0.03-300mgkg-1, i.v.) induced a dose-dependent increase in mean systemic arterial blood pressure accompanied by bradycardia.
5L-NMMA, L-NIO and L-NAME (100 mgkg-',i.v.) inhibited significantly the hypotensive responses to ACh and bradykinin. 6 The increase in blood pressure and bradycardia produced by these compounds were reversed by L-arginine (30-100 mg kg-1, i.v.) in a dose-dependent manner. 7 All of these effects were enantiomer specific. 8 These results indicate that L-NMMA, L-NIO and L-NAME are inhibitors of NO synthase in the vascular endothelium and confirm the important role of NO synthesis in the maintenance of vascular tone and blood pressure.
1 The role of L-arginine in the basal and stimulated generation of nitric oxide (NO) for endothelium-dependent relaxation was studied by use of NG-monomethyl L-arginine (L-NMMA), a specific inhibitor of this pathway. 2 L-Arginine (10-100MM), but not D-arginine (100 MM), induced small but significant endotheliumdependent relaxations of rings of rabbit aorta. In contrast, L-NMMA (1-300pM) produced small, endothelium-dependent contractions, while its enantiomer N -monomethyl-D-arginine (D-NMMA; 100M) had no effect.3 L-NMMA (1-300pM) inhibited endothelium-dependent relaxations induced by acetylcholine (ACh), the calcium ionophore A23187, substance P or L-arginine without affecting the endotheliumindependent relaxations induced by glyceryl trinitrate or sodium nitroprusside. 4 The inhibition of endothelium-dependent relaxation by L-NMMA (30Mm) was reversed by Larginine (3-300 yM) but not by D-arginine (300 gM) or a number of close analogues (100 pM).5 The release of NO induced by ACh from perfused segments of rabbit aorta was also inhibited by L-NMMA (3-300pM), but not by D-NMMA (1001Mm) and this effect of L-NMMA was reversed by L-arginine (3-300 Mm). 6 These results support the proposal that L-arginine is the physiological precursor for the basal and stimulated generation of NO for endothelium-dependent relaxation.
1Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes the endogenous nitric oxide synthase inhibitors N0-monomethyl-L-arginine and NG,NG-dimethy-L-arginine to citrulline, was identified by Western blotting in rat and human tissue homogenates.2 S-2-amino-4(3-methylguanidino)butanoic acid (4124W) inhibited the metabolism of ["4C]-N0-monomethyl-L-arginine to ["4C]-citrulline by rat liver homogenates (IC50 416 + 66 giM; n = 9), human cultured endothelial cells (IC50 250 + 34 gLM; n = 9) and isolated purified dimethylarginine dimethylaminohydrolase. 3 Addition of 4124W to culture medium increased the accumulation of endogenously-generated N',N0-dimethy-L-arginine in the supernatant of human cultured endothelial cells from 3.1 + 0.3 to 5+0.7 ,M (n= 15; P<0.005). 4 4124W (1 uM-1 mM) had no direct effect on endothelial nitric oxide synthase activity but caused endothelium-dependent contraction of rat aortic rings (1 mM 4124W increased tone by 81.5 + 9.6% of that caused by phenylephrine 100 nM). This effect was reversed by L-arginine (100 giM). 4124W reversed endothelium-dependent relaxation of human saphenous vein (19.2 + 6.7% reversal of bradykinin-induced relaxation at 1 mM 4124W). 5 These data suggest that inhibition of dimethylarginine dimethylaminohydrolase increases the intracellular concentration of N',N0-dimethyl-L-arginine sufficiently to inhibit nitric oxide synthesis.Inhibiting the activity of DDAH may provide an alternative mechanism for inhibition of nitric oxide synthases and changes in the activity of DDAH could contribute to pathophysiological alterations in NO generation.
1The fungal metabolite, wortmannin, has recently been shown to inhibit fMet-Leu-Phe-stimulated superoxide production and phospholipase D (PLD) activation in the human neutrophil. 2 We have found that a close structural analogue of wortmannin, demethoxyviridin, has a similar inhibitory profile but in addition blocks phosphatidylinositol 4,5-bisphosphate-specific phospholipase C and hence inositol 1,4,5-trisphosphate (1P3) formation. 3 Inhibition of fMet-Leu-Phe-stimulated PLD by demethoxyviridin was characteristically noncompetitive (IC50 = 31 + 10nM).4 Inhibition of fMet-Leu-Phe-stimulated 1P3 formation required concentrations almost 10 times higher (IC50 = 250 + 130 nM).5 Surprisingly, demethoxyviridin only inhibited fMet-Leu-Phe-induced intracellular calcium mobilization at concentrations 100 times greater than those needed to block IP3 formation. 6 Demethoxyviridin also inhibited PLD activation induced by sodium fluoride or phorbol myristate acetate (PMA) but the concentrations required were 100 times those needed to block fMet-Leu-Phestimulated PLD. 7 These observations support the contention that PLD plays an important role in signal transduction in the human neutrophil and indicate that wortmannin and demethoxyviridin inhibit PLD activation at a common step in the signalling pathway. 8 Furthermore, these results suggest that demethoxyviridin may block the interaction between the chemotactic peptide receptor and a GTP-binding protein that is intimately involved in PLD activation.
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