Kimiko Oishi

Expression of hepatic calcium-binding protein regucalcin mRNA is elevated by refeeding of fasted rats: Involvement of glucose, insulin and calcium as stimulating factors

The effect of refeeding on the expression of Ca2+-binding protein regucalcin mRNA in the liver of fasted rats was investigated. When rats were fasted overnight, the hepatic regucalcin mRNA level was reduced about 70% of that in feeding rats. Refeeding produced a remarkable elevation of hepatic regucalcin mRNA level (about 150–170% of fasted rats). Liver regucalcin concentration was appreciably increased by refeeding, although it was not altered by fasting. The oral administration of glucose (2 g/kg body weight) to fasted rats caused a significant increase in hepatic regucalcin mRNA level. Moreover, hepatic regucalcin mRNA level was clearly elevated by a single subcutaneous administration of insulin (10 and 100 U/kg) to fasted rats. The hormonal effect was not further enhanced by the simultaneous administration of calcium chloride (250 mg Ca/kg) to fasted rats, although calcium administration stimulated regucalcin mRNA expression in the liver. The present study suggests that the expression of hepatic regucalcin mRNA stimulated by refeeding is significantly involved in the action of insulin and/or calcium as stimulating factors.

17β-Estradiol stimulates the expression of hepatic calcium-binding protein regucalcin mRNA in rats

The effect of nuclear receptor-related hormones on the expression of hepatic calcium-binding protein regucalcin mRNA in rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb of open-reading frame). A single subcutaneons administration of 17β-estradiol (0.5, 1.0 and 2.0 mg/kg body weight) in rats induced a remarkable increase of regucalcin mRNA in liver; the level was about 200% of control at 24 h after the administration of 2.0 mg/kg. The increase showed about 350% even at 6 h after the administration. Meanwhile, hepatic regucalcin mRNA level was not appreciably altered by a single subcutaneous administration of thyroxine (T4) (20, 40 and 80 mg/kg) or hydrocortisone (10 and 30 mg/kg) in rats. The present study demonstrates that the expression of hepatic regucalcin mRNA is stimulated by estrogen action in the liver nuclei of rats.

Serum release of hepatic calcium-binding protein regucalcin by liver injury with galactosamine administration in rats

Whether calcium-binding protein regucalcin, which mainly localizes in liver, is released into the serum by liver injury was investigated in rats administered galactosamine. Galactosamine (25 mg/100 g body weight) was intraperitoneally administered 3 times at 2 h intervals in rats, and the animals were sacrificed at 10, 24 and 48 h after the first administration of galactosamine. Liver regucalcin mRNA levels were clearly reduced at 24 and 48 h after galactosamine administration with estimating for Northern blotting assay. When hepatic regucalcin concentration was estimated by enzyme-linked immunoadsorbent assay (ELISA) with rabbit-anti-regucalcin IgG, liver regucalcin concentration was not significantly altered by galactosamine administration. Serum regucalcin concentration was markedly elevated at 10 and 24 h after the first administration of galactosamine. Serum transaminases (GOT and GPT) activities were significantly increased by galactosamine administration, indicating that liver injury was induced. The present study demonstrates that liver regucalcin is released into the serum by liver injury with galactosamine administration in rats.

Characterization of Ca2+-stimulated adenosine 5′-triphosphatase and Ca2+ sequestering in rat liver nuclei

The role of Ca2+-stimulated adenosine 5′-triphosphatase (Ca2+-ATPase) in Ca2+ sequestering of rat liver nuclei was investigated. Ca2+-ATPase activity was calculated by subtracting Mg2+-ATPase activity from (Ca2+−Mg2+)-ATPase activity. Ca2+ uptake and release were determined with a Ca2+ electrode. Nuclear Ca2+-ATPase activity increased linearly in the range of 10–40 μM Ca2+ addition. With those concentrations, Ca2+ was completely taken up by the nuclei dependently on ATP (2 mM). Nuclear Ca2+-ATPase activity was decreased significantly by the presence of arachidonic acid (25 and 50 μM), nicotinamide-adenine dinucleotide (NAD+; 2 mM) and zinc sulfate (2.5 and 5.0 μM). These reagents caused a significant decrease in the nuclear Ca2+ uptake and a corresponding elevation in Ca2+ release from the nuclei. Moreover, calmodulin (10 μg/ml) increased significantly nuclear Ca2+-ATPase activity, and this increase was not seen in the presence of trifluoperazine (10 μM), an antogonist of calmodulin. The present findings suggest that Ca2+-ATPase plays a role in Ca2+ sequestering by rat liver nuclei, and that calmodulin is an activator. Moreover, the inhibition of Ca2+-ATPase may evoke Ca2+ release from the Ca2+-loaded nuclei.

Expression of hepatic calcium-binding protein regucalcin mRNA is decreased by phenobarbital administration in rats.

The effect of phenobarbital on the expression of calcium-binding protein regucalcin mRNA in rat liver was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin cDNA (0.9 kb of open reading frame). Phenobarbital (4, 8 and 12 mg/ 100 g body weight) was intraperitoneally administered to rats 3 times with 24 h intervals, and the animals were sacrificed by bleeding at 24 h after the last administration. The hepatic regucalcin mRNA levels were markedly reduced by phenobarbital administration. This decrease was about 50% of control level with the 12 mg/100 g dose. Moreover, the hepatic regucalcin concentration was significantly decreased by the administration of phenobarbital (12 mg/100 g), although the serum regucalcin concentration was not altered appreciably. Meanwhile, serum transaminases (GOT and GPT) activities were not increased by the administration of phenobarbital (4 and 12 mg/100 g). The present study demonstrates that the expression of hepatic regucalcin mRNA is decreased by phenobarbital administration in rats, suggesting that regucalcin does not have a role in drug metabolism related to phenobarbital.

