Effect of elastase on phospholipase activity in aortic smooth muscle cells.

We have studied the effects of elastase on phospholipase activity in aortic smooth muscle cells and have found that when added to cells prelabeled with [3H]arachidonic acid, elastase induced rapid phospholipid hydrolysis, resulting in release of up to 18% of incorporated [3H]arachidonic acid into the medium. Maximum stimulation by elastase without any cellular damage was observed at a concentration of 50 units/ml. At higher concentrations (75-100 units/ml), release of arachidonic acid was still observed, but cells were damaged. After the addition of elastase, degradation of phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine was observed and it was found that their loss was comparable to the amount of [3H]arachidonic acid released. In aortic smooth muscle cells biosynthetically labeled by the incorporation of [3H]choline, [3H]inositol and [3H]ethanolamine into cellular phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine, respectively, the amount of phosphatidylcholine and phosphatidylethanolamine hydrolyzed following elastase-treatment was not equal to the amounts of lysophosphatidylcholine and lysophosphatidylethanolamine produced. We also observed a transient rise in diacylglycerol after the addition of elastase. To test for phospholipase C activity, the release of incorporated [3H]choline, [3H]inositol and [3H]ethanolamine into the culture medium was determined. The levels of radioactive choline and ethanolamine showed increases, but the change in inositol was comparatively small. An increase in inositol was detectable within 1 min after elastase addition, and peaked after 15 min, whereas increases in choline and ethanolamine continued for up to 60 min. These results indicate that elastase stimulated the activities of phospholipases A2 and C. Both were shown to be Ca2+-dependent, and it was found that, moreover, elastase enhanced Ca2+ influx. These results suggest increased cell-membrane permeability to Ca2+-stimulated phospholipases A2 and C. Prostaglandin biosynthesis in these cells was also enhanced by elastase.