Pharmacological modulation of nitric oxide synthase activity has been achieved using structural analogs of arginine. In the present studies, we demonstrated that the minimal amidine structure required for enzymatic inhibition is formamidine. We found that the production of nitric oxide by primary cultures of rat hepatocytes and several mouse and human cell lines, including RAW 264.7 macrophages, PAM 212 keratinocytes, G8 myoblasts, S180 sarcoma, CX-1 human colon cells, and GH3 rat pituitary cells, was inhibited in a concentration- and time-dependent manner by formamidine. Formamidine was 2- to 6-fold more effective in inhibiting nitric oxide production in cells expressing inducible nitric oxide synthase (NOS2) than in a cell line expressing calcium-dependent neuronal nitric oxide synthase (NOS1). Whereas formamidine had no effect on γ-interferon-induced expression of nitric oxide synthase protein, its enzymatic activity was blocked. Kinetic analysis revealed that formamidine acts as a simple competitive inhibitor with respect to arginine (Ki formamidine 800 μM). Using a polarographic microsensor to measure real-time flux of nitric oxide release from RAW 264.7 macrophages, formamidine was found to require 30-90 min to inhibit enzyme activity, suggesting that cellular uptake of the drug may limit its biological activity. Our data indicate that formamidine is an effective inhibitor of nitric oxide production. Furthermore, its low toxicity may make it useful as a potential therapeutic agent in diseases associated with the increased production of nitric oxide.
Salicylic acid (SA) induced nitric oxide (NO) generation, Phenylalanine ammonia-lyase (PAL) activation, and salvianolic acid B (Sal B) biosynthesis. To determine the role of NO in SA-induced Sal B biosynthesis, the effects of NO donor sodium nitroprusside (SNP), NO synthase inhibitor L-NNA(Nω-nitro-L-arginine), NO scavenger carboxy-2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPITO), and PAL inhibitor L-AOPP (L-2-aminooxygen-3-phenyl acrylic acid) on SA-induced NO generation, PAL activation, and Sal B accumulation were studied individually. Pretreatment of the cells with SNP increased SA-induced NO generation, PAL activation and Sal B accumulation, which suggested that NO activated PAL and was involved in SA-induced Sal B biosynthesis. L-AOPP suppressed PAL activity and Sal B accumulation, but did not affect SA-induced NO generation, indicating that NO acted as an upstream signal of PAL. Results indicated that there was a causal relationship between SA-induced NO generation, PAL activation, and Sal B biosynthesis in Salvia miltiorrhiza suspension cell culture. Via activation of PAL, NO mediated the SA-induced Sal B biosynthesis.
Biosynthesis Of Nitric Oxide - mutant mass ua online
Nitric oxide (NO) is an important molecular mediator of numerous physiological processes in virtually every organ. In the kidney, NO plays prominent roles in the homeostatic regulation of glomerular, vascular, and tubular function. Differential expression and regulation of the NO synthase (NOS) gene family contribute to this diversity of action. This review explores recent advances in the molecular and cell biology of the NOS isoforms and relates these findings to functions of NO in the control of normal renal hemodynamics, the glomerular microcirculation, and renal salt excretion. Newly recognized molecular diversity of the NOS gene products, factors governing NOS isozyme gene expression and catalytic activity, and the intrarenal distribution of the NOS isoforms are examined. Physiological data regarding the complex roles of NO in the control of renal hemodynamics and the glomerular microcirculation are analyzed, and, the effects of chronic NOS inhibition on glomerular function and structure are presented. The contributions of NO to renal salt excretion as well as functional and molecular biological evidence for adaptive changes in NOS isoform expression during variations in dietary salt balance are discussed. Current investigative challenges and goals for future research of renal NO biology are presented.