Nature of taurodehydrocholic acid uptake in rat hepatocytes

1988 ◽  
Vol 254 (2) ◽  
pp. G269-G274 ◽  
Author(s):  
W. G. Hardison ◽  
P. J. Lowe ◽  
E. Gosink
Keyword(s):  
Membrane Potential ◽  
Rat Hepatocytes ◽  
Competitive Inhibitor ◽  
Acid Uptake ◽  
Isolated Rat Hepatocytes ◽  
Acid Analogue ◽  
Sodium Gradient ◽  
Sodium Dependent ◽  
Independent Process ◽  
Transport Site

We studied uptake into isolated rat hepatocytes of the bile acid analogue taurodehydrocholate (TDHC) over a concentration range of 2.5-4,000 microM. Uptake was mainly by a saturable sodium-dependent process with a Km of approximately 50 microM and a Vmax of 0.036 nmol.s-1.mg protein-1. A lesser sodium-independent process was evident but was linear in the range studied. Both processes were inhibited by incubation at 37 degrees C under nitrogen in the presence of 3 mM sodium cyanide or by incubation at 0 degrees C. A single transport site was suggested by the Eadie-Hofstee plot of TDHC uptake from 2.5 to 750 microM. TDHC was a weak competitive inhibitor of taurocholic acid (TCA) uptake (Ki = 236 microM) but was not itself taken up by the TCA transport site. TCA exhibited moderately potent mixed inhibition of TDHC uptake. Uptake of both compounds was strongly inhibited by bromosulfophthalein (BSP) and Rose Bengal, whereas 0.5 mM alanine uptake was not affected. BSP exhibited a complex pattern of inhibition of TDHC uptake: mixed partial inhibition. Degree of inhibition of both TDHC and TCA uptake did not increase as BSP concentrations were increased from 50 to 100 microM. BSP did not exert its inhibitory effects by alteration of membrane potential or sodium gradients; 50 microM BSP changed membrane potential less than 10% and sodium gradient not at all. The data indicate that despite close structural analogy between TDHC and TCA, the two compounds are taken up by different sodium-dependent mechanisms. Nonetheless, the similar qualitative and quantitative effects of BSP on their uptakes suggests the mechanisms are related.

Threonine metabolism in isolated rat hepatocytes

2001 ◽  
Vol 281 (6) ◽  
pp. E1300-E1307 ◽  
Author(s):  
James D. House ◽  
Beatrice N. Hall ◽  
John T. Brosnan
Keyword(s):  
Potent Inhibitor ◽  
Rat Hepatocytes ◽  
Competitive Inhibitor ◽  
Acid Uptake ◽  
Keto Acid ◽  
Glycine Cleavage System ◽  
Isolated Rat Hepatocytes ◽  
Threonine Dehydratase ◽  
Threonine Dehydrogenase ◽  
Glycine Cleavage

The removal of the 1-carbon of threonine can occur via threonine dehydrogenase or threonine aldolase, this carbon ending up in glycine to be liberated by the mitochondrial glycine cleavage system and producing CO2. Alternatively, in the threonine dehydratase pathway, the 1-carbon ends up in α-ketobutyrate, which is oxidized in the mitochondria to CO2. Rat hepatocytes, incubated in Krebs-Henseleit medium, were incubated with 0.5 mMl-[1-14C]threonine, and14CO2 production was measured. Added glycine (0.3 mM) marginally suppressed threonine oxidation. Cysteamine (0.5 mM), a potent inhibitor of the glycine cleavage system, reduced threonine oxidation to 65% of controls. However, α-cyanocinnamate (0.5 mM), a competitive inhibitor of mitochondrial α-keto acid uptake, reduced threonine oxidation to 35% of controls. These data provided strong evidence that ∼65% of threonine oxidation occurs through the glycine-independent threonine dehydratase pathway. Glucagon (10−7 M) increased threonine oxidation and stimulated threonine uptake by these cells. In summary, the majority of threonine oxidation occurs through the threonine dehydratase pathway in rat hepatocytes, and threonine oxidation is increased by glucagon, which also increases threonine's transport.

