Biomedical subjects

Masayoshi Yamaguchi

Publications and source records attributed to Masayoshi Yamaguchi.

At least 19 recordsLinked to original sources

PubMed2003

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.

Animals↗

PubMed2003

Enhancement of albumin expression in bone tissues with healing rat fractures.

The characterization of 66 kDa protein molecule, a major protein component which is produced from femoral-diaphyseal tissues with fracture healing (Igarashi and Yamaguchi [2002] Int. J. Mol. Med. 9:503-508), was investigated. Weaning rats were killed at 7 and 14 days after femoral fracture. When the femoral-diaphyseal tissues with fracture healing were cultured for 48 h in a serum-free medium, many proteins in the bone tissues were released into the medium. Analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a protein molecule of approximately 66 kDa was markedly increased in culture medium from bone tissues with fracture healing. N-terminal sequencing of 66 kDa protein indicated that its N-terminus was identical to that of rat albumin. Western blot analysis of medium 66 kDa protein showed expression of albumin. This expression was significantly enhanced by fracture healing. The expression of albumin was seen in the diaphyseal (cortical bone) and metaphyseal (trabecular bone) tissues of rat femur. When the femoral-diaphyseal tissues obtained at 7 days after femoral fracture were cultured in a serum-free medium containing either vehicle, parathyroid hormone (1-34) (10(-7) M), insulin-like growth factor-I (10(-8) M) or zinc acexamate (10(-4) M), medium albumin was significantly increased in the presence of those bone-stimulating factors. The addition of albumin (0.5 or 1.0 mg/ml of medium) caused a significant increase in calcium and deoxyribonucleic acid contents in the femoral-diaphyseal and -metaphyseal tissues obtained from normal rats in vitro. The present study demonstrates that fracture healing induces a remarkable production of albumin which is a major protein component produced from femoral-diaphyseal tissues of rats, and that albumin has an anabolic effect on bone components.

Albumins↗

PubMed2003

Inhibitory role of regucalcin in the regulation of nitric oxide synthase activity in rat brain cytosol: involvement of aging.

Regucalcin is shown to play a regulatory role in intracellular signaling system in many cells. The protein has been reported to be present in rat brain neurons. The role of regucalcin in the regulation of nitric oxide (NO) synthase activity in the brain cytosol of female young and aged rats was investigated. The presence of regucalcin (10(-9) and 10(-8) M) in the enzyme reaction mixture caused a significant decrease in NO synthase activity in the absence or presence of both calcium chloride (10 micro M) and calmodulin (2.5 micro g/ml). The effect of regucalcin (10(-8) M) in decreasing brain cytosolic NO synthase activity was not seen in the presence of Nw-nitro-L-argine methyl ester (NAME) (10(-4) M), trifluoperazine (20 micro M) or EGTA (1 mM). Regucalcin protein levels were significantly lowered in the brain cytosol of aged (50 weeks old) rats as compared with that of young (5 weeks old) rats. Brain cytosolic NO synthase activity was significantly reduced with increasing age. In aged rat brain cytosol, regucalcin caused a significant decrease in NO synthase activity. The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) caused a significant increase in NO synthase activity in the brain cytosol of young and aged rats. This elevation was completely blocked by the addition of regucalcin (10(-8) M). The present study demonstrates that endogenous regucalcin has an inhibitory effect on NO synthase activity in the brain cytosol of young and aged rats.

Age Factors↗

PubMed2003

Novel protein RGPR-p117: the gene expression in physiologic state and the binding activity to regucalcin gene promoter region in rat liver.

The binding activity of a novel regucalcin gene promoter region-related protein (RGPR-p117) to the TTGGC sequence of the rat regucalcin gene promoter region was investigated. The expression of RGPR-p117 mRNA was seen in the liver tissues of male and female rats. The sexual difference of this expression was not found. Liver RGPR-p117 mRNA expression was not changed with increasing age (1-50 weeks old), and its expression was not altered by fasting or refeeding. Nuclear factor I-A1 (NF1-A1) has been identified to be a transcription factor in stimulating the rat regucalcin gene promoter activity (Misawa and Yamaguchi [2002a] J Cell Biochem 84:795-802]. Recombinant nuclear factor I-A1 (NF1-A1) and RGPR-p117 proteins were used gel mobility shift assay. RGPR-p117 could not bind to TTGGC motif of the sequence between -525 and -504, which has been defined as a functional promoter element II-b. NF1-A1 was specifically bound to the II-b oligonucleotide. Moreover, RGPR-p117 was not bound to the II-b oligonucleotide in the presence of NF1-A1 or rat liver nuclear protein. The binding of NF1-A1 to the II-b oligonucleotide was not altered in the presence of RGPR-p117. This study demonstrates that RGPR-p117 mRNA, is expressed stably for physiologic change in rat liver, and that recombinant the protein does not directly bind to the TTGGC motif in rat regucalcin gene promoter.

