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

被引:30
作者
Combet, S
Balligand, JL
Lameire, N
Goffin, E
Devuyst, O
机构
[1] Univ Catholique Louvain, Sch Med, Div Nephrol, B-1200 Brussels, Belgium
[2] Univ Catholique Louvain, Sch Med, Div Pharmacotherapy, B-1200 Brussels, Belgium
[3] State Univ Ghent, Div Nephrol, B-9000 Ghent, Belgium
[4] CEA, Div Cell Biol, Saclay, France
关键词
nitric oxide; peritoneum; L-arginine; L-citrulline assay; infection; neurotransmission; dialysis membrane permeability;
D O I
10.1046/j.1523-1755.2000.00839.x
中图分类号
R5 [内科学]; R69 [泌尿科学(泌尿生殖系疾病)];
学科分类号
1002 ; 100201 ;
摘要
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-[H-3]-citrulline from L-[H-3]-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.
引用
收藏
页码:332 / 338
页数:7
相关论文
共 20 条
[1]   NITRIC OXIDE-DEPENDENT PARASYMPATHETIC SIGNALING IS DUE TO ACTIVATION OF CONSTITUTIVE ENDOTHELIAL (TYPE-III) NITRIC-OXIDE SYNTHASE IN CARDIAC MYOCYTES [J].
BALLIGAND, JL ;
KOBZIK, L ;
HAN, XQ ;
KAYE, DM ;
BELHASSEN, L ;
OHARA, DS ;
KELLY, RA ;
SMITH, TW ;
MICHEL, T .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (24) :14582-14586
[2]  
Breborowicz A, 1998, PERITON DIALYSIS INT, V18, P188
[3]   NITRIC-OXIDE - A PHYSIOLOGICAL MESSENGER MOLECULE [J].
BREDT, DS ;
SNYDER, SH .
ANNUAL REVIEW OF BIOCHEMISTRY, 1994, 63 :175-195
[4]   Arginase modulates nitric oxide production in activated macrophages [J].
Chang, CI ;
Liao, JC ;
Kuo, L .
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY, 1998, 274 (01) :H342-H348
[5]   The upstream regulatory region of the carbamoyl-phosphate synthetase I gene controls its tissue-specific, developmental, and hormonal regulation in vivo [J].
Christoffels, VM ;
vandenHoff, MJB ;
Lamers, MC ;
vanRoon, MA ;
deBoer, PAJ ;
Moorman, AFM ;
Lamers, WH .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (49) :31243-31250
[6]  
Combet S, 1999, J AM SOC NEPHROL, V10, P2185
[7]   Aquaporin-1 and endothelial nitric oxide synthase expression in capillary endothelia of human peritoneum [J].
Devuyst, O ;
Nielsen, S ;
Cosyns, JP ;
Smith, BL ;
Agre, P ;
Squifflet, JP ;
Pouthier, D ;
Goffin, E .
AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY, 1998, 275 (01) :H234-H242
[8]   The nitric oxide donor nitroprusside intraperitoneally affects peritoneal permeability in CAPD [J].
Douma, CE ;
deWaart, DR ;
Struijk, DG ;
Krediet, RT .
KIDNEY INTERNATIONAL, 1997, 51 (06) :1885-1892
[9]   Icodextrin with nitroprusside increases ultrafiltration and peritoneal transport during long CAPD dwells [J].
Douma, CE ;
Hiralall, JK ;
de Waart, DR ;
Struijk, DG ;
Krediet, RT .
KIDNEY INTERNATIONAL, 1998, 53 (04) :1014-1021
[10]  
Douma CE, 1995, ADV PERIT D, V11, P36