L. Vittone et al., MECHANISMS INVOLVED IN THE ACIDOSIS ENHANCEMENT OF THE ISOPROTERENOL-INDUCED PHOSPHORYLATION OF PHOSPHOLAMBAN IN THE INTACT HEART, The Journal of biological chemistry, 273(16), 1998, pp. 9804-9811
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.