It is commonly believed that the activity of NO synthase (NOS) solely controls NO production from its substrates, L-Arg and O(2). The Michaelis-Menten constant (K(m)) of NOS for L-Arg is in the micromolar range; cellular levels of L-Arg are much higher. However, evidence strongly suggests that cellular supply of L-Arg may become limiting and lead to reduced NO and increased superoxide anion (O(-)(2)*) formation, promoting cardiovascular dysfunction. Uptake of L-Arg into cells occurs primarily (approximately 85%) through the actions of a Na(+)-independent, carrier-mediated transporter (system y(+)). We have examined the effects of NOS agonists (substance P, bradykinin, and ACh) and NO donors (S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport of L-Arg into bovine aortic endothelial cells (BAEC). Our results demonstrate that NOS agonists increase y(+) transporter activity. A rapidly acting NO donor initially increases L-Arg uptake; however, after longer exposure, L-Arg uptake is suppressed. Exposure of BAEC without L-Arg to substance P and a Ca(2+) ionophore (A-23187) increased O(-)(2)* formation, which was blocked with concurrent presence of L-Arg or the NOS antagonist N(omega)-nitro-L-arginine methyl ester. We conclude that factors including NO itself control y(+) transport function and the production of NO and O(-)(2)*.

Tolerance to glyceryl trinitrate (GTN) involves superoxide (O2.−) production by endothelial cells. Nitric oxide synthase (NOS) produces O2.− when L‐arginine (L‐arg) is limited. The purpose of this study was to test the hypothesis that GTN stimulates NOS to increase O2.− synthesis in endothelial cells when L‐arg is limited. Production of O2.− by bovine aortic endothelial cells (BAEC, passages 3–5) was determined by spectrophotometrically measuring superoxide dismutase‐inhibited reduction of ferricytochrome C to ferrocytochrome C. Cells were incubated in buffer without L‐arg. O2.− production was measured using BAEC either untreated or treated with L‐NAME or L‐arg alone or following treatment with GTN (10−9 to 10−6 M) for 30 min or DPTA NONOate (10−7 and 10−6 M) alone or with GTN or DPTA NONOate after pretreatment with nitro‐L‐arginine methyl ester (L‐NAME), L‐arg or their inactive enantiomers, D‐NAME or D‐arg (all 5×10−4 M) (n=6–7/group). L‐NAME alone produced a 69% reduction in O2.− levels. Treatment with L‐arg alone had no effect. Cells treated with GTN alone exhibited an increase in O2.−. This effect was prevented by pretreatment with either L‐NAME or L‐arg, and was unaffected by D‐NAME or D‐arg. We observed a dose‐response relationship in O2.− production to GTN over a range of 10−9 to 10−7 M. The NO donor, DPTA‐NONOate, unlike GTN, did not have a significant effect on O2.− production. In conclusion, endothelial NOS is a site of O2.− synthesis in endothelial cells activated by GTN. British Journal of Pharmacology (2000) 131, 1019–1023; doi:10.1038/sj.bjp.0703665

Substance P (SP) and calcitonin gene-related peptide (CGRP) are well established mediators of inflammation. Therefore, inhibition of the biosynthesis of these neuropeptides is an attractive potential strategy for pharmacological intervention against a number of inflammatory diseases. The final step in the biosynthesis of SP and CGRP is the conversion of their glycineextended precursors to the active amidated peptide, and this process is catalyzed by sequential action of the enzymes peptidylglycine ␣-monooxygenase (PAM) and peptidylamidoglycolate lyase. We have demonstrated previously that 4-phenyl-3-butenoic acid (PBA) is a PAM inhibitor, and we have also shown that in vivo inhibition of serum PAM by PBA correlates with this compound's ability to inhibit carrageenan-induced edema in the rat. Here we demonstrate the ability of PBA to inhibit all three phases of adjuvant-induced polyarthritis (AIP) in rats; this represents the first time that an amidation inhibitor has been shown to be active in a model of chronic inflammation. We recently introduced 5-(acetylamino)-4-oxo-6-phenyl-2-hexenoic acid (AOPHA) as one of a new series of mechanismbased amidation inhibitors. We now report for the first time that AOPHA and its methyl ester (AOPHA-Me) are active inhibitors of serum PAM in vivo, and we show that AOPHA-Me correspondingly inhibits carrageenan-induced edema in rats in a dose-dependent manner. Neither PBA nor AOPHA-Me exhibits significant cyclooxygenase (COX) inhibition in vitro; thus, the anti-inflammatory activities of PBA and AOPHA-Me are apparently not a consequence of COX inhibition. We discuss possible pharmacological mechanisms that may account for the activities of these new antiinflammatory compounds.A number of neuropeptides, such as calcitonin gene-related peptide (CGRP), neuropeptide Y, substance P (SP), and vasoactive intestinal polypeptide, are initially synthesized as glycine-extended precursors, which are then converted to the bioactive C-terminal amidated form. Amidation is a two-step process catalyzed by the sequential actions of peptidylglycine ␣-monooxygenase (PAM; EC 1.14.17.3) and peptidylamidoglycolate lyase (EC 4.3.2.5). The monooxygenase first catalyzes ␣-hydroxylation of the glycine-extended precursor, and the lyase then catalyzes conversion of this ␣-hydroxyglycine derivative to the C-terminally amidated peptide (Katopodis et al., , 1991Ping et al., 1992). In the case of SP and CGRP, their release from nerves, as well as inflammatory cells, has been shown to facilitate a number of inflammatory events, including increased vascular permeability, chemotaxis, and release of inflammatory mediators, such as cytokines, eicosanoids, and histamine (Pernow, 1983;Matucci-Cernic and Partsch, 1992;Haines et al., 1993;Holzer and Holzer-Petsche, 1997). Further evidence demonstrating the inflammatory capacity of SP was noted when SP receptor (neurokinin-1 receptor) knockout mice developed significantly less inflammation than controls after treatment with a phlogistic agent (Bozic et al., 1996)....

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