Abstract. The implication of advanced glycation end products (AGE) in the pathogenesis of atherosclerosis and of diabetic and uremic complications has stimulated a search for AGE inhibitors. This study evaluates the AGE inhibitory potential of several well-tolerated hypotensive drugs. Olmesartan, an angiotensin II type 1 receptor (AIIR) antagonist, as well as temocaprilat, an angiotensin-converting enzyme (ACE) inhibitor, unlike nifedipine, a calcium blocker, inhibit in vitro the formation of two AGE, pentosidine and N ⑀ -carboxymethyllysine (CML), during incubation of nonuremic diabetic, nondiabetic uremic, or diabetic uremic plasma or of BSA fortified with arabinose. This effect is shared by all tested AIIR antagonists and ACE inhibitors. On an equimolar basis, they are more efficient than aminoguanidine or pyridoxamine. Unlike the latter two compounds, they do not trap reactive carbonyl precursors for AGE, but impact on the production of reactive carbonyl precursors for AGE by chelating transition metals and inhibiting various oxidative steps, including carbon-centered and hydroxyl radicals, at both the pre-and post-Amadori steps. Their effect is paralleled by a lowered production of reactive carbonyl precursors. Finally, they do not bind pyridoxal, unlike aminoguanidine. Altogether, this study demonstrates for the first time that widely used hypotensive agents, AIIR antagonists and ACE inhibitors, significantly attenuate AGE production. This study provides a new framework for the assessment of families of AGE-lowering compounds according to their mechanisms of action.Advanced glycation and oxidation irreversibly modify proteins over the years and thus contribute to aging phenomena (1). Their local or generalized acceleration is associated with atherosclerosis (2-6) as well as with various diabetic (7-10) and uremic complications (11-13). Inhibition of advanced glycation end products (AGE) formation has thus become a therapeutic goal.Aminoguanidine, the first AGE inhibitor discovered in 1986 (14), and (Ϯ)-2-isopropylidenehydrazono-4-oxo-thiazolidin-5-ylacetanilide (OPB-9195) (15) are both hydrazine-derivatives. They inhibit in vitro the formation of AGE, pentosidine (16), and N ⑀ -carboxymethyllysine (CML) (17) from a variety of individual precursors, such as ribose, glucose, and ascorbate, as well as that of advanced lipoxidation end products (ALE), malondialdehyde-lysine and 4-hydroxynonenal-protein adduct (18), from arachidonate (19). They also inhibit pentosidine generation in diabetic and uremic plasma incubated for 4 wk (20).As expected, both compounds correct several biologic effects that are associated with AGE formation. In murine thymocyte and fibroblasts, they inhibit the phosphorylation of tyrosine residues of a number of intracellular proteins induced by cell surface Schiff base formation (21). Given to diabetic animal models, such as Otsuka-Long-Evans-Tokushima-Fatty (OLETF) or streptozotocin-treated rats, they reduce urinary albumin excretion and improve glomerular morphology (15,22). Oral admi...

For many diseases, mediation of pathogenesis by nitric oxide (NO) has been suggested. In this study, we explored NO-induced viral pathogenesis with a focus on nucleic acid damage as evidenced by 8-nitroguanosine formation in vivo. Wild-type mice and littermate mice deficient in inducible NO synthase (iNOS) were infected with influenza or Sendai virus. Formation of 8-nitroguanosine in virusinfected lungs was assessed immunohistochemically with an antibody specific for 8-nitroguanosine. Extensive nitration of RNA either treated with peroxynitrite or obtained from cultured RAW 264 cells expressing iNOS was readily detected by this antibody. Strong 8-nitroguanosine immunostaining was evident primarily in the cytosol of bronchial and bronchiolar epithelial cells of virus-infected wild-type mice but not iNOS-deficient mice. This staining colocalized with iNOS immunostaining in the lung. 8-Nitroguanosine staining disappeared after addition of exogenous authentic 8-nitroguanosine during the antibody reaction and after pretreatment of tissues with sodium hydrosulfite, which reduces 8-nitroguanosine to 8-aminoguanosine. NO was generated in excess in lungs of wild-type mice but was eliminated in iNOSdeficient mice after virus infection; this result also correlated well with formation of 8-nitroguanosine and 3-nitrotyrosine. One consequence of the lack of iNOS expression was marked improvement in histopathological changes in the lung and the lethality of the infection without effects on cytokine responses and viral clearance. It is intriguing that 8-nitroguanosine markedly stimulated superoxide generation from cytochrome P450 reductase and iNOS in vitro. The present data constitute a demonstration of 8-nitroguanosine formation in vivo and suggest a potential role for NO-induced nitrative stress in viral pathogenesis.

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We studied steps that make up the initial and steadystate phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0.13 to 86 s ؊1 upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N -hydroxy-L-arginine (0.005 and 0.014 s ؊1 , respectively, at 10°C). O 2 binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nM), which were easily achieved with N -hydroxy-L-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K m for O 2 to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology. Nitric-oxide synthases (NOSs)1 catalyze a stepwise oxidation of L-arginine (Arg) to citrulline and nitric oxide (NO) (1-3). In mammals, three NOSs are expressed that differ in their primary sequence, post-translational modifications, cellular location, and tissue expression (4 -6), consistent with their participating in a range of physiologic and pathologic systems. Two NOSs (neuronal, nNOS or NOS-I; and endothelial, eNOS or NOS-III) are constitutively expressed and participate in signal cascades by synthesizing NO in response to Ca 2ϩ -dependent CaM binding. A third NOS (cytokine-inducible, iNOS or NOS-II) is primarily regulated by transcriptional mechanisms, binds CaM irrespective of the Ca 2ϩ concentration to be always active, and functions as both a regulator and effector of the immune response.Although NO synthesis activities of the NOS isoforms differ considerably, how and why this occurs is unclear. A comparison of published steady-state rates shows that eNOS is about four to eight times slower than either nNOS or iNOS (7-14). Because NO synthesis is actually the result of many steps, it is imperative to identify which steps limit the activity of a particular NOS isoform. Work with NOS chimeras containing swapped reductase domains has suggested heme reduction could be responsible for the low activity of eNOS (30). However, it seems that NOS catalysis is comprised of two parts (15, 16): an active component that includes...