Richard J. Bing

Nitric Oxide Donors

Nitric oxide (NO) donors are pharmacologically active substances that release NO in vivo or in vitro. NO has a variety of functions such as the release of prostanoids, inhibition of platelet aggregation, effect on angiogenesis, and production of oxygen free radicals. This report discusses the chemical and pharmacological characteristics of NO donors, their effect on platelet function and cyclooxygenase, their cardiac action including myocardial infarction, and release of superoxide anions. This review stresses NO tolerance and the effect of NO donors on angiogenesis in myocardial infarction and in solid tumors.

Microbubble dynamics visualized in the intact capillary circulation

The potential for the use of contrast echocardiography to study myocardial perfusion has generated efforts to develop standardized echo contrast agents. The two methods used in this laboratory to generate microbubbles in solutions serving as contrast agents included the widely used hand-agitation method and the newer ultrasonic microcavitation (sonication) method. The latter has been demonstrated to generate smaller and more uniform microbubbles in an in vitro system. The present study was designed to observe, by direct microscopic examination of a cat mesentery preparation, the behavior and fate of the microbubbles in an in vivo system. The in vivo mesentery observations confirm the critical role of microbubble size in its unhindered passage through the capillary vasculature. The smaller and more uniform sonicated microbubbles passed rapidly through the microcirculation along with the red blood cells, whereas the larger microbubbles were observed to coalesce and interrupt the flow of blood and subsequently collapse or shrink.

Involvement of inducible nitric oxide synthase in the inflammatory process of myocardial infarction

Inducible nitric oxide synthase (iNOS), which catalyzes the reaction of L-arginine to L-citrulline and nitric oxide (NO), plays an important role in immune-mediated cardiac disorders. The present report summarizes and discusses findings on the induction of NOS in myocardial infarction of rabbits. iNOS was significantly increased in infarcted myocardium 48 h after coronary artery ligation. The effect persisted for 14 days and declined thereafter. Immunohistochemical localization revealed macrophages as a major source of iNOS expression; iNOS expression was also present in infarcted human myocardium. Increased iNOS activity appeared to be related to the induction of apoptosis in infiltrating macrophages and cardiomyocytes. Moreover, preferential inhibition of iNOS by S-methylisothiourea sulfate (SMT) resulted in significant improvement of left ventricular performance and increased regional myocardial blood flow. These findings suggest that selective inhibition of iNOS activity may provide a therapeutic strategy in cardiac disorders such as myocardial infarction.

Why do cyclo-oxygenase-2 inhibitors cause cardiovascular events?

This report confirms evidence that selective nonsteroidal anti-inflammatory drugs (NSAIDs), such as celecoxib, can lead to thrombotic cardiovascular events. Aspirin, a nonselective COX-1 (cyclo-oxygenase) and COX-2 inhibitor may result in gastric toxicity. For this reason, selective COX-2 inhibitors have been developed to reduce erosion of the gastric mucosa. Both selective and nonselective NSAIDs reduce prostacyclin formation in the infarcted heart; they accomplish this by tipping the balance of prostacyclin/thromboxane in favor of thromboxane, a prothrombotic eicosanoid. The relative increase in thromboxane, coupled with a diminution in prostacyclin in infarcted heart muscle, can lead to the development of thrombotic cardiovascular events. This may be prevented by the addition of a nitric oxide donor to NSAIDs.

Immunohistochemistry in the identification of nitric oxide synthase isoenzymes in myocardial infarction

