Nahid Seyedi

Chronic exercise in dogs increases coronary vascular nitric oxide production and endothelial cell nitric oxide synthase gene expression.

Recently, we have shown that chronic exercise increases endothelium-derived relaxing factor (EDRF)/nitric oxide (NO)-mediated epicardial coronary artery dilation in response to brief occlusion and acetylcholine. This finding suggests that exercise can provide a stimulus for the enhanced production of EDRF/NO, thus possibly contributing to the beneficial effects of exercise on the cardiovascular system. Therefore, the purpose of the present study was to examine whether chronic exercise could influence the production of NO (measured as the stable degradation product, nitrite) and endothelial cell NO synthase (ECNOS) gene expression in vessels from dogs after chronic exercise. To this end, dogs were exercised by running on a treadmill (9.5 km/h for 1 hour, twice daily) for 10 days, and nitrite production in large coronary vessels and microvessels and ECNOS gene expression in aortic endothelial extracts were assessed. Acetylcholine (10(-7) to 10(-5) mol/L) dose-dependently increased the release of nitrite (inhibited by nitro-L-arginine) from coronary arteries and microvessels in control and exercised dogs. Moreover, acetylcholine-stimulated nitrite production was markedly enhanced in large coronary arteries and microvessels prepared from hearts of dogs after chronic exercise compared with hearts from control dogs. One potential mechanism that may contribute to the enhanced production of nitrite in vessels from exercised dogs may be the induction of the calcium-dependent ECNOS gene. Steady-state mRNA levels for ECNOS were significantly higher than mRNA levels for von Willebrands factor (vWF, a specific endothelial cell marker) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a constitutively expressed gene) in exercised dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Coronary Kinin Generation Mediates Nitric Oxide Release After Angiotensin Receptor Stimulation

Our goal was to determine whether angiotensin II (Ang II) and its metabolic fragments release nitric oxide and the mechanisms by which this occurs in blood vessels from the canine heart. We incubated 20 mg of microvessels or large coronary arteries in phosphate-buffered saline for 20 minutes and measured nitrite release. Nitrite release increased from 27 +/- 2 up to 103 +/- 5, 145 +/- 17, 84 +/- 4, 107 +/- 16, and 54 +/- 4 pmol/mg (P < .05) in response to 10(-5) mol/L of Ang I, II, III, IV, and Ang-(1-7), respectively. The effects of all angiotensins were blocked by N omega-nitro-L-arginine methyl ester (100 mumol/L), indicating that nitrite was a product of nitric oxide metabolism, and by Hoe 140 (10 mumol/L), a specific bradykinin B2 receptor antagonist, indicating a potential role for local kinin formation. The protease inhibitors aprotinin (10 mumol/L) and soybean trypsin inhibitor, which block local kinin formation, inhibited nitrite release by all of the angiotensins. Angiotensin nonselective (saralasin), type 1-specific (losartan), and type 2-specific (PD 123319) receptor antagonists abolished the nitrite released in response to all the fragments. Angiotensin type 1 and type 2 and receptors mediate nitrite release after Ang I, II, III, and Ang-(1-7), whereas only type 2 receptors mediate nitrite release after Ang IV. Similar results were obtained in large coronary arteries. In summary, formation of nitrite from coronary microvessels and large arteries in the normal dog heart in response to angiotensin peptides is due to the activation of local kinin production in the coronary vessel wall.

Pharmacodynamics of Plasma Nitrate/Nitrite as an Indication of Nitric Oxide Formation in Conscious Dogs

BACKGROUNDnThe 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.nnnMETHODS AND RESULTSnPlasma 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.nnnCONCLUSIONSnThese 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.

Regulation of Nitric Oxide Production in Human Coronary Microvessels and the Contribution of Local Kinin Formation

