A specific method for measurement of nitric oxide synthase enzymatic activity in peritoneal biopsies

Background. Nitric oxide (NO) is synthesized by NO synthase (NOS) isoforms that are expressed in the peritoneum. Thus far, NOS activity in the periton eum has been assessed by nonspecific methods. We describe the application o f a specific method for determination of NOS activity in rat and human peri toneal biopsies. Methods. The L-citrulline assay is based on the stoechiometric production o f NO and L-[H-3]-citrulline from L-[H-3]-arginine by NOS. The assay is tech nically difficult when applied on small samples with relatively low levels of NOS activity, which required specific procedures for extraction and samp les processing. Reaction parameters ensuring assay linearity in the periton eum were defined. Peritoneum lysates were also used for immunoblot analysis to identify the NOS isoforms involved. Results. A significant NOS activity is detected in the normal peritoneum be cause of both Ca2+-dependent and Ca2+-independent NOS. The specificity of N OS activity has been demonstrated by various controls, including the NOS in hibitor L-NMMA. Competition experiments with L-valine and amino acid analys es have reasonably excluded the interference of endogenous arginase and L-a rginine, which both might underestimate NOS activity. The procedure is sens itive; it detects a high range of NOS activities as well as the appropriate NOS isoforms in various tissues and conditions, as shown by correlations w ith immunoblot studies. Conclusions. We have adapted and characterized the L-citrulline assay to me asure specific NOS activities within the peritoneum. The peritoneum lysate assayed for NOS activity can also be used for characterizing NOS isoform ex pression by immunoblot analysis.