Abstract-Our objective was to determine the precise role of endothelial nitric oxide synthase (eNOS) as a modulator of cardiac O 2 consumption and to further examine the role of nitric oxide (NO) consumption in tissues taken from iNOS (Ϫ/Ϫ) (Ϫ28Ϯ4%), wild-type eNOS (ϩ/ϩ) (Ϫ22Ϯ4%), and heterozygous eNOS(ϩ/Ϫ) (Ϫ22Ϯ5%) but not homozygous eNOS (Ϫ/Ϫ) (Ϫ3Ϯ4%) mice. Responses to bradykinin in iNOS (Ϫ/Ϫ) and both wild-type and heterozygous eNOS mice were attenuated after NOS blockade with N-nitro-L-arginine methyl ester (L-NAME) (Ϫ2Ϯ5%, Ϫ3Ϯ2%, and Ϫ6Ϯ5%, respectively, PϽ0.05). In contrast, S-nitroso-N-acetyl-penicillamine (SNAP, 10Ϫ4 mol/L), which releases NO spontaneously, induced decreases in myocardial O 2 consumption in all groups of mice, and such responses were not affected by L-NAME. In addition, pretreatment with bacterial endotoxin elicited a reduction in basal O 2 consumption in tissues taken from normal but not iNOS (Ϫ/Ϫ)-deficient mice. Our results indicate that the pivotal role of eNOS in the control of myocardial O 2 consumption and modulation of mitochondrial respiration by NO may have an important role in pathological conditions such as endotoxemia in which the production of NO is altered. . Their initial observations demonstrated that activated mouse peritoneal macrophages severely inhibited O 2 consumption in numerous tumor cell lines obtained from different tissues and animal species in cultures by an unknown mechanism. Evidence now suggests that the macrophage-induced cytotoxic effect on mitochondrial metabolism is NO related. 2,3 NO inhibits 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. 4 -6 Recently, we and others have provided direct evidence to suggest that under physiological conditions NO plays a modulatory role on mitochondrial respiration and tissue O 2 consumption. For instance, L-arginine analogues, which are nonspecific inhibitors of the 3 isoforms of nitric oxide synthase (NOS), 7 increase O 2 consumption in whole body, 8 heart, skeletal muscle, and kidney both in vivo 9 -12 and in vitro. [12][13][14] We have interpreted our previous studies to suggest that endothelial nitric oxide synthase (eNOS), the most highly expressed isoform of NOS in vascular tissue under physiological conditions, is responsible for the control of tissue O 2 consumption by NO. However, we have yet to determine which isoform of NOS regulates mitochondrial O 2 consumption, because almost all cells are capable of expressing all 3 different NOS isoforms. Studies of the effects of bacterial endotoxins have attributed a substantial role for inducible nitric oxide synthase (iNOS) in the development of shock and perhaps other pathological states. To address the role of NO in both physiological and pathophysiological states in the control of mitochondrial respiration, we used tissues from mice deficient in iNOS and eNOS and 3 additional groups, ie, control C57B...

Background-Our objective for this study was to investigate whether nitric oxide (NO) modulates tissue respiration in the failing human myocardium. Methods and Results-Left ventricular free wall and right ventricular tissue samples were taken from 14 failing explanted human hearts at the time of transplantation. Tissue oxygen consumption was measured with a Clark-type oxygen electrode in an airtight stirred bath containing Krebs solution buffered with HEPES at 37°C (pH 7.4). Rate of decrease in oxygen concentration was expressed as a percentage of the baseline, and results of the highest dose are indicated. Bradykinin (10 Ϫ4 mol/L, Ϫ21Ϯ5%), amlodipine (10 Ϫ5 mol/L, Ϫ14Ϯ5%), the ACE inhibitor ramiprilat (10 Ϫ4 mol/L, Ϫ21Ϯ2%), and the neutral endopeptidase inhibitor thiorphan (10 Ϫ4 mol/L, Ϫ16Ϯ5%) all caused concentrationdependent decreases in tissue oxygen consumption. Responses to bradykinin (Ϫ2Ϯ6%), amlodipine (Ϫ2Ϯ4%), ramiprilat (Ϫ5Ϯ6%), and thiorphan (Ϫ4Ϯ7%) were significantly attenuated after NO synthase blockade with N-nitro-L-arginine methyl ester (10 Ϫ4 mol/L; all PϽ0.05). Ϫ4 mol/L, Ϫ34Ϯ5%) and nitroglycerin (10 Ϫ4 mol/L, Ϫ21Ϯ5%), also decreased tissue oxygen consumption in a concentration-dependent manner. However, the reduction in tissue oxygen consumption in response to S-nitroso-Nacetyl-penicillamine (Ϫ35Ϯ7%) or nitroglycerin (Ϫ16Ϯ5%) was not significantly affected by N-nitro-L-arginine methyl ester. Conclusions-These results indicate that the modulation of oxygen consumption by both endogenous and exogenous NOis preserved in the failing human myocardium and that the inhibition of kinin degradation plays an important role in the regulation of mitochondrial respiration. (Circulation. 1999;100:1291-1297.)Key Words: nitric oxide Ⅲ oxygen Ⅲ heart failure T he vast amount of ATP produced by the cardiac mitochondria is used mainly for cardiac muscle contraction. Abnormalities of mitochondria in cardiomyocytes in heart failure have been well documented in both animals [1][2][3][4] and human studies, 5-7 which provided structural and metabolic evidence of mitochondrial dysfunction. Mitochondrial DNA damage with increased mitochondrial DNA deletion in patients with heart failure has also been reported, 8,9 a defect associated with the impairment of oxidative phosphorylation. 10 On the other hand, normal mitochondrial metabolism has also been documented in chronic heart failure. 11,12 However, the role of nitric oxide (NO) in the control of mitochondrial metabolism is not well established. Our laboratory and others have demonstrated that attenuation of NO production increases whole-body or organ oxygen consumption. [13][14][15][16] The initial observation of the interaction between NO and mitochondrial enzymes was reported in cell culture studies of macrophage-induced cytotoxicity of neoplastic cells. 17,18 The activated macrophage induced reduction of electron transfer by inactivating iron-sulfur-containing complexes I and II of the respiratory chain and aconitase in the Krebs cycle. This effect was shown to b...

