Role of membrane-associated Ca~(2+) dependent matrix metalloprotease-2 in the oxidant activation of Ca~(2+) ATPase by tertiary butylhydroperoxide

摘要

Treatment of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant tert-butylhydroperoxide (t-buOOH) increases Ca~(2+)ATPase activity. The smooth muscle plasma membrane possesses a Ca~(2+) dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA and the tissue inhibitor of metalloprotease-2 (TIMP-2). Since 72 kDa is the molecular mass of MMP-2 and since in our present study the 72 kDa protease in the gelatin containing zymogram is inhibited by matrix metalloprotease inhibitors, EGTA and TIMP-2, it may be suggested that the 72 kDa protease is the MMP-2. In addition to the increasing Ca~(2+)ATPase activity, t-buOOH also enhances the activity of the membrane associated Ca~(2+) dependent protease that degrades ~(14)C-gelatin. The oxidant triggered protease activity and the Ca~(2+)ATPase activity were found to be prevented by the antioxidant vitamin E, and also by the Ca~(2+) dependent matrix metalloprotease inhibitors: EGTA and TIMP-2. Adding MMP-2 to the smooth muscle plasma membrane suspension caused an increase in Ca~(2+)ATPase activity and pretreatment with TIMP-2 prevents the increase in Ca~(2+)ATPase activity. Combined treatment of the smooth muscle plasma membrane with low doses of MMP-2 and t-buOOH augments further the Ca~(2+)ATPase activity caused by the respective doses of either t-buOOH or MMP-2. Pretreatment with TIMP-2 prevents the increase in Ca~(2+)ATPase activity elicited by the low doses of MMP-2 and/or t-buOOH.