N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was a slow, tight binding inhibitor of human inducible nitricoxide synthase (iNOS). The slow onset of inhibition by 1400W showed saturation kinetics with a maximal rate constant of 0.028 s ؊1 and a binding constant of 2.0 M. Inhibition was dependent on the cofactor NADPH. LArginine was a competitive inhibitor of 1400W binding with a K s value of 3.0 M. Inhibited enzyme did not recover activity after 2 h. Thus, 1400W was either an irreversible inhibitor or an extremely slowly reversible inhibitor of human iNOS with a K d value 7 nM. In contrast, inhibition of human neuronal NOS and endothelial NOS (eNOS) was relatively weaker, rapidly reversible, and competitive with L-arginine, with K i values of 2 M and 50 M, respectively. Thus, 1400W was at least 5000-fold selective for iNOS versus eNOS. This selectivity was similar to that observed in rat aortic rings, in which 1400W was greater than 1000-fold more potent against rat iNOS than eNOS. Finally, 1400W was greater than 50-fold more potent against iNOS than eNOS in a rat model of endotoxin-induced vascular injury. Thus, the potency and selectivity of 1400W inhibition of iNOS both in vitro and in vivo were far greater than of any previously described iNOS inhibitor.

1 The effect of endotoxin (E. coli lipopolysaccharide) on the induction of nitric oxide synthase (NOS) and the changes in vascular permeability in the colon and jejunum over a 5 h period have been investigated in the rat. 2 Under resting conditions, a calcium-dependent constitutive NOS, determined by the conversion of radiolabelled L-arginine to citrulline, was detected in homogenates of both colonic and jejunal tissue. 3 Administration of endotoxin (3 mg kg-', i.v.) led, after a 2 h lag period, to the appearance of calcium-independent NOS activity in the colon and jejunum ex vivo, characteristic of the inducible NOS enzyme. 4 Administration of endotoxin led to an increase in colonic and jejunal vascular permeability after a lag period of 3 h, determined by the leakage of radiolabelled albumin. 5 Pretreatment with dexamethasone (1 mg kg-' s.c., 2 h prior to challenge) inhibited both the induction of NOS and the vascular leakage induced by endotoxin. 6 Administration of the NO synthase inhibitor NG-monomethyl-L-arginine (12.5-50mg kg-', s.c.) 3 h after endotoxin injection, dose-dependently reduced the subsequent increase in vascular permeability in jejunum and colon, an effect reversed by L-arginine (300 mg kg-', s.c.). 7 These findings suggest that induction of NOS is associated with the vascular injury induced by endotoxin in the rat colon and jejunum.

1 The role of nitric oxide (NO) formed by the inducible isoform of NO synthase (NOS) in the generation of indomethacin-induced intestinal microvascular leakage was investigated in the rat. 2 Indomethacin (10 mg kg-', s.c.) provoked an elevation of vascular leakage of radiolabelled human serum albumin in the jejunum over 48 h, commencing 18 h after its administration. This was associated with the induction of a calcium-independent NOS, as assessed by the conversion of radiolabelled Larginine to citrulline. 3 Pretreatment with the glucocorticoid, dexamethasone (1 mg kg-' day'-, s.c.) inhibited the induction of NOS and reduced jejunal microvascular leakage, determined 24 and 48 h after indomethacin. 4 Administration of the broad-spectrum antibiotic, ampicillin (800 mg kg-' day-', p.o.) likewise inhibited both the induction of NOS and the plasma leakage observed 24 and 48 h after indomethacin. 5 Ampicillin pretreatment did not, however, inhibit the induction of NOS, determined 5 h following endotoxin (3 mg kg-' i.v.) challenge. Furthermore, incubation with ampicillin (1 mM, 10 min) did not inhibit the activity of the calcium-independent isoform in vitro.6 Administration of the NOS inhibitor, N0-nitro-L-arginine methyl ester (L-NAME, 2-10 mg kg-', s.c.), at the time of the detectable expression of the inducible NOS (18 h after indomethacin), dosedependently attenuated the plasma leakage, determined 6 later. This effect was reversed by pretreatment with L-arginine (300 mg kg-', s.c.) 15 min before L-NAME. 7 These findings suggest that induction of a calcium-independent NOS following indomethacin administration involves gut bacteria and leads to microvascular injury in the rat jejunum.

1 The effects of the nitric oxide (NO) synthase inhibitors, N0-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA), on the vascular damage induced by the endotoxin, E. coli lipopolysaccharide (LPS), in the ileum and colon were investigated in the conscious rat over a 5 h period. 2 Administration of LPS (3 mg kg-', i.v.) increased ileal and colonic vascular injury after a lag period of 2 h, as determined by the leakage of radiolabelled albumin. 3 Administration of L-NAME (1-5 mg kg-', s.c.) concurrently with LPS, produced a dose-dependent increase in vascular albumin leakage in the intestinal tissues, when determined over a 5 h period. Vascular albumin leakage with LPS and L-NAME (5 mg kg-') was substantially increased after 1 h, reached maximal levels 3 h after administration, and then slowly declined. 4 L-NMMA (50 mg kg-1, s.c.), likewise elevated intestinal albumin leakage when administered concurrently with LPS, but this reached maximal levels after 1 h and rapidly declined over the subsequent 2 h. 5 In control rats, in the absence of LPS challenge, neither L-NAME (5 mg kg-', s.c.) nor L-NMMA (50 mg kg-', s.c.) increased intestinal vascular leakage of albumin over a 5 h period. 6 By contrast, when L-NAME (1-5 mg kg-', s.c.) or L-NMMA (12.5-50mg kg-', s.c.) was injected 3 h after LPS, a dose-dependent reduction in the LPS-provoked vascular albumin leakage was observed. 7 Pretreatment with L-arginine (300 mg kg-', s.c.) 15 min prior to the NO synthase inhibitors, reversed either the potentiation or the inhibition by L-NAME (5 mg kg-', s.c.) or L-NMMA (50 mg kg-', s.c.) of the LPS-induced intestinal vascular damage. 8 These findings indicate that initial suppression of the constitutive NO synthase by L-NAME or L-NMMA following challenge with LPS aggravates the acute vascular injury in the ileum and colon, suggesting a defensive role of NO. By contrast, the delayed administration of NO synthase inhibitors, at a time of known expression of the inducible NO synthase, provides protection against the subsequent damage to the intestinal vasculature.

No abstract

Most chronic calcifying pancreatitis patients with common bile duct strictures respond to the increasing numbers of endoscopic stents, and remain stent free for medium term periods. Less patients (30%) does not benefit of biliary stenting, who are candidates for surgery.

Endogenous NO is involved in the formation of pancreatic edema in Arg-induced acute pancreatitis by increasing the vascular permeability and protein extravasation. L-NAME treatment decreased the cNOS activity and edema formation but did not prevent the histologic damage in Arg-induced acute pancreatitis.

No abstract