Regucalcin increases Ca2+-ATPase activity in the heart mitochondria of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein in intracellular signaling system, in the regulation of Ca2+-ATPase activity in rat heart mitochondria was investigated. Mitochondrial Ca2+-ATPase activity was significantly increased by increasing concentrations of CaCl2 (2.5-50 microM). An increase in the enzyme activity was saturated at 50 microM CaCl2. The addition of regucalcin (10(-11)-10(-8) M) in the enzyme reaction mixture caused a significant increase in Ca2+-ATPase activity in heart mitochondria in the presence of 50 microM CaCl2. Regucalcin did not have a significant effect on mitochondrial Mg2+-ATPase activity. Regucalcin (10(-9) M) did not have a significant effect on Ca2+-ATPase activity in the presence of digitonin (10(-3) or 10(-2) %), which is a solubilization effect on membranous lipids. The effect of regucalcin in increasing mitochondrial Ca2+-ATPase activity was not observed in the presence of ruthenium red (10(-7) M) or lanthanum chloride (10(-7) M), which is an inhibitor of Ca2+ uniporter. The effect of regucalcin (10(-9) M) in increasing mitochondrial Ca2+-ATPase activity was not significantly enhanced in the presence of calmodulin (5 microg/ml) or dibutyryl cyclic AMP (10(-4) M), which is an intracellular signaling factor that can cause a significant increase in the enzyme activity. Mitochondrial regucalcin localization was significantly increased in the heart of regucalcin transgenic rats as compared with that of normal rats using Western blot analysis. Ca2+-ATPase activity was significantly increased in the heart mitochondria of regucalcin transgenic rats. This study demonstrates that regucalcin has an activating effect on Ca2+-ATPase in rat heart mitochondria, suggesting its role in the regulation of heart mitochondrial function.     

Suppressive effect of regucalcin on protein phosphatase activity in the heart cytosol of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein in intracellular signaling pathway, in the regulation of protein phosphatase activity in the heart muscle cytosol was investigated by using normal (wild-type) and regucalcin transgenic (TG) rats. Protein phosphatase activity was assayed in a reaction mixture containing the cytosolic protein in the presence of phosphotyrosine, phosphoserine, and phosphothreonine. The addition of calcium chloride (10 and 20 microM) in the enzyme reaction mixture caused a significant increase in protein phosphatase activity toward three phosphoaminoacids. Trifluoperazine (10 and 20 microM), an antagonist of calmodulin, completely inhibited calcium (10 microM) addition-increased protein phosphatase activity toward three phosphoaminoacids. Moreover, the calcium (10 microM)-increased enzyme activity toward phosphoserine and phosphothreonine was significantly enhanced by the addition of calmodulin (2.5 or 5 microg/ml). Such an enhancement was not seen in the presence of phosphotyrosine. Regucalcin (10(-9) and 10(-8) M) significantly inhibited protein phosphatase activity toward three phosphoaminoacids in the presence of ethylene glycol bis (2-aminoethlether) N,N,N',N'-tetraacetic acid (EGTA; 1 mM), without Ca2+ addition. The inhibitory effect of regucalcin (10(-10)-10(-8) M) was also seen in the presence of calcium chloride (10 microM). Western blot analysis showed a remarkable expression of regucalcin protein in the cytosol of heart of regucalcin TG female rats as compared with that of wild-type female rats. Protein phosphatase activity toward three phosphoaminoacids was significantly decreased in the heart cytosol of TG rats. The enhancing effect of calcium (10 microM) addition on protein phosphatase activity toward three phosphoaminoacids was not seen in the heart cytosol of TG rats. This study demonstrates that endogenous regucalcin plays a suppressive role in the regulation of protein phosphatase activity in rat heart cytoplasm.     

