METHYLXANTHINES AND CALCIUM-MOBILIZING AGENTS INHIBIT THE EXPRESSION OF CYTOKINE-INDUCIBLE NITRIC-OXIDE SYNTHASE AND VASCULAR CELL-ADHESIONMOLECULE-1 IN MURINE MICROVASCULAR ENDOTHELIAL-CELLS

In response to exposure to the inflammatory cytokines tumor necrosis f actor-ct (TNF) and interferon-gamma (IFN-gamma), murine brain microvas cular endothelial cells (MME) synthesize the cell surface molecule, va scular cell adhesion molecule-1 (VCAM-1), and the intracellular enzyme , inducible nitric oxide synthase (iNOS). However, iNOS synthesis requ ires the presence of both TNF and IFN-gamma, while VCAM-1 can be induc ed by either cytokine alone. We examined the induction of VCAM-1 and i NOS under a variety of conditions to better define the regulation of T NF and LFN-gamma signal transduction pathways in MME. We utilized the analysis of steady-state levels of iNOS mRNA as well as the measuremen t of MME-released NO-EDRF (nitric oxide as an endothelium-derived rela xing factor) activity and accumulation of nitrite in the culture mediu m to define iNOS expression and activity. VCAM-1 expression was determ ined by flow cytometric analysis. Our data indicate that low density l ipoproteins inhibited cytokine-induced iNOS activity by affecting the steady-state levels of iNOS mRNA. Methylxanthines (caffeine and theoph ylline) as well as several calcium-mobilizing agents inhibited the exp ression/activity of both iNOS and VCAM-1 in MME. The effectiveness of these agents was dependent upon the degree of disruption in cell calci um homeostasis during cytokine treatment. Cells which had been pretrea ted with calcium-modulating drugs and then washed and allowed to retur n to normal calcium homeostasis showed little to no effect from these agents. In addition, our results suggest that NO produced by iNOS acts as a metabolic switch during inflammation by inhibiting oxidative pho sphorylation and forcing vascular endothelial cells to temporarily uti lize anaerobic energy metabolism. (C) 1994 Academic Press,Inc.