The acute inhibition of NO synthase by NLA causes a switch from fatty acids to lactate and glucose utilization by the heart which can be reversed by a NO donor, suggesting an important regulatory action of NO on cardiac metabolism.

Although the role of nitric oxide (NO) in the modulation of vascular tone has been studied and well understood, its potential role in the control of myocardial metabolism is only recently evident. Several lines of evidence indicate that NO regulates myocardial glucose metabolism; however, the details and mechanisms responsible are still unknown. The aim of this study was to further define the role of NO in the control of myocardial glucose metabolism and the nitric oxide synthase (NOS) isoform responsible using transgenic animals lacking endothelial NOS (ecNOS). In the present study, we examined the regulation of myocardial glucose uptake using isometrically contracting Langendorff-perfused hearts from normal mice (C57BL/6J), mice with defects in the expression of ecNOS [ecNOS (-/-)], and its heterozygote [ecNOS (+/-)], and wild-type mice [ecNOS (+/+)] (n=6, respectively). In hearts from normal mice, little myocardial glucose uptake was observed. This myocardial glucose uptake increased significantly in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME). Similarly, in the hearts from ecNOS (-/-), glucose uptake was much greater than in normal mice, whereas myocardial glucose uptake of ecNOS (+/-) and ecNOS (+/+) mice was not different from normal mice. In addition, myocardial glucose uptake of ecNOS (+/-) and ecNOS (+/+) mice increased significantly in the presence of L-NAME. At a workload of 800 g. beats/min, L-NAME increased glucose uptake from 0.1+/-0.1 to 3+/-0.4 microg/min x mg in ecNOS (+/-) mice and from 0.2+/-0.1 to 2.7+/-0.7 microg/min x mg in ecNOS (+/+) mice. Furthermore, in the hearts from ecNOS (-/-) mice, 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP), a cGMP analog or S-nitroso-N-acetylpenicillamine (SNAP), a NO donor essentially shut off glucose uptake, and in hearts from ecNOS (+/-) mice, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), an inhibitor of cGMP, increased the glucose uptake significantly. These results indicate clearly that cardiac NO production regulates myocardial glucose uptake via a cGMP-dependent mechanism and strongly suggest that ecNOS plays a pivotal role in this regulation. These findings may be important in the understanding of the pathogenesis of the diseases such as ischemic heart disease, heart failure, diabetes mellitus, hypertension, and hypercholesterolemia, in which NO synthesis is altered and substrate utilization by the heart changes.

Amlodipine is a mixture of two enantiomers, one having L-type channel blocking activity (S-) and the other about 1,000-fold weaker activity and of little known other activity (R+). To determine whether the R+ enantiomer releases nitric oxide, the ability of amlodipine, its enantiomers, and nitrendipine to release nitric oxide in isolated coronary microvessels and to regulate cardiac tissue oxygen consumption via nitric oxide release was studied in vitro. Amlodipine and the R+ enantiomer released nitric oxide in a concentration-dependent fashion, increasing nitrite release from coronary microvessels by 57 +/- 12 and 45 +/- 5 pmol/mg/20 min at 10(-6) M (p < 0.05 from control). Nitrite release was entirely blocked by N(omega)-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, and HOE-140, a B2-kinin receptor antagonist. The S- enantiomer had no effect on nitrite release at any concentration. Amlodipine and the R+ enantiomer also reduced oxygen consumption in canine cardiac tissue in vitro and this was in an L-NAME-blockable manner. The S- enantiomer of amlodipine had no effect. This study shows that the R+ enantiomer of amlodipine is responsible for the release of nitric oxide and not the S- enantiomer (the L-type calcium channel blocking portion of amlodipine). Interestingly, nitric oxide release is dependent on the production of kinins because it is blocked by HOE-140. This study defines a potentially important property by which calcium channel blockers may release nitric oxide and may contribute to their use in the treatment of cardiovascular disease.