Suppressive role of endogenous regucalcin in the regulation of nitric oxide synthase activity in heart muscle cytosol of normal and regucalcin transgenic rats

The role of regucalcin, a regulatory protein of Ca2+ signaling, in the regulation of nitric oxide (NO) synthase activity in the cytosol of rat heart muscle was investigated. The addition of calcium chloride (5-20 microM) into the enzyme reaction mixture containing the heart cytosolic protein caused a significant increase in NO synthase activity. The Ca2+ effect was significantly inhibited by trifluoperazine (TFP; 20 or 50 microM), an antagonist of calmodulin, indicating the existence of Ca2+/calmodulin-dependent NO synthase activity in rat heart muscle cytosol. NO synthase activity was significantly decreased by the addition of regucalcin (10(-9) or 10(-8) M). This effect was also seen in the presence of calcium chloride (10 microM), TFP (50 microM) or EGTA (1 mM), a chelator of Ca2+. Meanwhile, the effect of regucalcin (10(-8) M) in decreasing NO synthase activity was not seen in the presence of Nw-nitro-L-arginine methylester (NAME; 10(-6) or 10(-5) M), an inhibitor of the enzyme. The presence of anti-regucalcin monoclonal antibody (25 or 50 ng/ml) in the enzyme reaction mixture caused a significant increase in NO synthase activity. This effect was completely abolished by the addition of regucalcin (10(-7) M). NO synthase activity was not significantly changed in the heart muscle cytosol of transgenic rats overexpressing endogenous regucalcin as compared with that of wild-type rats. However, the effect of calcium (10 micro M) addition in increasing NO synthase activity was significantly weakened in the heart muscle cytosol of regucalcin transgenic rats. The present study demonstrates that endogenous regucalcin has a suppressive effect on NO synthase activity in the heart muscle cytosol of rats.     

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.     

Region specific increases in oxidative stress and superoxide dismutase in the hippocampus of diabetic rats subjected to stress

Oxidative stress and modulation of anti-oxidant enzymes may contribute to the deleterious consequences of diabetes mellitus and to the effects of chronic (i.e. 21 day) stress in the CNS. We therefore compared the effects of short- and long-term exposure to diabetes-induced hyperglycemia, restraint stress and the combined effects of restraint stress and diabetes upon parameters of oxidative stress in the rat hippocampus. Whereas 7 days of restraint stress or hyperglycemia, or the combination, produced similar increases in oxidative stress markers 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) throughout the hippocampus, 21 days of stress or hyperglycemia did not increase these markers in the dentate gyrus. In contrast, Ammon's horn still showed elevated levels of these lipid peroxidation products, especially in diabetic rats subjected to 21 days of restraint stress. The expression of two anti-oxidant enzymes, copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese SOD, was also differentially regulated by stress and hyperglycemia in a time- and region-specific manner in the rat hippocampus. Although long-term stress decreased both SOD isoforms, diabetes increased Cu/Zn-SOD expression in DG with or without 21 days of repeated stress. These increases may account for the finding that protein-conjugated HNE and MDA levels returned to control levels between 7 days and 21 days of hyperglycemia or the combination of diabetes and stress. These results suggest that while other anti-oxidant pathways may account for decreases in oxidative stress in the long-term stress paradigm, increases in Cu/Zn-SOD expression may contribute to the region-specific attenuation of oxidative stress in the diabetic rat hippocampus.     

Hyperlipidemia is induced in regucalcin transgenic rats with increasing age

Regucalcin plays an important role as a regulatory protein in intracellular signaling pathway in many cells. Regucalcin transgenic (TG) rats have been shown to induce a remarkable increase in serum triglyceride and HDL-cholesterol concentrations at the age of 36 weeks (35). Furthermore, this was investigated in regucalcin TG rats with increasing age (14, 25, 36 or 50 weeks). Serum triglyceride or HDL-cholesterol concentration was markedly increased in regucalcin TG male and female rats at 14, 25, 36 or 50 weeks of age. Serum-free fatty acid concentration was significantly elevated in regucalcin TG male and female rats at 25, 36 or 50 weeks. In the TG female rats, a significant increase in serum free fatty acid concentration was also observed at 14 weeks of age, while it was not seen in the TG male rats. Serum-free cholesterol concentration was significantly increased in regucalcin TG female rats at 14, 25, 36 or 50 weeks. Such an increase was not induced in the TG male rats. Moreover, serum calcium concentration was significantly raised in regucalcin TG male and female rats at 50 weeks of age. Also, serum albumin concentration was significantly elevated in regucalcin TG female rats at 25, 36, or 50 weeks of age. Such an increase was not observed in the TG male rats. Serum zinc, glucose or urea nitrogen concentration was not significantly altered in TG male and female rats. This study demonstrates that hyperlipidemia is uniquely induced in regucalcin TG rats with increasing age.     