BACKGROUNDnThe goal of this study was to define the regulation of nitric oxide release by coronary microvessels from the failing and nonfailing human heart and to determine the role of local kinin production in the elaboration of nitric oxide by human coronary microvascular endothelium.nnnMETHODS AND RESULTSnTen hearts from humans with end-stage heart failure and two hearts from patients without heart failure were harvested at the time of orthotopic cardiac transplantation. Microvessels were sieved and the production of nitrite was determined by the Griess reaction. Microvessels were incubated in the presence of agonists for nitric oxide production (acetylcholine and bradykinin), which caused dose-dependent increases in nitrite, a response that was blocked by NG-nitro-L-arginine methyl ester and receptor-specific antagonists (atropine and HOE 140, respectively). In addition, the production of nitrite by microvessels from the failing heart appeared to be less than that produced by microvessels from the nonfailing heart. Incubation with norepinephrine or the alpha2-adrenergic agonist BHT 920 also caused dose-dependent increases in nitrite production, which were blocked by the B2-receptor antagonist HOE 140. This implicated local kinin synthesis as an intermediate step in the production of nitric oxide in response to alpha2-adrenoceptor stimulation. The production of nitric oxide was also prevented by the addition of serine protease inhibitors, which blocked the action of local kallikrein, again suggesting a role for local kinin synthesis.nnnCONCLUSIONSnOur results indicate that nitric oxide is produced by human coronary microvessels, that nitric oxide production may be reduced but certainly not increased in microvessels from the failing human heart, and that there is active local kinin generation in these blood vessels.

Functional Identification of Histamine H3-Receptors in the Human Heart

Norepinephrine release contributes to ischemic cardiac dysfunction and arrhythmias. Because activation of histamine H3-receptors inhibits norepinephrine release, we searched for the presence of H3-receptors directly in sympathetic nerve endings (cardiac synaptosomes) isolated from surgical specimens of human atria. Norepinephrine was released by depolarization with K+. The presence of H3-receptors was ascertained because the selective H3-receptor agonists (R) alpha-methylhistamine and imetit reduced norepinephrine release, and the specific H3-receptor antagonist thioperamide blocked this effect. Norepinephrine release was exocytotic, since it was inhibited by the N-type Ca(2+)-channel blocker omega-conotoxin and the protein kinase C inhibitor Ro31-8220. Functional relevance of these H3-receptors was obtained by showing that transmural electrical stimulation of sympathetic nerve endings in human atrial tissue increased contractility, an effect blocked by propranolol and attenuated in a concentration-dependent manner by (R) alpha-methylhistamine. Also, thioperamide antagonized the effect of (R) alpha-methylhistamine. Our findings are the first demonstration that H3-receptors are present in sympathetic nerve endings in the human heart, where they modulate adrenergic responses by inhibiting norepinephrine release. Since myocardial ischemia causes intracardiac histamine release, H3-receptor-induced attenuation of sympathetic neurotransmission may be clinically relevant.

Role of EDRF/NO in Chronic High Coronary Blood Flow States During Myocardial Dysfunction and Failure

Historically, the changes in cardiovascular function and particularly those that occur in the heart during the development of heart failure have been attributed to alterations in the contractile state of the myocardium (4, 68). Thus either chronic pressure or volume overload are characterized by a reduced inotropic state as shown in papillary muscles removed from failing hearts (77, 78), reduced cardiac contractile indices in experimental animals in vivo (73), and reduced ejection fraction or other measures of myocardial contractility in man (4). At the cellular level, there may be disruption of mitochondria, disarray of myofilaments, focal or diffuse ischemic lesions, concentric or eccentric hypertrophy, depending upon the cause of the myocardial dysfunction (50). This large body of evidence has suggested that the primary insult in heart failure of various etiologies is an alteration in contractile function due primarily to myocyte dysfunction (77, 78). In fact, one of the most active areas of recent investigation is the use of relaxation indexes to uncover diastolic dysfunction in the hope of early detection of potential functional abnormalities in the myocardium (29). All of the above underscore the emphasis on the contractile failure of the myocardium as the progenitor of heart failure.

