scholarly journals Study of fluxes at low concentrations of l-tri-iodothyronine with rat liver cells and their plasma-membrane vesicles. Evidence for the accumulation of the hormone against a gradient

1981 ◽  
Vol 198 (3) ◽  
pp. 457-466 ◽  
Author(s):  
Govind S. Rao ◽  
Marie Luise Rao ◽  
Astrid Thilmann ◽  
Hans D. Quednau
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Liver Homogenate ◽  
Membrane Vesicles ◽  
Liver Cells ◽  
Free Form ◽  
Plasma Membrane Vesicles ◽  
Cytosol Fraction ◽  
Low Concentrations ◽  
Rat Liver Cells

1. Influx and efflux of l-tri-[125I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23°C and in the concentration range that included the concentration of free l-tri-iodothyronine in rat plasma (3–5pm) influx into cells was saturable; an apparent Kt value of 8.6±1.6pm was obtained. 3. At 5pm-l-tri-[125I]iodothyronine in the external medium the ratios of the concentrations inside to outside in cells and plasma-membrane vesicles were 38:1 and 366:1 respectively after 7s of incubation. At equilibrium (60s at 23°C) uptake of l-tri-[125I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a Kd value of 6.1±1.3pm. 4. Efflux of l-tri-[125I]iodothyronine from cells equilibrated with the hormone (5–123pm) was constant up to 21 s; the amount that flowed out was 17.7±3.8% when cells were equilibrated with 5pm-hormone. When plasma-membrane vesicles were equilibrated with l-tri-[125I]iodothyronine (556–1226pm) 66.8±5.8% flowed out after 21 s. 5. From a consideration of the data on efflux from cells and binding of l-tri-[125I]iodothyronine to the liver homogenate, as studied by the charcoal-adsorption and equilibrium-dialysis methods, it appears that 18–22% of the hormone exists in the free form in the cell. 6. Vinblastine and colchicine diminished the uptake of l-tri-[125I]iodothyronine by cells but not by plasma-membrane vesicles; binding to the cytosol fraction was not affected. Phenylbutazone, 6-n-propyl-2-thiouracil, methimazole and corticosterone diminished the uptake by cells, plasma-membrane vesicles and binding to the cytosol fraction to different extents. 7. These results suggest that at low concentrations of l-tri-[125I]iodothyronine rat liver cells and their plasma-membrane vesicles accumulated the hormone against an apparent gradient by a membrane-mediated process. Contribution of cytoplasmic proteins to uptake by plasma-membrane vesicles was negligible. The amount of l-tri-[125I]iodothyronine required to achieve half-maximal uptake agrees with that occurring in the free form in the blood, conferring physiological importance to the transporting system in the plasma membrane of the liver cell.

scholarly journals Uptake of L-tri-iodothyronine by isolated rat liver cells. A process partially inhibited by metabolic inhibitors; attempts to distinguish between uptake and binding to intracellular proteins

1979 ◽  
Vol 182 (2) ◽  
pp. 473-491 ◽  
Author(s):  
J Eckel ◽  
G S Rao ◽  
M L Rao ◽  
H Breuer
Keyword(s):  
Rat Liver ◽  
External Medium ◽  
Membrane Vesicles ◽  
Thiol Group ◽  
Liver Cells ◽  
Metabolic Inhibitors ◽  
Hill Coefficient ◽  
Plasma Membrane Vesicles ◽  
Rat Liver Cells ◽  
Sigmoidal Curve

1. Rat liver cells obtained by dispersion with collagenase were used to investigate the mode of entry of L-tri-iodothyronine into the cell. 2. The hormone was taken up very rapidly at 23 degrees C; the linear phase of uptake lasted for up to approx. 20 s. 3. A plot of the initial rates of uptake against different concentrations of L-tri-iodothyronine yielded a sigmoidal curve. The Eadie–Hofstee plot (v/[S]2 versus v) yielded two straight lines. The uptake component with an apparent Kt value of 86 +/- 15 pM was designated as system I, and the second uptake component with an apparent Kt of 726 +/- 11 pM as system II. The Hill plot for system I was not linear; the apparent Hill coefficient for system II was calculated to be 2.1.4. Uptake of L-tri-iodothyronine by system I was higher at pH 6.4 than at pH 7.4; system II was relatively insensitive to changes in the pH of the external medium. 5. Both systems exhibited a transition temperature at about 16 degrees C in the Arrhenius plot. The activation energies of the two systems below and above 16 degrees C were 72.8 and 47.7 and 54.4 and 33.1 J/mol respectively. 6. Inhibitors of cellular energy reduced the uptake by system I to a larger extent than that by system II. 7. Replacement of Na+ in the external medium by either K+ or choline led to uptake that followed normal Michaelis–Menten kinetics. 8. Thiol-group-blocking agents reduced the uptake of the hormone by both systems. 9. Treatment of liver cells with beta-glucosidase, Pronase and neuraminidase led to a decrease in the uptake of L-tri-iodothyronine by system I, whereas uptake by system II was decreased after treatment with phospholipase A2, beta-galactosidase. Pronase and neuraminidase. 10. The stereoisomer D-tri-iodothyronine (100–3000 pM) did not affect system I, but uptake by system II decreased with increasing concentration of D-tri-iodothyronine. Reverse L-tri-iodothyronine (2–100 pM) and L-thyroxine (100–3000 pM) did not influence uptake by either system. 11. Under identical conditions of incubation, the uptake of L-tri-iodothyronine was 3.7 times higher than binding to cytosol proteins. The binding was insensitive to metabolic inhibitors. The results suggest that cytosol proteins are not directly involved in the uptake of L-tri-iodothyronine. 12. Plasma-membrane vesicles also take up the hormone rapidly at 23 degrees C. Increasing the osmolarity of the external medium led to a decrease in the uptake of L-tri-iodothyronine by vesicles. 13. Uptake as a function of L-tri-iodothyronine concentration exhibited a sigmoidal curve. The Eadie–Hofstee plot showed two uptake components with apparent Kt values of 96.8 and 1581 pM. 14. The results of our study are consistent with a carrier-mediated translocation of the hormone into the cell.

