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.

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.

scholarly journals The kinetics of transport of lactate and pyruvate into isolated cardiac myocytes from guinea pig. Kinetic evidence for the presence of a carrier distinct from that in erythrocytes and hepatocytes

1989 ◽  
Vol 264 (2) ◽  
pp. 409-418 ◽  
Author(s):  
R C Poole ◽  
A P Halestrap ◽  
S J Price ◽  
A J Levi
Keyword(s):  
Guinea Pig ◽  
Cardiac Myocytes ◽  
Ventricular Myocytes ◽  
Silicone Oil ◽  
Free Acid ◽  
Competitive Inhibitor ◽  
Isolated Cardiac Myocytes ◽  
Lactate And Pyruvate ◽  
Time Courses ◽  
Kinetics Of

1. Time courses for the uptake of L-lactate, D-lactate and pyruvate into isolated cardiac ventricular myocytes from guinea pig were determined at 11 degrees C or 0 degrees C (for pyruvate) in a citrate-based buffer by using a silicone-oil-filtration technique. These conditions enabled initial rates of transport to be measured without interference from metabolism of the substrates. 2. At a concentration of 0.5 mM, transport of all these substrates was inhibited by approx. 90% by 5 mM-alpha-cyano-4-hydroxycinnamate; at 10 mM-L-lactate a considerable portion of transport could not be inhibited. 3. Initial rates of L-lactate and pyruvate uptake in the presence of 5 mM-alpha-cyano-4-hydroxycinnamate were linearly related to the concentration of the monocarboxylate and probably represented diffusion of the free acid. The inhibitor-sensitive component of uptake obeyed Michaelis-Menten kinetics, with Km values for L-lactate and pyruvate of 2.3 and 0.066 mM respectively. 4. Pyruvate and D-lactate inhibited the transport of L-lactate, with Ki values (competitive) of 0.077 and 6.6 mM respectively; the Ki for pyruvate was very similar to its Km for transport. The Ki for alpha-cyano-4-hydroxycinnamate as a non-competitive inhibitor was 0.042 mM. 5. These results indicate that L-lactate, D-lactate and pyruvate share a common carrier in guinea-pig cardiac myocytes; the low stereoselectivity for L-lactate over D-lactate and the high affinity for pyruvate distinguish it from the carrier in erythrocytes and hepatocytes. The metabolic roles for this novel carrier in heart 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.

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.

Free fatty-acid uptake by isolated rat hepatocytes

1985 ◽  
Vol 93 (4) ◽  
pp. 313-319 ◽  
Author(s):  
G. Renaud ◽  
M. E. Bouma ◽  
A. Foliot ◽  
R. Infante
Keyword(s):  
Fatty Acid ◽  
Free Fatty Acid ◽  
Rat Hepatocytes ◽  
Fatty Acid Uptake ◽  
Acid Uptake ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

Ascorbic acid uptake by isolated rat hepatocytes

1993 ◽  
Vol 46 (8) ◽  
pp. 1333-1338 ◽  
Author(s):  
Marie Christine Cornu ◽  
Gregory Adam Moore ◽  
Yoshio Nakagawa ◽  
Peter Moldéus
Keyword(s):  
Ascorbic Acid ◽  
Rat Hepatocytes ◽  
Acid Uptake ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat
Sign in / Sign up

Export Citation Format

Share Document