Animals↗

PubMed2003

Suppressive role of endogenous regucalcin in the enhancement of nitric oxide synthase activity in liver cytosol of normal and regucalcin transgenic rats.

The suppressive role of endogenous regucalcin, which is a regulatory protein of calcium signaling, in the enhancement of nitric oxide (NO) synthase activity in the liver cytosol of rats was investigated. The enzyme activity was measured in a reaction mixture containing either vehicle or calcium chloride (1-20 microM) in the absence or presence of regucalcin (0.1, 0.25, or 0.5 microM). NO synthase activity was significantly increased by the addition of calcium (5-20 microM). This increase was completely abolished in the presence of trifluoperazine (TFP; 10-50 microM), an antagonist of Ca(2+)/calmodulin. The addition of regucalcin (0.1-0.5 microM) caused a significant fall in the calcium-increased enzyme activity. The effect of regucalcin (0.25 microM) in decreasing NO synthase activity was seen in the presence of ethylene glycol bis-(2-aminoethylether) N,N,N',N'-tetraacetic acid (EGTA, 1 mM) or TFP (20 microM), indicating that regucalcin acts independent on Ca(2+)/calmodulin. NO synthase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture. The effect of the antibody (50 ng/ml) or calcium (10 microM) in elevating NO synthase activity in the liver cytosol of normal rats was not seen in the liver cytosol obtained from regucalcin transgenic rats. Moreover, the increase in NO synthase activity in the liver cytosol of normal rats induced by a single intraperitoneal administration of calcium (5.0 mg/100 g body weight) was significantly enhanced in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture. The administration of calcium caused a significant increase in regucalcin level in the liver cytosol of normal rats. The present study demonstrated that endogenous regucalcin plays a suppressive role in the enhancement of NO synthase activity in the liver cytosol of rats.

Animals↗

PubMed2003

Inhibitory effect of menaquinone-7 (vitamin K2) on the bone-resorbing factors-induced bone resorption in elderly female rat femoral tissues in vitro.

The inhibitory effect of menaquinone-7 (MK-7; vitamin K2) on osteoclast-like cell formation and osteoclastic bone resorption in vitro is found (Mol Cell Biochem 228: 39-47, 2001). This study, furthermore, was undertaken to determine the effect of MK-7 on the bone-resorbing factor-induced bone resorption using the femoral-diaphyseal and -metaphyseal tissues obtained from elderly female rats in vitro. Femoral-diaphyseal and -metaphyseal tissues were cultured for 48 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The experimental cultures contained MK-7 (10(-7)-10(-5) M). The bone-resorbing factors, parathyroid hormone (1-34) (PTH; 10(-7) M) and prostaglandin E2 (PGE2; 10(-5) M), caused a significant decrease in calcium content in the diaphyseal and metaphyseal tissues. The PTH or PGE2-induced decrease in bone calcium content was completely inhibited in the presence of MK-7 (10(-7)-10(-5) M). In addition, MK-7 (10(-7)-10(-5) M) completely prevented the PTH (10(-7) M)- or PGE2 (10(-5) M)-induced increase in medium glucose consumption and lactic acid production by bone tissues. These results support the view that MK-7 has a direct inhibitory effect on the bone-resorbing factor-induced bone resorption in bone culture using female aged femoral tissues in vitro.

Aging↗

PubMed2003

Effect of carotenoid on calcium content and alkaline phosphatase activity in rat femoral tissues in vitro: the unique anabolic effect of beta-cryptoxanthin.

