Stimulatory effect of regucalcin on mitochondrial ATP-dependent calcium uptake activity in rat kidney cortex

The effect of regucalcin, which is a regulatory protein of Ca2+ signaling, on Ca2+-ATPase activity in isolated rat renal cortex mitochondria was investigated. The presence of regucalcin (50, 100, and 250 nM) in the enzyme reaction mixture led to a significant increase in Ca2+-ATPase activity. Regucalcin significantly stimulated ATP-dependent Ca-45(2+) uptake by the mitochondria. Ruthenium red (10(-6) M) or lanthunum chloride (10(-6) M), an inhibitor of mitochondrial Ca2+ uptake, markedly inhibited regucalcin (100 nM)-increased mitochondrial Ca2+-ATPase activity and Ca-45(2+) uptake. The effect of regucalcin (100 nM) in elevating Ca2+-ATPase activity was completely prevented by the presence of digitonin (10-2%), a solubilizing reagent of membranous lipids, vanadate, an inhibitor of phosphorylation of ATPase, or dithiothreitol (50 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme. The activating effect of regucalcin (100 nM) on Ca2+-ATPase activity was not further enhanced by calmodulin (0.30 muM) or dibutyryl cyclic AMP (10(-4) M), which could increase Ca2+-ATPase activity. Trifluoperazine (TFP; 50 muM), an antagonist of calmodulin, significantly decreased Ca2+-ATPase activity. The activating effect of regucalcin on the enzyme was also seen in the presence of TFP, indicating that regucalcin's effect is not involved in mitochondrial calmodulin. The present study demonstrates that regucalcin can stimulate Ca2+-pump activity in rat renal cortex mitochondria, and that the protein may act on an active site (SH group) related to phosphorylation of mitochondrial Ca2+-ATPase. J. Cell. Biochem. 80:285-292, 2000. (C) 2000 Wiley-Liss, Inc.