ATP-dependent Ca2+ transport in vesicles isolated from the bile canalicular region of the hepatocyte plasma membrane

Three plasma membrane subfractions have been isolated and characterized from rat liver cells. The high affinity Ca2+-stimulated ATPase is highly enriched in the bile canalicular subfraction. Taking into account cross-contamination by the blood sinusoidal and lateral membranes it is suggested that the high-affinity Ca2+-ATPase is located exclusively in this fraction. The high-affinity Ca2+-ATPase is coupled to Ca2+ transport, is calmodulin-insensitive, sensitive to vanadate under appropriate experimental conditions and is strongly inhibited by La3+. In the presence of Ca2+ and ATP the ATPase forms a phosphorylated intermediate of molecular mass about 200 kDa.     

Effect of 2,5-anhydro-D-mannitol on membrane potential in rat hepatocyte couplets and hepatocyte monolayer cultures

The fructose analogue 2,5-anhydro-D-mannitol (2,5-AM), which depletes liver cells of ATP, has been shown to alter liver cell membrane potential (V(m)) in situ and in superfused liver slices. To study this effect of 2,5-AM on hepatocytes in more detail, patch-clamp experiments in the current-clamp mode were performed using two established models, rat hepatocyte couplets and confluent rat hepatocytes in primary culture. 2,5-AM, which has previously been shown to hyperpolarize hepatocytes in superfused liver slices and in vivo, failed to alter V(m) of hepatocyte couplets. Increasing intracellular Ca(2+) by addition of thapsigargin or ionomycin also did not evoke a change of V(m). This is most likely due to a lack of Ca(2+)-dependent K(+) channels in rat hepatocyte couplets. In contrast, 2,5-AM depolarized the cells in confluent hepatocyte monolayers. This depolarization was mimicked after inhibition of Na(+)/K(+) ATPase by ouabain. Ouabain was also able to block 2, 5-AM's effect on monolayer V(m). Thus, 2,5-AM affects the membrane potential of isolated and cultured hepatocytes in a way not comparable with cells integrated in the liver.     

Bile duct ligation-induced redistribution of canalicular antigen in rat hepatocyte plasma membranes demonstrated by immunogold quantitation

Extrahepatic obstructive cholestasis has been demonstrated to induce a redistribution of domain specific membrane proteins in rat hepatocytes reflecting loss or even reversal of cell polarity. In order to further characterize the redistribution of canalicular antigens, we used the Lowicryl K4M immunogold technique for examination of the effects of bile duct ligation (50 h) on the distribution of antigen in rat hepatocytes at the ultrastructural level and quantitated immuno-gold density in the three domains of the plasma membrane. In normal hepatocytes, antigen was localized almost exclusively in the canalicular domain while the sinusoidal and lateral membranes showed only weak immunoreactivity. Other localizations included organelles compatible with known pathways of biosynthesis and degradation. Bile duct ligation markedly reduced immunolabel in the canalicular and increased it slightly in the sinusoidal domain. The number and staining intensity of immunoreactive subcanalicular lysosomes and vesicles probably representing endosomes was augmented. Number of immunogold particles per micron of plasma membrane were 7.86 vs 2.46 (P less than 0.005) in the canalicular, 1.16 vs 1.38 (n.s.) in the sinusoidal, and 1.23 vs 1.08 (n.s.) in the lateral domain resulting in a canalicular decrease by 68.7% and a sinusoidal increase of 19.0%. Overall decrease in total plasma membranes was by 29.7% (P less than 0.05). Thus, our data show that the sinusoidal and lateral domains behave differently. Furthermore, quantitative immunocytochemistry demonstrates a decrease in the canalicular antigen density and suggests a sinusoidal increase. The present data agree with the concept that bile duct ligation results in a loss or even reversal of cell polarity in hepatocytes.     

