Factors involved in the uptake of corticosterone by rat liver cells

Isolated rat liver cells take up corticosterone rapidly; the initial rates increase with increasing temperature. A plot of the initial rates against the concentration of corticosterone indicated the presence of saturable and nonsaturable uptake systems. The Eadie-Hofstee plot showed the presence of two saturable and one nonsaturable uptake components. The apparent K_t values of the saturable systems were 64 ± 40 nM (n =3) and 1085 ± 313 nM (n =12). The nonsaturable system, probably diffusion, contributed 12% to the total uptake between 15 and 72 nM corticosterone, the physiological concentration of the free corticosterone in rat serum. Metabolic inhibitors did not influence the uptake of corticosterone. N-Ethylmaleimide, 1-fluor-2,4-dinitrobenzene and sodium ethyl mercurithiosalicylate (1 mM each) decreased the uptake by 40%. Iodoacetate did not have any influence. Treatment of cells with phospholipase A inhibited the uptake 35–45%. In the presence of cortisone, cortisol, dexamethasone, aldosteron, testosterone, estradiol-17β and estrone (2 μM each) the uptake decreased 30–50%. The presence of serum proteins in the external medium inhibits the uptake of corticosterone. These results suggest that corticosterone is transported into the cell and is accumulated. Only the free hormone is available for uptake which in turn may be regulated by protein and lipid components in the plasma membrane of the liver cell.     

Adipocyte differentiation of 3T3-L1 cells involves augmented expression of a 43-kDa plasma membrane fatty acid-binding protein.

A previously described 43-kDa plasma membrane fatty acid-binding protein (FABPPM) was not observed by immunohistochemical methods in proliferating 3T3-L1 fibroblasts. However, it was detectable in plasma membranes by the second day of confluent growth, prior to accumulation of visible lipid droplets, and was strongly expressed in 8-day differentiated adipocytes. These observations were confirmed by extraction of plasma membrane proteins and subsequent immunoblotting. Kinetics of initial [3H]oleate uptake by both fibroblasts and adipocytes consisted of the sum of a saturable and a non-saturable component. During differentiation the saturable component increased progressively. Vmax increased from 3 to 25 to 110 pmol.s-1.mg cell protein-1 between the fibroblast, the 4-day, and 8 day adipocyte stages; Km was 24 nM in fibroblasts and approximately 55 nM in both 4- and 8-day differentiated adipocytes. By contrast, the rate constant for nonsaturable oleate influx decreased progressively from 0.026 to 0.010 ml.s-1.mg protein-1 between the fibroblast and 8 day adipocyte stages. In 8-day adipocytes saturable oleate uptake was inhibited by up to 55% by antibodies against rat liver FABPPM; these antibodies had no effect on uptake of 2-deoxyglucose or the medium chain fatty acid octanoate. They also had no effect on oleate uptake by fibroblasts. These studies support the hypothesis that FABPPM is a component of a saturable transport mechanism for long chain fatty acids.     

The inhibitory effect of choline and other quaternary ammonium compounds on thiamine transport in isolated rat hepatocytes

Quaternary ammonium compounds, such as choline and acetylcholine significantly inhibited thiamine uptake in isolated rat hepatocytes. Kinetic analysis using Lineweaver-Burk and Dixon plots of inhibition experiments revealed that choline and acetylcholine were purely competitive inhibitors for thiamine uptake with Ki values of 0.61 mM and 0.31 mM, respectively. Among quaternary ammonium compounds, hemicholinium-3 and curare were the strongest inhibitors, and kinetic studies showed that these compounds were also purely competitive inhibitors with Ki values of 12.5 microM and 4.3 microM, respectively. These results indicate that choline, acetylcholine and their structural analogs share a common binding site with thiamine in isolated rat hepatocytes. On the other hand, choline uptake by isolated rat hepatocytes occurred by a saturable mechanism with a Kt of 162 +/- 3.85 microM and Vmax of 80.1 +/- 1.30 pmol/10(5) cells per min as well as by a nonsaturable mechanism. Thiamine, pyrithiamine, oxythiamine, chloroethylthiamine and dimethialium inhibited choline uptake, while thiamine phosphates such as thiamine monophosphate and thiamine pyrophosphate insignificantly inhibited uptake. Although a Lineweaver-Burk plot of choline uptake in the presence of thiamine showed that thiamine also competitively inhibited choline uptake, a Dixon plot of the inhibition experiment was hyperbolic and indicated that the inhibition of choline uptake by thiamine was 'pseudo-competitive'. On the basis of these results, it is suggested that in isolated rat hepatocytes thiamine and choline do not share common transport sites.     

