Hepatic uptake of bilirubin diglucuronide: analysis by using sinusoidal plasma membrane vesicles

In order to characterize the mechanism for bilirubin transport in the liver, the uptake of bilirubin diglucuronide (BDG) into purified sinusoidal plasma membrane vesicles was investigated. BDG uptake was saturable, and was inhibited by sulfobromophthalein and unconjugated bilirubin, but was not affected by sodium taurocholate. BDG uptake was sodium-independent and was stimulated by intravesicular bilirubin or BDG (trans-stimulation). BDG transport showed strong potential sensitivity; vesicle inside-negative membrane potential created by different anion gradients inhibited BDG uptake whereas vesicle inside-positive membrane potential generated by potassium gradients and valinomycin markedly stimulated BDG transport. These data suggest that BDG, sulfobromophthalein, and probably unconjugated bilirubin share a common transporter in liver cells which is sodium independent, membrane-potential-dependent and capable of exchange. The direction of transport in vivo may be governed by the intracellular concentration of BDG and of other yet unidentified organic anions sharing this transporter.     

Bilitranslocase is the protein responsible for the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver: immunochemical evidence using mono- and poly-clonal antibodies

Monoclonal antibodies raised against bilitranslocase, may display either inhibitory or enhancing activity on the electrogenic transport of sulfobromophthalein, evoked in rat liver plasma-membrane vesicles by the addition of valinomycin in the presence of K+. In both cases, the target protein is identified with a 37 kDa band in SDS-mercaptoethanol gel electrophoresis of solubilized membranes. The electrophoretically homogeneous protein isolated by ion-exchange chromatography, corresponds in all respects to the 37 kDa protein band of bilitranslocase, obtained in the past by different techniques. Using this protein as antigen, a polyclonal monospecific antibody preparation has been obtained. As expected, the antibody preparation inhibits the electrogenic movement of sulfobromophthalein in plasma membrane vesicles from rat liver. It is concluded that the 37 kDa protein of bilitranslocase is at least a necessary component of the transport system involved in the sulfobromophthalein movement in plasma membrane.     

Hepatocellular transport of cyclosomatostatins: evidence for a carrier system related to the multispecific bile acid transporter.

The uptake of the cyclopeptide c(Phe-Thr-Lys-Trp-Phe-D-Pro) (008), an analog of somatostatin with retro sequence, was studied in isolated hepatocytes. 008 is taken up by hepatocytes in a concentration-, time-, energy- and temperature- dependent manner. Since 008 is hydrophobic, it binds rapidly to liver cells. This is evident by the positive intercept at the gamma-axis in the uptake curves. At higher concentrations, a minor part of the transport occurs by diffusion at a rate of 8.307.10(-6) cm/s. This part of diffusion is measured at 4 degrees C and can be subtracted from the uptake at 37 degrees C resulting in the carrier mediated part of uptake which is saturable. Kinetic parameters for the saturable part of uptake are Km 1.5 microM and Vmax 40.0 pmol/mg per min. The transport is decreased in the absence of oxygen and in the presence of metabolic inhibitors. Uptake is accelerated at temperatures above 20 degrees C. The activation energy was determined to be 30.77 kJ/mol. The membrane potential and not a sodium gradient is the main driving force for 008 transport. Cholate (a typical substrate of the multispecific bile acid transporter) and taurocholate are mutual competitive inhibitors of 008 uptake. Phalloidin, antamanide and iodipamide, typical foreign substrates of the transporter, interfere with the uptake of 008. AS 30D ascites hepatoma cells, known to be unable to transport bile acids, phalloidin and iodipamide, are also unfit to transport 008. Interestingly, sulfobromophthalein (BSP) but not rifampicin, both foreign substrates of the bilirubin carrier, inhibits the transport of 008 in a competitive manner.     