Bile duct ligation-induced redistribution of canalicular antigen in rat hepatocyte plasma membranes demonstrated by immunogold quantitation

Summary Extrahepatic obstructive cholestasis has been demonstrated to induce a redistribution of domain specific membrane proteins in rat hepatocytes reflecting loss or even reversal of cell polarity. In order to further characterize the redistribution of canalicular antigens, we used the Lowicryl K4M immunogold technique for examination of the effects of bile duct ligation (50 h) on the distribution of antigen in rat hepatocytes at the ultrastructural level and quantitated immuno-gold density in the three domains of the plasma membrane. In normal hepatocytes, antigen was localized almost exclusively in the canalicular domain while the sinusoidal and lateral membranes showed only weak immunoreactivity. Other localizations included organelles compatible with known pathways of biosynthesis and degradation. Bile duct ligation markedly reduced immunolabel in the canalicular and increased it slightly in the sinusoidal domain. The number and staining intensity of immunoreactive sub-canalicular lysosomes and vesicles probably representing endosomes was augmented. Number of immunogold particles per m of plasma membrane were 7.86 vs 2.46 (P<0.005) in the canalicular, 1.16 vs 1.38 (n.s.) in the sinusoidal, and 1.23 vs 1.08 (n.s.) in the lateral domain resulting in a canalicular decrease by 68.7% and a sinusoidal increase of 19.0%. Overall decrease in total plasma membranes was by 29.7% (P<0.05). Thus, our data show that the sinusoidal and lateral domains behave differently. Furthermore, quantitative immunocytochemistry demonstrates a decrease in the canalicular antigen density and suggests a sinusoidal increase. The present data agree with the concept that bile duct ligation results in a loss or even reversal of cell polarity in hepatocytes.This study was supported by the Swiss National Science Foundation grants 3.846.0.87 (to L.L.) and 3.992.0.87 (to P.J.M.)     

ATP-dependent transport of taurocholate across the hepatocyte canalicular membrane mediated by a 110-kDa glycoprotein binding ATP and bile salt

Direct photoaffinity labeling of liver plasma membrane subfractions enriched in sinusoidal and canalicular membranes using [35S]adenosine 5'-O-(thiotriphosphate) ([35S]ATP gamma S) allows the identification of ATP-binding proteins in these domains. Comparative photoaffinity labeling with [35S]ATP gamma S and with the photolabile bile salt derivative (7,7-azo-3 alpha, 12 alpha-dihydroxy-5 beta-[3 beta-3H]-cholan-24-oyl-2'- aminoethanesulfonate followed by immunoprecipitation with a monoclonal antibody (Be 9.2) revealed the identity of the ATP-binding and the bile salt-binding canalicular membrane glycoprotein with the apparent Mr of 110,000 (gp110). The isoelectric point of this glycoprotein was 3.7. Transport of bile salt was studied in vesicles enriched in canalicular and sinusoidal liver membranes. Incubation of canalicular membrane vesicles with [3H] taurocholate in the presence of ATP resulted in an uptake of the bile salt into the vesicles which was sensitive to vanadate. ATP-dependent taurocholate transport was also observed in membrane vesicles from mutant rats deficient in the ATP-dependent transport of cysteinyl leukotrienes and related amphiphilic anions. Substrates of the P-glycoprotein (gp170), such as verapamil and doxorubicin, did not interfere with the ATP-dependent transport of taurocholate. Reconstitution of purified gp110 into liposomes resulted in an ATP-dependent uptake of [3H]taurocholate. These results demonstrate that gp110 functions as carrier in the ATP-dependent transport of bile salts from the hepatocyte into bile. This export carrier is distinct from hitherto characterized ATP-dependent transport systems.     

Phosphate transport,membrane potential,and movements of calcium in rat liver mitochondria

The membrane potential and calcium accumulation of mitochondria were followed by ion-specific electrodes in the presence of the proton-donor anions phosphate, acetate, glutamate, and beta-hydroxybutyrate. Phosphate was the only anion which allowed rapid and complete restoration of both the membrane potential and the steady-state extramitochondrial calcium concentration after the uptake of 100–200 nmol calcium per mg protein. If there was no influx of any proton-donor anion, the extent of calcium uptake depended on the intramitochondrial phosphate content. Both the fall of the membrane potential and the increase of the external calcium concentration brought about by a given amount of uncoupler were counteracted by phosphate transported into the mitochondria.     