Characterization of protein tyrosine phosphatase activity in rat liver microsomes: suppressive effect of endogenous regucalcin in transgenic rats

The role of regucalcin, a regulatory protein in intracellular signaling system, in the regulation of protein phosphatase activity in rat liver microsomes was investigated. Protein phosphatase activity torward phosphotyrosine, phosphoserine, and phosphothreonine was assayed in a reaction mixture containing the microsomal protein. Protein phosphatase activity toward phosphotyrosine was strong as compared with that of the enzyme activity toward phosphoserine and phosphothreonine, indicating the existence of protein tyrosine phosphatase. Protein phosphatase activity toward three phosphoaminoacids was significantly enhanced by the addition of both calcium chloride (10 micro M) and calmodulin (2.5 or 5 micro g/ml) in the reaction mixture. The presence of ethylene glycol bis (2-amino-ethylether) N, N, N', N'-tetracetic acid (EGTA; 0.1, 1 or 2 mM) or trifluoperazine (TFP; 10, 20 or 50 micro M), an antagonist of calmodulin, did not have a significant effect on protein phosphatase activity toward phosphotyrosine without calcium addition. Microsomal protein tyrosine phosphatase activity was not changed by okadaic acid (10(-6)-10(-4) M). The enzyme activity was significantly decreased by vanadate (10, 50 or 100 micro M). The addition of regucalcin (0.25 or 0.5 micro M) in the reaction mixture caused a significant inhibition of protein tyrosine phosphatase activity in liver microsomes. Western blot analysis showed a remarkable increase in regucalcin protein level in the liver microsomes of regucalcin transgenic (TG) rats. Protein tyrosine phosphatase activity was significantly suppressed in the liver microsomes of TG rats. This study demonstrates that protein tyrosine phosphatase activity is found in the liver microsomes, and that the enzyme activity is suppressed by regucalcin.     

Superoxide dismutase activity and superoxide dismutase-1 gene methylation in normal and tumoral human breast tissues.

The superoxide dismutase (SOD) activities in normal and tumor breast tissues from 14 human females were determined by the epinephrine autoxidation assay. SOD levels showed a marked interindividual variability in normal and malignant cells. However, each donor had a higher SOD activity in cancer than in normal tissue samples. In three cases in which Mn- and CuZnSOD activities were determined, it was found that tumoral increases in SOD were due to increases in both enzymatic forms. Therefore, it seems reasonable to assume a similar situation for all cases in our series. The level of DNA methylation in the SOD-1 gene was assessed in the first four donors. The four cases exhibited full methylation of SOD-1 genes corresponding to normal as well as to cancer cells. It is concluded that the variability in CuZnSOD activities is not related with the state of methylation of the SOD-1 gene. MspI restriction fragment polymorphisms between DNA samples from normal and malignant cells were detected in the four DNA donors. This phenomenon may be due to point mutations changing the frequency of MspI sites or to methylation of the external C in CCGG sequences.     

Identification of superoxide dismutase activity in Borrelia burgdorferi.

Infective and noninfective strains of Borrelia burgdorferi, along with Borrelia afzelii and Borrelia garinii, possessed a single iron-containing superoxide dismutase (SOD). None of the Lyme disease spirochetes tested possessed catalase or peroxidase activities. The borrelial SOD was not inducible by growth with increased oxygen concentrations and thus appeared to be produced constitutively.     

Erythrocyte superoxide dismutase activity in rats subjected to a toxic regime of intermittent hyperbaric oxygenation

The development of toxic manifestations in rats exposed to hyperoxia is accompanied by a significant decrease in the activity of erythrocytic superoxide dismutase (SOD). Incubation of hemolysates from control animals with H₂O₂ (10^–3 M) or cumene peroxide (1.6\10^–4 M) also led to a marked decrease in the initial SOD activity. The decrease in the initial SOD activity during hyperoxia is thus evidently connected with the formation of peroxidation products in the cells.Department of Hyperbaric Oxygenation, All-Union Research Institute of Clinical and Experimental Surgery. Department of Biochemistry, Medico-Biological Faculty, Second Moscow Medical Institute. (Presented by Academician B. V. Petrovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 8, pp. 959–961, August, 1976.     

