Carl I. Thompson

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.

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.

Endogenous Endothelial Nitric Oxide Synthase–Derived Nitric Oxide Is a Physiological Regulator of Myocardial Oxygen Consumption

Our objective was to determine the precise role of endothelial nitric oxide synthase (eNOS) as a modulator of cardiac O2 consumption and to further examine the role of nitric oxide (NO) in the control of mitochondrial respiration. Left ventricle O2 consumption in mice with defects in the expression of eNOS [eNOS (-/-)] and inducible NOS [iNOS (-/-)] was measured with a Clark-type O2 electrode. The rate of decreases in O2 concentration was expressed as a percentage of the baseline. Baseline O2 consumption was not significantly different between groups of mice. Bradykinin (10(-4) mol/L) induced significant decreases in O2 consumption in tissues taken from iNOS (-/-) (-28+/-4%), wild-type eNOS (+/+) (-22+/-4%), and heterozygous eNOS(+/-) (-22+/-5%) but not homozygous eNOS (-/-) (-3+/-4%) mice. Responses to bradykinin in iNOS (-/-) and both wild-type and heterozygous eNOS mice were attenuated after NOS blockade with N-nitro-L-arginine methyl ester (L-NAME) (-2+/-5%, -3+/-2%, and -6+/-5%, respectively, P<0.05). In contrast, S-nitroso-N-acetyl-penicillamine (SNAP, 10(-4) mol/L), which releases NO spontaneously, induced decreases in myocardial O2 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 O2 consumption in tissues taken from normal but not iNOS (-/-)-deficient mice. Our results indicate that the pivotal role of eNOS in the control of myocardial O2 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.

Role of Nitric Oxide in the Control of Renal Oxygen Consumption and the Regulation of Chemical Work in the Kidney

Inhibition of NO synthesis has recently been shown to increase oxygen extraction in vivo, and NO has been proposed to play a significant role in the regulation of oxygen consumption by both skeletal and cardiac muscle in vivo and in vitro. It was our aim to determine whether NO also has such a role in the kidney, a tissue with a relatively low basal oxygen extraction. In chronically instrumented conscious dogs, administration of an inhibitor of NO synthase, nitro-L-arginine (NLA, 30 mg/kg i.v.), caused a maintained increase in mean arterial pressure and renal vascular resistance and a decrease in heart rate (all P<0.05). At 60 minutes, urine flow rate and glomerular flow rate decreased by 44+/-12% and 45+/-7%, respectively; moreover, the amount of sodium reabsorbed fell from 16+/-1.7 to 8.5+/-1.1 mmol/min (all P<0.05). At this time, oxygen uptake and extraction increased markedly by 115+/-37% and 102+/-34%, respectively (P<0.05). Oxygen consumption also significantly increased from 4.5+/-0.6 to 7.1+/-0.9 mL O2/min. Most important, the ratio of oxygen consumption to sodium reabsorbed increased dramatically from 0.33+/-0.07 to 0.75+/-0.11 mL O2/mmol Na+ (P<0.05), suggesting a reduction in renal efficiency for transporting sodium. In vitro, both a NO-donating agent and the NO synthase-stimulating agonist bradykinin significantly decreased both cortical and medullary renal oxygen consumption. In conclusion, NO plays a role in maintaining a balance between oxygen consumption and sodium reabsorption, the major ATP-consuming process in the kidney, in conscious dogs, and NO can inhibit mitochondrial oxygen consumption in canine renal slices in vitro.

Phasic release of adenosine during steady state metabolic stimulation in the isolated guinea pig heart.

If adenosine is the major factor responsible for myocardial metabolic vasodilation, its release should be sustained as long as oxygen consumption and coronary flow are augmented. To see if adenosine meets this criterion, we examined the time course of its release during norepinephrine infusion in isolated, non-working guinea pig hearts (n = 8). During an 11-minute infusion period (steady state perfusate concentration = 6 X 10(-8) M), the coronary effluent was collected over 30-second intervals for measurements of coronary flow (ml/min per g), and adenosine and inosine release (pmol/min per g). Myocardial oxygen consumption (MVO2 = microliter O2/min per g) was measured at 1, 4, 6.5, and 11 minutes. Control values of coronary flow, myocardial oxygen consumption, and adenosine and inosine release were 7.5 +/- 0.4, 85 +/- 5, 22 +/- 5, and 431 +/- 39, respectively. During norepinephrine infusion, coronary flow, myocardial oxygen consumption, and adenosine release attained maximal levels within one minute (inosine within 2 minutes). These values were 10.6 +/- 0.4, 125 +/- 9, 849 +/- 110, and 2595 +/- 581, respectively. Thereafter, coronary flow and myocardial oxygen consumption values were sustained. In contrast, adenosine and inosine release significantly declined to nadirs by 9.5 minutes. Thereafter, steady state levels were maintained at 117 +/- 24 and 960 +/- 294, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Bilateral atrial appendectomy abolishes increased plasma atrial natriuretic peptide release and blunts sodium and water excretion during volume loading in conscious dogs.