Reconstitution in vitro of sulfobromophthalein transport by bilitranslocase

Liposomes containing 150 mM KCl and 0.48 mM sulfobromophthalein have been prepared. The internal pH was set at 6.5, a value at which sulfobromopthalein is colorless. When brought to alkaline pH a certain amount of the dye is deprotonated and can be read spectrophotometrically as external sulfobromophthalein. Upon addition of Triton X-100 the membrane is dissolved and all sulfobromophthalein present in the preparation may be measured. Addition of bilitranslocase to such a preparation of liposomes causes the internal sulfobromophthalein to leave the internal compartment. The rate of this phenomenon may be followed directly and shown to be greatly accelerated by the addition of valinomycin. The latter finding indicates that sulfobromophthalein transport occurs in response to a membrane diffusion potential created by permeabilisation to K⁺ of liposomes brought about by valinomycin (uniport). The permeability change induced by bilitranslocase is specific and does not reflect an alteration of the normal impermeability of liposomes to small ions such as protons or Ca²⁺.     

The interaction of the anionic fluorescence probe, 1-anilinonaphthalene-8-sulfonate, with hepatocytes and hepatoma tissue culture cells

The fluorescence probe 1-anilinonaphthalene-8-sulfonate (ANS) has been used to characterize the anion transport properties of normal hepatocytes and hepatoma tissue culture cells. Incubation of hepatocytes in the presence of ANS (20 micron) resulted in a 35-fold enhancement of fluorescence and a 50 nm blue shift. The time course of this process is biphasic. A rapid initial fluorescence enhancement suggests ANS binding to the plasma membrane, and a slower component reflects the uptake of ANS into intracellular compartments. Analysis of ANS uptake showed this latter process to be saturable, with a Km of 10 micron, to be temperature dependent and to occur only in viable cells. The above observations suggest a carrier-mediated anion transport mechanism. Incubation of hepatoma tissue culture cells with ANS (20 micron) gave a fluorescence emission spectrum similar to that obtained from purified plasma membranes. The kinetics of this interaction only exhibited a rapid initial binding of ANS. The second slow component was now absent, suggesting that ANS transport by the malignant cell system was greatly reduced. Transport of ANS could, however, be stimulated in the presence of the local anesthetic tetracaine. The observed transport was now saturable, temperature dependent, and as in normal hepatocytes, required viable cells, again indicating a carrier-mediated transport system. These studies suggest a significant alteration in membrane function in hepatoma tissue culture cells resulting in a major defect in anion transport.     

Hepatocellular uptake of 3H-dihydromicrocystin-LR, a cyclic peptide toxin

The cellular uptake of microcystin-LR, a cyclic heptapeptide hepatotoxin from the cyanobacterium Microcystis aeruginosa, was studied by means of a radiolabelled derivative of the toxin. 3H-dihydromicrocystin-LR. The uptake of 3H-dihydromicrocystin-LR was shown to be specific for freshly isolated rat hepatocytes whereas the uptake in the human hepatocarcinoma cell line Hep G2 as well as the mouse fibroblast cell line NIH-3T3, and the human neuroblastoma cell line SH-SY5Y, was negligible. By means of a surface barostat technique it was shown that the membrane penetrating capacity (surface activity) of microcystin-LR was low, indicating that the toxin requires an active uptake mechanism. The hepatocellular uptake of microcystin-LR could be inhibited in the presence of bile acid transport inhibitors such as antamanide (5 microM), sulfobromophthalein (50 microM) and rifampicin (30 microM). The uptake was also reduced in a concentration dependent manner when the hepatocytes were incubated in the presence the bile salts cholate and taurocholate. A complete inhibition of the hepatocellular uptake was achieved by 100 microM of either bile salt. The overall results indicate that the uptake of microcystin-LR is through the multispecific transport system for bile acids. This mechanism of cell entry would explain the previously observed cell specificity and organotropism of microcystin-LR.     

Phenylalanine uptake in isolated renal brush border vesicles.

