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The effect of ethanol on thiamine uptake and metabolism in kidney tissue
1343
ipwo031 Molecular function of a novel calci~m-binding protein from porcine cardiac muscle Sasaki, T., Naka, M., Kise, H., Hiraoka *, O., Furuichi *, Y. and Tanaka, T. Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Tsu, Mie 514 and * Subdivision of Biotechnology, Nippon Roche Research Center, Kamakura, Kanagawa 247, Japan
Intracellular calcium ions play a crucial role as intracellular messengers mediating the effects of a variety of extracellular signals in the regulation of cardiac function and their movements may be profoundly affected in various pathophysiological conditions. An increase in intracellular calcium ion concentration triggers a variety of events in cardiac muscle cells and the effects of calcium ions are mediated by a number of calcium-binding proteins. The dominant molecular mechanism for calcium activation of contractile proteins in cardiac muscle is due to a thin-filament calcium-dependent regulatory system, troponm-tropomyosin and calcium-binding molecule in this system is troponin C. Moreover, it has been reported that there are more calcium-binding proteins such as calmodulin, S100ao protein and others in cytosolic fraction of cardiac muscle. However, their cellular function in additional calcium-dependent regulatory systems in the heart is not known. Then we tried to purify calcium-binding proteins from porcine cardiac muscle and characterize their molecular properties. A low molecular weight, calcium-binding protein which we term CaBPc was isolated from porcine heart. The protein has been purified to homogeneity by calcium-dependent affinity chromatography on immobilized reactive red 120 and phenyl-Sepharose. The molecular weight of this protein is about 11 kDa as estimated by polyacrylamide gel electrophoresis in the presence of SDS. When we subjected CaBPc to analytical gel filtration on a calibrated Sephacryl S-200 column, it eluted much earlier than would be expected for a CaBPc monomer. This suggests that it may exist as a self associated complex. The ability of CaBPc to bind calcium ion was studied by the technique of equilibrium dialysis. CaBPc binds s_toichiometric amounts of calcium ion. This protein posseses some of the properties of S100 proteins such as self association and exposure of hydrophobic regions upon calcium-saturation, but is different from $100 proteins in isoelectric point ( p l - 6.2), amino acid composition and its calcium-dependent interaction with the immobilized dye. On an immunoblot, CaBPc did not crossreact with anti-S100ao anti serum, and S100ao did not crossreact with anti-CaBPc anti serum. The partial amino acid sequence of CaBPc was compared to other calcium-binding proteins and high degree of similarity was found between C~BPc and calpactin I light chaia, troponin C, cystic fibrosis-associated antigen, calcyclin, calmodulin, or S100 proteins. Moreover~ we studied calcium-dependent interaction of CaBPc with cardiac cytoskeletal proteins or various phospholipids. These results suggest that this protein may be a new member of calcium-binding proteins in porcine cardiac muscle and involved in the calcium-dependent regulatory system. I P.we.2dl
The effect of ethanol on thiamine uptake and metabolism in kidney tissue Pochal, M,A. and Acara, M. Department of Pharmacology and Therapeutics, School of Medicine and Biomedical Sciences, State University of New York at Buffalo. Buffalo, N Y 14214, U.S.A.
Thiamine deficiency is associated with chronic alcoholism and leads to the development of neurological and cardiovascular disturbances recognized as Wernicke-Korsakoff syndrome, peripheral neuritis, and beri-beri heart disease. While the focus of the pathogenesis of thiamine deficiency in chronic alcoholism has been on thiamine malabsorption, there have been few studies on the renal disposition of thiamine. These experiments were performed to determine whether ethanol interferes with renal transport and/or metabolism of thiamine contributing to its depletion and disturbing its bioavailability.
1344 Adult, male Sprague-Dawley rats were euthanized with sodium pentobarbital. Kidneys were removed and dissected into cortex, outer medulla, and inner medulla. Approximately 100 mg of tissue was placed in 2 ml of Cross-Taggert incubation medium containing 14C-thiamine (0.01 /tCi) and unlabeled thiamine to achieve concentrations over the range of 10 nM-10 mM. After 45 rain. incubation at 37°C, tissues were removed, quickly blotted, transferred to vials for digestion followed by counting of radioactivity along with vials containing aliquots of medium. All counts were corrected to dpm/g or ml and tissue to medium (T/M) ratios calculated. The ability of the tissues to phosphorylate thiamine was tested by identifying the metabolites of 14C-thiamine formed during 14C-thiamine uptake. Aliquots of solubiliTed tissue and medium were analyzed by thin layer chromatography ano measurement of radioactivity by scintillation counting. The following R~s were obtained: thiamine = 0.74, thiamine monophosphate ( T M P ) - 0.35, thiamine pyrophosphate ( T I P ) = 0.25. These results demonstrate that ~4C-thiamine accumulates against a concentration gradient in all three zones of the kidn~ as indicated by T / M > 1.0: cortex = 3.42, outer medulla = 2.22 and inner medulla = 1.99. When thiamine uptake was studied in cortical tissue over a large concentration range, a constant T / M was obtained until thiamine concentration of 11 microM was reached and a decrease in T / M then demonstrated saturation of the uptake system. Addition of ethanol at concentrations of 25 mM, 100 mM, 200 mM, and 500 mM yielded T / M ratios of 1.818, 1.885, 1.926, and 2.581, respectively. When these ratios were compared to the control (no ethanol) T / M ratio of 2.066, a biphasic effect of ethanol on T / M ratio was seen whereby the low concentrations (25 mM, 100 M) significantly (p < 0.05) decreased the ratio and the highest concentration (500 raM) significantly increased the ratio. Chromatographic analysis of cortical tissue and medium revealed 3 peaks with R t values the same as thiamine, TMP and TPP standards indicating thiamine phosphorylation had occurred. The following table illustrates the percen*.ages ef free thiamine (T) and total phosphorylated thiamine (T-PO4) in the tissue and medium. Tissue Control (n = 2) w125 mM Ethanol (n = 2)
T 64.45 83.55
Medium T-PO 4
37.45 15.15
T 69.05 4.8.17¢
T-PO 4
31.0% 51.95
These ethanol experiments demonstrate that kidney tissue accumulates and phosphorylates thiamine and that low ethanol concentrations decrease thiamine uptake while high ethanol concentrations enhance uptake of thiamigie into cortical tissue. These data also suggest that although total phosphate formation is unaffected, the intra and extracellular distribution of phosphorylated thiamine is altered by ethanol.