The high affinity (Ca2+-Mg2+)-ATPase in liver plasma membranes is a Ca2+ pump. Reconstitution of the purified enzyme into phospholipid vesicles

The purified (Ca2+-Mg2+)-ATPase from rat liver plasma membranes (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215) was incorporated into soybean phospholipid vesicles, together with its activator. In the presence of millimolar concentrations of Mg2+, the reconstituted proteoliposomes displayed a rapid, saturable, ATP-dependent Ca2+ uptake. Half-maximal Ca2+ uptake activity was observed at 13 +/- 3 nM free Ca2+, and the apparent Km for ATP was 16 +/- 6 microM. Ca2+ accumulated into proteoliposomes (2.8 +/- 0.2 nmol of Ca2+/mg of protein/90 s) was totally released upon addition of the Ca2+ ionophore A-23187. Ca2+ uptake into vesicles reconstituted with enzyme alone was stimulated 2-2.5-fold by the (Ca2+-Mg2+)-ATPase activator, added exogenously. The (Ca2+-Mg2+)-ATPase activity of the reconstituted vesicles, measured using the same assay conditions as for ATP-dependent Ca2+ uptake activity (e.g. in the presence of millimolar concentrations of Mg2+), was maximally activated by 20 nM free Ca2+, half-maximal activation occurring at 13 nM free Ca2+. The stoichiometry of Ca2+ transport versus ATP hydrolysis approximated 0.3. These results provide a direct demonstration that the high affinity (Ca2+-Mg2+)-ATPase identified in liver plasma membranes is responsible for Ca2+ transport.     

Oleic acid uptake and binding by rat adipocytes define dual pathways for cellular fatty acid uptake

Oleic acid (OA) uptake by rat adipocytes and the proportions of intracellular unesterified [3H]OA and its 3H-labeled esters were determined over 300 s. Uptake was linear for 20;-30 s, with rapid esterification indicating entry into normal metabolic pathways. Initial rates of OA uptake and its binding to plasma membranes were studied over a spectrum of oleic acid:bovine serum albumin (BSA) ratios, and expressed as functions of unbound OA concentrations calculated with both the 1971 OA:BSA association constants of Spector, Fletcher, and Ashbrook and more recent constants (e.g., the 1993 constants of Richieri, Anel, and Kleinfeld), which generate concentrations 10- to 100-fold lower. In either case, uptake was the sum of saturable and linear processes, with > or =90% occurring via the saturable pathway when the OA:BSA molar ratio was within the physiologic range (0.5;-3.0). Within this range, rate constants for saturable transmembrane influx (k(s)), calculated from both sets of constants, were similar (2.9 s(-1)) and were 10- to 30-fold faster than those for nonsaturable uptake (k(ns) = 0.26;-0.10 s(-1), t1/2 = 2.7;-6.6 s, based on the constants of Spector et al. and Richieri et al., respectively). The rate of oleic acid flip-flop into rat adipocytes (k(ff) = 0.16 +/- 0.02 s(-1), t1/2 = 4.3 +/- 0.5 s), computed from published data, was similar to k(ns). Thus, OA uptake occurs by both a saturable mechanism and passive flip-flop. This conclusion is independent of the OA:BSA association constants used to analyze the experimental measurements.     

Effect of 25-hydroxy-cholesterol on the uptake of corticosterone in isolated rat liver cells

Isolated rat liver cells are able to take up corticosterone. The Arrhenius plot of the uptake shows a biphasic course with a change in the slope around 25 degrees C. 25-OH-cholesterol is also taken up; this phenomenon reaches a maximum at 10-15 minutes. After preincubating liver cells at 37 degrees C in the presence of this sterol the phase transition is shifted to a higher temperature (32 degrees C) as shown in the Arrhenius plot of the corticosterone uptake. At the same time the uptake of corticosterone is diminished. This cannot readily be explained by direct competition. The mechanism might involve an inhibition of an active uptake mechanism caused by a change in the plasma membrane.     

