EFFECTS OF TNF-ALPHA ON THE PRODUCTION OF VASOACTIVE SUBSTANCES BY CEREBRAL ENDOTHELIAL AND SMOOTH-MUSCLE CELLS IN CULTURE

The effects of tumor necrosis factor-alpha. (TNF-alpha) on the production of the vasoactive substances nitric oxide (NO) and endothelin-1 (ET-1) were investigated in cerebrovascular cells in culture. Bovine cerebral endothelial cells (BCEC) stained positively for NADPH-diaphorase/NO synthase activity and spontaneously produced nitrite, a stable NO oxidation product, which accumulated in the culture medium in a linear way for 48 h. Low concentrations of TNF-alpha (0.5-2 ng/ml) significantly enhanced nitrite production after a 24-h incubation. Higher concentrations or longer exposure times resulted in a cytotoxic effect that altered cell morphology, released lactate dehydrogenase (LDH) to the culture medium, and reduced the protein content. Dexamethasone, but not the NO synthase inhibitor N-iminoethyl-L-ornithine (L-NIO), prevented the cytotoxic effect of TNF-alpha in BCEC. TNF-alpha also significantly enhanced nitrite production in bovine cerebral smooth muscle cells (BCSMC). The enhancement was detected at all times between 8 and 72 h and at all concentrations tested (2-100 ng/ml). Signs of cytotoxicity were not observed in BCSMC after incubation with TNF-alpha, ET-1 was constitutively secreted by BCEC. The production of ET-1 was stimulated by thrombin. TNF-alpha enhanced the release of ET-1 in BCEC, and this enhancement was not modified by the simultaneous addition of interferon-gamma (IFN-gamma). BCSMC did not produce ET-1, either spontaneously or in the presence of TNF-alpha, IFN-gamma, or of both together. These effects of TNF-alpha on cerebrovascular cells may explain the microvascular alterations found in some inflammatory or traumatic conditions in which this cytokine is increased in the cerebral tissue.