Attenuation of Endotoxin-Induced Cytotoxicity and Prostacyclin Production in Cultured Bovine Pulmonary Artery Endothelial Cells by Phosphodiesterase Inhibition

Abstract

Exposure of cultured bovine pulmonary endothelial cells to endotoxin (lipopolysaccharide, LPS) causes cytotoxicity and increased prostacyclin production. Since cyclic nucleotides have been proposed as modulators of inflammation, we wondered whether they were involved in LPS-induced endothelial damage. Bovine pulmonary endothelial cells were exposed for 24 h to LPS and the effects of 1-methyl-3-isobutylxanthine (MIX), a phosphodiesterase inhibitor, dibutyryl cyclic AMP (db-cAMP), forskolin (an adenylate cyclase activator), and sodium nitroprusside (an agent known to stimulate intracellular cyclic GMP generation) on LPS-induced injury were determined. Injury was assessed by measurement of lactate dehydrogenase (LDH) (activity) and prostacyclin (6-keto-PGF1 alpha) in the bathing medium. Incubation with MIX attenuated LPS-induced endothelial cytotoxicity and prostacyclin production in a dose-dependent manner (ANOVA, p less than 0.001). Dibutyryl cyclic AMP also inhibited LPS-stimulated LDH release from the endothelial cells but did not suppress increased prostacyclin production. The combinations of MIX and dibutyryl cyclic AMP produced protection similar to that of MIX alone. Neither nitroprusside nor forskolin affected LPS-induced endothelial injury. Measurements of intracellular cyclic nucleotide concentrations showed that MIX caused marked increases in both cyclic AMP and cyclic GMP within 30 min of incubation, while forskolin and nitroprusside failed to cause such early elevations. Thus, phosphodiesterase inhibition protects endothelial cells from the effects of LPS. Increased intracellular concentrations of cyclic AMP also protect endothelial cells from LPS-induced cytotoxicity but do not alter the prostanoid response. We conclude that increased intracellular concentrations of cyclic AMP protect against LPS-induced endothelial cytotoxicity if present early in the exposure. We further conclude that LPS-mediated endothelial cytotoxicity can be separated from increased prostacyclin production.