Saturable and nonsaturable process of sugar uptake: effect of oncogenic transformation in transport and uptake of nutrients.

Uptake of sugars into cells by a saturable process increased enormously during and after transformation, and uptake by a nonsaturable process increased significantly but less remarkably compared to controls. The drastic change of uptake rates, observed at around 5 x 10(-3) M sugar during and after transformation, emphasizes the significant observation that transition of the sugar uptake system from a saturable to a nonsaturable process occurs near the physiological concentration of D-glucose normally seen in animal blood. At concentrations below higher than 5 x 10(-3) M, where a saturable process is barely involved, nonsaturable uptakes of D-glucose, D-mannose, D-galactose, 2-deoxy-D-glucose and 3-O-methyl-D-glucose proceed tens to hundreds fold faster than the rate of simple diffusion of L-glucose. These findings suggest that nonsaturable uptake of the sugars known to be substrates for the saturable transport carrier system may not be a physical process or simple diffusion, as observed for L-glucose uptake. Rather, the nonsaturable uptake might be part of the total physiological process which, along with the saturable process, is controlled by a membrane-coordination mechanism. A plausible mechanism is discussed in which negative cooperativity of nutrient uptake, such as that found in bacteria, is involved.     

Uptake of thyroid hormone by isolated rat liver cells

L-Triiodothyronine is taken up by isolated rat liver cells by a process which is saturable and exhibits sigmoidity. Two uptake systems make themselves evident: A system with high affinity with an apparent K_t value of 52±22 nM and a system with low affinity with an apparent K_t value of 1446±764 nM. Cells heated at 60°C or after freezing do not show saturability of uptake. KCN inhibits the uptake by the low affinity system. In the presence of L-thyroxine and L-tyrosine the uptake of L-triiodothyronine is increased. The results suggest transport of L-triiodothyronine by proteins in the plasma membrane of the liver cell.     

Effect of insulin and glucagon on the uptake of carnitine by perfused rat liver

The possible direct effects of insulin and glucagon on carnitine uptake by perfused rat liver were studied with L-[3H]carnitine of an initial concentration of 50 microM in the perfusate. Insulin (10 nM) did not significantly affect the uptake by livers from fed animals. However, insulin could reverse the stimulated transport by livers from 24-h fasted animals, reducing the uptake rate from 852 +/- 54.1 to 480 +/- 39.9 (mean +/- S.E.), P less than 0.01 (rates are expressed as nmol per h per 100 g body wt). Glucagon (50 nM) stimulated the uptake rate when livers were either from fed (551 +/- 40.1 vs. 915 +/- 55.3, P less than 0.01) or from fasted animals (852 +/- 54.1 vs. 1142 +/- 88.1, P less than 0.02). Based on these and earlier observations, we propose that the carnitine concentration in rat liver is controlled by insulin and glucagon via cellular transport processes.     

Glucocorticoid-recognizing and -effector sites in rat liver plasma membrane. Kinetics of corticosterone uptake by isolated membrane vesicles--I. Binding and transport.

To gain insight into the mechanisms governing cellular uptake of glucocorticoids, we studied the binding and membrane transport of corticosterone (B) on a highly purified plasma membrane fraction from rat liver that was homogenized using a gentle, isotonic procedure. The fraction was mostly in the form of right-side out and osmotically active vesicles that were free of intracellular glucocorticoid receptors (GCR), transcortin (CBG) and ATP. Our uptake and binding studies carried out at 22 degrees C with [3H]B in physiological concentrations resulted in the following findings: (1) unlabeled B competed with [3H]B for uptake by the membrane vesicles; half-maximal competition of specific uptake was achieved with a 10- to 11-fold molar excess of unlabeled B. (2) [3H]B uptake was a saturable process of unusual kinetics (multiple sigmoidity); modified Scatchard plots revealed three significantly different apparent Kd-values of 1.3, 4.7 and 17.3 nM, corresponding to free B in the blood of non-stressed rats (4-16 nM). (3) Osmotic shrinkage of the vesicles led to a linear decrease in specific uptake, while non-specific uptake was independent of vesicle volume. Passive diffusion of [3H]B took place in leaky, but not in intact, vesicles. Reversible binding to, and mediated transport through, the membrane were interdependent parts of a strongly linked process. B was accumulated inside the vesicle up a concentration gradient by an active transport that followed first-order kinetics (Kt:3.9 nM); for its statistically reliable mathematical formulation and kinetic analysis, a replot was developed that revealed that relative accumulation increased with decreasing external hormone concentration. (4) Comparative binding studies disclosed that the apparent Kd-values (86.5 +/- 7.3 and 77.0 +/- 14.3 nM, respectively) of the [3H]B interactions with CBG and GCR did not differ (P greater than 0.3). These findings permit the conclusion that a plasma membrane-inserted carrier for B, effectively operating at physiological concentrations in the blood, is involved in a functional and regulatory manner in the biological action of glucocorticoids.     

