Activation of Human Neutrophil Procollagenase by Nitrogen Dioxide and Peroxynitrite: A Novel Mechanism for Procollagenase Activation Involving Nitric Oxide

The involvement of nitric oxide (NO) and its reactive intermediates such as nitrogen dioxide (NO2) and peroxynitrite (ONOO−) in the activation of matrix metalloproteinase was investigated. The human neutrophil procollagenase (matrix metalloproteinase-8) (Mr, 85 kDa) was purified to homogeneity from human neutrophils by using column chromatography. After incubation of human neutrophil procollagenase with various nitrogen oxide-generating systems, collagenolytic activity in each reaction system was measured. In addition, neutrophil collagenase activity was determined by assessment of proteolysis of human α1-protease inhibitor. NO was formed by the propylamine NONOate, and NO2was generated by oxidation of NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO). NO2, formed by NONOate and carboxy-PTIO, and the synthetic ONOO−exhibited strong activation of the procollagenase at 1–20 μm. Significant activation of the procollagenase was observed with use of authentic NO2gas as well. Constant flux infusion of ONOO−into the procollagenase solution resulted in stronger procollagenase activation than did a bolus addition of ONOO−to the reaction mixture. However, NO showed only weak activating potential under the aerobic (ambient) condition; an NO concentration of more than 10 mmwas needed for appreciable activation of the procollagenase. Of considerable importance was the fact that NO participates in activation of the neutrophil collagenase through its conversion to NO2or ONOO−in human neutrophils. These results suggest that NO2and ONOO−may be potent activators of human neutrophil procollagenase.