Simvastatin upregulates coronary vascular endothelial nitric oxide production in conscious dogs.

Statin drugs can upregulate endothelial nitric oxide (NO) synthase (eNOS) in isolated endothelial cells independent of lipid-lowering effects. We investigated the effect of short-term simvastatin administration on coronary vascular eNOS and NO production in conscious dogs and canine tissues. Mongrel dogs were instrumented under general anesthesia to measure coronary blood flow (CBF). Simvastatin (20 mg. kg(-1). day(-1)) was administered orally for 2 wk; afterward, resting CBF was found to be higher compared with control (P < 0.05) and veratrine- (activator of reflex cholinergic NO-dependent coronary vasodilation) and ACh-mediated coronary vasodilation were enhanced (P < 0.05). Response to endothelium-independent vasodilators, adenosine and nitroglycerin, was not potentiated. After simvastatin administration, plasma nitrate and nitrite (NO(x)) levels increased from 5.22 +/- 1.2 to 7. 79 +/- 1.3 microM (P < 0.05); baseline and agonist-stimulated NO production in isolated coronary microvessels were augmented (P < 0.05); resting in vivo myocardial oxygen consumption (MVO(2)) decreased from 6.8 +/- 0.6 to 5.9 +/- 0.4 ml/min (P < 0.05); NO-dependent regulation of MVO(2) in response to NO agonists was augmented in isolated myocardial segments (P < 0.05); and eNOS protein increased 29% and eNOS mRNA decreased 50% in aortas and coronary vascular endothelium. Short-term administration of simvastatin in dogs increases coronary endothelial NO production to enhance NO-dependent coronary vasodilation and NO-mediated regulation of MVO(2).

Endogenous nitric oxide in the control of skeletal muscle oxygen extraction during exercise.

Our previous studies uncovered an inhibitory effect of nitric oxide (NO) on leg skeletal muscle respiration in dogs at rest. The role of NO in the modulation of O2 consumption and O2 extraction in hindlimb muscle during elevated metabolic states was investigated in chronically instrumented dogs while walking and at three exercise intensities which markedly increased hindlimb blood flow. Walking resulted in increased O2 consumption by 17 +/- 4 mL min-1 and O2 extraction from 24 +/- 1 to 37 +/- 8%, with no alteration in hindlimb blood flow (BFLeg) and vascular resistance (VRLeg). Running at the highest speed (9.1 mph) resulted in an increase in BFLeg from 0.67 +/- 0.05 to 2.2 +/- 0.1 L min-1, a reduction of VRLeg and elevation of hindlimb O2 consumption from 33 +/- 3 to 226 +/- 21 mL min-1 and O2 extraction from 29 +/- 2 to 61 +/- 5%, with a decrease in leg venous PO2 from 38 +/- 1 to 25 +/- 1 mmHg. After nitro-L-arginine (NLA) (35 mg kg-1, i.v.) to inhibit endogenous NO synthesis, walking caused greater increases in hindlimb O2 consumption (29 +/- 5 mL min-1) and O2 extraction (43 +/- 1 to 60 +/- 3%) (both P < 0.05), with no significant change in BFLeg. During running at the highest speed, BFLeg was 1.9 +/- 0.1 L min-1 (P < 0. 05) and VRLeg was higher, accompanied by increases in hindlimb O2 consumption from 49 +/- 7 to 318 +/- 24 mL min-1 and O2 extraction from 41 +/- 2 to 79 +/- 4% (both P < 0.05), with a greater decrease in leg venous PO2 from 33 +/- 1 to 20 +/- 1 mmHg (P < 0.05). Similar results were found for intermediate levels of exercise. Our results indicate that NO modulates hindlimb skeletal muscle O2 extraction and O2 usage whether blood flow increased or not during exercise.

Cytokines are not a requisite part of the pathophysiology leading to cardiac decompensation.

An increase in circulating levels of proinflammatory cytokines has been proposed as an important pathogenic factor contributing to cardiac injury during chronic heart failure. To determine whether plasma levels of the cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) increase during pacing-induced heart failure, we paced the hearts of seven dogs at 210 beats/min for 3 weeks and at 240 beats/min for an additional week to induce severe clinical signs of cardiac decompensation. Hemodynamic measurements and blood samples from the aorta and coronary sinus (CS) were taken at control, at 3 weeks, and in end-stage failure. Decompensated heart failure occurred at 29 +/- 1.8 days, when left ventricular (LV) end-diastolic pressure was 25 +/- 1.3 mmHg, LV systolic pressure was 92 +/- 4 mmHg, mean arterial pressure was 77 +/- 3 mmHg, and dP/dtmax was 1219 +/- 73 (all P < 0.05 vs control). Arterial concentration of IL-6 was 12 +/- 4.0 U/ml at control, 11 +/- 2.7 U/ml at 3 weeks, and 10 +/- 1.7 U/ml in end-stage failure (NS). At the same time points, IL-6 in CS plasma was 12 +/- 3.5, 13 +/- 2.8 and 11 +/- 2.4 U/ml, respectively (NS vs control and vs arterial concentrations). TNF-alpha did not reach detectable concentrations in arterial or CS blood at any time. TNF-alpha and IL-6 concentrations did not increase in arterial blood, were not released in the CS from the heart during the development of pacing-induced heart failure, and can not universally be implicated in the pathogenesis of all forms of cardiac dysfunction. Our findings are consistent with other data from patients in which severe heart failure was not associated with increased levels of circulating cytokines.