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.

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.     

Derepression of amino Acid-h cotransport in developing soybean embryos

The uptake of the unnatural amino acid α-aminoisobutyric acid (AIB) and glutamine by developing soybean (Glycine max Merr. cv Chippewa 64) embryos was investigated. In freshly excised embryos, the accumulation ratio (cytoplasmic concentration/external concentration) of AIB did not exceed 1.0. After an 18-hour preincubation in nitrogen-free medium the accumulation ratio of AIB exceeded 4.5 at an external AIB concentration of 10 micromolar. This indicates the derepression of an active amino acid uptake mechanism operative at low external amino acid concentration. The presence of sucrose, NH₄NO₃, or glutamine during a 21-hour preincubation prior to measuring glutamine uptake inhibited the enhancement of uptake by 43%, 51%, and 96%, respectively. The time course of the decline in free amino acids and the time course of enhancement of amino acid uptake was not consistent with enhanced uptake resulting from relief of transinhibition, but suggested instead the derepression of synthesis of new carriers. The time course of enhancement of amino acid uptake was paralleled by an increase in glutamine-induced depolarization of the membrane potential. The kinetics of glutamine uptake indicated the presence of a saturable and a nonsaturable component of uptake. The saturable component of uptake is attributed to a mechanism of amino acid-H⁺ cotransport which is derepressed by nitrogen and/or carbon starvation. At physiological concentrations of amino acids, uptake through the saturable system in freshly excised embryos is negligible. Thus, uptake through the nonsaturable system is of primary importance in the nitrogen nutrition of developing soybean embryos.     

alpha-aminoisobutyrate transport into cells from R3230AC mammary adenocarcinoma. Evidence for sodium ion-dependent and -independent carrier-mediated entry and effects of diabetes.

In the presence of Na+, alpha-aminoisobutyrate was transported by saturable and non-saturable processes into R3230AC mammary tumour cells isolated by enzymic treatment. Eadie-Hofstee analysis for the saturable process gave a curvilinear plot, suggesting that transport occurred by more than one carrier. In the absence of Na+, alpha-aminoisobutyrate was also transported by both saturable and non-saturable processes. This Na+-independent saturable process gave a linear plot according to Eadie-Hofstee analysis: V, 708 +/- 105 pmol/min per 5 X 10(6) cells; Km, 0.36 +/- 0.33 mM (mean +/- S.E.M.). Subtracting alpha-aminoisobutyrate entry in the absence of Na+ from total alpha-aminoisobutyrate uptake (in the presence of Na+) showed the presence of another saturable process (Na+-dependent), accounting for 75% of total alpha-aminoisobutyrate uptake. This component gave a linear Eadie-Hofstee plot: V, 2086 +/- 213; Km, 1.75 +/- 0.16 alpha-(Methylamino)isobutyrate, a substrate specifically taken up by the A system, inhibited 80% of alpha-aminoisobutyrate entry. The presence of both alhpa-(methylamino)isobutyrate and phenylalanine inhibited alpha-aminoisobutyrate entry completely. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate, an analogue specifically taken up by the Na+-independent system, inhibited completely the Na+-independent entry of alpha-aminoisobutyrate. In the presence of Na+, the distribution ratio, which is defined as the amino acid concentration in the intracellular space divided by that in the incubation medium for alpha-aminoisobutyrate, at 90 min was 19, and in the absence of Na+ at 60 min was 5. These concentrative processes were sensitive to the metabolic inhibitor pentachlorophenol. The Na+-dependent, but not the Na+-independent, alpha-aminoisobutyrate uptake was increased in cells from diabetic rats. This was primarily due to an increase in the V for the Na+-dependent component (164%) with no effect on the Km. We conclude, therefore, that alpha-aminoisobutyrate entry into cells from this mammary tumour is mediated by two transport systems, one Na+-dependent and another Na+-independent. Furthermore, the Na+-dependent component of alpha-aminoisobutyrate is sensitive to alterations of insulin in vivo.     