Mechanisms of taurocholate transport in canalicular and basolateral rat liver plasma membrane vesicles. Evidence for an electrogenic canalicular organic anion carrier

The driving forces for taurocholate transport were determined in highly purified canalicular (cLPM) and basolateral rat liver plasma membrane (LPM) vesicles. Alanine transport was also examined for comparison. Inwardly directed Na+ but not K+ gradients transiently stimulated [3H]taurocholate (1 microM) and [3H]alanine (0.2 mM) uptake into basolateral LPM 3-4- fold above their respective equilibrium values (overshoots). Na+ also stimulated [3H]taurocholate countertransport and tracer exchange in basolateral LPM whereas valinomycin-induced inside negative K+ diffusion potentials stimulated alanine uptake but had no effect on taurocholate uptake. In contrast, in the "right-side out" oriented cLPM vesicles, [3H]taurocholate countertransport and tracer exchange were not dependent on Na+. Efflux of [3H]taurocholate from cLPM was also independent of Na+ and could be trans-stimulated by extra-vesicular taurocholate. Furthermore, an inside negative valinomycin-mediated K+ diffusion potential inhibited taurocholate uptake into and stimulated taurocholate efflux from the cLPM vesicles. These studies provide direct evidence for a "carrier mediated" and potential-sensitive conductive pathway for the canalicular excretion of taurocholate. In addition, they confirm the presence of a possibly electroneutral Na+-taurocholate cotransport system in basolateral membranes of the hepatocyte.     

Isolation of a rat liver plasma membrane fraction of probable canalicular origin Preparative technique,enzymatic profile,composition, and solute transport

A technique currently used for isolation of brush border membranes from renal and intestinal epithelium that involves vigorous tissue homogenization and sedimentation of non-luminal membranes in the presence of Mg²⁺ has been adapted to rat liver. Liver plasma membranes so prepared consisted almost exclusively of vesicles by electron microscopy, showed some contamination with endoplasmic reticulum and minimal contamination with mitochondria or Golgi by marker enzymes, were highly enriched in alkaline phosphatase, Mg²⁺-ATPase, and 5′-nucleotidase activity compared with homogenate, and showed little enrichment in (Na⁺,K⁺)-ATPase. Comparison of this enzymatic profile with cytochemical studies localizing (Na⁺,K⁺)-ATPase and alkaline phosphatase to the sinusoidal/lateral and canalicular membranes, respectively, suggested that these membranes were predominantly of canalicular origin. They had a lower ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ specific activity, lower lipid content, and higher cholesterol to phospholipid molar ratio than a conventional plasma membrane preparation believed to be enriched in canaliculi. Moreover, it was possible to measure movement of d-[³H]glucose into an osmotically sensitive space bounded by these membrane vesicles.     

BJ-HCC-20, a potential novel cancer-testis antigen.

In an effort to identify novel Cancer-Testis genes, we analyzed the sequence in the q26-28 region of human X chromosome by several on-line tools. The candidate sequences were then confirmed by experiments. We have obtained a novel Cancer-Testis gene, BJ-HCC-20. In vivo, it was found to have two isoforms. In samples of liver, colon, gastric and lung cancer tested, the expression frequency of BJ-HCC-20 is 25%, 17%, 21% and 15%, respectively. Full-length cDNAs of both BJ-HCC-20 isoforms were isolated and their gene structures and promoter regions were characterized. BJ-HCC-20 might have implications in theoretical and practical tumor biology.     

ATP-dependent S-(2,4-dinitrophenyl)glutathione transport in canalicular plasma membrane vesicles from rat liver

Uptake of the thioether S-(2,4-dinitrophenyl)glutathione (DNPSG) in canalicular plasma membrane vesicles from rat liver is enhanced in the presence of ATP and exhibits an overshoot with a transient 5.5-fold accumulation of DNPSG. Stimulation by ATP is not caused by the generation of a membrane potential, based on responses of the indicator dye oxonol V. ATP-dependent uptake has an apparent Km of 71 microM for DNPSG and a Vmax of 0.34 nmol.min-1.mg of vesicle protein-1. Protein thiol groups are essential for transport activity as indicated by the sensitivity of DNPSG transport to sulfhydryl reagents. There is competitive inhibition with other thioethers, S-hexylglutathione (Ki = 66 microM), the photoaffinity label S-(4-azidophenacyl)glutathione (Ki = 56 microM), as well as with glutathione disulfide (Ki = 0.44 mM) and with the bile acid taurocholate (Ki = 0.61 mM). GSH (2 mM) or cholate (0.4 mM) does not inhibit. Both glutathione disulfide and taurocholate show ATP-dependent transport in the canalicular membrane vesicles which is inhibited by DNPSG. No ATP-dependent transport is found for GSH. Transport of DNPSG is also inhibited competitively by alpha-naphthyl-beta-D-glucuronide (Ki = 0.42 mM) but not by alpha-naphthylsulfate (2 mM), and there is substantial inhibition with the glucuronides from ebselen and p-nitrophenol. The results indicate that the canalicular transport system for DNPSG is directly driven by ATP and that the biliary transport of other classes of compounds may also proceed via this system.     

