Suppressive effect of endogenous regucalcin on nitric oxide synthase activity in cloned rat hepatoma H4-II-E cells overexpressing regucalcin.
The role of endogenous regucalcin, which is a regulatory protein in calcium signaling, in the regulation of nitric oxide (NO) synthase activity in the cloned rat hepatoma H4-II-E cells was investigated. Hepatoma cells were cultured for 24-72 h in the presence of fetal bovine serum (FBS; 10%). NO synthase activity in the 5,500 g supernatant of cell homogenate was significantly increased by the addition of calcium chloride (10 microM) and calmodulin (2.5 microg/ml) in the enzyme reaction mixture. The presence of trifluoperazine (TFP; 50 microM), an antagonist of calmodulin, inhibited the effect of calcium (10 microM) addition in increasing NO synthase activity, indicating the existence of Ca(2+)/calmodulin-dependent NO synthase in hepatoma cells. NO synthase activity was significantly decreased by the addition of regucalcin (10(-8) or 10(-7) M) in the reaction mixture without or with Ca(2+)/calmodulin addition. The effect of regucalcin (10(-7) M) in decreasing NO synthase activity was also seen in the presence of TFP (50 microM) or EGTA (1 mM). The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant elevation of NO synthase activity. NO synthase activity was significantly suppressed in the hepatoma cells (transfectants) overexpressing regucalcin. This decrease was completely abolished in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. Moreover, the effect of Ca(2+)/calmodulin addition in increasing NO synthase activity in the hepatoma cells (wild-type) was completely prevented in transfectants. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the cloned rat hepatoma H4-II-E cells.
Izumi
Takako
T
Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.
Tsurusaki
Yoshinori
Y
Yamaguchi
Masayoshi
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002265:Carboxylic Ester Hydrolases; D002999:Clone Cells; D004533:Egtazic Acid / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008114:Liver Neoplasms, Experimental / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015238:Sulfotransferases; D014162:Transfection; D014268:Trifluoperazine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; D014268:Trifluoperazine; D004533:Egtazic Acid; D019001:Nitric Oxide Synthase; D015238:Sulfotransferases; C028152:alcohol sulfotransferase; D002265:Carboxylic Ester Hydrolases; C513325:Rgn protein, rat; D002118:Calcium