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 product ion by examining the effects of Amadori products (AP-BSA) and advanced glyc ation end products (AGE-BSA) on primary cultures of rabbit proximal tubular epithelial (PTE) cells. Nitric oxide synthase activity was assessed by mea surement 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. Sho rt incubations of PTE cells with either 200 mug/ml AP-BSA or 40 mug/ml ACE- BSA significantly decreased NO production. AP-BSA (3000 mug/ml) inhibited t he Ca2+-dependent NOS activity even though above 50 mug/ml it increased Ca2 +-independent NOS activity. In contrast, 40 mug/ml AGE-BSA inhibited both i soforms of NOS. Longer incubations with 200 mug/ml AP-BSA or 250 mug/ml AGE -BSA decreased NO release and inhibited Ca2+-dependent and -independent NOS activities. APs did not affect NO release by S-nitroso-N-acetyl-penicillam ine (SNAP), while 250 mug/ml AGEs decreased it. After 3 days incubation, gl ycation products had no effect on the NOS cell content. Cell viability and proliferation were not modified under these experimental conditions, sugges ting 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 a dditionally that AGEs quench released NO. Thus, both types of glycated prot eins alter the production of NO by PTE cells and could participate in the r enal tubule dysfunction associated with aging and diabetes. (C) 2000 Elsevi er Science B.V. All rights reserved.