Heterogeneous distribution of the sodium-dependent alanine transport activity in the rat hepatocyte plasma membrane

The localization of the sodium-dependent alanine uptake activity in rat liver cells was studied. Fractions representative of the canalicular, the contiguous (lateral) and the blood-sinusoidal surface of the hepatocyte were isolated by means of centrifugal fractionation and density gradient centrifugation. The distribution of various marker-enzyme activities in conjunction with the occurrence of alanine transport activity was studied both in fractions obtained after zonal density gradient centrifugation, and in the subcellular fractions mentioned above.It is concluded that the sodium-dependent alanine transport activity is primarily located in the blood-sinusoidal plasma membrane of the hepatocyte.     

Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles.

P-glycoprotein (P-gp) is believed to function as an ATP-dependent efflux pump for natural product anti-cancer drugs in multidrug-resistant (MDR) tumor cells and in certain normal tissues. P-gp has been localized to the apical plasma membrane of the bile canaliculus where it has been shown to transport [3H]daunomycin. In this study, we investigated whether alterations in membrane lipid fluidity of canalicular membrane vesicles (CMV) could modulate the P-gp-mediated accumulation of [3H]daunomycin and [3H]vinblastine. Accumulation of both cytotoxic agents was stimulated by ATP, exhibited temperature dependence and osmotic sensitivity, and followed Michaelis-Menten kinetics. Alterations in CMV lipid fluidity were induced by the known fluidizers, 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) and benzyl alcohol, and were assessed by fluorescence polarization techniques using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). Both A2C (2.5-5.0 microM) and benzyl alcohol (10-20 mM) produced a dose-dependent increase in CMV lipid fluidity. Moreover, both fluidizers, at the above doses, significantly inhibited (p < 0.05) the ATP-dependent accumulation of [3H]daunomycin. [3H]Vinblastine accumulation was also inhibited by A2C (p < 0.05). Lower doses of A2C (0.6 microM) and benzyl alcohol (1 mM) failed to influence either lipid fluidity or P-gp-mediated drug accumulation. Kinetic analysis revealed that A2C (5.0 microM) noncompetitively inhibited [3H]daunomycin accumulation and uncompetitively inhibited [3H]vinblastine accumulation with apparent Ki values of approximately 1.5 and approximately 1.2 microM, respectively. Verapamil competitively inhibited P-gp-mediated accumulation of [3H]daunomycin but failed to alter the fluidity of CMV. Taken together, the present results demonstrate that while increases in membrane fluidity of CMV are not necessarily required to inhibit P-gp-mediated drug accumulation, they can inhibit these processes, at least in CMV. Alterations in the physical state of CMV, therefore, appear to be at least one important modulator of P-gp function.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

Chloroquine,a novel inhibitor of amino acid transport by rat renal brush border membrane vesicles

Summary Chloroquine is an antimalarial and antirheumatic lysosomotropic drug which inhibits taurine uptake into and increases efflux from cultured human lymphoblastoid cells. It inhibits taurine uptake by rat lung slices and affects the uptake and release of cystine from cystinotic fibroblasts. Speculations on its mode of action include a proton gradient effect, a non-specific alteration in membrane integrity, and membrane stabilization. In this study, the effect of chloroquine on the uptake of several amino acids by rat renal brush border membrane vesicles (BBMV) was examined. Chloroquine significantly inhibited the secondary active, NaCl-dependent component of 10µM taurine uptake at all concentrations tested, but did not change equilibrium values. Analysis of these data indicated that the inhibition was non-competitive. Taurine uptake was reduced at all osmolarities tested, but inhibition was greatest at the lowest osmolarity. Taurine efflux was not affected by chloroquine, nor was the NaCl-independent diffusional component of taurine transport. Chloroquine (1 mM) inhibited uptake of the imino acids L-proline and glycine, and the dibasic amino acid L-lysine. It inhibited the uptake of D-glucose, but not the neutral-amino acids L-alanine or L-methionine. Uptake of the dicarboxylic amino acids, L-glutamic acid and L-aspartic acid, was slightly enhanced. With regard to amino acid uptake by BBMV, these findings may support some of the currently proposed mechanisms of the action of chloroquine but further studies are indicated to determine why it affects the initial rate of active amino acid transport.     

