IMPAIRED ARGININE METABOLISM AND NO SYNTHESIS IN CORONARY ENDOTHELIAL-CELLS OF THE SPONTANEOUSLY DIABETIC BB RAT

Arginine metabolism via nitric oxide (NO) synthase and other pathways was studied in coronary endothelial cells (EC) from the spontaneously diabetic BE rat, an animal model of human type I diabetes mellitus (IDDM). EC were prepared from insulin-treated diabetic BE (BBd) and non-diabetes-prone BE (BBn) rats. Basal NO synthesis was studied in EC cultured for 48 h in medium containing 0.4 mM L-arginine. At the end of the culture period, the medium was analyzed for nitrite and nitrate (two major end stable oxidation products of NO), and the cells were used to determine arginine uptake and metabolism and the activities of some arginine-degrading enzymes. For studies of arginine metabolism, cells were incubated at 37 degrees C for 1 h in Krebs-Henseleit bicarbonate buffer (pH 7.4) containing 1 mM L-[1-C-14]arginine or L-[1-C-14]ornithine. The rates of production of nitrite plus nitrate by BBd EC were only 15% of those of BBn cells. This impaired NO synthesis in BBd EC was not due to alterations in arginine uptake, NO synthase activity, or intracellular arginine concentrations but might have resulted from a limited intracellular availability of cofactors of NO synthase. In addition to the arginine-NO pathway, arginine was found to be metabolized to urea, ornithine, and, to a much lesser extent, CO2 via arginase and ornithine aminotransferase. The activities of arginase and the formation of ornithine and urea from arginine were decreased by 90% in BBd compared with BBn cells. These results, coupled with the reduced NO synthesis, indicate metabolic defects in arginine metabolism in BBd EC. We suggest that arginine degradation in coronary EC occurs predominantly via the arginase path way. Our findings may offer a biochemical basis for the hitherto unexplained observations that endothelium-dependent relaxation is impaired in BBd rats and IDDM patients.