OBJECTIVE Inducible nitric oxide synthase (iNOS) activity, as measured by conversion of L-14C-arginine to L-14C-citrulline is significantly increased in infarcted rabbit myocardium as compared to healthy myocardium 48 h after coronary occlusion. The aim of this study was to localise the nitric oxide synthase (NOS) isoforms in normal myocardium and compare these findings with NOS activity in cells of the infarcted region. METHODS Activities of NOS isoforms were enzymatically assayed in normal and infarcted myocardium. Localisation of NOS was performed on identical sections using specific monoclonal IgG antibodies against endothelial constitutive (cNOS) and macrophage inducible (iNOS) nitric oxide synthase. In addition, macrophages were identified using fluorescein conjugated ChromPure rabbit IgG, Fc fragment. RESULTS iNOS activity increased significantly in infarcted as compared to normal myocardium [0.42(SEM 0.03) v 0.85(0.08) fmol.microgram-1.min-1, P = 0.02, respectively]. However, cNOS did not increase significantly in infarcted regions [0.18(0.04) v 0.24(0.06) fmol.microgram-1.min-1; P = 0.16, respectively]. cNOS was immunohistochemically localised in endothelial and endocardial cells in normal and infarcted tissues. The presence of iNOS activity in macrophages in infarcted myocardium was identified immunohistochemically. Cardiomyocytes and neutrophils did not label with the antibodies to cNOS and iNOS. CONCLUSIONS (1) Infiltrating macrophages are the main site of increased iNOS activity in infarcted rabbit myocardium. (2) cNOS activity is not significantly increased in infarcted tissues as compared to normal myocardium. (3) Neutrophils and cardiomyocytes do not express NOS immunoreactivity in infarcted and normal rabbit myocardium.

INDUCTION OF APOPTOSIS IN MYOCARDIAL INFARCTION AND ITS POSSIBLE RELATIONSHIP TO NITRIC OXIDE SYNTHASE IN MACROPHAGES

Activated macrophages produce nitric oxide through the inducible form of nitric oxide synthase (iNOS). Previously, a significant increase of iNOS activity in macrophages in infarcted rabbit heart tissue was observed. The present study is concerned with the induction of apoptosis in macrophages and cardiomyocytes in infarcted rabbit heart tissue. The left anterior descending artery of rabbits was ligated. The heart was excised five hours, one, two, three, ten and twenty days later, and DNA was extracted from infarcted and non-infarcted region and subjected to electrophoresis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) was carried out, and iNOS activity was measured by conversion of L-[14C]-arginine to L-[14C]-citrulline. Positive staining by TUNEL was seen in some cardiomyocytes five hours after coronary ligation and on postoperative day (POD) 1; internucleosomal DNA fragmentation was not noted. On POD 2 and 3, many infiltrating cells, immunohistochemically identified as macrophages, were positively stained by TUNEL; DNA fragmentation was also present. Apoptosis was not found on POD 10 and 20. The peak activity of iNOS was noted on POD 3, which corresponded with the induction of apoptosis. It is tempting to speculate that a causal relationship exists between increased iNOS formation and induction of apoptosis in macrophages in infarcted rabbit heart tissue.

Production of oxidative products of nitric oxide in infarcted human heart

OBJECTIVES We sought to assess whether oxidation products of nitric oxide (NO), nitrite (NO2-) and nitrate (NO3-), referred to as NOx, are released by the heart of patients after acute myocardial infarction (AMI) and whether NOx can be determined in peripheral blood of these patients. BACKGROUND Previously we reported that in experimental myocardial infarction (rabbits) NOx is released mainly by inflammatory cells (macrophages) in the myocardium 3 days after onset of ischemia. NOx is formed in heart muscle from NO; NO originates through the activity of the inducible form of nitric oxide synthase (iNOS). METHODS Eight patients with acute anterior MI and an equal number of controls were studied. Coronary venous blood was obtained by coronary sinus catheterization; NOx concentrations in coronary sinus, in arterial and peripheral venous plasma were measured. Left ventricular end-diastolic pressure was determined. Measurements were carried out 24, 48 and 72 h after onset of symptoms. The type and location of coronary arterial lesions were determined by coronary angiography. Plasma NO3- was reduced to NO2- by nitrate reductase before determination of NO2- concentration by chemiluminescence. RESULTS The results provided evidence that in patients with acute anterior MI, the myocardial production of nitrite and nitrate (NOx) was increased, as well as the coronary arterial-venous difference. Increased NOx production by the infarcted heart accounted for the increase of NOx concentration in arterial and the peripheral venous plasma. The peak elevation of NOx occurred on days 2 and 3 after onset of the symptoms, suggesting that NOx production was at least in part the result of production of NO by inflammatory cells (macrophages) in the heart. CONCLUSIONS The appearance of oxidative products of NO (NO2- and NO3-) in peripheral blood of patients with acute MI is the result of their increased release from infarcted heart during the inflammatory phase of myocardial ischemia. Further studies are needed to define the clinical value of these observations.