The uptake of L-phenylalanine into brush border microvilli vesicles and basolateral plasma membrane vesicles isolated from rat kidney cortex by differential centrifugation and free flow electrophoresis was investigated using filtration techniques. Brush border microvilli but not basolateral plasma membrane vesicles take up L-phenylalanine by an Na+-dependent, saturable transport system. The apparent affinity of the transport system for L-phenylalanine is 6.1 mM at 100 mM Na+ and for Na+ 13mM at 1 mM L-phenylalanine. Reduction of the Na+ concentration reduces the apparent affinity of the transport system for L-phenylalanine but does not alter the maximum velocity. In the presence of an electrochemical potential difference of Na+ across the membrane (etaNao greater than etaNai) the brush border microvilli accumulate transiently L-phenylalanine over the concentration in the incubation medium (overshoot pheomenon). This overshoot and the initial rate of uptake are markedly increased when the intravesicular space is rendered electrically more negative by membrane diffusion potentials induced by the use of highly permeant anions, of valinomycin in the presence of an outwardly directed K+ gradient and of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence of an outward-directed proton gradient. These results indicate that the entry of L-phenylalanine across the brush border membrane into the proximal tubular epithelial cells involves cotransport with Na+ and is dependent on the concentration difference of the amino acid, on the concentration difference of Na+ and on the electrical potential difference. The exit of L-phenylalanine across the basolateral plasma membranes is Na+-independent and probably involves facilitated diffusion.     

Effects of ionophores and metabolic inhibitors on the mitochondrial membrane potential within isolated hepatocytes as measured with the safranine method.

A difference spectrum with a peak of absorbance at 526nm appears slowly upon addition of valinomycin or KCN in combination with oligomycin to a hepatocyte suspension in the presence of safranine. When the cells are incubated at 37 degrees C in a medium containing safranine, a slow decrease in the absorbance occurs at the wavelength pair 524-484 nm. The change in absorbance is completed within 20-30 min after additions of cells to a medium containing safranine. At this time the safranine concentration of the outer medium is considerably decreased. The safranine signal is completely reversed by valinomycin, carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone or KCN in combination with oligomycin. None of these treatments have any immediate effect on cellular ATP concentrations or the 36Cl- equilibrium potential across the plasma membrane. In the presence of iodoacetate a slow reversal of the trace can be induced upon addition of KCN, but not of oligomycin alone. Rotenone, in combination with oligomycin, does not reverse the safranine signal except when both KF and iodoacetate are present, in which case a slow reversal is seen. A subsequent addition of duroquinone brings back the signal to the same level as in the presence of rotenone alone. The results indicate that the spectral response of safranine in the presence of isolated hepatocytes is a result of a slow penetration of safranine into intracellular mitochondria, where aggregation of safranine molecules occurs as a response to the mitochondrial membrane potential.     

Down-regulation by extracellular ATP of rat hepatocyte organic anion transport is mediated by serine phosphorylation of oatp1

Recent studies implicate a role in hepatocyte organic anion transport of a plasma membrane protein that has been termed oatp1 (organic anion transport protein 1). Little is known regarding mechanisms by which its transport activity is modulated in vivo. In previous studies (Campbell, C. G., Spray, D. C., and Wolkoff, A. W. (1993) J. Biol. Chem. 268, 15399-15404), we demonstrated that hepatocyte uptake of sulfobromophthalein was down-regulated by extracellular ATP. We have now found that extracellular ATP reduces the V(max) for transport of sulfobromophthalein by rat hepatocytes; K(m) remains unaltered. Reduced transport also results from incubation of hepatocytes with the phosphatase inhibitors okadaic acid and calyculin A. Immunoprecipitation of biotinylated cell surface proteins indicates that oatp1 remains on the cell surface after exposure of cells to ATP or phosphatase inhibitor, suggesting that loss of transport activity is not caused by transporter internalization. Exposure of (32)P-loaded hepatocytes to extracellular ATP results in serine phosphorylation of oatp1 with the appearance of a single major tryptic phosphopeptide; oatp1 from control cells is not phosphorylated. This phosphopeptide comigrates with one of four phosphopeptides resulting from incubation of cells with okadaic acid. These studies indicate that the phosphorylation state of oatp1 must be an important consideration when assessing alterations of its functional expression in pathobiological states.     

