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Sodium-dependent high-affinity binding of carnitine to human placental brush border membranes
Abstracts:
R.T.C.
and T.G.WM.S.
Canada
1996
Permeability and Biochemical Properties of BeWo Trophoblast Cell Monolayers. Fei Liu, Michael J. Soares* and Kenneth L. Audus, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas. *Department of Physiology,University of KansasMedical Center, KansasCity, Kansas,USA. The human choriocarcinoma cell line, BeWo (b30), has been cultured on semipermeable membranes, both in Snapwell-TranswellB inserts and on polycarbonate membranes. The cells grew and formed confluent monolayers in about 5 days and both structurally and hmctionally were similar to human placental primary cultures. The transcellular permeability characteristicsof the monolayers were confirmed by using various dextrans and fluorescein in permeation studies.The transmonolayer resistancewas eliminated when EDTA was presented and was only partially attenuated with amiloride. Cytochrome ~450 1Al (CYPlAl) activity in the cultures has been measured and compared favorably with the activities in human placentaltissue and primary placental cell cultures. An inducer, 8-naphthoflavone, significantly increased CYPl Al activity in the cultures. Because of these properties, the BeWo trophoblast cell monolayersmay be examined as a simple, in vitro model of the human placental barrier. We would expect that this model will prove to have applications in the study of xenobiotic and nutrient transport and metabolism, and chemical compound screening.(Support-MDA NOlDA-4-7405)
Sodium-dependent High-a@@ Binding of Carnitine to Human Placental Brush Border Membranes. V.Gananathv, A.S.Roque, P.D.Prasad, J.S.Bhatia, and F. H.Leibach. Dept. of Biochemistry & Molecular Biology, Medical College of Georgia, Augusta, GA, USA. The interaction of carnitine with human placental brush border membrane vesicleswas investigated. Carnitine was found to associate with the membrane vesicles in a Na+-dependent manner. The time course of this association did not exhibit an overshoot, which is typical of a Na+ gradient-driven transport process. The absolute requirement for Na+ was noticeable whether the associationof carnitine with the vesicleswas measuredwith a short time incubation or under equilibrium conditions, indicating Na+-dependent binding of carnitine to the human placental brush border membranes. The binding was saturable and was of a highaffinity type with a dissociationconstant of 1.37kO.03 PM. Anions had little or no influence on the binding process. The binding process was specific for carnitine and its acyl derivatives. Betaine also competed for the binding process, but other structurally related compounds did not. Kinetic analysis revealed that Na+ increased the affinity of the binding process for carnitine and the Na+/carnitine coupling ratio for the binding process was 1. The dissociation constant for the interaction of Na+ with the binding of carnitine was 24&4 mM. This constitutes the first report on the identification of Nat-dependent high affinity carnitine binding in the plasma membrane of a mammalian cell.
A.25
Effekt of hyperglycemia on the glucose transport system of isolated human term trophoblast cells in vitro. T. Hahn, S. Barth’, G. Desoye, Department of Obstetrics and Gynecology and 1Institute of Medical Biochemistry, University of Graz, A-8036 Graz, Austria The human trophoblast (TC) constitutes the main barrier for maternal-fetal glucose transport. The present study investigated the effect of hyperglycemia in vitro on the trophoblast glucose transport system. TC were isolated by a standard procedure and subsequently immunopurified using an HLA class-I antibody. Cells were cultured for 24, 48 and 96 hrs in DMEM containing either 5.5 (control) or 25 mmol/l D-glucose (hyperglycemia) or 25 mmol/l L-glucose (osmotic control), respectively. 3-O-[14C]methyl-D-glucose uptake was measured in 5 set intervals at 4 “C! using concentrations from l-25 mmol/l. Experimental data were fitted to a first order rate Tation yielding KM= 72 mM and V max= 29 fmol s- per cell. At 20 and 25 mmol/l exogenous glucose concentrations uptake rates of cells cultured under hyperglycemic conditions were decreased by 25% @< 0.05). This effect was due to lower (pcO.05) Vmax (- 50%) and higher (pzO.77) KM (+lO%) values. The levels of GLUT1 transcripts (Northern blotting) were lower by 20% (pCO.05) in TC cultured under hyperglycemic as compared to control conditions. We conclude that (a) hyperglycemia may slow down placental glucose transfer at substrate concentrations cotiesponding to blood levels of poorly-controlled diabetic oatients and 0~) this effect is mediated bv diminished t&scription ofGLUT in the trophoblast. ’ [Grant HA 2064/2-l of the GermanResearchFoundation.]
Nutritional Role for the Fetal-stage Rat Visceral Yolk Sac? D.A.Beckman, R.L.Brent and J.B.Lloyd, Jefferson Medical College, Philadelphia, PA, USA.
A
The rat visceral yolk sac persists to term, and in the fetal stage continues to pinocytose and digest maternally derived proteins. We calculate that the quantity of amino acids thus generated is almost sufficient to meet the entire net needs of the fetus, as judged by its rate of protein accretion. We reasoned that, if yolk-sac proteolysis generates much of the fetal amino acid supply, a radiolabeled amino acid injected into the maternal bloodstream would display substantially lower specific radio&t1 ivity in the fetal blood. Gestational day-i7.5 rats received a single i.v. injection of [ 3H] leucine. Maternal and fetal blood samples were obtained at intervals from 15 to 180 minutes after injection. The concentration of free leucine was some 5 times higher, and its specific radioactivity was some 5 times lower, in fetal than in maternal plasma. These findings are consistent with the role proposed above for the yolk sac. As turnover of fetal proteins could also contribute to the isotope dilution we report, future work will seek to determine the relative importance of the two mechanisms. Supported by NIH HD-29902.