Myocardial ischemia and infarction: growth of ideas

This report reviews the authors involvement in the growth of ideas and basic concepts in myocardial ischemia resulting in the histological changes of myocardial infarction. Concepts arising from the study of myocardial substrate utilization, activation of the inducible form of nitric oxide synthase and production of prostacyclin and thromboxane in the infarcted heart are presented. New approaches are discussed dealing with the effects of nonsteroidal anti-inflammatory drugs on myocardial production of nitric oxide and prostanoids, and with the relevance of the inducible form of cyclooxygenase. The review also records a number of significant similarities between angiogenesis in the ischemic heart and some cancers. Angiogenesis in both instances originates from inflammatory reactions, illustrating how different tissues and organs such as ischemic heart muscle and cancer react to similar pathological stimuli in an identical manner. This multifocal approach opens new concepts on myocardial ischemia and cancer.

Cardiac performance in pediatric near-drowning.

Between July 1985 and December 1986, 29 near-drowned children ranging in age between 6 months and 13 yr were admitted to the Pediatric ICU of Huntington Memorial Hospital. Eight patients suffered cardiopulmonary arrest and had an admission Glasgow Coma Score of 3 or 4. Hemodynamic monitoring was performed on five of these patients. Three received cerebral resuscitation with controlled hyperventilation, hypothermia, pentobarbital, and mannitol because of intracranial hypertension. After CPR, a low cardiac index (CI) and high systemic vascular resistance index (SVRI) were found. When cerebral resuscitative therapy was initiated later, it caused a reduction of SVRI, CI, mean arterial pressure, and cerebral perfusion pressure. Fluid volume therapy and inotropic cardiac support was necessary to maintain adequate cerebral perfusion pressure. These observations indicate that cerebral resuscitative therapy can affect cardiovascular function. The hemodynamic depressive effects might even worsen the outcome. For this reason, it is advisable to obtain CI and pulmonary capillary wedge pressure to optimize cerebral perfusion and potentially neurologic outcome.

Reduction of biological effluents in purge and trap micro reaction vessels and detection of endothelium-derived nitric oxide (edno) by chemiluminescence

Various analytical approaches have been used to measure endothelium-derived nitric oxide (NO). We have detected NO in perfusates with a sample size as low as 2 ml after acidification with 4 N HC1 to pH less than 2 at 25 degrees C by using a Nitric Oxide Analyser (Sievers, Colorado). This procedure had the advantage that the detectable level of NO was enhanced by the self-decomposition of HNO2 when the PH less than pKa of NHO2 (pKa = 3.15) and also the reaction temperature of 25 degrees C substantially increased the half-line of NO. Palmer, et al., measured NO released by cultured porcine endothelial cells by chemiluminescence after passing cell effluents continuously at a rate of 5 ml/min into 75 ml of 1% sodium iodide in glacial acetic acid. The larger volumes involved in this method for continuous refluxing, made it less desirable for the detection of endothelium-derived nitric oxide. Feelisch et al. utilized the activation of soluble guanylate cyclase, as well as, the quantitative oxidation of oxyhemoglobin to methemoglobin in aqueous solutions by NO as a means of measuring nitric oxide. We describe here a modification of our earlier micromethod which now enables us to detect NO after complete reduction with glacial acetic acid and sodium iodide. A comparison of the two procedures indicate that while freshly prepared NO standard solutions gave identical chemiluminescence response with and without reduction, effluents from bovine intrapulmonary artery under basal conditions gave substantially higher values upon reduction.