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The sexual difference of calcium-binding protein regucalcin mRNA expression in the liver of rats was investigated by Northern blot analysis. Liver regucalcin cDNA (0.9 kb of open reading frame) was used as a probe. The analysis of total RNAs extracted from various tissues of rats indicated that regucalcin mRNA was present primarily in the liver with a size of 1.8 kb. Hepatic regucalcin mRNA was expressed in male rats more than in females. This sexual difference was also seen in aged rats (50 weeks old), although the expression was decreased with increasing age. Ovariectomy did not cause a significant alteration in hepatic regucalcin mRNA levels. The subcutaneous administration of 17beta-estradiol (0.2 mg/100 g body weight) in ovariectomized rats did not cause an appreciable increase in hepatic mRNA levels. The results demonstrate that regucalcin mRNA expression in rat liver is based on sex, and that this difference may not be related to estrogen.

The expression of hepatic calcium-binding protein regucalcin mRNA in fetal rats was investigated. The alteration in regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb with complete open reading frame). Hepatic regucalcin mRNA levels were progressively increased with fetal development; the mRNA was clearly expressed at 15 and 21 days of pregnancy but only slightly at the 8 days. Meanwhile, beta-actin mRNA levels in the fetal liver were remarkable at 8 and 15 days of pregnancy. The fetal liver regucalcin mRNA levels at 15 days of pregnancy were significantly decreased by overnight-fasting of maternal rats. The oral administration of calcium chloride (50 mg Ca/100 g body weight) to maternal rats at 15 days of pregnancy caused a remarkable elevation (about 2 fold) of regucalcin mRNA levels in the fetal liver; this increase was seen 60 and 180 min after the calcium administration. After birth, regucalcin mRNA was increasingly expressed in the livers of newborn and weanling rats, while hepatic beta-actin mRNA expression was not appreciably altered with increasing ages. These findings demonstrate that the expression of hepatic regucalcin mRNA is increased with fetal development, and that the gene expression may be stimulated by the ingestion of dietary calcium.

The effect of regucalcin, a Ca2+-binding protein isolated from rat liver cytosol, on ribonucleic acid (RNA) synthesis in the nuclei of normal rat liver and of regenerating rat liver was investigated. The liver weight at 1 day after partial hepatectomy was increased about 50% of that of sham-operated (control) rats. Calcium chloride (1.0-20 microM Ca2+ as final concentration) was added into the reaction mixture of nuclear RNA synthesis. RNA synthesis was established by incorporation of [3H]-uridine 5'-triphosphate (UTP) into the nuclear RNA. Addition of Ca2+ (5 and 10 microM) caused a significant increase of RNA synthesis in the nuclei from control rat liver. Such effect of Ca2+ was potentiated in the nuclei of regenerating liver; nuclear RNA synthesis was increased about 2 fold by the 1.0 and 2.5 microM Ca2+ addition. The stimulatory effect of Ca2+ was significantly inhibited by the presence of alpha-amanitin (10(-8) M), an inhibitor of RNA polymerase II. The presence of regucalcin (0.25 and 0.5 microM) significantly inhibited RNA synthesis in the nuclei from control rat liver and from regenerating rat liver. The inhibitory effect of regucalcin was remarkable in the presence of EGTA (0.5 mM), and it was weakened by the addition of Ca2+ (5 microM). Such regucalcin effect was not seen in the presence of alpha-amanitin. The presence of anti-regucalcin IgG in the reaction mixture significantly increased RNA synthesis in the nuclei from control rat liver, indicating that the endogenous regucalcin may be involved in nuclear RNA synthesis. The present results demonstrate that regucalcin can inhibit nuclear RNA synthesis in rat liver. Regucalcin may have an inhibitory role in the regulation of liver nuclear RNA synthesis.