Iodipamide uptake by rat liver plasma membrane vesicles enriched in the sinusoidal fraction: evidence for a carrier-mediated transport dependent on membrane potential

Iodipamide, a cholecystographic agent, is known to be taken up by isolated hepatocytes by a mechanism similar or identical with the inward transport of bile salts (Petzinger, E., Joppen, C. and Frimmer, M. (1983) Naunyn-Schmiedeberg's Arch. Pharmacol. 322, 174-179). To elucidate its mode of transport, uptake of iodipamide was studied by rapid-filtration techniques on plasma membrane vesicles enriched in the sinusoidal fraction. Uptake was found to be dependent upon the temperature, the intravesicular volume, a gradient of monovalent cations (Na+, K+ or Li+) and the substrate concentration (saturation kinetics with respect to iodipamide: apparent Km = 70 microM, Vmax = 0.31 nmol per mg protein per min at 100 mM NaCl and 25 degrees C). Countertransport and transstimulation in tracer exchange experiments indicate that in vesicles, iodipamide uptake rather than binding occurs. Na+ could be replaced by K+ or Li+ in our system without any effect. However, in the presence of choline chloride a slight, but distinct reduction occurred. Iodipamide uptake was inhibited by cholate, phalloidin, 4,4'-diisothiocyanato-1,2-diphenylethane-2,2'-disulfonic acid and by bromosulfophthalein with inhibition being competitive in the case of cholate and non-competitive in the case of bromosulfophthalein. Alteration of the membrane potential by addition of NO3-, SCN- or SO4(2-) modified the uptake rate for iodipamide. The above results support our earlier hypothesis that the hepatocellular uptake of iodipamide is due to a carrier-mediated transport, probably similar to that of bile acids. However, translocation of iodipamide is assumed to be driven by the membrane potential only and not by Na+ contransport.     

The function of Gp170, the multidrug resistance gene product, in rat liver canalicular membrane vesicles

Gp170 (also known as P-glycoprotein) is a transmembrane glycoprotein which is overexpressed in multidrug-resistant tumor cells and is also found in the apical plasma membrane domain of several normal human and animal tissues. Gp170 has been postulated to function as an energy-dependent efflux pump for cytotoxic drugs. In rat liver, Gp170 is restricted to the bile canalicular domain of the plasma membrane. Canalicular membrane vesicles (CMV), but not sinusoidal membrane vesicles, contained a approximately 160-kDa protein which reacts with anti-Gp170 monoclonal antibody and manifest ATP-dependent [3H]daunomycin transport which is temperature dependent, osmotically sensitive, and saturable. Among several nucleotides, ATP was a potent stimulator of transport whereas non- or slowly hydrolyzable analogues (adenosin-5-O-(3-thiotriphosphate, adenyl-5-yl-imidodiphosphate) were ineffective. ATP-dependent daunomycin transport was inhibited by cytotoxic drugs (vinblastine, vincristine, and adriamycin) and other drugs, such as verapamil and quinidine, which restore anti-cancer drug sensitivity in resistant cells. Inside-out CMV were separated from right side-out CMV by antibody-induced affinity density perturbation. Only inside-out CMV manifested ATP-dependent daunomycin transport. These results suggest that Gp170 is an ATP-dependent efflux pump which is responsible for the undirectional, energy-dependent transport of daunomycin and other drugs by rat liver into the bile.     

Cevadine-induced changes of membrane potential and sodium transport of muscle membrane in a chloride-free solution