The effects of various carotenoids and rutin on calcium content and alkaline phosphatase activity in the femoral-diaphyseal and femoral-metaphyseal tissues of young rats in vitro were investigated. Bone tissues were cultured for 48 h in serum-free Dulbecco's modified Eagle's medium containing either vehicle or a compound (10(-8)-10(-6) M). The presence of beta-cryptoxanthin (10(-7) or 10(-6) M) caused a significant increase in calcium content and alkaline phosphatase activity in the femoral-diaphyseal and femoral-metaphyseal tissues. Also, the lowest concentration (10(-8) M) of beta-cryptoxanthin used caused a significant increase in diaphyseal and metaphyseal calcium content. Lutein (10(-8)-10(-6) M) had no effect on diaphyseal and metaphyseal calcium contents and diaphyseal alkaline phosphatase activity, while 10(-7) and 10(-6) M lutein significantly decreased metaphyseal alkaline phosphatase activity. Lycopene (10(-8)-10(-6) M) or rutin (10(-8)-10(-6) M) did not have a significant effect on bone calcium content and alkaline phosphatase activity. The present study suggests that the carotenoid beta-cryptoxanthin has a unique anabolic effect on bone calcification in vitro.

Alkaline Phosphatase↗

PubMed2003

Suppressive effect of endogenous regucalcin on protein tyrosine phosphatase activity in the nucleus of rat brain: attenuation with increasing age.

The effect of endogenous regucalcin, which is a regulatory protein of Ca2+ signaling, on protein tyrosine phosphatase activity in the nucleus of brain tissues of young and aged rats was investigated. Phosphotyrosine was used as the substrate for assay of protein tyrosine phosphatase activity. Protein tyrosine phosphatase activity in the brain nucleus of young (5 weeks old) rats was significantly increased in the presence of calcium chloride (5-50 micro M) in the enzyme reaction mixture. The increase was completely blocked by the addition of trifluoperazine (10-50 micro M), an antagonist of calmodulin, indicating that the enzyme is activated by endogenous Ca2+/ calmodulin. The addition of regucalcin (10(-4)-10(-8) M) in the enzyme reaction mixture caused a significant decrease in protein tyrosine phosphatase activity in the absence or presence of calcium chloride (20 micro M). Brain nuclear protein tyrosine phosphatase activity was significantly raised in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the enzyme reaction mixture. The increase was completely prevented by the addition of regucalcin (10(-6) M). In the brain nucleus of aged (50 weeks old) rats, protein tyrosine phosphatase activity was elevated significantly as compared with that of 5 weeks old rats. The effect of anti-regucalcin monoclonal antibody in increasing the enzyme activity in the brain nucleus of aged rats was seen in the presence of 50 ng/ml of the antibody. Such an effect was not found by the antibody of 10 and 25 ng/ml. Regucalcin protein in brain nucleus was detected by Western blot analysis. This level was significantly decreased by increasing age. The present study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein tyrosine phosphatase activity in the nucleus of rat brain, and that this regulation is attenuated with increasing age.

Aging↗

PubMed2003

Great increase in bone 66 kDa protein and osteocalcin at later stages with healing rat fractures: effect of zinc treatment.

Fracture healing has been demonstrated to increase production of bone growth factors, and this elevation has been shown to be enhanced by zinc treatment. Moreover, the effect of zinc treatment on production of bone osteocalcin, which is a kind of Ca2+-binding protein localized in bone matrix, at the later stages with bone fracture was investigated. Rats were sacrificed 7 (earlier stage) or 21 (later stage) days after fracture of femoral diaphysis. Femoral-diaphyseal tissues with fracture healing were cultured in a serum-free medium for 24 h. Many proteins in the bone tissues were released into the medium. Bone protein production was markedly elevated 21 days after bone fracture as compared with that of 7 days. A approximately 66 kDa protein molecule, a major protein component which was produced by the diaphyseal tissues during fracture healing, was predominantly increased at the later stages with fracture healing. Bone osteocalcin production was significantly increased during fracture healing. This increase was enhanced at the later stages with fracture healing. The presence of zinc acexamate (10(-4) M) in culture medium caused a significant increase in bone protein and osteocalcin production at 7 or 21 days after bone fracture. The effect of zinc acexamate in increasing bone total protein and osteocalcin production was remarkable at the later stages with fracture healing. Moreover, zinc treatment caused a significant increase in alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium content in the femoral-diaphyseal tissues of the later stages with fracture healing in vitro. The present study demonstrates that bone protein production is markedly increased at the later stages with fracture healing, and that zinc treatment can enhance production of bone protein components including osteocalcin in vitro. Zinc treatment may stimulate the healing of femoral fracture at earlier and later stages.

