The objective of this study was to determine whether nitric oxide (NO) is responsible for the vascular smooth muscle relaxation elicited by endothelium-derived relaxing factor (EDRF). EDRF is an unstable humoral substance released from artery and vein that mediates the action of endothelium-dependent vasodilators. NO is an unstable endothelium-independent vasodilator that is released from vasodilator drugs such as nitroprusside and glyceryl trinitrate. We have repeatedly observed that the actions of NO on vascular smooth muscle closely resemble those of EDRF. In the present study the vascular effects of EDRF released from perfused bovine intrapulmonary artery and vein were compared with the effects of NO delivered by superfusion over endotheliumdenuded arterial and venous strips arranged in a cascade. EDRF was indistinguishable from NO in that both were labile (1i/2 = 3-5 sec), inactivated by pyrogallol or superoxide anion, stabilized by superoxide dismutase, and inhibited by oxyhemoglobin or potassium. Both EDRF and NO produced comparable increases in cyclic GMP accumulation in artery and vein, and this cyclic GMP accumulation was inhibited by pyrogallol, oxyhemoglobin, potassium, and methylene blue. EDRF was identified chemically as NO, or a labile nitroso species, by two procedures. First, like NO, EDRF released from freshly isolated aortic endothelial cells reacted with hemoglobin to yield nitrosylhemoglobin. Second, EDRF and NO each similarly promoted the diazotization of sulfanilic acid and yielded the same reaction product after coupling with N-(1-naphthyl)-ethylenediamine. Thus, EDRF released from artery and vein possesses identical biological and chemical properties as NO.Both artery and vein are capable of releasing endotheliumderived relaxing factor (EDRF) in response to chemically diverse vasodilators (1-9). Endothelium-dependent relaxation of artery and vein appears to be mediated by increases in tissue cyclic GMP levels (10-13), and such effects are inhibited by methylene blue, hemoglobin, and myoglobin (13-15). Nitroso compounds, organic nitrate and nitrite esters, and inorganic nitrite cause vascular smooth muscle relaxation and cyclic GMP accumulation by endotheliumindependent mechanisms, and these actions are attributed to the release of nitric oxide (NO) (16)(17)(18)(19). NO itself is a labile substance that causes transient relaxation and cyclic GMP accumulation in both artery and vein (16)(17)(18)(19)(20) MATERIALS AND METHODSReagents. Acetylcholine chloride, phenylephrine hydrochloride, A23187, pyrogallol, hemoglobin (human), and superoxide dismutase (bovine liver) were obtained from Sigma. Glyceryl trinitrate (10% wt/wt triturate in lactose) was a gift from Imperial Chemical Industries (Macclesfield, England), and propylbenzylylcholine mustard was provided by the National Institute for Medical Research (Mill Hill, London). NO (99o pure) was obtained from Matheson. A saturated solution of NO (1-2 mM) in oxygen-free water (prepared by vacuum evacuation and nitrogen flushing) was pre...

The objective of this study was to elucidate the close similarity in properties between endothelium-derived relaxing factor (EDRF) and nitric oxide radical (NO). Whenever possible, a comparison was also made between arterial and venous EDRF. In vascular relaxation experiments, acetylcholine and bradykinin were used as endothelium-dependent relaxants of isolated rings of bovine intrapulmonary artery and vein, respectively, and NO was used to relax endothelium-denuded rings. Oxyhemoglobin produced virtually identical concentration-dependent inhibitory effects on both endothelium-dependent and NO-elicited relaxation. Oxyhemoglobin and oxymyoglobin lowered cyclic guanosine monophosphate (cGMP) levels, increased tone in unrubbed artery and vein, and abolished the marked accumulation of vascular cGMP caused both by endothelium-dependent relaxants and by NO. The marked inhibitory effects of oxyhemoglobin on arterial and venous relaxant responses and cGMP accumulation as well as its contractile effects were abolished or reversed by carbon monoxide. These observations indicate that EDRF and NO possess identical properties in their interactions with oxyhemoproteins. Both EDRF from artery and vein and NO activated purified soluble guanylate cyclase by heme-dependent mechanisms, thereby revealing an additional similarity in heme interactions. Spectrophotometric analysis disclosed that the characteristic shift in the Soret peak for hemoglobin produced by NO was also produced by an endothelium-derived factor released from washed aortic endothelial cells by acetylcholine or A23187. Pyrogallol, via the action of superoxide anion, markedly inhibited the spectral shifts, relaxant effects, and cGMP accumulating actions produced by both EDRF and NO. Superoxide dismutase enhanced the relaxant and cGMP accumulating effects of both EDRF and NO. Thus, EDRF and NO are inactivated by superoxide in a closely similar manner. We conclude, therefore, that EDRF from artery and vein is either NO or a chemically related radical species.