Nitric oxide and oxygen utilization: exercise, heart failure and diabetes.

In addition to regulating vascular tone, there is increasing evidence for the involvement of NO in the modulation of oxygen consumption. Our in-vitro studies indicated that exogenous and endogenous NO reduces the consumption of oxygen in isolated canine skeletal and cardiac muscle, which is probably related to its direct effect on mitochondria, i.e. cytochrome oxidase. In resting, conscious dogs, the blockade of NO synthesis results in an increase in total oxygen consumption. During exercise, there is a significant increase in the release of NO from the coronary circulation in conscious dogs, and there are greater increases in total oxygen consumption, and oxygen consumption in skeletal muscle and in the heart when NO synthesis is blocked. Our results suggest that NO plays a role in matching blood flow to tissue metabolism at rest and during exercise. The modulation of the consumption of O2 by endogenous NO in skeletal or cardiac muscle is blunted after the development of heart failure or diabetes. After heart failure, the heart switches from fatty acid to glucose metabolism, suggesting that NO also plays a role in the regulation of metabolism in the heart.

Reduced nitric oxide production and altered myocardial metabolism during the decompensation of pacing-induced heart failure in the conscious dog.

The aim of the present study was to determine whether cardiac nitric oxide (NO) production changes during the progression of pacing-induced heart failure and whether this occurs in association with alterations in myocardial metabolism. Dogs (n=8) were instrumented and the heart paced until left ventricular end-diastolic pressure reached 25 mm Hg and clinical signs of severe failure were evident. Every week, hemodynamic measurements were recorded and blood samples were withdrawn from the aorta and the coronary sinus for measurement of NO metabolites, O2 content, free fatty acids (FFAs), and lactate and glucose concentrations. Cardiac production of NO metabolites or consumption of O2 or utilization of substrates was calculated as coronary sinus-arterial difference times coronary flow. In end-stage failure, occurring at 29+/-1.6 days, left ventricular end-diastolic pressure was 25+/-1 mm Hg, left ventricular systolic pressure was 92+/-3 mm Hg, mean arterial pressure was 75+/-2.5 mm Hg, and dP/dtmax was 1219+/-73 mm Hg/s (all P<0.05). These changes in hemodynamics were associated with a fall of cardiac NO metabolite production from 0.37+/-0.16 to -0.28+/-0.13 nmol/beat (P<0.05). O2 consumption and lactate uptake did not change significantly from control, while FFA uptake decreased from 0.16+/-0.03 to 0.05+/-0.01 microEq/beat and glucose uptake increased from -2.3+/-7.0 to 41+/-10 microgram/beat (P<0.05). The cardiac respiratory quotient also increased significantly by 28%. In 14 normal dogs the same measurements were performed at control and 1 hour after we injected 30 mg/kg of nitro-L-arginine, a competitive inhibitor of NO synthase .O2 consumption increased from 0.05+/-0.002 mL/beat at control to 0.071+/-0.003 mL/beat after nitro-L-arginine, while FFA uptake decreased from 0.1+/-0.01 to 0.06+/-0.01 microEq/beat, lactate uptake increased from 0.15+/-0.04 to 0.31+/-0.03 micromol/beat, glucose uptake increased from 8.2+/-5.0 to 35.4+/-9.5 microgram/beat, and RQ increased by 23% (all P<0.05). Our results indicate that basal cardiac production of NO falls below normal levels during cardiac decompensation and that there are shifts in substrate utilization. This switch in myocardial substrate utilization also occurs after acute pharmacological blockade of NO production in normal dogs.

Mechanisms of nitrate accumulation in plasma during pacing-induced heart failure in conscious dogs.

