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The involvement of L-arginine-dependent nitric oxide (NO) production in the vascular failure observed in endotoxemia was investigated in male Wistar rats treated with Escherichia coli lipopolysaccharide (LPS). Contractile responses to norepinephrine (NE) were measured ex vivo in aortas isolated from rats treated with LPS (20 mg/kg ip, 4 h before experiments) and pressor responses to NE were recorded in vivo in rats infused with LPS (5 mg.kg-1.h-1 iv). LPS pretreatment induced a rightward shift of the concentration-response curve to NE and a reduction of the maximal contraction by approximately 43% and 54% (P less than 0.05) in aortic rings with and without functional endothelium, respectively. This was not modified by the presence of indomethacin (10 microM) during the contractile experiments. In contrast, in the presence of NG-monomethyl-L-arginine (L-NMMA, 300 microM) or methylene blue (10 microM), maximal contractions to NE were restored to control values whether functional endothelium was present or not. The effects of L-NMMA were reversed by L- but not by D-arginine. Additionally, the effects of LPS pretreatment on vascular contractility were potentiated by L-arginine. In vivo, LPS infusion produced a reduction in pressor responsiveness to NE (0.1-10 mg/kg), which was also abolished by L-NMMA (30 mg/kg iv). This effect of L-NMMA was reversed by L- but not by D-arginine (100 mg/kg iv). These results demonstrate that activation of the L-arginine pathway has a major role in the production of vascular hyporeactivity in endotoxemia, ex vivo as well as in vivo. Additionally, they suggest that endothelium-independent vascular production of NO may be involved.

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Our method for producing tissue-engineered blood vessels based exclusively on the use of human cells, i.e., without artificial scaffolding, has previously been described (1). In this report, a tissue-engineered vascular media (TEVM) was specifically produced for pharmacological studies from cultured human vascular smooth muscle cells (VSMC). The VSMC displayed a differentiated phenotype as demonstrated by the re-expression of VSMC-specific markers and actual tissue contraction in response to physiological stimuli. Because of their physiological shape and mechanical strength, rings of human TEVM could be mounted on force transducers in organ baths to perform standard pharmacological experiments. Concentration-response curves to vasoconstrictor agonists (histamine, bradykinin, ATP, and UTP) were established, with or without selective antagonists, allowing pharmacological characterization of receptors (H1, B2, and P2Y1, and pyrimidinoceptors). Sustained agonist-induced contractions were associated with transient increases in cytosolic Ca2+ concentration, suggesting sensitization of the contractile machinery to Ca2+. ATP caused both Ca2+ entry and Ca2+ release from a ryanodine- and caffeine-sensitive store. Increased cyclic AMP or cyclic GMP levels caused relaxation. This human TEVM displays many of functional characters of the normal vessel from which the cells were originally isolated, including contractile/relaxation responses, cyclic nucleotide sensitivity, and Ca2+ handling mechanisms comparable to those of the normal vessel from which the cells were originally isolated. These results demonstrate the potential of this human model as a versatile new tool for pharmacological research.