Nitric oxide (NO) regulates renal O2 consumption, but the source of NO mediating this effect is unclear. We explored the effects of renal NO production on O2 consumption using renal cortex from mice deficient (-/-) in endothelial (e) nitric oxide synthase (NOS). O2 consumption was determined polarographically in slices of cortex from control and eNOS-/- mice. NO production was stimulated by bradykinin (BK) or ramiprilat (Ram) in the presence or absence of an NOS inhibitor. Basal O2 consumption was higher in eNOS-/- mice than in heterozygous controls (919 +/- 46 vs. 1,211 +/- 133 nmol O(2). min(-1). g(-1); P < 0.05). BK and Ram decreased O2 consumption significantly less in eNOS-/- mice [eNOS-/-: BK -19.0 +/- 2.8%, Ram -20.5 +/- 3.3% at 10(-4) M; control: BK -29.5 +/- 2.5%, Ram -34 +/- 1.6% at 10(-4) M]. The NO synthesis inhibitor nitro-L-arginine methyl ester (L-NAME) attenuated this decrease in control but not eNOS-/- mice. An NO donor inhibited O2 consumption similarly in both groups independent of the presence of L-NAME. These results demonstrate that NO production by eNOS is responsible for regulation of renal O2 consumption in mouse kidney.

Abstract-The aim of this study was to determine whether bradykinin, the angiotensin-converting enzyme inhibitor ramiprilat, and the calcium-channel antagonist amlodipine reduce myocardial oxygen consumption (MV O 2 ) via a B 2 -kinin receptor/nitric oxide-dependent mechanism. Left ventricular free wall and septum were isolated from normal and B 2 -kinin receptor knockout (B 2 Ϫ/Ϫ) mice. Myocardial tissue oxygen consumption was measured in an airtight chamber with a Clark-type oxygen electrode. Baseline MV O 2 was not significantly different between normal (to 10 Ϫ4 mol/L) reduced oxygen consumption in a concentration-dependent manner in both normal (maximum, 36Ϯ3%) and B 2 Ϫ/Ϫ mice (28Ϯ3%). This was also true for the endothelium-dependent vasodilator substance P (10 Ϫ10 to 10 Ϫ7 mol/L; 22Ϯ7% in normal mice and 20Ϯ4% in B 2 Ϫ/Ϫ mice). Bradykinin (10 Ϫ7 to 10 Ϫ4 mol/L), ramiprilat (10 Ϫ7 to 10 Ϫ4 mol/L), and amlodipine (10 Ϫ7 to 10 Ϫ5 mol/L) all caused concentration-dependent decreases in MV O 2 in normal mice. At the highest concentration, tissue O 2 consumption was decreased by 18Ϯ3%, 20Ϯ5%, and 28Ϯ3%, respectively. The reduction in MV O 2 to all 3 drugs was attenuated in the presence of N G -nitro-L-arginine-methyl ester. However, in the B 2 Ϫ/Ϫ mice, bradykinin, ramiprilat, and amlodipine had virtually no effect on MV O 2 . Therefore, nitric oxide, through a bradykinin-receptor-dependent mechanism, regulates cardiac oxygen consumption. This physiological mechanism is absent in B 2 Ϫ/Ϫ mice and may be evidence of an important therapeutic mechanism of action of angiotensin-converting enzyme inhibitors and amlodipine. (Hypertension. 1999;34:563-567.) Key Words: heart Ⅲ oxygen Ⅲ angiotensin-converting enzyme inhibitors Ⅲ amlodipine N itric oxide synthase (NOS) metabolizes L-arginine into nitric oxide (NO) and citrulline. NOS is a Ca 2ϩ /calmodulin-dependent enzyme present in endothelial cells that is inhibited by analogs of L-arginine such as N G -nitro-Larginine-methyl ester (L-NAME). [1][2][3][4] NO is involved in numerous physiological mechanisms, including the inhibition of platelet aggregation and neuronal communication and the regulation of vascular tone. 1,2 We and others have shown that NO can modulate mitochondrial respiration in vivo 5,6 and in vitro. 7 NO attenuates mitochondrial respiration by inhibiting complexes I and II of the electron transport chain 8 -10 and by interactions with cytochrome oxidase. 9 Bradykinin, an endogenous vasodilator, activates B 2 -kinin receptors, which are primarily on endothelial cells, 11,12 to augment the release of NO. 13 The angiotensin-converting enzyme (ACE) converts bradykinin into an inactive form; hence, ACE inhibitors, such as ramiprilat, are vasodilators, inhibit the inactivation of bradykinin, and augment the effects of bradykinin on NO release. 14,15 Recent studies using the calcium-channel antagonist amlodipine, a dihydropyridine, have shown improvement in the morbidity and mortality of patients with severe, chronic, nonischemic heart failure. 16 We have als...

Chronic LVAD support potentiates endogenous NO-mediated regulation of mitochondrial respiration. Use of medical or surgical interventions that augment NO bioavailability may promote myocardial recovery in end-stage heart failure.