Identification and properties of renal mineralocorticoid receptors in relation to glucocorticoid binders in rat liver and kidney.

The binding of the natural mineralocorticoid aldosterone and the glucocorticoid corticosterone to macromolecules in rat liver and kidney cytoplasmic fractions was compared by various chromatographic procedures. Equilibration of kidney cytosol with 10nM-aldosterone, either alone or in the presence of a competing steroid, was ideal for ionexchange chromatography of DEAE-cellulose DE-52, and revealed the presence of four sorts of binding components. One of these, eluted in the 0.001M-phosphate pre-wash, and another, less abundant, forming a peak at 0.006M-phosphate, did not bind corticosterone at equimolar concentrations, and appear to constitute the mineralocorticoid-specific 'MR' receptor in rat kidney. They could not be detected in the liver. Radioactivity eluted in the 0.02 and 0.06M-phosphate regions on DEAE-cellulose DE-52 appears to be due to [3H]aldosterone binding to glucocorticoid-specific 'GR' receptors and to transcortin respectively, since labelling was greater with corticosterone even at 10 nM than with the mineralocorticoid at 100nM and since [14C]corticosterone bound to blood serum transcortin was always co-chromatographed in the 0.06M-phosphate region. These two components appear to be identical with those in the liver and could be labelled maximally only by 100nM-corticosterone. The separation between specific mineralo- and glucocorticoid-binding species was less clear when chromatography was attempted on DEAE-Sephadex A-50 columns, possibly because of disaggregation into subunits in the presence of the high KC1 concentrations required for elution. Competitive binding followed by filtration through Sephadex G-200 gel indicated that cellular MR binders, unlike GR receptors, exist mostly as high-molecular-weight aggregates, although both appear to exhibit a comparable monomeric molecular weight of approx. 67000.     

Uptake of corticosterone into isolated rat liver cells: Possible involvement of Na/K-ATPase

Isolated rat hepatocytes posses a saturable glucocorticoid uptake system with high affinity (K_d value = 2.8 ± 0.7 × 10⁻⁸ M; 318,000 ± 80,000 binding sites per cell; 317 fmol/mg protein). The initial rates of uptake decrease by about 30–40% if the cells are incubated simultaneously with [³H]corticosterone and either SH-reagents (N-ethylmaleimide and p-chloromercuriphenylsulphonate, 1 mM), metabolic inhibitors (2,4-dinitrophenol, 1 mM; and antimycin, 0.1 mM) or the Na⁺/K⁺-ATPase-inhibitors, ouabain and quercetine. These Na⁺/K⁺-ATPase-blockers exert half-maximal inhibition at 3 × 10⁻⁷ and 3 × 10⁻⁶ M, respectively. A slight increase in K⁺ concentration and a corresponding decrease in Na⁺ in the medium leads to a significant reduction in the initial uptake rate. The uptake system from the rat hepatocytes shows a clear steroid specificity, being different from the intracellular receptor. Corticosterone and progesterone are the strongest competitors, cortisol, 5- and 5β-dihydrocorticosterone, 11-deoxycorticosterone, cortisone and testosterone have an intermediate effect and only weak competition is exerted by dexamethasone and by the mineralocorticoid, aldosterone. Estradiol and estrone sulphate as well as the synthetic glucocorticoid triamcinolone acetonide are unable to inhibit initial corticosterone uptake.     

