A specific method for measurement of nitric oxide synthase enzymatic activity in peritoneal biopsies.
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 peritoneum has been assessed by nonspecific methods. We describe the application of a specific method for determination of NOS activity in rat and human peritoneal biopsies.


METHODS
The L-citrulline assay is based on the stoechiometric production of NO and L-[3H]-citrulline from L-[3H]-arginine by NOS. The assay is technically difficult when applied on small samples with relatively low levels of NOS activity, which required specific procedures for extraction and samples processing. Reaction parameters ensuring assay linearity in the peritoneum 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 because of both Ca2+-dependent and Ca2+-independent NOS. The specificity of NOS activity has been demonstrated by various controls, including the NOS inhibitor L-NMMA. Competition experiments with L-valine and amino acid analyses have reasonably excluded the interference of endogenous arginase and L-arginine, which both might underestimate NOS activity. The procedure is sensitive; it detects a high range of NOS activities as well as the appropriate NOS isoforms in various tissues and conditions, as shown by correlations with immunoblot studies.


CONCLUSIONS
We have adapted and characterized the L-citrulline assay to measure specific NOS activities within the peritoneum. The peritoneum lysate assayed for NOS activity can also be used for characterizing NOS isoform expression by immunoblot analysis.
Combet
S
S
Divisions of Nephrology and Pharmacotherapy, Université Catholique de Louvain Medical School, Brussels, Belgium.
Balligand
J L
JL
Lameire
N
N
Goffin
E
E
Devuyst
O
O
D000328:Adult; D000818:Animals; D001706:Biopsy; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002956:Citrulline / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010537:Peritoneum / Q000201:enzymology* / Q000473:pathology; D051381:Rats; D017207:Rats, Sprague-Dawley
D007527:Isoenzymes; D002956:Citrulline; D019001:Nitric Oxide Synthase; D002118:Calcium