Animals↗

PubMed2003

Regulatory effect of regucalcin on nitric oxide synthase activity in rat kidney cortex cytosol: Role of endogenous regucalcin in transgenic rats.

The effect of regucalcin, a regulatory protein in Ca2+ signaling, on nitric oxid (NO) synthase activity in the cytosol of kidney cortex of rats was investigated. The presence of calcium chloride (10 micro M) in the enzyme reaction mixture caused a significant increase in NO synthase activity. This increase was significantly prevented by the addition of trifluoperazine (TFP; 20 or 50 micro M), an antagonist of calmodulin, supporting the existence of Ca2+/calmodulin-dependent NO synthase in rat kidney cortex cytosol. NO synthase activity was significantly decreased by the addition of regucalcin (10(-10)-10(-8) M) in the reaction mixture in the absence or presence of calcium chloride (10 micro M). The regucalcin (10(-8) M) effect was not seen in the presence of Nw-nitro-L-argine metylester (NAME; 10(-6) or 10(-5) M), an inhibitor of NO synthase. Regucalcin significantly reduced NO synthase activity in the presence of TFP (50 micro micro M) or EGTA (1 mM) which has a significant inhibitory effect on the enzyme activity. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the reaction mixture caused a significant increase in NO synthase activity. This increase was completely abolished by the addition of regucalcin (10(-7) M). NO synthase activity was not significantly changed in the kidney cortex cytosol of regucalcin transgenic rats overexpressing endogenous regucalcin as compared with that of wild-type rats. However, the effect of calcium chloride (10 micro M) in increasing NO synthase activity in the kidney cortex cytosol of wild-type rats was significantly weakened in regucalcin transgenic rats. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the kidney cortex cytosol of rats.

Animals↗

PubMed2003

Role of endogenous regucalcin in transgenic rats: suppression of protein tyrosine phosphatase and ribonucleic acid synthesis activities in liver nucleus.

The role of endogenous regucalcin, a regulatory protein of Ca2+ signaling, in the regulation of liver nuclear function was investigated by using regucalcin transgenic (TG) rats. Regucalcin levels were significantly increased in the liver nuclei of regucalcin TG male and female rats. Nuclear protein tyrosine phosphatase activity was significantly elevated in the presence of anti-regucalcin monoclonal antibody (50 ng/ml) in the reaction mixture containing liver nuclear protein obtained from normal (wt) rats. This increase was significantly prevented in the liver nuclei of regucalcin TG rats. Moreover, nuclear ribonucleic acid (RNA) synthesis was significantly suppressed in the liver nuclei of regucalcin TG rats as compared with that of normal rats. The effect of calcium chloride (10 micro M) or anti-regucalcin monoclonal antibody (50 ng/ml) in increasing RNA synthesis was completely blocked in the liver nuclei of the TG rats. These results suggest that endogenous regucalcin plays a suppressive role in the regulation of liver nuclear function in rats.

Animals↗

PubMed2003

Decrease in regucalcin level and enhancement of protein tyrosine phosphatase activity in rat brain microsomes with increasing age.

The role of regucalcin, a regulatory protein in Ca2+ signaling, in the microsomes of brain tissue in rats has not been clarified so far. Western blot analysis showed that regucalcin was present in the brain microsomes. Regucalcin levels were significantly decreased in the brain microsomes obtained from 50-week-old rats as compared with that of 5-week-old rats. Meanwhile, protein tyrosine phosphatase activity was seen in the brain microsomes. The enzyme activity was significantly increased with increasing age. The presence of regucalcin (10(-9) M) in the enzyme reaction mixture containing the brain microsomes obtained from 50-week-old rats caused a significant decrease in protein tyrosine phosphatase activity. Such a decrease was not seen in the brain microsomes from 5-week-old rats. Moreover, the presence of anti-regucalcin monoclonal antibody (10 ng/ml) in the enzyme reaction mixture containing the brain microsomes from young and aged rats caused a significant increase in protein tyrosine phosphatase activity, indicating a suppressive role of microsomal endogenous regucalcin. The present study demonstrates that regucalcin is present in the brain microsomes, and that its level is decreased with increasing age. This decrease may be partly involved in the enhancement of protein tyrosine phosphatase activity in the brain mirosomes with increasing age.

