ENDOTOXIN IMPAIRS AGONIST-INDUCED CALCIUM MOBILIZATION IN RAT MESANGIAL CELLS

We hypothesized that endotoxin would impair agonist-induced calcium (C a2+) mobilization in rat mesangial cells, owing to the induction of ni tric oxide synthase (NOS) and augmented nitric oxide (NO) synthesis. W e measured basal and bradykinin-induced peak free cytosolic Ca2+ conce ntrations through microspectrofluorimetry with fura-2 in confluent mes angial cells, and assayed conditioned medium for nitrite accumulation. Prior to measurement, cells were incubated overnight in serum-supplem ented medium, with or without endotoxin, L-arginine, indomethacin, mec lofenamate, or N-(sic)-nitro-Larginine methyl ester (L-NAME). Endotoxi n (1 mg/ml) decreased bradykinin-induced peak Ca2+ responses by 35 to 60% (p < 0.0001) and increased nitrite accumulation > B-fold (p < 0.01 ). Arginine supplementation further (> 9-fold, p < 0.0001) increased n itrite accumulation without changing the effect on Ca2+. Inhibition of NOS abolished increments in nitrite concentration but had no effect o n impaired Ca2+ responses. Cyclooxygenase (COX) inhibitors, present du ring incubation with endotoxin, but not afterward, normalized bradykin in-stimulated calcium responses. Thrombin-stimulated Ca2+ responses we re similarly affected. We conclude that neither NO nor prostaglandins act directly to impair agonist-induced Ca2+ mobilization following end otoxin exposure; however, this effect may be an indirect effect of COX products, including reactive oxygen intermediates.