Giovanni E. Mann

Identification of inhibitors of nitric oxide synthase that do not interact with the endothelial cell L-arginine transporter.

The effects of inhibitors of nitric oxide (NO) synthase and other cationic amino acids on unidirectional l‐arginine transport were studied in porcine aortic endothelial cells cultured in microwell plates or perfused in microcarrier columns. l‐Homoarginine, l‐lysine and l‐ornithine inhibited transport of l‐arginine. The NO synthase inhibitors NG‐monomethyl‐l‐arginine and NG‐iminoethyl‐l‐ornithine also reduced l‐arginine uptake, whereas NG‐nitro‐l‐arginine and its methyl‐ester had no inhibitory effect. The ability to modulate selectively endothelial cell l‐arginine transport or NO synthase activity will allow further characterization of the arginine transporter and its role in regulating NO biosynthesis.

Induction of the antioxidant stress proteins heme oxygenase-1 and MSP23 by stress agents and oxidised LDL in cultured vascular smooth muscle cells

Enhanced expression of the antioxidant stress proteins heme oxygenase‐1 (HO‐1) and macrophage stress protein (MSP23) by oxidative stress agents and oxidatively modified low density lipoproteins (LDL) was investigated in cultured porcine aortic smooth muscle cells. Treatment of smooth muscle cells with glucose oxidase, CdCl2 or diethylmaleate resulted in a time‐dependent (6–48 h) induction of HO‐1 and MSP23 expression. Exposure of cells to 100 μg protein/ml highly oxidised LDL increased the expression of HO‐1 and MSP23 within 24 h, and the induction was dependent on the degree of LDL oxidation. The induction of HO‐1 and MSP23 may thus play an important cytoprotective role against oxidative stress in atherogenesis.

Selective inhibition by dexamethasone of induction of NO synthase, but not of induction of L-arginine transport, in activated murine macrophage J774 cells.

1 Effects of dexamethasone on induction of nitric oxide (NO) synathase and l‐arginine transport by lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line J774. Metabolism of l‐arginine to l‐citrulline and subsequent changes in intracellular amino acids pools were correlated with changes in nitrite production. 2 Despite a high intracellular concentration of arginine in activated J774 cells, LPS (1 μg ml−1, 8 h) induced a 2.4 fold increase in arginine transport. Treatment of cells with cycloheximide (1 μg ml−1) inhibited the time‐dependent (1–8 h) induction of NO synthase and arginine transport mediated by LPS. 3 Induction of NO synthase by LPS (1 μg ml−1, 24 h) alone was accompanied by a marked increase in arginine utilisation leading to decreased intracellular arginine levels and elevated intracellular and extracellular l‐citrulline levels. These changes were further enhanced in the presence of interferon‐γ (IFN‐γ, 100 units ml−1, 24 h). 4 Dexamethasone (1 μm) abolished the increases in both nitrite and citrulline production induced by LPS alone but only partially reversed the combined effects of LPS and IFN‐γ. In contrast, treatment of cells with dexamethasone (10 μm) had no effect on the LPS‐mediated induction of arginine transport or the decrease in intracellular arginine concentration. 5 We conclude that induction of arginine transporter activity in LPS‐stimulated J774 cells involves de novo synthesis of carrier proteins, which increases transport of exogenous arginine during enhanced NO production. Moreover, the intracellular signalling pathways mediating induction of arginine transport and of NO synthase by LPS in activated macrophages diverge, since only the latter is sensitive to dexamethasone.

Discrimination between citrulline and arginine transport in activated murine macrophages: inefficient synthesis of NO from recycling of citrulline to arginine

1 The kinetics, specificity, pH‐ and Na+‐dependency of l‐citrulline transport were examined in unstimulated and lipopolysaccharide (LPS)‐activated murine macrophage J774 cells. The dependency of nitric oxide production on extracellular arginine or citrulline was investigated in cells activated with LPS (1 μg ml−1) for 24 h. 2 In unstimulated J774 cells, transport of citrulline was saturable (Kt = 0.16 mm and Vmax = 32 pmol μg−1 protein min−1), pH‐insensitive and partially Na+‐dependent. In contrast to arginine, transport of citrulline was unchanged in LPS‐activated (1 μg ml−1, 24 h) cells. 3 Kinetic inhibition experiments revealed that arginine was a relatively poor inhibitor of citrulline transport, whilst citrulline was a more potent inhibitor (Ki = 3.4 mm) of arginine transport but only in the presence of extracellular Na+. Neutral amino acids inhibited citrulline transport (Ki = 0.2–0.3 mm), but were poor inhibitors of arginine transport. 4 Activated J774 cells did not release nitrite in the absence of exogenous arginine. Addition of citrulline (0.01 − 10 mm), in the absence of exogenous arginine, could only partially restore the ability of cells to synthesize nitrite, which was abolished by 100 μm NG‐nitro‐l‐arginine methyl ester or NG‐iminoethyl‐l‐ornithine. 5 Intracellular metabolism of l‐[14C]‐citrulline to l‐[14C]‐arginine was detected in unstimulated J774 cells and was increased further in cells activated with LPS and interferon‐γ. 6 We conclude that J774 macrophage cells transport citrulline via a saturable but nonselective neutral carrier which is insensitive to induction by LPS. In contrast, transport of arginine via the cationic amino acid system y+ is induced in J774 cells activated with LPS. 7 Our findings also confirm that citrulline can be recycled to arginine in activated J774 macrophage cells. Although this pathway provides a mechanism for enhanced arginine generation required for NO production under conditions of limited arginine availability, it cannot sustain maximal rates of NO synthesis.

