The Cytosolic Phospholipase A sub(2) Pathway, a Safeguard of beta sub(2)-Adrenergic Cardiac Effects in Rat

We have recently demonstrated that in human heart, beta sub(2)-adrenergic receptors ( beta sub(2)-ARs) are biochemically coupled not only to the classical adenylyl cyclase (AC) pathway but also to the cytosolic phospholipase A sub(2) (cPLA sub(2)) pathway (Pavoine, C., Behforouz, N., Gauthier, C., Le Gouvello, S., Roudot-Thoraval, F., Martin, C. R., Pawlak, A., Feral, C., Defer, N., Houel, R., Magne, S., Amadou, A., Loisance, D., Duvaldestin, P., and Pecker, F. (2003) Mol. Pharmacol. 64, 1117-1125). In this study, using Fura-2-loaded cardiomyocytes isolated from adult rats, we showed that stimulation of beta sub(2)-ARs triggered an increase in the amplitude of electrically stimulated [Ca super(2+)] sub(i) transients and contractions. This effect was abolished with the PKA inhibitor, H89, but greatly enhanced upon addition of the selective cPLA sub(2) inhibitor, AACOCF sub(3). The beta sub(2)-AR/cPLA sub(2) inhibitory pathway involved G sub(i) and MSK1. Potentiation of beta sub(2)-AR/AC/PKA-induced Ca super(2+) responses by AACOCF sub(3) did not rely on the enhancement of AC activity but was associated with eNOS phosphorylation (Ser super(1177)) and L-NAME-sensitive NO production. This was correlated with PKA-dependent phosphorylation of PLB (Ser super(16)). The constraint exerted by the beta sub(2)-AR/cPLA sub(2) pathway on the beta sub(2)-AR/AC/PKA-induced Ca super(2+)responses required integrity of caveolar structures and was impaired by Filipin III treatment. Immunoblot analyses demonstrated zinterol-induced translocation of cPLA and its cosedimentation with MSK1, eNOS, PLB, and sarcoplasmic reticulum Ca super(2+) pump (SERCA) 2a in a low density caveolin-3-enriched membrane fraction. This inferred the gathering of beta sub(2)-AR signaling effectors around caveolae/sarcoplasmic reticulum (SR) functional platforms. Taken together, these data highlight cPLA as a cardiac beta sub(2)-AR signaling pathway that limits beta sub(2)-AR/AC/PKA-induced Ca super(2+) responses in adult rat cardiomyocytes through the impairment of eNOS activation and PLB phosphorylation.