Effects of TNF-α on the Production of Vasoactive Substances by Cerebral Endothelial and Smooth Muscle Cells in Culture

Abstract

The effects of tumor necrosis factor-α (TNF-α) 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-α (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-α in BCEC. TNF-α 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-α. ET-1 was constitutively secreted by BCEC. The production of ET-1 was stimulated by thrombin. TNF-α enhanced the release of ET-1 in BCEC, and this enhancement was not modified by the simultaneous addition of interferon-γ (IFN-γ). BCSMC did not produce ET-1, either spontaneously or in the presence of TNF-α, IFN-γ, or of both together. These effects of TNF-α 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.