ABSTRACT
Cardiovascular diseases involve critical mechanisms including impaired nitric oxide (NO) levels and abnormal matrix metalloproteinase (MMP) activity. While NO downregulates MMP expression in some cell types, no previous study has examined whether NO downregulates MMP levels in endothelial cells. We hypothesized that NO donors could attenuate MMP-9 production by human umbilical vein endothelial cells (HUVECs) as a result of less NFκB activation or cyclic GMP (cGMP)-mediated mechanisms. We studied the effects of DetaNONOate (10-400 µM) or SNAP (50-400 µM) on phorbol 12-myristate 13-acetate (PMA; 10 nM)-induced increases in MMP-9 activity (by gel zymography) or concentrations (by ELISA) as well as on a tissue inhibitor of MMPs' (TIMP)-1 concentrations (by ELISA) in the conditioned medium of HUVECs incubated for 24 h with these drugs. We also examined whether the irreversible inhibitor of soluble guanylyl cyclase ODQ modified the effects of SNAP or whether 8-bromo-cGMP (a cell-permeable analog of cGMP) influenced PMA-induced effects on MMP-9 expression. Total and phospho-NFκB p65 concentrations were measured in HUVEC lysates to assess NFκB activation. Both NO donors attenuated PMA-induced increases in MMP-9 activity and concentrations without significantly affecting TIMP-1 concentrations. This effect was not modified by ODQ, and 8-bromo-cGMP did not affect MMP-9 concentrations. While PMA increased phospho-NFκB p65 concentrations, SNAP had no influence on this effect. In conclusion, this study shows that NO donors may attenuate imbalanced MMP expression and activity in endothelial cells independent of cGMP- or NFκB-mediated mechanisms. Our results may offer an important pharmacological strategy to approach cardiovascular diseases.
Subject(s)
Cyclic GMP/physiology , Human Umbilical Vein Endothelial Cells/enzymology , Matrix Metalloproteinase 9/metabolism , Nitric Oxide/physiology , Transcription Factor RelA/metabolism , Cells, Cultured , Culture Media, Conditioned , Enzyme Repression/drug effects , Gene Expression , Humans , Matrix Metalloproteinase 9/genetics , Nitric Oxide/pharmacology , Nitric Oxide Donors/pharmacology , Nitriles/pharmacology , Nitroso Compounds/pharmacology , Phosphorylation , Protein Processing, Post-Translational , Sulfones/pharmacology , Tetradecanoylphorbol Acetate/pharmacology , Tissue Inhibitor of Metalloproteinase-1/genetics , Tissue Inhibitor of Metalloproteinase-1/metabolism , Transcription Factor RelA/antagonists & inhibitors , Transcription, GeneticABSTRACT
Malathion is an organophosphate (OP) pesticide whose toxicity depends on its bioactivation to malaoxon. Human malathion poisoning has been treated with oximes (mainly pralidoxime) in an attempt to reactivate OP-inhibited acetylcholinesterase (AChE). However, pralidoxime has shown unsatisfactory therapeutic effects in malathion poisoning and its routine use has been questioned. In this study, we evaluated the in vitro potency of standards and newly developed oximes in reactivating malaoxon-inhibited AChE derived from mouse brain supernatants. Malaoxon displayed a concentration-dependent inhibitory effect on mouse brain AChE (IC(50) = 2.36 microM), and pralidoxime caused a modest reactivating effect (30% of reactivation at 600 microM). Obidoxime and trimedoxime, as well as K047 and K075, displayed higher reactivating effects (from 55% to 70% of reactivation at 600 muM) when compared with pralidoxime. The results show that obidoxime, trimedoxime, K074 and K075 present higher reactivating effects on malaoxon-inhibited AChE under in vitro conditions when compared with pralidoxime. Taking into account the unsatisfactory effects of pralidoxime as antidotal treatment in malathion poisonings, the present results suggest that obidoxime, trimedoxime, K074 and K075 might be interesting therapeutic strategies to reactivate malaoxon-inhibited AChE in malathion poisonings.