An Unsuspected Property of Natriuretic PeptidesClinical Perspective

Background— Although natriuretic peptides are considered cardioprotective, clinical heart failure trials with recombinant brain natriuretic peptide (nesiritide) failed to prove it. Unsuspected proadrenergic effects might oppose the anticipated benefits of natriuretic peptides.nnMethods and Results— We investigated whether natriuretic peptides induce catecholamine release in isolated hearts, sympathetic nerve endings (cardiac synaptosomes), and PC12 cells bearing a sympathetic neuron phenotype. Perfusion of isolated guinea pig hearts with brain natriuretic peptide elicited a 3-fold increase in norepinephrine release, which doubled in ischemia/reperfusion conditions. Brain natriuretic peptide and atrial natriuretic peptide also released norepinephrine from cardiac synaptosomes and dopamine from nerve growth factor–differentiated PC12 cells in a concentration-dependent manner. These catecholamine-releasing effects were associated with an increase in intracellular calcium and abolished by blockade of calcium channels and calcium transients, demonstrating a calcium-dependent exocytotic process. Activation of the guanylyl cyclase-cyclic GMP-protein-kinase-G system with nitroprusside or membrane-permeant cyclic GMP analogs mimicked the proexocytotic effect of natriuretic peptides, an action associated with an increase in intracellular cyclic AMP (cAMP) and protein-kinase-A activity. Cyclic AMP enhancement resulted from an inhibition of phosphodiesterase type 3–induced cAMP hydrolysis. Collectively, these findings indicate that, by inhibiting phosphodiesterase type 3, natriuretic peptides sequentially enhance intracellular cAMP levels, protein kinase A activity, intracellular calcium, and catecholamine exocytosis.nnConclusions— Our results show that natriuretic peptides, at concentrations likely to be reached at cardiac sympathetic nerve endings in advanced congestive heart failure, promote norepinephrine release via a protein kinase G–induced inhibition of phosphodiesterase type 3–mediated cAMP hydrolysis. We propose that this proadrenergic action may counteract the beneficial cardiac and hemodynamic effects of natriuretic peptides and thus explain the ineffectiveness of nesiritide as a cardiac failure medication.nn# Clinical Perspective {#article-title-55}Background— Although natriuretic peptides are considered cardioprotective, clinical heart failure trials with recombinant brain natriuretic peptide (nesiritide) failed to prove it. Unsuspected proadrenergic effects might oppose the anticipated benefits of natriuretic peptides. Methods and Results— We investigated whether natriuretic peptides induce catecholamine release in isolated hearts, sympathetic nerve endings (cardiac synaptosomes), and PC12 cells bearing a sympathetic neuron phenotype. Perfusion of isolated guinea pig hearts with brain natriuretic peptide elicited a 3-fold increase in norepinephrine release, which doubled in ischemia/reperfusion conditions. Brain natriuretic peptide and atrial natriuretic peptide also released norepinephrine from cardiac synaptosomes and dopamine from nerve growth factor–differentiated PC12 cells in a concentration-dependent manner. These catecholamine-releasing effects were associated with an increase in intracellular calcium and abolished by blockade of calcium channels and calcium transients, demonstrating a calcium-dependent exocytotic process. Activation of the guanylyl cyclase-cyclic GMP-protein-kinase-G system with nitroprusside or membrane-permeant cyclic GMP analogs mimicked the proexocytotic effect of natriuretic peptides, an action associated with an increase in intracellular cyclic AMP (cAMP) and protein-kinase-A activity. Cyclic AMP enhancement resulted from an inhibition of phosphodiesterase type 3–induced cAMP hydrolysis. Collectively, these findings indicate that, by inhibiting phosphodiesterase type 3, natriuretic peptides sequentially enhance intracellular cAMP levels, protein kinase A activity, intracellular calcium, and catecholamine exocytosis. Conclusions— Our results show that natriuretic peptides, at concentrations likely to be reached at cardiac sympathetic nerve endings in advanced congestive heart failure, promote norepinephrine release via a protein kinase G–induced inhibition of phosphodiesterase type 3–mediated cAMP hydrolysis. We propose that this proadrenergic action may counteract the beneficial cardiac and hemodynamic effects of natriuretic peptides and thus explain the ineffectiveness of nesiritide as a cardiac failure medication.

An Unsuspected Property of Natriuretic PeptidesClinical Perspective: Promotion of Calcium-Dependent Catecholamine Release Via Protein Kinase G-Mediated Phosphodiesterase Type 3 Inhibition

