Release of phospholipase A 2 activity from rat vascular smooth muscle cells mediated by cAMP

Primary cultures of smooth muscle cells (SMC) derived from rat aorta release a phospholipase A 2 activity into the culture medium. Phospholipase A 2 activity was determined with [1‐ 14 C]oleate‐labelled Escherichia coli as substrate. The enzyme has a neutral pH optimum and the activity is critically dependent on the free calcium concentration, with significant activity in the micromolar range of free calcium. Treatment of SMC with the β agonist salbutamol, forskolin or cholera toxin, which all activate adenylate cyclase and increase intracellular cAMP concentration, increase the release of phospholipase A 2 activity in a dose‐dependent manner. Likewise, the addition of the membrane‐permeable cAMP analogues, ( S p)‐adenosine 3′,5′‐[thio]phosphate and N 6 , O ‐2′ ‐dibutyryladenosine 3′,5′‐phosphate, enhance the release of phospholipase A 2 activity from SMC in a dosedependent manner. There is a lag period of about 4 h before a significant secretion of phospholipase A 2 can be detected under basal, as well as under stimulated conditions. The forskolin analogue 1,9‐dideoxyforskolin, which is inactive as a stimulator of adenylate cyclase, has no effect on phospholipase A 2 secretion. Likewise, the potent vasoconstrictive peptide angiotensin II activates inositol phospholipid turnover in SMC, but has no effect on phospholipase A 2 release. Pretreatment of SMC with actinomycin D or cycloheximide completely suppresses basal and cAMP‐stimulated secretion of phospholipase A 2 activity, thus demonstrating that transcription and protein synthesis are necessary for enzyme release.