Phosphate transport into brush-border membrane vesicles isolated from rat small intestine.

Uptake of Pi into brush-border membrane vesicles isolated from rat small intestine was investigated by a rapid filtration technique. The following results were obtained. 1. At pH 7.4 in the presence of a NaCl gradient across the membrane (sodium concentration in the medium higher than sodium concentration in the vesicles), phosphate was taken up by a saturable transport system, which was competitively inhibited by arsenate. Phosphate entered the same osmotically reactive space as D-glucose, which indicates that transport into the vesicles rather than binding to the membranes was determined. 2. The amount of phosphate taken up initially was increased about fourfold by lowering the pH from 7.4 to 6.0.3. When Na+ was replaced by K+, Rb+ or Cs+, the initial rate of uptake decreased at pH 7.4 but was not altered at pH 6.0.4. Experiments with different anions (SCN-,Cl-, SO42-) and with ionophores (valinomycin, monactin) showed that at pH 7.4 phosphate transport in the presence of a Na+ gradient is almost independent of the electrical potential across the vesicle membrane, whereas at pH 6.0 phosphate transport involves the transfer of negative charge. It is concluded that intestinal brush-border membranes contain a Na+/phosphate co-transport system, which catalyses under physiological conditions an electroneutral entry of Pi and Na+ into the intestinal epithelial cell. In contrast with the kidney, probably univalent phosphate and one Na+ ion instead of bivalent phosphate and two Na+ ions are transported together.     

Hepatocellular uptake of aflatoxin B1 by non-ionic diffusion. Inhibition of bile acid transport by interference with membrane lipids

Aflatoxin B1 permeates isolated rat hepatocytes by non-ionic diffusion. Its uptake is neither saturable nor influenced by metabolic energy and not inhibited by treatment of cells with proteases. The initial rate of aflatoxin B1 uptake measured at 7 degrees C is between 40 and 50% compared to that at 37 degrees C. However, after an incubation period of 7 minutes identical equilibrium uptake is reached at both temperatures. The apparent activation energies, calculated for aflatoxin B1 uptake by Arrhenius diagrams ranged between 1.69 and 4.5 kcal/mol. A Q10 value of 1.34 was calculated for a temperature interval of 7-17 degrees C but decreased to 1.05 for the interval of 27-37 degrees C. Liposomes or lipoproteins added to the cell suspension inhibited the aflatoxin B1 uptake into hepatocytes. Liposomes mainly composed of unsaturated fatty acids bind twice as much aflatoxin B1 as those composed of saturated ones, indicating that the lipophilicity of the mycotoxin is crucial in the determination of its uptake into liver cells. At concentrations above 5 micrograms/ml, aflatoxin B1 inhibited the carrier-mediated uptake of cholic acid and of phalloidin into hepatocytes. This effect was reversible and abolished by washing the cells after preincubation with aflatoxin. In concentrations below 5 micrograms/ml the uptake of phallotoxin and cholic acid was however stimulated by 15-25%. These results indicate, that a carrier-mediated uptake into hepatocytes via the multispecific bile salt transporter is not responsible for the organoselective clearance of aflatoxins by the liver. On the other hand, the cholestatic effect of aflatoxin B1 results at least partially from the inhibition of the multispecific bile acid transport system. This inhibition may arise from affinity of aflatoxins to lipid domains of the cell membrane.     