Effect of glucagon on hepatic taurocholate uptake: relationship to membrane potential

1985 ◽  
Vol 249 (4) ◽  
pp. G427-G433
Author(s):  
J. W. Edmondson ◽  
B. A. Miller ◽  
L. Lumeng
Keyword(s):  
Plasma Membrane ◽  
Bile Acid ◽  
Membrane Potential ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Hill Coefficient ◽  
Isolated Rat Hepatocytes ◽  
Plasma Membrane Potential ◽  
Sodium Dependent

Since glucagon can hyperpolarize hepatic plasma membrane and stimulate biliary bile acid secretion in vitro, we studied the effect of glucagon on taurocholate uptake and its relationship to plasma membrane potential in isolated rat hepatocytes. [14C]taurocholate uptake was linear through 1 min and contained a saturable sodium-dependent and a nonsaturable sodium-independent component. Km of taurocholate uptake by the sodium-dependent system was 18.4 microM. Hill coefficient for Na+ was 2.59 and for taurocholate was 1.1, suggesting that the stoichiometry is 2 Na+:1 bile acid. Stimulation of taurocholate uptake by glucagon was limited to the sodium-dependent component, detected within 5 min of hormone exposure, and was maximum at 30 min. Glucagon, from 10(-8) to 10(-5) M, stimulated taurocholate uptake and hyperpolarized concurrently the plasma membrane potential. Because valinomycin produced a dose-related depolarization of plasma membrane potential, this agent was used to counteract the effects of glucagon. With 10(-6) M glucagon, valinomycin (10(-10) M) depolarized membrane potential from -35.50 to -28.00 mV and inhibited taurocholate uptake from 60% above the control rate to 5% below. These data strongly suggest that taurocholate uptake by isolated hepatocytes is an electrogenic process, and its stimulation by glucagon may be mediated by changes in plasma membrane potential.

scholarly journals The kinetics of transport of lactate and pyruvate into rat hepatocytes. Evidence for the presence of a specific carrier similar to that in erythrocytes

1988 ◽  
Vol 249 (1) ◽  
pp. 117-126 ◽  
Author(s):  
G L Edlund ◽  
A P Halestrap
Keyword(s):  
Silicone Oil ◽  
Significant Proportion ◽  
Rat Hepatocytes ◽  
Competitive Inhibitor ◽  
Acid Uptake ◽  
Lactate Transport ◽  
Diffusion Kinetic ◽  
Isolated Rat Hepatocytes ◽  
Carrier Mediated Transport ◽  
Lactate And Pyruvate

Time courses of L-lactate and pyruvate uptake into isolated rat hepatocytes were measured in a citrate-based medium to generate a pH gradient (alkaline inside), by using the silicone-oil-filtration technique at 0 degrees C to minimize metabolism. At low concentrations of lactate and pyruvate (0.5 mM), transport was inhibited by over 95% by 5 mM-alpha-cyano-4-hydroxycinnamate, whereas at higher concentrations (greater than 10 mM) a significant proportion of transport could not be inhibited. The rate of this non-inhibitable transport was linearly related to the substrate concentration, was less with pyruvate than with L-lactate, and appeared to be due to diffusion of undissociated acid. Uptake of D-lactate was not inhibited by alpha-cyano-4-hydroxycinnamate and occurred only by diffusion. Kinetic parameters for the carrier-mediated transport process were obtained after correction of the initial rates of uptake of lactate and pyruvate in the absence of 5 mM-alpha-cyano-4-hydroxycinnamate by that in the presence of inhibitor. Under the conditions used, the Km values for L-lactate and pyruvate were 2.4 and 0.6 mM respectively and the Ki for alpha-cyano-4-hydroxycinnamate as a competitive inhibitor was 0.11 mM. Km values for the transport of L-lactate and pyruvate into rat erythrocytes under similar conditions were 3.0 and 0.96 mM. The Vmax. of lactate and pyruvate transport into hepatocytes at 0 degrees C was 3 nmol/min per mg of protein. Carrier-mediated transport of 0.5 mM-L-lactate was inhibited by 0.2 mM-p-chloromercuribenzenesulphonate (greater than 90%), 0.5 mM-quercetin (80%), 0.6 mM-isobutylcarbonyl-lactyl anhydride (70%) and 0.5 mM-4,4′-di-isothiocyanostilbene-2,2′-disulphonate (50%). A similar pattern of inhibition of lactate transport is seen in erythrocytes. It is suggested that the same or a similar carrier protein exists in both tissues. The results also show that L-lactate transport into rat hepatocytes is very rapid at physiological temperatures and is unlikely to restrict the rate of its metabolism. Differences between our results and those of Fafournoux, Demigne & Remesy [(1985) J. Biol. Chem. 260, 292-299] are discussed.