Background— Although natriuretic peptides are considered cardioprotective, clinical heart failure trials with recombinant brain natriuretic peptide (nesiritide) failed to prove it. Unsuspected proadrenergic effects might oppose the anticipated benefits of natriuretic peptides.nnMethods and Results— We investigated whether natriuretic peptides induce catecholamine release in isolated hearts, sympathetic nerve endings (cardiac synaptosomes), and PC12 cells bearing a sympathetic neuron phenotype. Perfusion of isolated guinea pig hearts with brain natriuretic peptide elicited a 3-fold increase in norepinephrine release, which doubled in ischemia/reperfusion conditions. Brain natriuretic peptide and atrial natriuretic peptide also released norepinephrine from cardiac synaptosomes and dopamine from nerve growth factor–differentiated PC12 cells in a concentration-dependent manner. These catecholamine-releasing effects were associated with an increase in intracellular calcium and abolished by blockade of calcium channels and calcium transients, demonstrating a calcium-dependent exocytotic process. Activation of the guanylyl cyclase-cyclic GMP-protein-kinase-G system with nitroprusside or membrane-permeant cyclic GMP analogs mimicked the proexocytotic effect of natriuretic peptides, an action associated with an increase in intracellular cyclic AMP (cAMP) and protein-kinase-A activity. Cyclic AMP enhancement resulted from an inhibition of phosphodiesterase type 3–induced cAMP hydrolysis. Collectively, these findings indicate that, by inhibiting phosphodiesterase type 3, natriuretic peptides sequentially enhance intracellular cAMP levels, protein kinase A activity, intracellular calcium, and catecholamine exocytosis.nnConclusions— Our results show that natriuretic peptides, at concentrations likely to be reached at cardiac sympathetic nerve endings in advanced congestive heart failure, promote norepinephrine release via a protein kinase G–induced inhibition of phosphodiesterase type 3–mediated cAMP hydrolysis. We propose that this proadrenergic action may counteract the beneficial cardiac and hemodynamic effects of natriuretic peptides and thus explain the ineffectiveness of nesiritide as a cardiac failure medication.nn# Clinical Perspective {#article-title-55}Background— Although natriuretic peptides are considered cardioprotective, clinical heart failure trials with recombinant brain natriuretic peptide (nesiritide) failed to prove it. Unsuspected proadrenergic effects might oppose the anticipated benefits of natriuretic peptides. Methods and Results— We investigated whether natriuretic peptides induce catecholamine release in isolated hearts, sympathetic nerve endings (cardiac synaptosomes), and PC12 cells bearing a sympathetic neuron phenotype. Perfusion of isolated guinea pig hearts with brain natriuretic peptide elicited a 3-fold increase in norepinephrine release, which doubled in ischemia/reperfusion conditions. Brain natriuretic peptide and atrial natriuretic peptide also released norepinephrine from cardiac synaptosomes and dopamine from nerve growth factor–differentiated PC12 cells in a concentration-dependent manner. These catecholamine-releasing effects were associated with an increase in intracellular calcium and abolished by blockade of calcium channels and calcium transients, demonstrating a calcium-dependent exocytotic process. Activation of the guanylyl cyclase-cyclic GMP-protein-kinase-G system with nitroprusside or membrane-permeant cyclic GMP analogs mimicked the proexocytotic effect of natriuretic peptides, an action associated with an increase in intracellular cyclic AMP (cAMP) and protein-kinase-A activity. Cyclic AMP enhancement resulted from an inhibition of phosphodiesterase type 3–induced cAMP hydrolysis. Collectively, these findings indicate that, by inhibiting phosphodiesterase type 3, natriuretic peptides sequentially enhance intracellular cAMP levels, protein kinase A activity, intracellular calcium, and catecholamine exocytosis. Conclusions— Our results show that natriuretic peptides, at concentrations likely to be reached at cardiac sympathetic nerve endings in advanced congestive heart failure, promote norepinephrine release via a protein kinase G–induced inhibition of phosphodiesterase type 3–mediated cAMP hydrolysis. We propose that this proadrenergic action may counteract the beneficial cardiac and hemodynamic effects of natriuretic peptides and thus explain the ineffectiveness of nesiritide as a cardiac failure medication.

Altered Oxygen Availability and the Role of Nitric Oxide in the Development of Heart Failure

Publisher Summary This chapter focuses on two aspects that determine the delivery and extraction of oxygen in the coronary circulation of the failing heart. As the mechanical function of the heart may limit blood flow to the myocardium during systole and as diastolic wall stress increases because of the disease process, alterations in coronary blood flow may include alterations in the transmural distribution of blood flow. Recently, an increasing number of studies in humans and in animals have suggested that the production of nitric oxide by the vascular endothelium of coronary blood vessels of the failing heart may be reduced and that this contributes to altered coronary blood flow regulation. The chapter discusses the factors that control oxygen delivery to the myocardium during the development of heart failure and the impact of the loss of nitric oxide production by the vascular endothelium on both the regulation of coronary vascular resistance and oxygen extraction/consumption during the development of heart failure.