Role of regucalcin as an activator of sarcoplasmic reticulum Ca2+-ATPase activity in rat heart muscle

Abstract

The expression of regucalcin, a regulatory protein of Ca2+ signaling, and its effect on Ca2+ pump activity in the microsomes (sarcoplasmic reticulum) of rat heart muscle was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in heart muscle using rat regucalcin-specific primers. Results with Western blot analysis showed that regucalcin protein was present in the cytoplasm, although it was not detected in the microsomes. Microsomal Ca2+-ATPase activity was significantly increased in the presence of regucalcin (10-10-10-8 M) in the enzyme reaction mixture. This increase was not seen in the presence of thapsigargin (TP) (10-5 M), a specific inhibitor of the microsomal Ca2+ pump enzyme. Regucalcin (10-10-10-8 M) significantly stimulated ATP-dependent 45Ca2+ uptake by the microsomes. The effect of regucalcin (10-8 M) in increasing microsomal Ca2+-ATPase activity was completely prevented in the presence of digitonin (10-3 or 10-2%), which has a solubilizing effect on membranous lipid, or N-ethylmaleimide (NEM), a modifying reagent of sulfhydryl (SH) groups. Dithiothreitol (DTT; 5 mM), a protecting reagent of SH groups, increased markedly Ca2+-ATPase activity. In the presence of DTT (5 mM), regucalcin could not significantly enhance the enzyme activity. Also, the effect of regucalcin in increasing Ca2+-ATPase activity was completely inhibited by the addition of vanadate (1 mM), an inhibitor of phosphorylation of enzyme. In addition, the effect of regucalcin on Ca2+-ATPase activity was not significantly modulated in the presence of dibutyryl cyclic AMP (10-4 M), inositol 1,4,5-trisphosphate (10-3 M), or calmodulin (5 μg/ml) which is an intracellular signaling factor. The present study demonstrates that regucalcin can activate Ca2+ pump activity in rat heart microsomes, and that the protein may act the SH groups of Ca2+-ATPase by binding to microsomal membranes. © 2002 Wiley-Liss, Inc.