Sarcoplasmic reticulum (SR) dysfunction is one of the multiple alterations that 
occurs in ischemia-reperfused hearts. Because SR function is regulated by 
phosphorylation of phospholamban (PLB), a SR protein phosphorylated by 
cAMP-dependent protein kinase (PKA) at Ser(16)and Ca(2+)-calmodulin-dependent 
protein kinase (CaMKII) at Thr(17), the phosphorylation of these residues during 
ischemia and reperfusion was examined in Langendorff-perfused rat hearts. 
Ser(16)phosphorylation increased significantly after 20 min of ischemia from 
2.5+/-0.6% to 99.8+/-25.5% of maximal isoproterenol-induced site-specific 
phosphorylation and decreased to control values immediately after reperfusion. 
Thr(17)phosphorylation transiently increased at 2-5 min of ischemia and at 1 min 
of reperfusion (R1, 166.2+/-28.2%). The ischemia-induced increase in 
Ser(16)phosphorylation was significantly diminished in hearts from 
catecholamine-depleted animals and/or after beta-blockade and abolished in the 
presence of the PKA-inhibitor, H-89. Thr(17)phosphorylation at the beginning of 
ischemia was blunted by nifedipine, whereas at R1 it was significantly 
diminished by perfusion with 0 m m Ca(2+)in the presence of EGTA and by the 
Na(+)/Ca(2+)exchanger inhibitor KB-R7943. KN-93, used to specifically inhibit 
CaMKII, decreased Thr(17)phosphorylation at R1 and significantly prolonged half 
relaxation time. The results demonstrated a dissociation between the 
phosphorylation of PLB sites, being phosphorylation of Ser(16)dependent on the 
beta-adrenergic cascade during ischemia and phosphorylation of Thr(17)on 
Ca(2+)influx both, at the beginning of ischemia and reperfusion. Phosphorylation 
of Thr(17)at the onset of reflow may provide the cell a mechanism to cope with 
Ca(2+)overload, transiently favoring the recovery of relaxation during early 
reperfusion.