Protein phosphatase inhibitor-1 (PPI-1) has been shown to be present in heart tissue and smooth muscle. Whether PPI-1 is present in cardiomyocytes is not known. The purpose of this study was to determine whether PPI-1 is present and is hormonally regulated in cardiomyocytes. A trichloroacetic acid (TCA) extract enriched in PPI-1 was isolated from guinea pig ventricular cardiomyocytes. The TCA extract inhibited the activity of type 1 protein phosphatase by 20 +/- 4% (n = 3 expts). On phosphorylation by the catalytic subunit of adenosine 3', 5'-cyclic monophosphate-dependent protein kinase, the extent of this inhibition was augmented to 4.5-fold. Dephosphorylation of the phosphorylated TCA extract by type 2 protein phosphatase reduced inhibition to 2 +/- 0.2% (n = 3 expts). To determine whether isoproterenol increases phosphorylation of PPI-1 in cardiomyocytes, the TCA extracts were prepared from cardiomyocytes treated with 1 microM isoproterenol and from untreated cardiomyocytes. The inhibitory activity of the TCA extract in untreated cardiomyocytes was 25 +/- 3% (n = 3 expts) and increased to 75 +/- 2% (n = 3 expts) in isoproterenol-treated cardiomyocytes. With the use of a rabbit skeletal muscle PPI-1 antibody, immunoblots of the TCA extract of cardiomyocytes identified a 28-kDa protein. A 28-kDa protein was also immunoprecipitated from a TCA extract isolated from isoproterenol-treated 32P-labeled cardiomyocytes. The immunoprecipitation was blocked by the addition of excess amounts of purified rabbit skeletal muscle PPI-1. Isoproterenol-treated cardiomyocytes increased the phosphorylation of the 28-kDa protein by 232 +/- 20% (n = 3 expts) compared with untreated cardiomyocytes. We conclude that 1) the 28-kDa protein is PPI-1, 2) PPI-1 is present in ventricular cardiomyocytes, and 3) PPI-1 is hormonally regulated. A decrease in type 1 protein phosphatase activity through phosphorylation of PPI-1 may be an important pathway for augmenting cardiac contractility.
Hormonal regulation of protein dephosphorylation. Identification and hormonal regulation of protein phosphatase inhibitor-1 in rat adipose tissue.