P.we.205 ! I
Demethylational and conjugational biotransformations in the renal tissue KvStina, J., Trejtna7 *, F. and Jindrovfi, O. Dept. of Pharmacology, Faculty of Pharmacy, Charles University, Hradec Krtilov6and * Institute of Experimental Biopharmacy, Czechoslovak Academy of Sciences, Hradec Kr~lov$, Czechoslovakia
In order to render it possible to utilize the differences in drug biotransformation between the individual organ system for the construction of novel agents with targeted biodistribution in the organism, the following selected biotransformational processes in the renal, or liver tissue were studied: one enzymatic process from the Ist biotransformational series (N-demethylation), two from the lind biotransformational series (conjugation with glycine and with glucuronic acid), N-demethylation was investigated by means of the substrate aminophenazone (on the basis of formaldehyde formation) in the kidney and fiver homogenates in experimental rats and rabbits. This demethylation activity in the renal tissue has been shown not to be negligible and it makes up circa 445 of the activity of the liver tissue in rats and circa 475 in rabbits. Further analysis (in the renal cortex and medulla), which was carried out due to technical reasons in rabbits only, has demonstrated that this enzymatic activity is localized solely in the cortex.
ipwo031 Molecular function of a novel calci~m-binding protein from porcine cardiac muscle Sasaki, T., Naka, M., Kise, H., Hiraoka *, O., Furuichi *, Y. and Tanaka, T. Department of Molecular and Cellular Pharmacology, Mie University School of Medicine, Tsu, Mie 514 and * Subdivision of Biotechnology, Nippon Roche Research Center, Kamakura, Kanagawa 247, Japan
Intracellular calcium ions play a crucial role as intracellular messengers mediating the effects of a variety of extracellular signals in the regulation of cardiac function and their movements may be profoundly affected in various pathophysiological conditions. An increase in intracellular calcium ion concentration triggers a variety of events in cardiac muscle cells and the effects of calcium ions are mediated by a number of calcium-binding proteins. The dominant molecular mechanism for calcium activation of contractile proteins in cardiac muscle is due to a thin-filament calcium-dependent regulatory system, troponm-tropomyosin and calcium-binding molecule in this system is troponin C. Moreover, it has been reported that there are more calcium-binding proteins such as calmodulin, S100ao protein and others in cytosolic fraction of cardiac muscle. However, their cellular function in additional calcium-dependent regulatory systems in the heart is not known. Then we tried to purify calcium-binding proteins from porcine cardiac muscle and characterize their molecular properties. A low molecular weight, calcium-binding protein which we term CaBPc was isolated from porcine heart. The protein has been purified to homogeneity by calcium-dependent affinity chromatography on immobilized reactive red 120 and phenyl-Sepharose. The molecular weight of this protein is about 11 kDa as estimated by polyacrylamide gel electrophoresis in the presence of SDS. When we subjected CaBPc to analytical gel filtration on a calibrated Sephacryl S-200 column, it eluted much earlier than would be expected for a CaBPc monomer. This suggests that it may exist as a self associated complex. The ability of CaBPc to bind calcium ion was studied by the technique of equilibrium dialysis. CaBPc binds s_toichiometric amounts of calcium ion. This protein posseses some of the properties of S100 proteins such as self association and exposure of hydrophobic regions upon calcium-saturation, but is different from $100 proteins in isoelectric point ( p l - 6.2), amino acid composition and its calcium-dependent interaction with the immobilized dye. On an immunoblot, CaBPc did not crossreact with anti-S100ao anti serum, and S100ao did not crossreact with anti-CaBPc anti serum. The partial amino acid sequence of CaBPc was compared to other calcium-binding proteins and high degree of similarity was found between C~BPc and calpactin I light chaia, troponin C, cystic fibrosis-associated antigen, calcyclin, calmodulin, or S100 proteins. Moreover~ we studied calcium-dependent interaction of CaBPc with cardiac cytoskeletal proteins or various phospholipids. These results suggest that this protein may be a new member of calcium-binding proteins in porcine cardiac muscle and involved in the calcium-dependent regulatory system. I P.we.2dl
The effect of ethanol on thiamine uptake and metabolism in kidney tissue Pochal, M,A. and Acara, M. Department of Pharmacology and Therapeutics, School of Medicine and Biomedical Sciences, State University of New York at Buffalo. Buffalo, N Y 14214, U.S.A.
