mitochondrial respiration and myocardial oxygen consumption (MVO 2 ). The role of nNOS in the regulation of MVO 2 remains to be elucidated.French et al 22 showed that the local production of NO by mitoch...

To date, the demonstration that the molecular components of the renin-angiotensin system (RAS) are present in adult ventricular myocytes is lacking. In addition, whether the RAS is upregulated under conditions of overload and myocyte hypertrophy in vivo remains to be determined. By employing an in vivo model of ischemic cardiomyopathy in rats, we document that adult myocytes express genes for renin, angiotensinogen, angiotensin-converting enzyme (ACE), and angiotensin II (ANG II) receptors. Moreover, renin, ACE, and ANG II receptor mRNAs increased in stressed myocytes undergoing cellular hypertrophy. At the protein level, the percentage of myocytes containing renin, ANG I, and ANG II was significantly increased in the overloaded heart. The number of binding sites for ANG II per myocyte also markedly increased under this setting. These results provide direct evidence of the existence of a myocyte RAS, which may be activated in pathological states of the heart to support myocyte growth and contractile function.