Nadia Manzoli

Effect of Native and Oxidized Low-Density Lipoprotein on Endothelial Nitric Oxide and Superoxide Production Key Role of l-Arginine Availability

BACKGROUND Native and oxidized LDLs (n-LDL and ox-LDL) are involved in the atherogenic process and affect endothelium-dependent vascular tone through their interaction with nitric oxide (NO). METHODS AND RESULTS In this study we evaluated directly, by using a porphyrinic microsensor, the effect of increasing lipoprotein concentrations on endothelial NO and superoxide (O(2)(-)) production. We investigated where lipoproteins may affect the L-arginine-NO pathway by pretreating cells with L-arginine, L-N-arginine methyl ester (L-NAME), and superoxide dismutase. Bovine aortic endothelial cells were exposed for 1 hour to increasing concentrations of n-LDL (from 0 to 240 mg cholesterol/dL) and ox-LDL (from 0 to 140 mg cholesterol/dL). A stimulated (calcium ionophore) NO concentration decreased to 29% of the control at n-LDL concentration of 80 mg cholesterol/dL and to 15% of the control at 20 mg cholesterol/dL of ox-LDL. L-Arginine partially neutralized the inhibitory effect of n-LDL and ox-LDL on the NO generation. Superoxide dismutase pretreatment did not modify NO production, whereas L-NAME blunted NO generation at all LDL concentrations. O(2)(-) production was increased at low n-LDL and very low ox-LDL concentrations; this was reversed by L-arginine. CONCLUSIONS These findings confirm the inhibitory role of n-LDL and ox-LDL on NO generation and suggest that lipoproteins may induce a decreased uptake of L-arginine. The local depletion of the L-arginine substrate may derange the NO synthase, leading to overproduction of O(2)(-) from oxygen, the other substrate of NO synthase.

Characterization of the endothelin receptor subtype mediating epithelium-derived relaxant nitric oxide release from guinea-pig trachea

1 The endothelin (ET) receptor subtype that mediates niric oxide (NO)‐dependent airway relaxation in tracheal tube preparations precontracted with carbachol and pretreated with indomethacin was investigated. The release of NO induced by ET from guinea‐pig trachea using a recently developed porphyrinic microsensor was also measured. 2 ET‐1 (1 pM–100 nM) contracted tracheal tube preparations pretreated with the NO‐synthase inhibitor, L‐NMMA, and relaxed, in an epithelium‐dependent manner, preparations pretreated with the inactive enantiomer D‐NMMA. The effect of L‐NMMA was reversed by L‐Arg, but not by D‐Arg. 3 The selective ETB receptor agonists, IRL 1620 or sarafotoxin S6c, both (1 pM–100 nM) contracted tracheal tube preparations in a similar manner either after treatment with D‐NMMA or with L‐NMMA. In the presence of the ETA receptor antagonist, FR139317 (10 μM), ET‐1 administration resulted in a contraction that was similar after either L‐NMMA or D‐NMMA. In the presence of the ETB receptor antagonist, BQ788 (1 μM), ET‐1 relaxed and contracted tracheas pretreated with D‐NMMA and L‐NMMA, respectively. 4 Exposure of tracheal segments to ET‐1 (1–1000 nM) caused a concentration‐dependent increase in NO release that was reduced by L‐NMMA. IRL1620 (1 μM) did not cause any significant NO release. FR139317 (10 μM), but not, BQ788 (1 μM), inhibited the NO release induced by ET‐1. 5 These results demonstrate that in the isolated guinea‐pig trachea activation of ETB receptors results in a contractile response, whereas activation of ETA receptors cause both a contraction, and an epithelium‐dependent relaxation that is mediated by NO release.