The role of the Ca2+-calmodulin dependent pathway of phospholamban phosphorylation on the relaxant effect of β-adrenergic agonists was studied in isolated perfused rat heart. Administration of the calmodulin antagonist W7 or lowering [Ca]0 from 1.35 mM (control) to 0.25 mM, were used as experimental tools to inhibit the Ca2+-calmodulin dependent protein kinase activity. 3×10−8 M isoproterenol increased cAMP levels from 0.613±0.109 pmol/mg wet weight to 1.581±0.123, phospholamban phosphorylation from 36±6 pmol32P/mg protein to 277±26 and decreased time to half relaxation (t1/2) from 61±2 msec to 39±2. Simultaneous perfusion of isoproterenol with 10−6 M W7, decreased phospholamban phosphorylation to 170±23 and prolongated t1/2 to 47±3 but did not affect the increase either in cAMP levels or myocardial contractility produced by isoproterenol. Similar effects on phospholamban phosphorylation and myocardial relaxation were obtained when isoproterenol was perfused in low [Ca]0. Low [Ca]0 did not affect the increase in cAMP elicited by isoproterenol but offset the positive inotropic effect of the β-agonist.Centro de Investigaciones Cardiovasculares