Copper transport kinetics by isolated rat hepatocytes

1983 ◽  
Vol 244 (2) ◽  
pp. G183-G191 ◽  
Author(s):  
R. C. Schmitt ◽  
H. M. Darwish ◽  
J. C. Cheney ◽  
M. J. Ettinger
Keyword(s):  
Trace Metals ◽  
Specific Inhibitor ◽  
Rat Hepatocytes ◽  
Competitive Inhibitor ◽  
Metabolic Inhibitors ◽  
Rapid Phase ◽  
Isolated Rat Hepatocytes ◽  
Simple Diffusion ◽  
Saturation Kinetics ◽  
Parenchymal Cells

Uptake and efflux of 64Cu were examined to determine whether hepatic parenchymal cells exhibit the kinetic criteria of a specific transport system for copper and related trace metals. Saturation kinetics were clearly indicated by both v versus [Cu] and 1/v versus 1/[Cu] plots (Km = 11 +/- 0.6 microM and Vmax = 2.7 nmol Cu X min-1 X mg prot-1). Identical results were obtained by cold-copper analyses, and contributions from simple diffusion or nonspecific binding were not detected. Virtually all of the accumulated 64Cu was intracellular by 0.5 min (the initial velocity period), with approximately 40% in the cytosolic fraction. Several related trace metals inhibited 64Cu uptake, but Ni(II) at a 10:1 molar excess did not. Zn(II) acted as a simple competitive inhibitor of 64Cu uptake (Ki = 16 microM). Efflux from preloaded cells was biphasic, with an initial rapid phase of approximately 5 min. Approximately 35% of preloaded 64Cu was transported out of the cells by 40 min, and little efflux occurred thereafter. Thus, hepatocytes exhibit saturation kinetics, competition by related substrates, and countertransport criteria of specific facilitated transport. A wide variety of metabolic inhibitors have no effect on 64Cu uptake under the same conditions that inhibit the active transport of bile acids. Specific inhibitor tests for electrogenic coupling were also negative. Because the identical kinetic parameters were obtained for free 64Cu and the 1:1 64Cu-histidine complex, it is inferred that copper is probably transported as the free ion. Cells incubated with greater than or equal to 10 microM 64Cu showed a net loss of copper after 40- to 60-min incubation, which may involve specific hepatic mechanisms in copper homeostasis.

The effects of potassium and membrane potential on sodium-dependent glutamic acid uptake

1980 ◽  
Vol 599 (1) ◽  
pp. 191-201 ◽  
Author(s):  
Gerhard Burckhardt ◽  
Rolf Kinne ◽  
Gertraud Stange ◽  
Heini Murer
Keyword(s):  
Membrane Potential ◽  
Glutamic Acid ◽  
Acid Uptake ◽  
Sodium Dependent

Interaction of bumetanide derivatives with hepatocellular bile acid uptake

1993 ◽  
Vol 265 (5) ◽  
pp. G942-G954
Author(s):  
E. Petzinger ◽  
W. Follmann ◽  
M. Blumrich ◽  
R. Schermuly ◽  
S. Schulz ◽  
...  
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Model Building ◽  
Organic Anion ◽  
Rat Hepatocytes ◽  
Salt Transport ◽  
Acid Uptake ◽  
Ligand Interaction ◽  
Isolated Rat Hepatocytes ◽  
Bile Acid Uptake

