Am. J. Respir. Crit. Care Med., Volume 156, Number 6, December 1997, 1846-1854

Endotoxin Impairs Agonist-induced Calcium Mobilization in Rat Mesangial Cells

PATRICK T. MURRAY, MARK E. WYLAM, and JASON G. UMANS

Departments of Anesthesia and Critical Care, Medicine, and Pediatrics; and Committee on Clinical Pharmacology, Division of the Biological Sciences, University of Chicago, Chicago, Illinois

We hypothesized that endotoxin would impair agonist-induced calcium (Ca²⁺) mobilization in rat mesangial cells, owing to the induction of nitric oxide synthase (NOS) and augmented nitric oxide (NO) synthesis. We measured basal and bradykinin-induced peak free cytosolic Ca²⁺ concentrations through microspectrofluorimetry with fura-2 in confluent mesangial cells, and assayed conditioned medium for nitrite accumulation. Prior to measurement, cells were incubated overnight in serum-supplemented medium, with or without endotoxin, L-arginine, indomethacin, meclofenamate, or N-nitro-L-arginine methyl ester (L-NAME). Endotoxin (1 mg/ml) decreased bradykinin-induced peak Ca²⁺ responses by 35 to 60% (p < 0.0001) and increased nitrite accumulation > 6-fold (p < 0.01). Arginine supplementation further (> 9-fold, p < 0.0001) increased nitrite accumulation without changing the effect on Ca²⁺. Inhibition of NOS abolished increments in nitrite concentration but had no effect on impaired Ca²⁺ responses. Cyclooxygenase (COX) inhibitors, present during incubation with endotoxin, but not afterward, normalized bradykinin-stimulated calcium responses. Thrombin-stimulated Ca²⁺ responses were similarly affected. We conclude that neither NO nor prostaglandins act directly to impair agonist-induced Ca²⁺ mobilization following endotoxin exposure; however, this effect may be an indirect effect of COX products, including reactive oxygen intermediates.

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