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This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 27, 1999.The objective of this study was to elucidate the mechanisms by which nitric oxide (NO) inhibits rat aortic smooth muscle cell (

Rat aortic endothelial cells were found to contain both constitutive and lipopolysaccharide (LPS)-inducible arginase activity. Studies were performed to determine whether induction of nitric oxide synthase (NOS) by LPS and cytokines is accompanied by sufficient arginase induction to render arginine concentrations rate limiting for high-output NO production. Unactivated cells contained abundant arginase activity accompanied by continuous urea formation. LPS induced the formation of both inducible NOS (iNOS) and arginase, and this was accompanied by increased production of NO, citrulline, and urea. Immunoprecipitation experiments revealed the constitutive presence of arginase-I in both unactivated and LPS-activated cells and arginase-II induction by LPS. Arginase-I and iNOS were verified by reverse transcriptase-polymerase chain reaction. Induction of large amounts of iNOS by LPS plus several cytokines resulted in large quantities of NO, citrulline, and NG-hydroxy-L-arginine (NOHA), but urea production was markedly diminished. Decreased urea production was attributed to increased formation of NOHA, the precursor to NO and citrulline and a potent inhibitor of arginase-I activity with an inhibitory constant of 10-12 microM. Inhibition of iNOS activity by NG-methyl-L-arginine decreased NO and NOHA production and increased urea production. This study reveals for the first time that substantial arginase activity is present constitutively in rat aortic endothelial cells, a different isoform of arginase is induced by LPS, and intracellular arginase activity can be markedly inhibited during cytokine induction of iNOS because of NOHA formation. The inhibition of arginase activity that occurs by NOHA during marked iNOS induction may be a mechanism to ensure sufficient arginine availability for high-output production of NO.

An in vitro bioassay system was developed to study endothelium-mediated, shear stressinduced, or flow-dependent generation of endothelium-derived relaxing factor (EDRF). Monolayers of aortic endothelial cells were grown on a rigid and large surface area of microcarrier beads and were packed in a small column perfused with Krebs bicarbonate solution. The perfusate was allowed to superfuse three endothelium-denuded target pulmonary arterial strips arranged in a cascade. T here are two components that make up the total force applied to the intimal surface of blood vessels during blood flow. One is a perpendicular pressure component and the other is a tangential component called the wall shear stress, a frictional force produced when the blood flows across the endothelial surface. The perpendicular pressure component is not appreciable at the endothelial surface because the force is borne primarily by structural proteins in the blood vessel wall. However, wall shear stress falls entirely on the endothelial cell layer. Thus, fluid shear stress refers to the mechanical forces generated at the endothelial cell surface by blood flowing under pressure.The vascular endothelium is capable of modulating the tone of the underlying smooth muscle in conduit and resistance vessels of the arterial bed in response to local changes in shear stress, pressure, and other mechanical factors.