Age Factors↗

PubMed2003

Role of endogenous regucalcin in brain function: suppression of cytosolic nitric oxide synthase and nuclear protein tyrosine phosphatase activities in brain tissue of transgenic rats.

The role of regucalcin, a regulatory protein of Ca2+ signaling, in the regulation of brain function was investigated by using regucalcin transgenic (TG) rats. Western blot analysis showed a remarkable expression of regucalcin protein in the cytosol and nucleus of the brain tissue of TG female rats (5-week-old) as compared with that of wild-type (wt) female rats. In TG male rats, the enhancement of regucalcin expression in the brain cytosol and nucleus was only slight. Nitric oxide (NO) synthase activity was significantly decreased in the brain cytosol of TG female rats. Protein tyrosine phosphatase activity was not significantly altered in the brain nucleus of TG female rats. The presence of calcium chloride (10 microM) or anti-regucalcin monoclonal antibody (50 ng/ml) in the enzyme reaction mixture caused a significant increase in cytosolic NO synthase and nuclear protein tyrosine phosphatase activities in the brain tissue of wt rats. The increase was significantly prevented in the brain cytosol and nucleus of TG rats. The present study supports the view that endogenous regucalcin plays a suppressive role in the regulation of brain function in rats.

Animals↗

PubMed2002

Identification of transcription factor in the promoter region of rat regucalcin gene: binding of nuclear factor I-A1 to TTGGC motif.

Hepatic nuclear protein has been reported to bind specifically to the TTGGC sequence of the rat regucalcin gene promoter region in stimulating the promoter activity (Misawa and Yamaguchi [2000] Biochem. Biophys. Res. Commun. 279: 275-281). The present study was undertaken to identify transcription factor, which binds to TTGGC motif in the rat regucalcin gene promoter, using the yeast one-hybrid system. The sequence between -525 and -504, which has been defined as a functional promoter element II-b, was used as bait to screen a rat liver cDNA library. Two cDNA clones were identified as a nuclear factor I-A1 (NF1-A1). The results of gel mobility shift assay and mutation analysis using recombinant NF1-A1 protein showed that this protein could specifically bind to TTGGC motif of the II-b oligonucleotide in promoter region. The expression of NF1-A1 mRNA was found in the liver, kidney, heart, spleen, and brain of rats. This study demonstrates that NF1-A1 is a transcription factor in stimulating the rat regucalcin gene promoter activity.

Animals↗

PubMed2002

Suppressive role of endogenous regucalcin in the enhancement of deoxyribonucleic acid synthesis activity in the nucleus of regenerating rat liver.

The role of endogenous regucalcin in the regulation of deoxyribonucleic acid (DNA) synthesis activity in the nuclei of regenerating rat liver after partial hepatectomy was investigated. The addition of regucalcin (0.25 and 0.5 microM) in the reaction mixture caused a significant decrease in the nuclear DNA synthesis activity of normal rat liver. This decrease was also seen in the presence of Ca2+-chelator EGTA (0.4 mM), indicating that the effect of regucalcin is not related to nuclear Ca2+. Nuclear DNA activity was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture. The effect was completely abolished by the addition of regucalcin (0.5 microM). Nuclear DNA synthesis activity was significantly increased at 24, 48, and 72 h after partial heptectomy. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing nuclear DNA synthesis activity was significantly enhanced at 24 and 48 h after partial hepatectomy. The presence of staurospone (10(-6) M), trifluoperazine (2 x 10(-5) M), or PD98059 (10(-5) M) in the reaction mixture caused a significant decrease in DNA synthesis activity in the nuclei obtained at 24 after partial hepateactomy. The effect of these inhibitors in the presence of anti-regucalcin monoclonal antibody (25 ng/ml) was greater than that in the absence of the antibody. The present study suggests that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis activity in regenerating liver with cell proliferation after partial hepatectomy in rats.

Animals↗

PubMed2002

Endogenous regucalcin suppresses the enhancement of protein phosphatase activity in the cytosol and nucleus of kidney cortex in calcium-administered rats.