scholarly journals Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles

1996 ◽  
Vol 316 (3) ◽  
pp. 999-1004 ◽  
Author(s):  
Lorella PASCOLO ◽  
Savino DEL VECCHIO ◽  
Ronald K. KOEHLER ◽  
J. Enrique BAYON ◽  
Cecile C. WEBSTER ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Experimental Conditions ◽  
Saturation Kinetics ◽  
Basolateral Plasma Membrane ◽  
Component C ◽  
Uptake Studies

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB–HSA affinity constants (K´f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 μM, K´f (litre/mol) was inversely related to [HSA], irrespective of the [Bt]/[HSA] ratio. K´f was 2.066×106+(3.258×108/[HSA]). When 50 mM KCl was iso-osmotically substituted for sucrose, the K´f value was significantly lower {2.077×106+(1.099×108/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] ⩽ 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with Km values of 28±7 and 57±8 nM respectively (mean±S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112±12 versus 45±4 pmol of UCB·mg-1 of protein·15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K´f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with Km values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K´f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

scholarly journals Carrier-mediated uptake of L-(+)-lactate in plasma membrane vesicles from rat liver

FEBS Letters ◽  
1988 ◽  
Vol 235 (1-2) ◽  
pp. 224-228 ◽  
Author(s):  
Irene Quintana ◽  
Antonio Felipe ◽  
Xavier Remesar ◽  
Marçal Pastor-Anglada
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles

Characterization of two Mg2+transporters in sealed plasma membrane vesicles from rat liver

1998 ◽  
Vol 275 (4) ◽  
pp. C995-C1008 ◽  
Author(s):  
Christie Cefaratti ◽  
Andrea Romani ◽  
Antonio Scarpa
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Channel Blockers ◽  
Transport Mechanisms ◽  
Plasma Membrane Vesicles ◽  
New Information

The plasma membrane of mammalian cells possesses rapid Mg2+ transport mechanisms. The identity of Mg2+ transporters is unknown, and so are their properties. In this study, Mg2+ transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg2+with negligible leak; 2) the addition of Na+ or Ca2+ induces a concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+ ⋅ μm−2 ⋅ min−1after Na+ and Ca2+ addition, respectively); 4) coaddition of maximal concentrations of Na+ and Ca2+ induces an additive Mg2+ efflux; 5) both Na+- and Ca2+-stimulated Mg2+ effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+ channel blockers; 6) extracellular Na+ or Ca2+ can stimulate Mg2+ efflux in the absence of Mg2+ gradients; and 7) Mg2+ uptake occurs in LPM loaded with Na+ but not with Ca2+, thus indicating that Na+/Mg2+but not Ca2+/Mg2+exchange is reversible. These data are consistent with the operation of two distinct Mg2+ transport mechanisms and provide new information on rates of Mg2+ transport, specificity of the cotransported ions, and reversibility of the transport.

Thiamine transport by basolateral rat liver plasma membrane vesicles

1992 ◽  
Vol 103 (3) ◽  
pp. 1056-1065 ◽  
Author(s):  
Richard H. Moseley ◽  
Pankaj G. Vashi ◽  
Suzanne M. Jarose ◽  
Chris J. Dickinson ◽  
Patricia A. Permoad
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Liver Plasma Membrane ◽  
Thiamine Transport

Taurocholate transport by basolateral plasma membrane vesicles isolated from developing rat liver

1985 ◽  
Vol 248 (6) ◽  
pp. G648-G654
Author(s):  
F. J. Suchy ◽  
S. M. Courchene ◽  
B. L. Blitzer
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Plasma Membrane Vesicles ◽  
Adult Rat ◽  
Basolateral Plasma Membrane ◽  
Taurocholate Transport

Taurocholate transport was characterized in basolateral plasma membrane vesicles prepared from the livers of 14-day-old Sprague-Dawley rats using a self-generating Percoll gradient method. Liver plasma membrane protein yield, intravesicular volume, and enrichments of various marker enzymes were similar to those obtained for vesicles from adult rat liver. The basolateral marker enzyme Na+-K+-ATPase was enriched 26-fold in the suckling rat basolateral membrane fraction while the bile canalicular marker enzymes alkaline phosphatase and Mg2+-ATPase were enriched only 3- and 5-fold, respectively. The activities of marker enzymes for endoplasmic reticulum, mitochondria, or lysosomes were not enriched compared with homogenate. In the presence of an inwardly directed 100 mM Na+ gradient, vesicle accumulation of taurocholate transiently reached a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot") in suckling and adult membrane vesicles, but the initial rate of taurocholate entry and peak intravesicular accumulation were markedly decreased in suckling compared with adult membrane vesicles. In the presence of an inwardly directed 100 mM K+ gradient, the rate of uptake was slower, and no overshoot occurred in either suckling or adult rat vesicles. The decreased rate of Na+-coupled taurocholate uptake by membrane vesicles from suckling rat liver could not be explained on the basis of more rapid dissipation of the transmembrane Na+ gradient. Kinetic studies demonstrated saturable, Na+-dependent taurocholate uptake for both suckling and adult vesicles. However, the Vmax for taurocholate uptake in suckling rat vesicles was less than half of the adult rate (2.46 +/- 0.13 vs. 5.25 +/- 0.22 nmol X mg prot-1 X min-1, respectively, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

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