R.T.C.
and T.G.WM.S.
Canada
1996
Permeability and Biochemical Properties of BeWo Trophoblast Cell Monolayers. Fei Liu, Michael J. Soares* and Kenneth L. Audus, Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas. *Department of Physiology,University of KansasMedical Center, KansasCity, Kansas,USA. The human choriocarcinoma cell line, BeWo (b30), has been cultured on semipermeable membranes, both in Snapwell-TranswellB inserts and on polycarbonate membranes. The cells grew and formed confluent monolayers in about 5 days and both structurally and hmctionally were similar to human placental primary cultures. The transcellular permeability characteristicsof the monolayers were confirmed by using various dextrans and fluorescein in permeation studies.The transmonolayer resistancewas eliminated when EDTA was presented and was only partially attenuated with amiloride. Cytochrome ~450 1Al (CYPlAl) activity in the cultures has been measured and compared favorably with the activities in human placentaltissue and primary placental cell cultures. An inducer, 8-naphthoflavone, significantly increased CYPl Al activity in the cultures. Because of these properties, the BeWo trophoblast cell monolayersmay be examined as a simple, in vitro model of the human placental barrier. We would expect that this model will prove to have applications in the study of xenobiotic and nutrient transport and metabolism, and chemical compound screening.(Support-MDA NOlDA-4-7405)
Sodium-dependent High-a@@ Binding of Carnitine to Human Placental Brush Border Membranes. V.Gananathv, A.S.Roque, P.D.Prasad, J.S.Bhatia, and F. H.Leibach. Dept. of Biochemistry & Molecular Biology, Medical College of Georgia, Augusta, GA, USA. The interaction of carnitine with human placental brush border membrane vesicleswas investigated. Carnitine was found to associate with the membrane vesicles in a Na+-dependent manner. The time course of this association did not exhibit an overshoot, which is typical of a Na+ gradient-driven transport process. The absolute requirement for Na+ was noticeable whether the associationof carnitine with the vesicleswas measuredwith a short time incubation or under equilibrium conditions, indicating Na+-dependent binding of carnitine to the human placental brush border membranes. The binding was saturable and was of a highaffinity type with a dissociationconstant of 1.37kO.03 PM. Anions had little or no influence on the binding process. The binding process was specific for carnitine and its acyl derivatives. Betaine also competed for the binding process, but other structurally related compounds did not. Kinetic analysis revealed that Na+ increased the affinity of the binding process for carnitine and the Na+/carnitine coupling ratio for the binding process was 1. The dissociation constant for the interaction of Na+ with the binding of carnitine was 24&4 mM. This constitutes the first report on the identification of Nat-dependent high affinity carnitine binding in the plasma membrane of a mammalian cell.
A.25
Effekt of hyperglycemia on the glucose transport system of isolated human term trophoblast cells in vitro. T. Hahn, S. Barth’, G. Desoye, Department of Obstetrics and Gynecology and 1Institute of Medical Biochemistry, University of Graz, A-8036 Graz, Austria The human trophoblast (TC) constitutes the main barrier for maternal-fetal glucose transport. The present study investigated the effect of hyperglycemia in vitro on the trophoblast glucose transport system. TC were isolated by a standard procedure and subsequently immunopurified using an HLA class-I antibody. Cells were cultured for 24, 48 and 96 hrs in DMEM containing either 5.5 (control) or 25 mmol/l D-glucose (hyperglycemia) or 25 mmol/l L-glucose (osmotic control), respectively. 3-O-[14C]methyl-D-glucose uptake was measured in 5 set intervals at 4 “C! using concentrations from l-25 mmol/l. Experimental data were fitted to a first order rate Tation yielding KM= 72 mM and V max= 29 fmol s- per cell. At 20 and 25 mmol/l exogenous glucose concentrations uptake rates of cells cultured under hyperglycemic conditions were decreased by 25% @< 0.05). This effect was due to lower (pcO.05) Vmax (- 50%) and higher (pzO.77) KM (+lO%) values. The levels of GLUT1 transcripts (Northern blotting) were lower by 20% (pCO.05) in TC cultured under hyperglycemic as compared to control conditions. We conclude that (a) hyperglycemia may slow down placental glucose transfer at substrate concentrations cotiesponding to blood levels of poorly-controlled diabetic oatients and 0~) this effect is mediated bv diminished t&scription ofGLUT in the trophoblast. ’ [Grant HA 2064/2-l of the GermanResearchFoundation.]
Nutritional Role for the Fetal-stage Rat Visceral Yolk Sac? D.A.Beckman, R.L.Brent and J.B.Lloyd, Jefferson Medical College, Philadelphia, PA, USA.
A
The rat visceral yolk sac persists to term, and in the fetal stage continues to pinocytose and digest maternally derived proteins. We calculate that the quantity of amino acids thus generated is almost sufficient to meet the entire net needs of the fetus, as judged by its rate of protein accretion. We reasoned that, if yolk-sac proteolysis generates much of the fetal amino acid supply, a radiolabeled amino acid injected into the maternal bloodstream would display substantially lower specific radio&t1 ivity in the fetal blood. Gestational day-i7.5 rats received a single i.v. injection of [ 3H] leucine. Maternal and fetal blood samples were obtained at intervals from 15 to 180 minutes after injection. The concentration of free leucine was some 5 times higher, and its specific radioactivity was some 5 times lower, in fetal than in maternal plasma. These findings are consistent with the role proposed above for the yolk sac. As turnover of fetal proteins could also contribute to the isotope dilution we report, future work will seek to determine the relative importance of the two mechanisms. Supported by NIH HD-29902.