The atrial appendages contain most of the atrial natriuretic factor (ANF) in the mammalian heart, and atrial appendage mechanical function predicts ANF secretion during volume loading. To demonstrate the crucial role of the atrial appendages in ANF release, we first measured hemodynamics and changes in plasma ANF after injection of 1,000 ml i.v. normal saline in conscious dogs and again after bilateral atrial appendectomy; we next measured changes in renal function using infusions of atriopeptin 24 to achieve plasma levels corresponding to levels achieved during volume loading; and we lastly measured renal function during acute volume expansion and also after atrial appendectomy. Plasma ANF increased from 65 +/- 11 to 246 +/- 54 pg/ml after volume loading but did not increase after atrial appendectomy. Atrial appendectomy did not alter the tachycardia or hemodynamic effects of volume loading. Infusion of 10 ng/kg/min atriopeptin 24 increased plasma ANF from 50 +/- 9 to 234 +/- 54 pg/ml, increased urine output 34 +/- 10%, and increased sodium excretion 62 +/- 10% in dogs with intact atrial appendages. Renal function was compared in dogs before atrial appendectomy: 20, 40, and 60 minutes after volume loading, urine flow rate increased by 5.9 +/- 0.5, 6.9 +/- 0.4, and 4.4 +/- 0.8 ml/min, while sodium excretion increased by 717 +/- 60, 839 +/- 84, and 582 +/- 57 mueq/min. After atrial appendectomy urine flow rate increased 2.1 +/- 0.7, 2.7 +/- 0.7, and 2.0 +/- 0.6 ml/min, and sodium excretion increased only by 327 +/- 110, 324 +/- 77, and 340 +/- 92 mueq/min (p less than 0.01) during volume loading.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of Prenatal Glucocorticoids on Cerebral Vasculature of the Developing Brain

Background and Purpose— Prenatal glucocorticoids prevent germinal matrix hemorrhage in premature infants. The underlying mechanism, however, is elusive. Germinal matrix is enriched with angiogenic vessels exhibiting paucity of pericytes and glial fibrillary acidic protein-positive astrocyte end feet. Therefore, we asked whether glucocorticoid treatment would suppress angiogenesis and enhance periendothelial coverage by pericytes and glial fibrillary acidic protein-positive end feet in the germinal matrix microvasculature. Methods— We treated pregnant rabbits with intramuscular betamethasone and delivered pups prematurely by cesarean section at E29 (term=32 days). Endothelial turnover, vascular density, pericyte coverage, glial fibrillary acidic protein-positive end feet, cell death, and growth factors orchestrating angiogenesis, including vascular endothelial growth factor, angiopoietins, transforming growth factor-&bgr;, and platelet-derived growth factor-B, were compared between betamethasone-treated and untreated pups. Similar comparisons were done between autopsy materials from premature infants exposed and unexposed to prenatal glucocorticoids. Results— Antenatal glucocorticoid treatment reduced endothelial proliferation, vascular density, and vascular endothelial growth factor expression in the germinal matrix of both rabbits and humans. The pericyte coverage was greater in glucocorticoid-treated rabbit pups and human infants than in controls, but not the glial fibrillary acidic protein-positive end feet coverage. Transforming growth factor-&bgr;, but not angiopoietins and platelet-derived growth factor-B, were elevated in glucocorticoid-treated rabbit pups compared with controls. Betamethasone treatment induced apoptosis, neuronal degeneration, and gliosis in rabbit pups. However, there was no evidence of increased cell death in glucocorticoid-exposed human infants. Conclusions— Prenatal glucocorticoid suppresses vascular endothelial growth factor and elevates transforming growth factor-&bgr; levels, which results in angiogenic inhibition, trimming of neovasculature, and enhanced pericyte coverage. These changes contribute to stabilizing the germinal matrix vasculature, thereby reducing its propensity to hemorrhage. Prenatal glucocorticoid exposure does not induce neural cell death in humans, unlike rabbits.

