Phosphorylation site-specific antibodies, quantification of 32 P incorporation into phospholamban, and simultaneous measurements of mechanical activity were used in Langendorff-perfused rat hearts to provide further insights into the underlying mechanisms of phospholamban phosphorylation. Immunological detection of phospholamban phosphorylation sites showed that the isoproterenol concentration-dependent increase in phospholamban phosphorylation was due to increases in phosphorylation of both 16 and Thr 17 , respectively (3). These phosphorylations are independent of each other, and when both are operating, they appear to have an additive action (4). In the intact heart, ␤-adrenergic stimulation phosphorylates phospholamban at both sites (5), which indicates that PKA-and CaMKII-dependent pathways are also working in the functioning heart. Whether these phosphorylation mechanisms are independent of each other and additive, as described in the isolated SR membranes, remains unknown. Different attempts to phosphorylate phospholamban by CaMKII in the intact heart have systematically failed unless cAMP levels within the cell increase (6 -11). This consistent finding strongly suggests an interaction between PKA and CaMKII pathways of phospholamban phosphorylation in the intact heart. The nature of this interaction as well as the cause for the difference between the in vivo and in vitro results have never been explored.The availability of phosphorylation-site specific antibodies to phospholamban, which precisely discriminate between Ser 16 and Thr 17 phosphorylation sites (12), prompted us to reexamine the issue. Combination of this technique with the quantitative assessment of phospholamban phosphorylation by radiochemical labeling of ATP pools and simultaneous measurements of mechanical parameters allowed us to characterize the PKA and CaMKII-dependent mechanisms of phospholamban phosphorylation in the intact heart and their relative physiological roles on cardiac performance. EXPERIMENTAL PROCEDURESHeart Perfusions-Experiments were performed in isolated hearts from male Wistar rats (250 -350 g body wt) perfused according to the Langendorff technique as described previously (8). The composition of the physiological salt solution (PSS) was (in mM): 128.3 NaCl, 4.7 KCl, 1.35 CaCl 2 , 20.2 NaHCO 3 , 0.4 NaH 2 PO 4 , 1.1 MgCl 2 , 11.1 glucose, and 0.04 Na 2 EDTA. This solution was equilibrated with 95% O 2 , 5% CO 2 to give a pH of 7.4. The mechanical activity of the heart was assessed by either sewing an isometric strain gauge arch (Micro Measurements, type MA-06 -030LB-120) to the left ventricular wall or passing into the left ventricle a latex balloon connected to a pressure transducer (Namic, perceptor DT disposable transducer). The initial length of the gauge was set by stretching the segment attached by approximately 30%. The balloon was filled with aqueous solution to achieve a left ventricular

The present work was undertaken with two main goals: 1) to further elucidate the physiological role of the adenosine 3',5'-cyclic monophosphate (cAMP) and Ca2(+)-calmodulin (Ca2(+)-Cm)-dependent mechanisms of phospholamban phosphorylation (32PiPHL), and 2) to study the possible interaction between these two systems in the intact heart. Interventions that increased twitch or tetanic tension without modifying cAMP levels [high extracellular Ca2+ concentration [( Ca2+]o) or BAY K 8644 in catecholamine-depleted hearts] failed to alter 32PiPHL. Moderate and high beta-adrenergic stimulation (3 x 10(-9) and 3 x 10(-8) M isoproterenol, respectively) increased cAMP from 0.345 +/- 0.032 to 0.636 +/- 0.069 and 0.772 +/- 0.060 pmol/mg wet wt, and 32PiPHL from 26.8 +/- 4.1 to 58.6 +/- 13.1 and 174.7 +/- 13.8 pmol 32Pi/mg sarcoplasmic reticular [SR] protein, respectively. Both doses of isoproterenol produced an enhanced myocardial relaxation. Reversal of the positive inotropic effect of isoproterenol by interventions that decrease intracellular Ca2+ supply failed to reduce the enhancement in 32PiPHL and myocardial relaxation elicited by 3 x 10(-9) M isoproterenol but diminished the increase in 32PiPHL induced by 3 x 10(-8) M isoproterenol to 116.3 +/- 10.9 without significant changes in cAMP. Changes in myocardial relaxation closely paralleled the changes in 32PiPHL. These results suggest that 1) 32PiPHL may be enhanced by the cAMP-dependent mechanism independently of the Ca2(+)-Cm system, and 2) 32PiPHL and myocardial relaxation may be modified by intracellular Ca2+ changes only at high-intracellular cAMP levels.

The contribution of endoplasmic reticulum (ER) and phosphorylation of phospholamban (PLB) to the relaxant effect of cGMP- and cAMP-elevating agents was studied in feline aorta. Sodium nitroprusside (NP, 100 microM) completely relaxed contracture induced by 10 microM norepinephrine. This NP-induced relaxation was partially prevented by tetraethylammonium, suggesting that a fraction of NP-induced relaxation was mediated by activation of K(+) channels. In the absence and presence of tetraethylammonium, the relaxant effect of NP was associated with a significant increase in Ser(16) phosphorylation of PLB immunodetected by phosphorylation site-specific antibodies. The relaxant effect of NP on aortic strips precontracted with 80 mM KCl was significantly reduced by 1 microM thapsigargin. This decrease, which represents the ER contribution to the relaxant effect of NP, reached 23 +/- 9% at 100 microM NP and was closely associated with a dose-dependent increase in Ser(16) phosphorylation (128 +/- 49% over control at 100 microM NP). Effects of NP were associated with a significant increase in activity of protein kinase G and were mimicked by 8-bromo-cGMP. Forskolin produced a dose-dependent relaxant effect on KCl-induced contracture, which reached 64 +/- 8% at 50 microM and was associated with an increase in phosphorylation of Ser(16) residue of PLB (88 +/- 18% over control). Thapsigargin reduced this relaxant effect by 38 +/- 9%. 8-Bromo-cAMP mimicked effects of forskolin. The ER-mediated relaxant effect and the increase in Ser(16) phosphorylation produced by forskolin were partially blocked by the protein kinase A inhibitor H-89 (5 microM). The results indicate that ER partially contributes to the relaxant effect of NP and forskolin in feline aorta. This effect may be mediated by the associated increase in Ser(16) phosphorylation of PLB.

The role of the Ca(2+)-calmodulin dependent pathway of phospholamban phosphorylation on the relaxant effect of beta-adrenergic agonists was studied in isolated perfused rat heart. Administration of the calmodulin antagonist W7 or lowering [Ca]o from 1.35 mM (control) to 0.25 mM, were used as experimental tools to inhibit the Ca(2+)-calmodulin dependent protein kinase activity. 3 x 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 pmol 32P/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]o. Low [Ca]o did not affect the increase in cAMP elicited by isoproterenol but offset the positive inotropic effect of the beta-agonist. The results suggest a physiological role of the Ca(2+)-calmodulin dependent phospholamban phosphorylation pathway as a mechanism that supports, in part, the beta-adrenergic cardiac relaxant effect.