R. Dudek

Selective Modulation of Inducible Nitric Oxide Synthase Isozyme in Myocardial Infarction

BACKGROUND Inducible nitric oxide synthase (iNOS) is activated in cardiac disorders. We investigated the contribution of increased iNOS activity to the development of left ventricular dysfunction after myocardial infarction by selective inhibition of the isozyme. METHODS AND RESULTS Male New Zealand rabbits were subjected to myocardial infarction. Animals were treated with either saline, S-methylisothiourea sulfate (SMT) (a selective iNOS inhibitor), or N(omega)-nitro-L-arginine (L-NNA) (a nonselective NOS inhibitor). Inducible and constitutive NOS (cNOS) activity, plasma NO(x), cGMP, hemodynamics, and myocardial blood flow were measured before and 5, 24, and 72 hours after coronary occlusion. Infarction 72 hours after occlusion resulted in increased myocardial iNOS activity, increased cardiac NO(x) production, and elevated cGMP levels. cNOS remained unchanged. Infarction increased left ventricular end-diastolic pressure (LVEDP) and decreased maximum +dP/dt and -dP/dt. L-NNA inhibited iNOS and cNOS activities and plasma NO(x) levels. L-NNA further increased LVEDP and reduced myocardial blood flow. Administration of SMT 72 hours after infarction significantly inhibited iNOS and cardiac NO(x) production but had no effects on cNOS. SMT improved left ventricular maximum +dP/dt and -dP/dt and decreased LVEDP. Myocardial blood flow in the remote myocardium increased. CONCLUSIONS These findings suggest that induction of iNOS activity 72 hours after infarction exerts negative inotropic effects and contributes to the development of myocardial dysfunction; selective modulation of increased iNOS activity by SMT improves cardiac performance, enhances myocardial blood flow, and may be beneficial in the treatment of acute myocardial infarction.

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.

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.

Intracellular Translocation of Endothelial Nitric Oxide Synthase by Lysophosphatidylcholine

The amphiphile lysophosphatidylcholine (LPC) modulates the activity of membrane-associated enzymes such as phospholipase A2, adenylate and guanylate cyclases and ATPase. LPC also relaxes vascular smooth muscle through production of nitric oxide. On the basis of reports that bradykinin translocates nitric oxide synthase (NOS) from the membrane to the cytosol, we investigated whether a similar translocation occurs with LPC. It was found that LPC translocated NOS from the membrane to the cytosolic fraction. Total NOS activity remained at the control level.

Expression of Nitric Oxide Svnthase Isoforms after Miocardial Infarction in Humans

The present study is concerned with the expression of inducible (iNOS) and constutive nitric oxide synthase (cNOS) in human myocardial infarction. This investigation is based on previous studies which showed a time related activation of iNOS derived from infiltrating macrophages in infarcted rabbit myocardium. Specific monoclonal antibodies to both human isoforms were used in order to localize iNOS and cNOS in tissue sections from 6 patients who died on days 2, 7 and 25 after myocardial infarction. In addition, infiltrating macrophages were identified using a monoclonal antibody against human macrophages (CD68). Results showed that, comparable to the animal model, iNOS was primarily located in macrophages infiltrating the infarcted region; this was observed at days 7 and 25 after clinical diagnosis. iNOS was also found in non-necrotic cardiomyocytes within or close to the infarcted region and closely associated with the intercalated discs (IC). In all infarcts, cNOS was expressed in endothelial cells of p...

Cytokine production from freshly harvested human mononuclear cells attached to plastic beads

The release of tumor necrosis factor (TNF), interleukin-1 beta (IL-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) from freshly harvested monocytes and lymphocytes attached to plastic beads was investigated. Previous studies had shown that freshly harvested endothelial cells attached to microcarrier beads release an endothelium-derived relaxing factor. Attachment of freshly harvested lymphocytes and monocytes to plastic beads created a dense network, consisting of 25% monocytes and 75% lymphocytes as shown by flow cytometry. Viability of cells was 90%. Monocytes were characterized by phagocytosis and non-specific esterase stain. Freshly harvested cells stimulated with lipoprotein lipase (LPS) released TNF and IL-1. Non-stimulated cells also produced GM-CSF five hours after collection of blood.

Lysophosphatidylcholine-induced vascular relaxation and production of cGMP are mediated by endothelium-derived relaxing factor.

Summary Abstract. Endothelial cells produce powerful vasorelaxant substances, among them an endothelium-derived relaxing factor that is believed to be nitric oxide. It relaxes vascular smooth muscle via activation of guanylate cyclase and a subsequent rise in cyclic GMP level. Lysophosphatidylcholine is a potent endothelium-dependent vascular smooth muscle relaxant. Its action, similar to that of endothelium-derived relaxing factor, mediates an increase of cGMP in smooth muscle cells. The experiments reported here demonstrate that inhibitors of nitric oxide formation, such as N-ω-nitro-L-arginine and its methyl ester, inhibit relaxation and cyclic GMP formation by lysophosphatidylcholine in bovine pulmonary artery strips with intact endothelium in a dose-dependent manner. N-ω-Nitro-D-arginine methyl ester does not inhibit relaxation; L-arginine, but not D-arginine, reverses the effect of N-ω-nitro-L-arginine and its methyl ester. It is concluded that lysophosphatidylcholine-induced endothelium-dependent vasorelaxation is endothelium-derived relaxing factor-mediated. [P.S.E.B.M. 1993, Vol 203]

The effect of immune mediators (cytokines) on the release of endothelium-derived relaxing factor (EDRF) and of prostacyclin by freshly harvested endothelial cells

The effect of recombinant tumor necrosis factor and other cytokines stimulated by LPS (lipopolysaccharide), on the release of endothelial-derived relaxing factor and of prostacyclin was investigated using freshly harvested endothelial cells attached to plastic microcarrier beads. The results show that the cytokines failed to interfere with the release of EDRF and prostacyclin under the conditions of these experiments.

The production of nitric oxide in endothelial cells by amphiphiles

Lysophosphatidylcholine, an endogenous detergent is an endothelium-dependent smooth muscle relaxant, which acts through the release of nitric oxide. It is known to activate a number of membrane-bound enzymes. Because of the relationship between detergent action, relaxation of endothelium-intact rabbit aortic strips and the release of nitric oxide, we considered the possibility that other amphiphiles also produce nitric oxide from endothelial cells. We therefore investigated the effect of digitonin on relaxation of precontracted rabbit aortic strips and the release of nitric oxide from freshly harvested bovine endothelial cells as determined by chemiluminescence. We found that both digitonin and LPC release nitric oxide and that this process is inhibited by the NO synthase inhibitor N omega-Nitro-L-Arginine Methyl Ester (L-NNAME).