Cevadine-induced changes in membrane potential, sodium transport, intracellular Na, K, and water content were investigated in sartorius muscles incubated in chloride-free (glutamate) Ringer. Cevadine sensitivity of muscles incubated in glutamate Ringer was about five times greater than that of muscles incubated in normal Ringer. Therefore, even 0.005 mmol/l cevadine could induce depolarization and membrane potential oscillations. The membrane potential oscillations were recorded much longer from muscles incubated in chloride-free Ringer (even in the 15th hour of treatment) than in normal Ringer. Depolarization and membrane potential oscillations reversed more slowly in cevadine-free glutamate Ringer than in alkaloid-free normal Ringer. The rhythmic activity could be recorded even in the 10th-15th hour of incubation in cevadine-free glutamate Ringer. Cevadine increased the 24Na uptake of muscles incubated in glutamate Ringer by an average of 230%. In comparison, the cevadine-induced increase of 24Na uptake of muscles incubated in normal Ringer was approximately 350%. In the presence of cevadine the 24Na loss of muscles incubated either in glutamate or in normal Ringer increased to the same degree, i.e. three times. The increase of 24Na loss developed faster in glutamate Ringer than in the presence of chloride. The water content of muscles incubated in cevadine containing, chloride-free (glutamate) Ringer did not increase significantly. Muscles incubated in normal Ringer with cevadine showed a 42.7% increase of water content in 2 hours. Intracellular Na content and Na concentration increased by about 60% during a 2-hour-treatment with cevadine in a chloride-free environment. At the same time, cevadine treatment increased the intracellular Na content and Na concentration of muscles incubated in normal Ringer by about 160% and 80%, respectively. The cevadine-induced decrease of intracellular K content and concentration of muscles incubated in glutamate Ringer was 5% and 10%, respectively, in 2 hours. On the other hand, the decrease of intracellular K concentration in muscles incubated in cevadine-containing normal Ringer occasionally reached 30% due to the increase of water content of the muscles. The cevadine-induced increase of the wet weight of muscles incubated in normal Ringer was practically irreversible. It was not possible to eliminate the increase of wet weight even by washout lasting for 10-15 hours.     

Plasma membrane transport of 2-ketoisocaproate by rat hepatocytes in primary culture

Others have shown that the branched chain 2-keto acids are generated in muscle, released into the bloodstream, and then removed by the liver where further catabolism occurs. The present investigation describes the plasma membrane transport systems for these metabolites in cultured rat hepatocytes. One of these systems in Na+-dependent, concentrates the 2-keto acids against a gradient, and is inhibited by pyruvate. The second process is Na+-independent, is less concentrative, and may be composed of two distinct systems as suggested by pyruvate inhibition studies. None of these systems accept neutral amino acids. For the transport of 2-ketoisocaproate, the Na+-dependent system exhibits a Km value of about 5 mM, whereas the corresponding value for the Na+-independent agency is 60 microM. The activity of the Na+-dependent system is moderately increased by insulin treatment of the cells, while neither agency is stimulated by glucagon, dexamethasone, or the combination of these two hormones. Hepatocytes from diabetic rats show enhanced transport by the Na+-dependent system and incubation of cultured hepatocytes for 24 h in the absence of 2-keto acids results in a 3-fold stimulation of the Na+-dependent system, but has no effect on the rate of Na+-independent transport. These results demonstrate the existence of at least two saturable transport systems for the branched chain 2-keto acids in the rat hepatocyte and the ability of the Na+-dependent system to respond to the extracellular environment.     

Identification of protein-bound riboflavin in rat hepatocyte plasma membrane as a source of autofluorescence

The presence of flavin compound(s) giving a yellowish-green autofluorescence in rat hepatocyte plasma membrane has recently been reported (Nokubo, M. et al. (1988) Biochim. Biophys. Acta 939, 441-448). The fluorophore can quantitatively be extracted with water at 80 degrees C from isolated plasma membranes. Gel filtration of the extract eluted with water showed two peaks, the fluorescence of which closely resembled that of riboflavin. The major peak comigrated with proteins and the minor one displayed a position identical to authentic riboflavin. When the components of the major peak were rechromatographed after acetic acid treatment and eluted with 20 mM of acetic acid, the fluorescent compound separated from the proteins and eluted at the same position as riboflavin. In paper chromatography and HPLC, the behavior of the fluorescent compound (separated by acid treatment from the proteins) was identical to that of riboflavin. SDS gel filtration of subcellular fractions of rat liver revealed that riboflavin was the dominant flavin, whereas FAD and FMN were not detectable in the plasma membrane. Microsomes and mitochondria contain predominantly FAD and FMN, and only minor quantities of riboflavin. The presence of riboflavin in the plasma membrane is a novel finding, the functional significance of which is still unclear; however, a hypothesis can be forwarded on the basis of the ability of flavins to generate superoxide anion radicals during their autoxidation.