Role of an aprotinin-sensitive protease in the activation of Ca2+-ATPase by superoxide radical (O-2(-center dot)) in microsomes of pulmonary vascular smooth muscle

We have investigated the role of an aprotinin-sensitive protease in regulating Ca2+-ATPase activity and Ca2+ uptake (ATP-dependent and Na+-dependent) in microsomes of bovine pulmonary vascular smooth muscle during treatment with the O-2(-.)-generating system hypoxanthine plus xanthine oxidase. Treatment of the smooth muscle microsomes with the O-2(-.)-generating system produced a protease in a gelatin-containing zymogram with an apparent molecular mass of 16 kDa. This 16 kDa proteolytic protein was found to be inhibited by superoxide dismutase (SOD) and aprotinin but not by PMSF. Using polyclonal antiserum to aprotinin, we found that it is an ambient antiprotease of the smooth muscle microsomes. Treatment of the microsomes with the O-2(-.)-generating system stimulated protease activity tested with a synthetic substrate N-benzoyl-DL-arginine p-nitroanilide and also enhanced Ca2+-ATPase activity. It also stimulated ATP-dependent Ca2+ uptake. In contrast, Na+-dependent Ca2+ uptake was found to be inhibited by the O-2(-.)-generating system. Pretreatment of the microsomes with SOD and aprotinin preserved the increase in protease activity, Ca2+-ATPase activity and ATP-dependent Ca2+ uptake. In addition, O-2(-.)-caused inhibition of the Na+-dependent Ca2+ uptake which was reversed by SOD and aprotinin. Pretreatment with PMSF did not cause any discernible alteration in the protease activity, Ca2+-ATPase activity, ATP-dependent Ca2+ uptake and Na+-dependent Ca2+ uptake in the microsomes caused by the O-2(-.)-generating system. These results suggest that an aprotinin-sensitive protease plays a pivotal role in regulating Ca2+-ATPase and Ca2+-uptake activities in microsomes of pulmonary vascular smooth muscle under oxidant O-2(-.)-triggered conditions.