Characteristics of tripeptide transport in human jejunal brush-border membrane vesicles

These studies are aimed at characterizing the transport of the tripeptide, glycylglycyl-L-proline (GlyGlyPro) across human jejunal brush-border membrane vesicles. GlyGlyPro (0.65 mM) was hydrolyzed by brush-border membrane vesicles with the extent of hydrolysis per mg protein being 23% at 0.5 min, 57% at 1 min and complete hydrolysis at 60 min. Treatment of the membrane vesicles with gel-complexed papain (to remove membrane peptidases) resulted in minimal hydrolysis of GlyGlyPro up to 10 min of incubation. Measurement of GlyGlyPro influx with papain-treated vesicles in the presence of increasing medium osmolarity showed that uptake occurred into an osmotically reactive intravesicular space. Transport of GlyGlyPro with normal and papain-treated membrane vesicles was similar in the presence of an inward Na+ or K+ gradient. No overshoot phenomenon was observed in the presence of an inward proton gradient (extravesicular pH 5.5; intravesicular pH 7.5). An interior negative membrane potential induced by a K+ diffusion potential in the presence of valinomycin stimulated the uptake of the peptide. The effect of increasing concentrations on initial rates of GlyGlyPro uptake revealed the presence of a saturable component as well as a diffusional component. Preloading the membrane vesicles with 20 mM glycylsarcosylsarcosine stimulated uptake by 4-fold. Uptake of GlyGlyPro was inhibited greater than 50% by dipeptides and tripeptides and less than 15% by free amino acids. These results indicate that GlyGlyPro uptake in jejunal brush-border membrane vesicles is not energized by a Na+ or proton gradient and that transport occurs by carrier-mediated and diffusional processes.     

Transport of dehydroepiandrosterone and dehydroepiandrosterone sulphate into rat hepatocytes

The purpose of the present study was to characterize the transport of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) into hepatocytes at physiological and pharmacological concentrations. Hepatocytes were isolated from female Sprague-Dawley rats by collagenase perfusion. Uptake of [³H]DHEA and [³H]DHEAS at increasing concentrations (3.5 nM-100 μM) was measured by the rapid filtration technique at 30 s intervals up to 120 s. The uptake of DHEAS by hepatocytes was saturable (K_m = 17.0 μM; V_max = 3.7 nmol/min/mg cell protein). In contrast, a specific saturable transport system for DHEA could not be detected in rat hepatocytes. It is suggested that DHEA enters the cell by diffusion. The uptake of DHEAS could be inhibited by antimycin A, carbonylcyanide-m-chlorophenylhydrazone, and dinitrophenol (inhibitors of the mitochondrial respiratory chain), by dinitrofluorobenzene and p-hydroxymercuribenzoate (NH₂- and SH-blockers, respectively), and by monensin (Na⁺-specific ionophore). No inhibition was seen in the presence of ouabain (inhibitor of Na⁺-K⁺-ATPase) and phalloidin (inhibitor of cholate transport and actin-blocker). Interestingly, DHEAS uptake was inhibited by bile acids (cholate, taurocholate and glycocholate). Conversely, [³H]cholate uptake was strongly inhibited by DHEAS, which indicates a competition for the same carrier. Replacement of sodium ion with choline markedly decreased uptake velocity at pharmacological DHEAS concentrations. The results suggest that DHEAS uptake is a saturable, energy-dependent, carrier-mediated, partially Na⁺-dependent process, and that DHEAS may be taken up via the multispecific bile acid transport system.     

A quantitative assessment of the use of 36Cl- distribution to measure plasma membrane potential in isolated hepatocytes

The plasma membrane potential of isolated rat hepatocytes was clamped at different values between 0 and -68 mV by addition of valinomycin in the presence of different extracellular concentrations of K+, and measured by the distribution of 86Rb+ between cells and medium. 36Cl- distribution came to steady state in 10-15 min. This steady-state distribution was compared to the plasma membrane potential over a range of values. 36Cl- distribution provided an accurate measurement of plasma membrane potential between -4 and -40 mV. At higher potentials intracellular chloride concentration is less than 20% of the extracellular concentration and errors due to uncertainties in the measurement of intracellular volume and of the contamination of cell pellets by extracellular medium precluded accurate determination of membrane potential: thus in our experiments 36Cl- underestimated the plasma membrane potential at -68 mV by 8 mV.     