Bacterial endotoxin rapidly stimulates prolonged endothelium-dependent vasodilatation in the rat isolated perfused heart

1 The effects of bacterial lipopolysaccharide (Escherichia coli 0111‐B4; LPS) on coronary vascular tone were examined in the isolated perfused heart of the rat. The role of nitric oxide and/or prostaglandin products of the cyclo‐oxygenase pathway in mediating the actions of LPS were also investigated. 2 Coronary vascular tone was raised and maintained by a continuous perfusion of the thromboxane‐mimetic U46619 (5 nm). LPS perfusion (0.1–100 μg ml−1) caused a concentration‐dependent fall in coronary tone without any significant change in the force of cardiac contractility. 3 At 5 μg ml−1, LPS reduced perfusion pressure by 38 ± 9 mmHg. This effect was rapid in onset, maximal within the first 5 min and sustained for 90 ± 10 min (n = 6). 4 The vasodilatation induced by LPS was dependent on the presence of an intact endothelium and abolished following endothelial damage caused by air embolism. 5 NG‐nitro‐l‐arginine methylester (l‐NAME; 50 μm) or NG‐nitro‐l‐arginine (l‐NOARG; 50 μm) blocked the vasodilatation induced by LPS (5 μg ml−1). The inhibition caused by these arginine analogues was partially reversed by 1 mm l‐ but not d‐arginine. 6 The vasodilator action of LPS was also completely blocked by the glucocorticoid, dexamethasone (10 μm) but unaffected by indomethacin (10 μm). 7 These results suggest that LPS evokes rapid release of nitric oxide (NO) in the microvasculature of the rat isolated heart presumably via activation of the constitutive l‐arginine‐NO pathway in the endothelium. Furthermore, the lack of effect of indomethacin suggests that prostaglandins released via the cyclo‐oxygenase pathway are not involved in mediating this action of LPS.

Mechanisms of acute vasodilator response to bacterial lipopolysaccharide in the rat coronary microcirculation

In this study the mechanisms of the acute vasodilator action of bacterial lipopolysaccharide (LPS) were investigated in the rat Langendorff perfused heart. Infusion of LPS (5 μg ml−1) caused a rapid and sustained fall in coronary perfusion pressure (PP) of 59±4 mmHg (n=12) and a biphasic increase in NO levels determined in the coronary effluent by chemiluminescent detection. Both the fall in PP and the increase in NO release were completely abolished (n=3) by pretreatment of hearts with the NO synthase inhibitor L‐NAME (50 μM). LPS‐induced vasodilatation was markedly attenuated to 5±4 mmHg (n=3) by pretreatment of hearts with the B2 kinin receptor antagonist Hoe‐140 (100 nM). Vasodilator responses to LPS were also blocked by brief pretreatment with mepacrine (0.5 μM, n=3) or nordihydroguaiaretic acid (0.1 μM, n=4) and markedly attenuated by WEB 2086 (3 μM, n=4). Thirty minutes pretreatment of hearts with dexamethasone (1 nM), but not progesterone (1 μM), significantly modified responses to LPS. The action of dexamethasone was time‐dependent, having no effect when applied either simultaneously with or pre‐perfused for 5 min before the administration of LPS but inhibiting the response to LPS by 91±1% (n=4) when pre‐perfused for 15 min. The inhibition caused by dexamethasone was blocked by 15 min pretreatment with the glucocorticoid receptor antagonist RU‐486 (100 nM) or by 2 min pre‐perfusion of a 1 : 200 dilution of LCPS1, a selective anti‐lipocortin 1 (LC1) neutralizing antibody. Treatment with the protein synthesis inhibitor, cycloheximide (10 μM, for 15 min) selectively blunted LPS‐induced vasodilatation, reducing the latter to 3±5 mmHg (n=3), while having no effect on vasodilator responses to either bradykinin or sodium nitroprusside. These results indicate that LPS‐induced vasodilatation in the rat heart is dependent on activation of kinin B2 receptors and synthesis of NO. In addition, phospholipase A2 (PLA2) is activated by LPS resulting in the release of platelet‐activating factor (PAF) and lipoxygenase but not cyclo‐oxygenase products. These effects are dependent on de novo synthesis of an intermediate protein which remains to be identified.