The suppressive role of endogenous regucalcin (RC), which is a regulatory protein of calcium signaling, in the enhancement of protein phosphatase activity (PPA) in the cytosol and nucleus of kidney cortex in calcium-administered rats was investigated. Calcium content in the kidney cortex was significantly increased at 0.5-5 h after a single intraperitoneal administration of calcium chloride solution (10 mg Ca/100 g body weight) to rats. The analysis with Western blotting of RC protein showed that RC levels in the cytosol and nucleus were significantly increased 0.5-5 h after the administration of calcium (10 mg/100 g). PPA toward phosphotyrosine, phosphoserine, and phosphothreonine was found in the cytosol and nucleus of kidney cortex. PPA toward three phosphoamino acids in the cytosol and nucleus was significantly increased by the administration of calcium (10 mg/100 g). The presence of anti-RC monoclonal antibody (25 ng/ml) in the enzyme reaction caused a significant increase in PPA toward phosphotyrosine, phosphoserine, and phosphothreonine in the cytosol and nucleus of kidney cortex in normal rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA toward three phosphoamino acids in the cytosol and nucleus was significantly enhanced in calcium-administered rats. The effect of anti-RC monoclonal antibody (25 ng/ml) in increasing PPA in the cytosol and nucleus of normal rats and calcium-administered rats was completely abolished by the addition of RC (10(- 6) M) in the enzyme reaction mixture. The present study suggests that endogenous RC suppresses the enhancement of PPA in the cytosol and nucleus of kidney cortex in calcium-administered rats.

Animals↗

PubMed2002

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

The expression of regucalcin, a regulatory protein of Ca(2+) signaling, and its effect on Ca(2+) 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 Ca(2+)-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 Ca(2+) pump enzyme. Regucalcin (10(-10)-10(-8) M) significantly stimulated ATP-dependent (45)Ca(2+) uptake by the microsomes. The effect of regucalcin (10(-8) M) in increasing microsomal Ca(2+)-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 Ca(2+)-ATPase activity. In the presence of DTT (5 mM), regucalcin could not significantly enhance the enzyme activity. Also, the effect of regucalcin in increasing Ca(2+)-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 Ca(2+)-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 microg/ml) which is an intracellular signaling factor. The present study demonstrates that regucalcin can activate Ca(2+) pump activity in rat heart microsomes, and that the protein may act the SH groups of Ca(2+)-ATPase by binding to microsomal membranes.

Animals↗

PubMed2002

Role of endogenous regucalcin in transgenic rats: suppression of kidney cortex cytosolic protein phosphatase activity and enhancement of heart muscle microsomal Ca2+-ATPase activity.

Rats were generated by pronuclear injection of the transgene with a cDNA construct encoding rat regucalcin that is a regulatory protein of Ca2+ signaling. Transgenic (TG) founders were fertile, transmitted the transgene at the expected frequency, and bred to homozygote. Western analysis of the cytosol prepared from the tissue of TG female rats (5-week-old) showed a remarkable expression of regucalcin (3.3 kDa) protein in the liver, kidney cortex, heart, lung, stomach, brain, spleen, muscle, colon, and duodenum. Regucalcin expression of TG male rats was seen in the liver, kidney cortex, heart, and lung. In wild-type (wt) male and female rats, regucalcin was mainly present in the liver and kidney cortex. Regucalcin inhibited protein phosphatase activity in rat kidney cortex cytosol and activated Ca2+-ATPase activity in rat heart muscle microsomes. The suppressive effect of regucalcin on protein phosphatase activity was significantly enhanced in the cytosol of kidney cortex of TG male and female rats as compared with those of wt rats. Likewise, heart muscle microsomal Ca2+-ATPase activity was significantly enhanced in TG rats. The changes in their enzyme's activities in TG rats were completely abolished in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture. Moreover, the body weight of TG female rats was significantly lowered as compared with that of wt rats. Serum inorganic phosphorus concentration was significantly increased in TG male and female rats, while serum calcium, glucose, triglyceride, free cholesterol, albumin, and urea nitrogen concentrations were not significantly altered in TG rats. Regucalcin TG rats should be a useful model to define a regulatory role of endogenous regucalcin in the tissues in vivo.

Animals↗