Selective inhibition of long-chain fatty acid uptake in short-term cultured rat hepatocytes by an antibody to the rat liver plasma membrane fatty acid-binding protein

Uptake of long-chain fatty acids by short-term cultured hepatocytes was studied. Rat hepatocytes, which were cultured for 16 h on plastic dishes (3.6 X 10(6) cells/dish), were incubated with [3H]oleate in the presence of various concentrations of bovine serum albumin as a function of the concentration of unbound [3H]oleate in the medium. At 37 degrees C initial uptake velocity (V0) was saturable (Km = 9 X 10(-8) M; Vmax = 835 pmol/min per mg protein). V0 was temperature dependent with an optimum at 37 degrees C and markedly reduced at 4 degrees C and 70 degrees C. To evaluate the biologic significance of a previously isolated rat liver plasma membrane fatty acid-binding protein as putative carrier protein in the hepatocellular uptake of fatty acids, cultured hepatocytes were treated with a monospecific rabbit antibody (IgG-fraction) to this membrane protein or the IgG-fraction of the pre-immune serum as controls. Uptake kinetics of [3H]oleate in antibody pretreated short-term cultured hepatocytes revealed a depression of Vmax by 70%, while Km was only reduced by 16% compared to controls, indicating a predominant non-competitive type of inhibition. V0 of a variety of long-chain fatty acids (oleic acid, arachidonic acid, palmitic acid, stearic acid) was reduced by 56-69%, while V0 of [35S]sulfobromophthalein, [3H]cholic acid and [14C]taurocholic acid remained unaltered. These data support the concept that in the system of cultured hepatocytes, uptake of long-chain fatty acids is mediated by the rat liver plasma membrane fatty acid-binding protein.     

Mechanisms of hepatic transport of cyclosporin A: an explanation for its cholestatic action?

The hepatic transport of the immunosuppressive Cyclosporin A (CyA) was studied using liposomal phospholipid membranes, freshly isolated rat hepatocytes and bile canalicular plasma membrane vesicles from rat liver. The Na(+)-dependent, saturable uptake of the bile acid 3H-taurocholate into isolated rat liver cells was apparently competitively inhibited by CyA. However, the uptake of CyA into the cells was neither saturable, nor temperature-dependent nor Na(+)-dependent, nor could it be inhibited by bile salts or CyA-derivatives, indicating passive diffusion. In steady state depolarization fluorescence studies, CyA caused a concentration-dependent decrease of anisotropy, indicating a membrane fluidizing effect. Ion flux experiments demonstrated that CyA dramatically increases the permeability of Na+ and Ca2+ across phospholipid membranes in a dose- and time-dependent manner, suggesting a iontophoretic activity that might have a direct impact on cellular ion homeostasis and regulation of bile acid uptake. Photoaffinity labeling with a [3H]-labeled photolabile CyA-derivative resulted in the predominant incorporation of radioactivity into a membrane polypeptide with an apparent molecular weight of 160,000 and a minor labeling of polypeptides with molecular weights of 85,000-90,000. In contrast, use of a photolabile bile acid resulted in the labeling of a membrane polypeptide with an apparent molecular weight of 110,000, representing the bile canalicular bile acid carrier. The photoaffinity labeling as well as CyA transport by canalicular membrane vesicles were inhibited by CyA and the p-glycoprotein substrates daunomycin and PSC-833, but not by taurocholate, indicating that CyA is excreted by p-glycoprotein. CyA uptake by bile canalicular membrane vesicles was ATP-dependent and could not be inhibited by taurocholate. CyA caused a decrease in the maximum amount of bile salt accumulated by the vesicles with time. However, initial rates of [3H]-taurocholate uptake within the first 2.5 min remained unchanged at increasing CyA concentrations. In summary, the data indicate that CyA does not directly interact with the hepatic bile acid transport systems. Its cholestatic action may rather be the result of alterations in membrane fluidity, intracellular effects and an interaction with p-glycoprotein.     