The loop diuretic bumetanide is an organic monocarboxylic organic anion assumed to be transported into hepatocytes by a transport system for bile acids. The structural requirements of 22 bumetanide analogues were analyzed for an interaction with bile acid uptake into isolated rat hepatocytes. Whereas bumetanide inhibited the hepatocellular uptake of [14C]cholate to the same degree as its own uptake, derivatization altered affinity and specificity and yielded compounds that selectively inhibited either cholate or taurocholate uptake or uptake of both. No correlation was found between the diuretic potency of bumetanide derivatives, reflecting the affinity to the Na(+)-K(+)-Cl- cotransporter, and their affinity to hepatic bile salt transport. Computer-aided model building combined with the calculation of potential energy maps showed a strictly amphipathic charge separation in bumetanide analogues as in bile acids. Ranking bumetanide compounds by their mean inhibitory concentration values, inhibition constants, and their type of competition, we conclude that at least three binding domains in the proteins are essential for recognition by bile acid transporters, namely two hydrophobic and an anionic side, and that for the anionic binding region a carbonyl atom in the ligands as an electron donor group is sufficient for ligand interaction.

Effect of membrane potential and cellular ATP on glutathione efflux from isolated rat hepatocytes

1988 ◽  
Vol 255 (4) ◽  
pp. G403-G408 ◽  
Author(s):  
J. C. Fernandez-Checa ◽  
C. Ren ◽  
T. Y. Aw ◽  
M. Ookhtens ◽  
N. Kaplowitz
Keyword(s):  
Membrane Potential ◽  
High Performance ◽  
Direct Relationship ◽  
Rat Hepatocytes ◽  
Antimycin A ◽  
Total Glutathione ◽  
Rat Hepatocyte ◽  
Isolated Rat Hepatocytes ◽  
Carbonyl Cyanide ◽  
Isolated Rat

total glutathione (GSH) efflux was studied in isolated rat hepatocyte suspensions at repleted GSH content (45-55 nmol/10(6) cells). The increase in concentrations of medium K+ in place of Na+ caused a parallel fall in membrane potential and total GSH efflux. Ouabain (1 mM) and replacement of Na+ with choline caused a gradual fall in membrane potential and GSH efflux. Hyperpolarization of hepatocytes with lipophilic anions, thiocyanate, and nitrate was associated with significantly increased efflux. Total GSH efflux was inhibited by increasing concentrations of fructose, antimycin A, and carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and there was a direct relationship between the rate of efflux and cellular ATP. Changes in total GSH efflux were paralleled by changes in GSH determined by high-performance liquid chromatography. Vanadate markedly inhibited efflux but caused only a modest decrease in cellular ATP. Fructose, antimycin A, and vanadate did not affect membrane potential or cell volume under the conditions at which efflux was inhibited. These results suggest independent requirements for both membrane potential and ATP in the transport of GSH.

scholarly journals Involvement of intracellular Ca2+ and K+ in dissipation of the mitochondrial membrane potential and cell death induced by extracellular ATP in hepatocytes

1992 ◽  
Vol 288 (1) ◽  
pp. 207-213 ◽  
Author(s):  
J P Zoeteweij ◽  
B van de Water ◽  
H J de Bont ◽  
G J Mulder ◽  
J F Nagelkerke
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Rat Hepatocytes ◽  
Extracellular Atp ◽  
Complete Loss ◽  
Isolated Rat Hepatocytes ◽  
Isolated Mitochondria ◽  
Isolated Rat

Isolated rat hepatocytes were incubated with extracellular ATP to induce a prolonged increase in intracellular Ca2+ ([Ca2+]i) and a loss of viability within 2 h. By using video-intensified fluorescence microscopy, the effects of exposure to extracellular ATP on [Ca2+]i, mitochondrial membrane potential (MMP) and cell viability were determined simultaneously in individual living hepatocytes. The increase in [Ca2+]i on exposure to ATP was followed by a decreasing MMP; there were big differences between individual cells. Complete loss of the MMP occurred before cell death was observed. Omission of K+ from the incubation medium decreased the cytotoxicity of ATP; under these conditions, intracellular K+ was decreased by more than 80%. Treatment with nigericin also depleted intracellular K+ and decreased ATP-induced toxicity. Protection against loss of viability by means of a decrease in intracellular [K+] was reflected by maintenance of the MMP. These observations suggest that ATP-induced cell death may be caused by a mechanism that has been described for isolated mitochondria: after an increase in Ca2+ levels, a K+ influx into mitochondria is induced, which finally disrupts the MMP and leads to cell death.

Sign in / Sign up

Export Citation Format

Share Document