Inhibition of nitric oxide synthase activity by early and advanced glycation end products in cultured rabbit proximal tubular epithelial cells.
Nitric oxide (NO) is important in the regulation of renal tubular function. We have investigated whether glycated proteins could impair the NO production by examining the effects of Amadori products (AP-BSA) and advanced glycation end products (AGE-BSA) on primary cultures of rabbit proximal tubular epithelial (PTE) cells. Nitric oxide synthase activity was assessed by measurement of the conversion of L-arginine to L-citrulline and by production of NO, after short-term (30 min) or long-term (1 or 3 days) incubation. Short incubations of PTE cells with either 200 microg/ml AP-BSA or 40 microg/ml AGE-BSA significantly decreased NO production. AP-BSA (3000 microg/ml) inhibited the Ca(2+)-dependent NOS activity even though above 50 microg/ml it increased Ca(2+)-independent NOS activity. In contrast, 40 microg/ml AGE-BSA inhibited both isoforms of NOS. Longer incubations with 200 microg/ml AP-BSA or 250 microg/ml AGE-BSA decreased NO release and inhibited Ca(2+)-dependent and -independent NOS activities. APs did not affect NO release by S-nitroso-N-acetyl-penicillamine (SNAP), while 250 microg/ml AGEs decreased it. After 3 days incubation, glycation products had no effect on the NOS cell content. Cell viability and proliferation were not modified under these experimental conditions, suggesting that the fall in NO production was not due to there being fewer cells. These data indicate that APs and AGEs directly inhibit NOS activity, and additionally that AGEs quench released NO. Thus, both types of glycated proteins alter the production of NO by PTE cells and could participate in the renal tubule dysfunction associated with aging and diabetes.
Verbeke
P
P
Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Université Paris 7-Denis Diderot, T23-33 1er étage CC 7128, 2 Place Jussieu, 75252, Paris, France.
Perichon
M
M
Friguet
B
B
Bakala
H
H
D000375:Aging / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002455:Cell Division; D002470:Cell Survival; D002478:Cells, Cultured; D003920:Diabetes Mellitus / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D017127:Glycation End Products, Advanced / Q000378:metabolism* / Q000494:pharmacology; D006023:Glycoproteins / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000201:enzymology / Q000378:metabolism*; D009569:Nitric Oxide / Q000032:analysis / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D011817:Rabbits
D002147:Calmodulin; D017127:Glycation End Products, Advanced; D006023:Glycoproteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D002118:Calcium