Effect of nicotinamide-adenine dinucleotides on Ca2+ transport system in rat liver nuclei: stimulation of Ca2+ release by NAD+

The effect of nicotinamide-adenine dinucleotides (NAD+ and NADP+) on Ca2+ transport in rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. Ca2+ uptake was dependent on adenosine triphosphate (ATP; 2mM). The presence of NAD+ (2mM) or NADP+ (1 and 2mM) caused a significant inhibition of Ca2+ uptake following addition of 2mM ATP. Ca2+, which accumulated in the nuclei during 6 min after ATP addition, was significantly released by the addition of NAD+ (0.5–2mM) or NADP+ (0.1–2mM). However, the effect of NADH (2mM) or NADPH (2mM) on Ca2+ uptake and release clearly weakened in comparison with the effects of NAD+ and NADP+. Meanwhile, ryanodine (10μM), thapsigargin (10μM) or oxalate (0.5mM) had no effect on Ca2+ uptake and release in rat liver nuclei. These reagents did not significantly alter the effects of 2mM NAD+ on Ca2+ uptake and release. Thus, NAD+ and NADP+ had a potent effect on Ca2+ transport in rat liver nuclei. The present findings suggest that the liver cytosolic NAD+ (NADP+) is a factor in the regulation of the nuclear Ca2+ concentration. (Mol Cell Biochem121: 127–133, 1993)

Regulatory effect of arachidonic acid on the calcium transport system in rat liver nuclei

The effect of arachidonic acid (AA) on Ca2+ transport in rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. Ca2+ uptake increased dependent on ATP (0.5-2.0 mM), while uptake was negligible in the presence of 2.0 mM ADP or AMP. AA (10-100 microM) caused a marked inhibition of Ca2+ uptake following the addition of 2.0 mM ATP. Also, Ca2+, which accumulated in the nuclei during 6 min after ATP addition, was clearly released by the addition of AA (10-100 microM). The alterations were concentration dependent. The nuclear Ca2+ uptake and release were not altered significantly by the presence of prostaglandin E2 (10 and 20 microM), prostaglandin H2 (1 and 4 microM), thromboxane B2 (1 and 4 microM), leukotriene A4 (1 and 4 microM), Ins(1,4,5)P3 (1 and 10 microM) or dibutyryl cAMP (10 and 50 microM). Only, 5-hydroxy-eicosatetraenoic acid (5-HETE) at 4 microM caused a significant inhibition of nuclear Ca2+ uptake and an appreciable increase in Ca2+ release; the 1 microM concentration had no effect. These results indicate that AA, one of the prostanoids, has a unique effect on Ca2+ uptake and release in rat liver nuclei. The finding suggests that AA has a regulatory effect on the Ca2+ transport system in liver nuclei.

Effect of nuclear Ca2+ uptake inhibitors on Ca2+-activated DNA fragmentation in rat liver nuclei

The effect of nuclear Ca2+ uptake inhibitors on the Ca2+-activated DNA fragmentation in rat liver nuclei was investigated. The addition of Ca2+ (40 μM) into the reaction mixture containing liver nuclei in the presence of 2.0 mM ATP caused a remarkable increase in nuclear DNA fragmentation. This Ca2+-activated DNA fragmentation was not seen in the absence of ATP, because nuclear Ca2+ uptake is not initiated without ATP addition. Moreover, the presence of various reagents (10 μM arachidonic acid, 2.0 mM NAD+, 10 μM zinc sulfate and 0.2 mM N-ethylmaleimide), which could inhibit Ca2+-ATPase activity and Ca2+ uptake in the nuclei, produced a significant inhibition of the Ca2+-activated DNA fragmentation in the nuclei. The results show that the Ca2+-activated DNA fragmentation is involved in the uptake of Ca2+ by the nuclei, suggesting a role of Ca2+ transport system in the regulation of liver nuclear functions.

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca2+-stimulated adenosine 5′-triphosphatase (Ca2+-ATPase) in Ca2+ transport system of rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. Ca2+-ATPase activity was calculated by subtracting Mg2+-ATPase activity from (Ca2+−Mg2+)-ATPase activity. The release of Ca2+ from the Ca2+-loaded nuclei was evoked progressively after Ca2+ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca2+ from the Ca2+-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca2+ uptake and induced a promotion of Ca2+ release from the Ca2+-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca2+-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca2+ uptake and release. Meanwhile, verapamil and diltiazem (10 μM), a blocker of Ca2+ channels, did not prevent the NAD+ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 μM) and arachidonic acid (10 μM)-induced increase in nuclear Ca2+ release, suggesting that Ca2+ channels do not involve on Ca2+ release from the nuclei. These results indicates that an inhibition of nuclear Ca2+-ATPase activity causes the decrease in nuclear Ca2+ uptake and the release of Ca2+ from the Ca2+-loaded nuclei. The present finding suggests that Ca2+-ATPase plays a critical role in the regulatory mechanism of Ca2+ uptake and release in rat liver nuclei.