The calcium-dependent association and functional coupling of calmodulin with myocardial phospholipase A₂. Implications for cardiac cycle-dependent alterations in phospholipolysis

Herein we demonstrate the calcium-dependent regulation of myocardial phospholipase A₂ activity, which is mediated by a cytosolic protein constituent that can be chromatographically resolved from, and subsequently reconstituted with, purified myocardial phospholipase A₂. Purification of this protein by sequential column chromatographies revealed an 18-kDa doublet, which was identified as calmodulin by Western blotting, calcium- dependent precipitation with W-7 agarose beads, and reconstitution of calcium-mediated phospholipase A₂ inhibition with authentic homogeneous calmodulin. Calcium-induced calmodulin-mediated inhibition of myocardial phospholipase A₂ was titrated by physiologic increments of calcium ion (K(d) ~200 nm). Moreover, ternary complex affinity chromatography with calmodulin- Sepharose demonstrated that inhibition of myocardial phospholipase A₂ activity by calmodulin resulted from the direct interaction of calmodulin with the myocardial phospholipase A₂ catalytic complex. Exposure of cultured A-10 muscle cells to three structurally disparate calmodulin antagonists (W- 7, trifluoperazine, and calmidazolium) resulted in the robust release of arachidonic acid, which was entirely ablated by pretreatment of cells with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2-H-tetrahydropyran-2-one. Collectively, this study identifies a novel mechanism whereby latent phospholipase A₂ activity can be released from tonic inhibition by alterations in the interactions between the phospholipase A₂ catalytic complex, calcium ion, and the intracellular calcium transducer, calmodulin.