摘要：RS-7897 is a novel antianginal nitrate containing an L -2-oxothiazolidine-4-carboxylic acid (OTCA) and unlike other organic nitrates does not induce nitrate tolerance. OTCA is known to be converted to L -cysteine ( L -Cys) by rat 5-oxo-prolinase (5-OPase) and was detected in the plasma of RS-7897-treated dogs. Nitrate tolerance is considered to develop mainly through sulfhydryl depletion. We hypothesized that RS-7897-derived OTCA was converted into L -Cys by 5-OPase and supplied sulfhydryl groups in vascular smooth muscle cells, the targets of the nitrate. As the initial step for clarifying the presumed role of 5-OPase in RS-7897 metabolism, we established a non-radiochemical assay method highly specific for 5-OPase. Using this assay method, we purified 5-OPase from bovine kidney cytosol until homogeneous results were obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The catalytic activity of bovine 5-OPase to convert OTCA to L -Cys was confirmed, as was the case for the rat enzyme. Then the cDNA encoding bovine 5-OPase was cloned. The deduced amino acid sequence revealed that bovine 5-OPase was highly homologous to rat 5-OPase. Based on the bovine cDNA sequence, oligonucleotide primers were synthesized and used for RT-PCR. 5-OPase mRNA was significantly detected in the RT-PCR product of the bovine coronary artery, a major target organ of RS-7897. These results suggest that OTCA may be converted to L -Cys by 5-OPase in the mammalian coronary artery when treated with RS-7897, and thus generated L -Cys may reduce sulfhydryl depletion and contribute to the prevention of the development of nitrate tolerance.
关键词：5-oxo- L -prolinase; L -2-oxothiazolidine-4-carboxylic acid;organic nitrate;nitrate tolerance;sulfhydryl depletion;coronary artery