Thiamine deficiency is associated with chronic alcoholism and leads to the development of neurological and cardiovascular disturbances recognized as Wernicke-Korsakoff syndrome, peripheral neuritis, and beri-beri heart disease. While the focus of the pathogenesis of thiamine deficiency in chronic alcoholism has been on thiamine malabsorption, there have been few studies on the renal disposition of thiamine. These experiments were performed to determine whether ethanol interferes with renal transport and/or metabolism of thiamine contributing to its depletion and disturbing its bioavailability.
1344 Adult, male Sprague-Dawley rats were euthanized with sodium pentobarbital. Kidneys were removed and dissected into cortex, outer medulla, and inner medulla. Approximately 100 mg of tissue was placed in 2 ml of Cross-Taggert incubation medium containing 14C-thiamine (0.01 /tCi) and unlabeled thiamine to achieve concentrations over the range of 10 nM-10 mM. After 45 rain. incubation at 37°C, tissues were removed, quickly blotted, transferred to vials for digestion followed by counting of radioactivity along with vials containing aliquots of medium. All counts were corrected to dpm/g or ml and tissue to medium (T/M) ratios calculated. The ability of the tissues to phosphorylate thiamine was tested by identifying the metabolites of 14C-thiamine formed during 14C-thiamine uptake. Aliquots of solubiliTed tissue and medium were analyzed by thin layer chromatography ano measurement of radioactivity by scintillation counting. The following R~s were obtained: thiamine = 0.74, thiamine monophosphate ( T M P ) - 0.35, thiamine pyrophosphate ( T I P ) = 0.25. These results demonstrate that ~4C-thiamine accumulates against a concentration gradient in all three zones of the kidn~ as indicated by T / M > 1.0: cortex = 3.42, outer medulla = 2.22 and inner medulla = 1.99. When thiamine uptake was studied in cortical tissue over a large concentration range, a constant T / M was obtained until thiamine concentration of 11 microM was reached and a decrease in T / M then demonstrated saturation of the uptake system. Addition of ethanol at concentrations of 25 mM, 100 mM, 200 mM, and 500 mM yielded T / M ratios of 1.818, 1.885, 1.926, and 2.581, respectively. When these ratios were compared to the control (no ethanol) T / M ratio of 2.066, a biphasic effect of ethanol on T / M ratio was seen whereby the low concentrations (25 mM, 100 M) significantly (p < 0.05) decreased the ratio and the highest concentration (500 raM) significantly increased the ratio. Chromatographic analysis of cortical tissue and medium revealed 3 peaks with R t values the same as thiamine, TMP and TPP standards indicating thiamine phosphorylation had occurred. The following table illustrates the percen*.ages ef free thiamine (T) and total phosphorylated thiamine (T-PO4) in the tissue and medium. Tissue Control (n = 2) w125 mM Ethanol (n = 2)
T 64.45 83.55
Medium T-PO 4
37.45 15.15
T 69.05 4.8.17¢
T-PO 4
31.0% 51.95
These ethanol experiments demonstrate that kidney tissue accumulates and phosphorylates thiamine and that low ethanol concentrations decrease thiamine uptake while high ethanol concentrations enhance uptake of thiamigie into cortical tissue. These data also suggest that although total phosphate formation is unaffected, the intra and extracellular distribution of phosphorylated thiamine is altered by ethanol.
P.we.205 ! I
Demethylational and conjugational biotransformations in the renal tissue KvStina, J., Trejtna7 *, F. and Jindrovfi, O. Dept. of Pharmacology, Faculty of Pharmacy, Charles University, Hradec Krtilov6and * Institute of Experimental Biopharmacy, Czechoslovak Academy of Sciences, Hradec Kr~lov$, Czechoslovakia
In order to render it possible to utilize the differences in drug biotransformation between the individual organ system for the construction of novel agents with targeted biodistribution in the organism, the following selected biotransformational processes in the renal, or liver tissue were studied: one enzymatic process from the Ist biotransformational series (N-demethylation), two from the lind biotransformational series (conjugation with glycine and with glucuronic acid), N-demethylation was investigated by means of the substrate aminophenazone (on the basis of formaldehyde formation) in the kidney and fiver homogenates in experimental rats and rabbits. This demethylation activity in the renal tissue has been shown not to be negligible and it makes up circa 445 of the activity of the liver tissue in rats and circa 475 in rabbits. Further analysis (in the renal cortex and medulla), which was carried out due to technical reasons in rabbits only, has demonstrated that this enzymatic activity is localized solely in the cortex.