Uptake of [(3)H]bilirubin in freshly isolated rat hepatocytes: role of free bilirubin concentration

Hepatocytic transport of physiological concentrations of unconjugated bilirubin (UCB) has not been determined in isolated liver cells. Initial uptake of highly purified [(3)H]UCB was measured in rat hepatocytes in the presence of human serum albumin at various free, unbound UCB concentrations, [UCB]. At [UCB]=42 nM (below aqueous solubility of 70 nM), uptake was strictly temperature dependent; this was much less evident at [UCB]=166 nM (supersaturated). At low, physiological UCB concentrations, specific UCB uptake showed saturative kinetics with an apparent K(m) of 41 nM, indicating carrier-mediated transport. With aqueous supersaturation, UCB entered hepatocytes mainly by passive diffusion.     

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

1. Influx and efflux of l-tri-[(125)I]iodothyronine with isolated rat liver parenchymal cells and their plasma-membrane vesicles were studied by a rapid centrifugation technique. 2. At 23 degrees 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 K(t) value of 8.6+/-1.6pm was obtained. 3. At 5pm-l-tri-[(125)I]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 degrees C) uptake of l-tri-[(125)I]iodothyronine by cells was linear with the hormone concentration, whereas that by plasma-membrane vesicles exhibited an apparent saturation with a K(d) value of 6.1+/-1.3pm. 4. Efflux of l-tri-[(125)I]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-[(125)I]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-[(125)I]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-[(125)I]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-[(125)I]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-[(125)I]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.     

Taurocholate uptake by adult rat hepatocytes in primary culture

Adult rat hepatocytes were cultured on Petri dishes for 25--30 h prior to measuring their ability to transport taurocholate. A rapid uptake of the bile acid (25 muM) was observed: about 20% was accumulated in the cells within 15 min. The taurocholate transport was saturable with an apparent Km of 28 +/- 10 muM and a maximal velocity V of 0.07 +/- 0.02 nmol/(micrograms DNA x min). Uptake was shown to be energy dependent as it was inhibited about 65% by antimycin A (20 micrograms/ml). The monohydroxylated bile acid taurolithocholate and the dihydroxylated taurochenodeoxycholate inhibited taurocholate transport to about 30 and 40% resp. of the control. The transport process was strongly dependent on sodium ions. It is concluded that the characteristics of taurocholate uptake into adult rat hepatocytes are very similar either in freshly prepared cells or in hepatocytes which are cultured on Petri dishes for 25--30 h.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Saturable, energy-dependent uptake of phenanthrene in aqueous phase by Mycobacterium sp. strain RJGII-135

The mechanism of uptake of phenanthrene by Mycobacterium sp. strain RJGII-135, a polycyclic hydrocarbon-degrading bacterium, was examined with cultures grown on phenanthrene (induced for phenanthrene metabolism) and acetate (uninduced). Washed cells were suspended in aqueous solutions of [9-(14)C]phenanthrene, and then the cells were collected by filtration. Low-level steady-state (14)C concentrations in uninduced cells were achieved within the first 15 s of incubation. This immediate uptake did not show saturation kinetics and was not susceptible to inhibitors of active transport, cyanide and carbonyl cyanide m-chlorophenylhydrazone. These results indicated that phenanthrene enters rapidly into the cells by passive diffusion. However, induced cells showed cumulative uptake over several minutes. The initial uptake rates followed saturation kinetics, with an apparent affinity constant (K(t)) of 26 +/- 3 nM (mean +/- standard deviation). Uptake of phenanthrene by induced cells was strongly inhibited by the inhibitors. Analysis of cell-associated (14)C-labeled compounds revealed that the concurrent metabolism during uptake was rapid and was not saturated at the substrate concentrations tested, suggesting that the saturable uptake observed reflects membrane transport rather than intracellular metabolism. These results were consistent with the presence of a saturable, energy-dependent mechanism for transport of phenanthrene in induced cells. Moreover, the kinetic data for the cumulative uptake suggested that phenanthrene is specifically bound by induced cells, based on its saturation with an apparent dissociation constant (K(d)) of 41 +/- 21 nM (mean +/- standard deviation). Given the low values of K(t) and K(d), Mycobacterium sp. strain RJGII-135 may use a high-affinity transport system(s) to take up phenanthrene from the aqueous phase.