MECHANISMS INVOLVED IN THE ACIDOSIS ENHANCEMENT OF THE ISOPROTERENOL-INDUCED PHOSPHORYLATION OF PHOSPHOLAMBAN IN THE INTACT HEART

Previous experiments have shown that acidosis enhances isoproterenol-i nduced phospholamban (PHL) phosphorylation (Mundina-Weilemann, C., Vit tone, L., Cingolani, H. E., Orchard, C. H. (1996) Am. J. Physiol. 270, C107-C114). In the present experiments, performed in isolated Langend orff perfused rat hearts, phosphorylation site-specific antibodies to PHL combined with the quantitative measurement of P-32 incorporation i nto PHL were used as experimental tools to gain further insight into t he mechanism involved in this effect. At all isoproterenol concentrati ons tested (3-300 nM), phosphorylation of Thr(17) of PHL was significa ntly higher at pH(o) 6.80 than at pH(o) 7.40, without significant chan ges in Ser(16) phosphorylation, This increase in Thr(17) phosphorylati on was associated with an enhancement of the isoproterenol-induced rel axant effect. In the absence of isoproterenol, the increase in [Ca](o) at pH(o) 6.80 (but not at pH(o) 7.40) evoked an increase in PHL phosp horylation that was exclusively due to an increase in Thr(17) phosphor ylation and that was also associated with a significant relaxant effec t. This effect and the phosphorylation of Thr(17) evoked by acidosis w ere both offset by the Ca2+/calmodulin-dependent protein kinase II inh ibitor KN-62. In the presence of isoproterenol, either the increase in [Ca](o) or the addition of a 1 mu M concentration of the phosphatase inhibitor okadaic acid was able to mimic the increase in isoproterenol -induced Thr(17) phosphorylation produced by acidosis. In contrast, th ese two interventions have opposite effects on phosphorylation of Ser( 16). Whereas the increase in [Ca](o) significantly decreased phosphory lation of Ser(16), the addition of okadaic acid significantly increase d the phosphorylation of this residue. The results are consistent with the hypothesis that the increase in phospholamban phosphorylation pro duced by acidosis in the presence of isoproterenol is the consequence of two different mechanisms triggered by acidosis: an increase in [Ca2 +](i) and an inhibition of phosphatases.