Hypoxic preconditioning enhances renal superoxide dismutase levels in rats

Renal ischaemia releases reactive oxygen species (ROS) in the kidneys. We hypothesized that the kidneys are more resistant to the insult of ROS in chronically hypoxic rats. We thus compared rats kept at sea level (SL) and those that had been adapted to hypoxia (hypoxia adapted, HA) by exposure to an altitude of 5500 m in an altitude chamber for 15 h day⁻¹ for 4 weeks. Xanthine (X, 0.75 mg kg⁻¹) and xanthine oxidase (XO, 24.8 mU kg⁻¹) were injected intrarenally. A lucigenin-enhanced chemiluminescence method was employed to detect the amount of free radicals in renal venous blood samples and on the kidney surface. In the renal venous blood samples, 26.05 (± 4.36) × 10⁴ and 10.98 (± 1.79) × 10⁴ counts were detected in the SL and HA rats, respectively, after X-XO treatment; these figures were significantly different. On the kidney surface of the SL rats, the free radical count amounted to 12.77 (± 1.64) × 10⁴, while that in the HA rats was 8.47 (± 0.42) × 10⁴; these figures were also significantly different. There was a significant increase in urine volume and urinary excretion of Na⁺, K⁺ and protein after X-XO administration in both groups of rats. However, the effect was greater for the SL rats than for the HA rats. The lipid peroxidation of the kidneys was not significantly different in the two groups of rats. Finally, we found that the activity of superoxide dismutase (SOD) and SOD mRNA were higher in the renal tissue of HA rats. We conclude that the renal response to free radicals is attenuated after chronic hypoxia in rats, and that SOD might play an important role in protecting HA rats from oxidative stress.     

Enhancement of protein kinase activity in the cytosol of regenerating rat liver: regulatory role of endogenous regucalcin

The effect of regucalcin, a Ca2+-binding protein, on protein kinase activity in the cytosol of regenerating rat liver was investigated. Protein phosphorylation was significantly increased in the liver cytosol obtained at 6, 24, and 48 h after a partial hepatectomy (about 65%) in comparison with that of sham-operated rats. This increase was significantly inhibited by the addition of trifluoperazine (2x10(-5) M), staurosporine (10(-7) M) or genistein (10(-5) M), which is an inhibitor of protein kinases, in the reaction mixture. The presence of regucalcin (0.1-0.5 microM) caused a significant decrease in protein phosphorylation in the cytosol from normal and regenerating rat livers. The effect of regucalcin (0.5 microM) was completely abolished by the addition of anti-regucalcin monoclonal antibody (50 ng/ml). The elevation of protein phosphorylation in regenerating rat liver was significantly enhanced by the presence of anti-regucalcin monoclonal antibody (50 ng/ml). The effect of regucalcin in decreasing protein phosphorylation in the cytosol of regenerating rat liver was not seen in the presence of the antibody. The present study demonstrates that protein kinase activity, is enhanced in the cytosol of regenerating rat liver, and that endogenous regucalcin has an inhibitory role in the enhancement of protein phosphorylation by various protein kinases.     

Characterization of transgenic mice that overexpress both copper zinc superoxide dismutase and catalase

Transgenic mice overexpressing both Cu/ZnSOD and catalase [Tg(SOD1/CAT) +/o] were used to evaluate the effects of overexpression of both genes against oxidative stress. Characterization of these transgenic mice revealed that catalase or Cu/ZnSOD activities were two- to fourfold higher in the tissues of transgenic mice compared to wild-type mice, and the activities of the other major antioxidant enzymes were not altered in the tissues of the transgenic mice. The murine embryonic fibroblasts (MEFs) from the Tg(SOD1/CAT) +/o and MEFs overexpressing Cu/ZnSOD were more resistant to paraquat cytotoxicity, relative to wild-type MEFs. The MEFs from Tg(SOD1/CAT) +/o tended to be more resistant (up to 2.25-fold) to paraquat cytotoxicity than MEFs overexpressing either Cu/ZnSOD or catalase alone. MEFs from Tg(CAT) +/o and Tg(SOD1/CAT) +/o were equally as resistant to hydrogen peroxide cytotoxicity. However, there were no significant differences in whole animal survival against either paraquat or gamma-radiation.