The objective of this study was to determine whether nitric oxide (NO) could function as a negative feedback modulator of endothelial cell function by inhibiting NO synthase in vascular endothelial cells. The rationale for this approach was a previous study from this laboratory, which revealed that NO inhibits neuronal NO synthase from rat cerebellum. In the present study, NO and NO-donor agents noncompetitively inhibited NO synthase derived from bovine aortic endothelial cells. Oxyhemoglobin blocked the inhibitory action of NO and by itself increased NO synthase activity. This finding suggests that NO acts as a negative feedback modulator of NO synthase. In intact aortic endothelial cells grown on microcarrier beads and perfused in a bioassay cascade system, pretreatment of cells with NO-donor agents caused a marked inhibition of endothelial NO biosynthesis in response to bradykinin and increased fluid shear or flow. When isolated bovine pulmonary arterial rings precontracted by phenylephrine were used, pretreatment of arterial rings with NO-donor agents diminished endothelium-dependent arterial relaxation involving the L-arginine-NO pathway without altering endothelium-independent relaxation to NO itself. On the basis of these studies, NO is suggested to play an important negative feedback regulatory role on endothelial NO synthase and, therefore, vascular endothelial cell function.

. Transgenic 6F tomatoes act on the small intestine to prevent systemic infl ammation and dyslipidemia caused by Western diet and intestinally derived lysophosphatidic acid. J. Lipid Res. 2013. 54: 3403-3418. Supplementary key words 6F peptide • apolipoprotein A-I mimetic peptides • atherosclerosis • lysophosphatidic acid • genetically engineered tomato plantsMimetics of apolipoprotein A-I (apoA-I) containing only 18 amino acids showed promise in animal models of disease ( 1, 2 ), and improved HDL function in humans when given orally at high doses despite achieving low plasma peptide levels ( 3 ). However, when high plasma levels were achieved with low doses of peptide given intravenously or by subcutaneous (SQ) injection, no improvement in HDL function was seen ( 4 ). Studies in mice surprisingly suggested that the major site of action for these peptides is in the Abstract We recently reported that levels of unsaturated lysophosphatidic acid (LPA) in the small intestine significantly correlated with the extent of aortic atherosclerosis in LDL receptor-null (LDLR ؊ / ؊ ) mice fed a Western diet (WD). Here we demonstrate that WD increases unsaturated (but not saturated) LPA levels in the small intestine of LDLR ؊ / ؊ mice and causes changes in small intestine gene expression. Confi rmation of microarray analysis by quantitative RT-PCR showed that adding transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to WD prevented many WD-mediated small intestine changes in gene expression. If instead of feeding WD, unsaturated LPA was added to chow and fed to the mice: i ) levels of LPA in the small intestine were similar to those induced by feeding WD; ii ) gene expression changes in the small intestine mimicked WD-mediated changes; and iii ) changes in plasma serum amyloid A, total cholesterol, triglycerides, HDLcholesterol levels, and the fast-performance liquid chromatography lipoprotein profi le mimicked WD-mediated changes. Adding Tg6F (but not control tomatoes) to LPA-supplemented chow prevented the LPA-induced changes. We conclude that: i ) WD-mediated systemic infl ammation and dyslipidemia may be in part due to WD-induced increases in small intestine LPA levels; and ii ) Tg6F reduces WD-mediated systemic infl ammation and dyslipidemia by preventing WDinduced increases in LPA levels in the small intestine. -Navab, M., G. Hough, G. M. Buga, F. Su, A. C. Wagner, D. Meriwether, A. Chattopadhyay, F. Gao, V. Grijalva, J. S. Danciger, B. J. Van Lenten, E. Org, A. J. Lusis, C. Pan, G. M. the Laubisch, Castera, and M. K. Grey Abbreviations: CXCL1, chemokine (CXC motif) ligand 1; EV, empty vector tomatoes (transgenic tomatoes constructed with the vector pBI121 containing the GUS gene; 6F, This work was supported in part bycontaining all L -amino acids; FPLC, fast-performance liquid chromatography; LDLR Ϫ / Ϫ , LDL receptor-null; LPA, lysophosphatidic acid; PA, phosphatidic acid; PPAR, peroxisome proliferatoractivated receptor; RT-qPCR, quantitative RT-PCR ; SAA, serum amyloid A; SQ, subcutaneous; Tg6F, t...