Mechanisms of coronary vasodilatation produced by ATP in guinea-pig isolated perfused heart

1 Isolated hearts of guinea‐pigs were perfused in vitro with a physiological salt solution via a retrograde aortic cannulation (Langendorff preparation) at constant perfusion pressure. Bolus intra‐arterial injections of various vasodilator drugs were made and the coronary flow responses were measured with an electromagnetic flow probe placed in the arterial inflow circuit. Inhibitory drugs were infused intra‐arterially. 2 Nitro‐l‐arginine (NLA; 500 μm), an NO synthesis inhibitor, decreased coronary baseline flow by 16 ± 0.8%, converted acetylcholine‐induced coronary vasodilatation to vasoconstriction and had no effect on coronary flow responses to adenosine or papaverine. Sodium nitroprusside‐induced responses were enhanced during NLA infusion by 46 ± 11%. 3 Adenosine 5′‐triphosphate (ATP) increased coronary flow but coronary flow responses to ATP were not altered by infusion of NLA. 4 ATP‐induced coronary dilatation was not significantly attenuated by infusion of the adenosine receptor antagonist XAC, (xanthine amine congener; 2 μm), whereas XAC decreased coronary flow responses to adenosine by 75% ± 5%. 5 ATP‐induced coronary flow responses were reduced by only 31 ± 4% during indomethacin infusion (2.8 μm) whereas indomethacin completely eliminated the initial vasoconstriction phase and greatly attenuated the peak flow and duration of the later vasodilatation phase seen in response to arachidonic acid (0.75 nmol). Indomethacin had no effect on vasodilatations produced by adenosine or prostaglandin I2. 6 These results indicate that ATP‐induced coronary dilatation in the isolated, perfused heart of the guinea‐pig is not dependent upon NO production or upon degradation of ATP to adenosine. The coronary dilator action of ATP may be partially dependent (∼30%) upon the production of vasodilator prostaglandins.

Elevated level of pro-inflammatory eicosanoids and EPC dysfunction in diabetic patients with cardiac ischemia

BACKGROUND Circulating endothelial progenitor cells (EPCs) are recruited from the blood system to sites of ischemia and endothelial damage, where they contribute to the repair and development of blood vessels. Since numerous eicosanoids including leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) have been shown to exert potent pro-inflammatory activities, we examined their levels in chronic diabetic patients with severe cardiac ischemia in conjunction with the level and function of EPCs. RESULTS Lipidomic analysis revealed a diabetes-specific increase (p<0.05) in inflammatory and angiogenic eicosanoids including the 5-lipoxygenase-derived LTB (4.11±1.17 vs. 0.96±0.27 ng/ml), the lipoxygenase/CYP-derived 12-HETE (117.08±35.05 vs. 24.34±10.03 ng/ml), 12-HETrE (17.56±4.43 vs. 4.15±2.07 ng/ml), and the CYP-derived 20-HETE (0.32±0.04 vs. 0.06±0.05 ng/ml) the level of which correlated with BMI (p=0.0027). In contrast, levels of the CYP-derived EETs were not significantly (p=0.36) different between these two groups. EPC levels and their colony-forming units were lower (p<0.05) with a reduced viability in diabetic patients compared with non-diabetics. EPC function (colony-forming units (CFUs) and MTT assay) also negatively correlated with the circulating levels of HgA1C. CONCLUSION This study demonstrates a close association between elevated levels of highly pro-inflammatory eicosonoids, diabetes and EPC dysfunction in patients with cardiac ischemia, indicating that chronic inflammation impact negatively on EPC function and angiogenic capacity in diabetes.

Inhibition of soluble epoxide hydrolase increases coronary perfusion in mice

Roles of soluble epoxide hydrolase (sEH), the enzyme responsible for hydrolysis of epoxyeicosatrienoic acids (EETs) to their diols (DHETs), in the coronary circulation and cardiac function remain unknown. We tested the hypothesis that compromising EET hydrolysis/degradation, via sEH deficiency, lowers the coronary resistance to promote cardiac perfusion and function. Hearts were isolated from wild type (WT), sEH knockout (KO) mice and WT mice chronically treated with t‐TUCB (sEH inhibitor), and perfused with constant flow at different pre‐loads. Compared to WT controls, sEH‐deficient hearts required significantly greater basal coronary flow to maintain the perfusion pressure at 100 mmHg and exhibited a greater reduction in vascular resistance during tension‐induced heart work, implying a better coronary perfusion during cardiac performance. Cardiac contractility, characterized by developed tension in response to changes in preload, was potentially increased in sEH‐KO hearts, manifested by an enlarged magnitude at each step‐wise increase in end‐diastolic to peak‐systolic tension. 14,15‐EEZE (EET antagonist) prevented the adaptation of coronary circulation in sEH null hearts whereas responses in WT hearts were sensitive to the inhibition of NO. Cardiac expression of EET synthases (CYP2J2/2C29) was comparable in both genotypic mice whereas, levels of 14,15‐, 11,12‐ and 8,9‐EETs were significantly higher in sEH‐KO hearts, accompanied with lower levels of DHETs. In conclusion, the elevation of cardiac EETs, as a function of sEH deficiency, plays key roles in the adaptation of coronary flow and cardiac function.