1 Endothelial dysfunction has been described with ageing but the mechanisms responsible have not been clearly elucidated and might be di erent from one vessel to the other. This study assesses the relative contribution of endothelial nitric oxide (NO) and cyclo-oxygenase (COX) metabolites in relaxation to acetylcholine with ageing in the aorta and the small mesenteric artery of the rat. 2 In the aorta and branch II or III of superior mesenteric artery (SMA), endothelium-dependent relaxation to acetylcholine was not di erent between 12 ± 14 (adult) and 32-week-old rats whereas it was reduced at 70 ± 100 (old) weeks of age. 3 Despite an increased endothelial NO-synthase protein expression, the NO-synthase inhibitor, N G -nitro-L-arginine-sensitive component of relaxation decreased with ageing. 4 In old rats, exposure to the COX inhibitor, indomethacin, but not the selective COX-2 inhibitor, NS-398, potentiated response to acetylcholine. The thromboxane A 2 /prostaglandin H 2 receptor antagonist, GR 32191B enhanced relaxation to acetylcholine in aorta but it had no e ect in SMA. Furthermore, acetylcholine increased thromboxane B 2 production (enzymeimmunoassay) in aorta but not in SMA. Finally, Western blot analysis showed enhanced expression of COX-1 and 2 in the two arteries with ageing. 5 These results suggest that the decrease in acetylcholine-induced relaxation with ageing involves reduced NO-mediated dilatation and increased generation of vasoconstrictor prostanoids most likely from COX-1. They also point out vascular bed heterogeneity related to the nature of prostanoids involved between the aorta (i.e., thromboxane A 2 ) and the SMA (unidenti®ed) arteries even though increased expression of COX occurs in both vessels.

1 The effects of selective inhibitors of adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and guanosine 3': 5'-cyclic monophosphate (cyclic GMP) phosphodiesterases (PDEs) were investigated on PDEs isolated from the rat aorta and on relaxation of noradrenaline (1 pM) precontracted rat aortic rings, with and without functional endothelium. 2 Four PDE forms were isolated by DEAE-sephacel chromatography from endothelium-denuded rat aorta: a calmodulin-activated PDE (PDE I) which hydrolyzed preferentially cyclic GMP, two cyclic AMP PDEs (PDE III and PDE IV) and one cyclic GMP-specific PDE (PDE V). The latter was selectively and potently inhibited by zaprinast. The two cyclic AMP PDEs were discriminated by specific inhibitors: one was inhibited by cyclic GMP (PDE III) and by new cardiotonic agents (milrinone, CI 930, LY 195115 and SK&F 94120); the other was inhibited by denbufylline and rolipram (PDE IV). None of these drugs significantly inhibited PDE I. 3 The PDE III inhibitors caused endothelium-independent relaxations of rat aortic rings with the following EC50 values (aM concentration producing 50% relaxation) : LY 195115: 3.4, milrinone: 5.7, CI 930; 7.8, SK&F 94120: 14.7. Neither N0-monomethyl-L-arginine (L-NMMA, 300pM), an inhibitor of the Larginine-NO pathway, nor L-arginine (1 mM) modified the effect of PDE III inhibitors. However, methylene blue (1OpM) an inhibitor of soluble guanylate cyclase abolished relaxation induced by PDE III inhibitors except in the case of compound CI 930. 4 The specific PDE IV and PDE V inhibitors both produced endothelium-dependent relaxations which were inhibited by L-NMMA and by methylene blue (10piM). In the presence of L-NMMA, relaxation was restored by subsequent addition of L-arginine. 5 The relaxant effects of denbufylline and rolipram were studied in the presence of drugs stimulating either adenylate cyclase (forskolin and isoprenaline) or soluble guanylate cyclase (sodium nitroprusside, SNP), or inhibiting PDE III (milrinone). In endothelium-denuded rings, a relaxing effect of both denbufylline and rolipram was found in the presence of milrinone (EC5o values 1.7 and 12puM, respectively) or SNP (EC50 values 12.3 and 124pM, respectively), but not in the presence of forskolin or isoprenaline. However in the presence of functional endothelium, relaxations produced by PDE IV inhibitors were significantly potentiated by forskolin, isoprenaline, milrinone and SNP (respective EC50 values for denbufylline: 2, 2, 0.4 and 0.7 JM and for rolipram: 7, 13, 7 and 1.2 pM). 6 These results indicate that the relaxant effects of inhibitors of the cyclic AMP-specific PDE IV are markedly enhanced by cyclic GMP elevating agents and by the PDE III inhibitor milrinone. They support the hypothesis that cyclic GMP enhances cyclic AMP-mediated relaxation, possibly through the inhibition of the cyclic GMP-inhibited PDE III.

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