The goal of this study was to understand the mechanisms behind the changes in plasma NOx during heart failure. Heart failure is associated with an increase in plasma nitrate levels, and yet most experimental evidence demonstrates a reduction in endothelial nitric oxide production during heart failure. Dogs were chronically instrumented for measurement of systemic hemodynamics and left ventricular (LV) dimensions. Hearts were paced at 210 bpm for 3 weeks (n = 14) and then 240 bpm for 1 week (n = 7). Hemodynamics, arterial blood gases, plasma NOx, and creatinine levels were monitored weekly. Heart failure was evidenced by cachexia, ascites, and hemodynamic alterations. Resting heart rate rose (94 +/- 6 to 135 +/- 9 bpm), and LV dP/dt fell (2810 +/- 82 to 1471 +/- 99 mm Hg/s), while LV end diastolic pressure quadrupled (5.8 +/- 0.7 to 25 +/- 0.8 mm Hg), and diastolic wall stress quadrupled (11 +/- 1.3 to 43 +/- 6.0 g/cm2, all P < 0.05). These changes occurred during a doubling in plasma NOx (5.5 +/- 1.5 to 10 +/- 1.6 microM, P < 0.05). There were no changes in plasma NOx through 3 weeks of pacing. Plasma creatinine levels increased 450% (from 0.27 +/- 0.32 to 1.21 +/- 0.63 mg%). Stimulated nitrite production by agonists in sieved coronary microvessels was unchanged after 3 weeks of pacing but was reduced after heart failure. Plasma NOx did not correlate with LV dP/dt or systolic wall stress but correlated directly with LV EDP or diastolic wall stress and inversely with cardiac work. Plasma NOx rose in direct relation to plasma creatinine levels (Y = 4.8X + 2.8, r2 = 0.84), suggesting that the rise in plasma NOx during heart failure is due to decreased renal function not increased NO production.

Function and production of nitric oxide in the coronary circulation of the conscious dog during exercise.

This study determined the changes in NO production from the coronary circulation of the conscious dog during exercise. The role of endogenous NO as it relates to coronary flow, myocardial work, and metabolism was also studied. Mongrel dogs were chronically instrumented for measurements of coronary blood flow (CBF), ventricular and aortic pressure, and ventricular diameter, with catheters in the aorta and coronary sinus. Acute exercise (5 minutes at 3.6, 5.9, and 9.1 mph) was performed, and hemodynamic measurements and blood samples were taken at each exercise level. Nitro-L-arginine (NLA, 35 mg/kg IV) was given to block NO synthesis, and the exercise was repeated. Blood samples were analyzed for oxygen, plasma nitrate/nitrite (an index of NO), lactate, glucose, and free fatty acid (FFA) levels. Acute exercise caused significant elevations in NO production by the coronary circulation (46 +/- 23, 129 +/- 44, and 63 +/- 32 nmol/min at each speed respectively, P < .05). After NLA, there was no measurable NO production at rest or during exercise. Blockade of NO synthesis resulted in elevations in myocardial oxygen consumption and reductions in myocardial FFA consumption for comparable levels of CBF and cardiac work. The metabolic changes after NLA occurred in the absence of alterations in myocardial lactate or glucose consumptions. NO production by the coronary circulation is increased with exercise and blocked by NLA. The absence of NO in the coronary circulation during exercise does not affect levels of CBF, because it shifts the relationship between cardiac work and myocardial oxygen consumption, suggesting that endogenous NO modulates myocardial metabolism.

Pharmacodynamics of plasma nitrate/nitrite as an indication of nitric oxide formation in conscious dogs.

BACKGROUND: The present investigation was undertaken to better understand the production of nitric oxide (NO) in vivo as measured by alterations in plasma nitrite or nitrate in blood samples from studies in experimental animals or clinical studies in humans. METHODS AND RESULTS: Plasma samples were taken from the aorta, the coronary sinus, a peripheral vein in the leg (skeletal muscle), or the right ventricle (mixed venous) in chronically instrumented conscious dogs. Plasma nitrite was converted to NO gas in an argon environment by use of hydrochloric acid, and plasma nitrate was converted first to nitrite with nitrate reductase and then to NO gas with acid. Standard curves were constructed, and the amount of nitrite and nitrate in plasma was determined. The primary metabolite was nitrate, whereas nitrate was approximately 10% of the total and remained constant. In the resting dog, the only vascular bed with a positive arterial-venous nitrate difference, evidence for production of NO, was the heart. Nitrate infusion into quietly resting dogs resulted in increases in plasma nitrate up to 38 +/- 3.4 mmol/L, increases in systemic arterial pressure, and a marked diuresis. The plasma half-life was calculated as 3.8 hours. The volume of distribution was calculated as 0.215 L/kg, or equivalent to the extracellular volume. CONCLUSIONS: These studies indicate that nitrate is a reliable measure of NO metabolism in vivo but that because of the long half-life, nitrate will accumulate in plasma once it is produced. Because of the large volume of distribution (21% of body weight versus the 4% of body weight usually attributed to plasma volume, the compartment in which nitrate is measured), simple measures of plasma nitrate underestimate by a factor of 4 to 6 the actual production of nitrate or NO by the body. In disease states, such as heart failure, in which renal function and extracellular volume are altered, caution should be exercised when increases in nitrate in plasma as an index of NO formation are evaluated.