Glycine transport by intestinal brush border vesicles of the amphibian Discoglossus pictus.

1. In order to determine the different components of glycine uptake by the intestine of the frog, Discoglossus pictus, we have used brush border membrane vesicles isolated by a classical precipitation technique. 2. Enzymatic tests showed that a good purification was obtained. The concentration ratio of alkaline phosphatase was 14.8. 3. Glycine entry in vesicles as a function of time, in presence or absence of sodium, indicated an overshoot which decreased when incubation time was prolonged. The overshoot was dependent on the presence of sodium. 4. The nature of the anion associated to sodium had little effect on glycine uptake. Nevertheless, chloride and thiocyanate appeared more efficient than glutarate. 5. The effect of transmembrane potential was studied by using valinomycin associated with a potassium gradient. The addition of this substance stimulated glycine transport by 43%. 6. The transport at different glycine concentrations showed two components: one non-saturable with weak affinity and the other saturable with strong affinity (Kt = 0.338 mM). 7. In conclusion, glycine transport by the brush border of D. pictus intestine presents a saturable component depending on sodium and on transmembrane electrical potential.     

Glutamine Transport in Rat Brain Synaptic and Non-Synaptic Mitochondria

Glutamine transport into rat brain mitochondria (synaptic and non-synaptic) was monitored by the uptake of [³H]glutamine as well as by mitochondrial swelling. The uptake is inversely correlated to medium osmolarity, temperature-dependent, saturable and inhibited by mersalyl, and glutamine is upconcentrated in the mitochondria. These results indicate that glutamine is transported into an osmotically active space by a protein catalyzed mechanism. The uptake is slightly higher in synaptic mitochondria than in non-synaptic ones. It is inhibited both by rotenone and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the latter at pH 6.5, showing that the transport is activated by an electrochemical proton gradient. The K⁺/H⁺ ionophore nigericin also inhibits the uptake at pH 6.5 in the presence of external K⁺, which indicates that glutamine, at least in part, is taken up by a proton symport transporter. In addition, glutamine uptake as measured by the swelling technique revealed an additional glutamine transport activity with at least 10 times higher K_m value. This uptake is inhibited by valinomycin in the presence of K⁺ and is thus also activated by the membrane potential. Otherwise, the two methods show similar results. These data indicate that glutamine transport in brain mitochondria cannot be described by merely a simple electroneutral uniport mechanism, but are consistent with the uptake of both the anionic and the zwitterionic glutamine.     

Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.     

Regulation of plasma membrane permeability to calcium in primary cultures of rat hepatocytes.

Experiments were designed to characterize the hormone sensitive transport of Ca2+ from the external media into rat hepatocytes maintained in culture. In the absence of added vasopressin, hepatocytes were nearly impermeable to Ca2+, whereas a significant and rapid influx of Ca2+ could be detected when external Ca2+ was added to hepatocytes after the addition of 20 nM vasopressin. The transport was measured as the initial rate of increase of free intracellular Ca2+ [( Ca2+]i) after Ca2+ addition to the external media. Most data were obtained from the majority of cells on a coverslip immersed in a spectrophotometric cuvette, but selected data were obtained by measuring Ca2+ changes in single cells. Ca2+ influx measured using a large number of cells was similar to data obtained using single cells. The Vmax of Ca2+ influx was 140 nM/s. Ca2+ transport was competitive with H+ so that the Km was 17.4 mM at pH 6.8, 3.7 mM at pH 7.4 and 1.8 mM at pH 7.8. Ca2+ influx was insensitive to external K+ (1 to 70 mM) and to the presence of 5 nM valinomycin, suggesting that it was independent of the electrical potential gradient across the plasma membrane. Transport also appeared to be insensitive to the activity of protein kinase C, which was varied by addition of the activator, 12-myristate 13-acetate phorbol ester, and by addition of the kinase inhibitor, staurosporine. Stimulation of transport following vasopressin addition exhibited a delay with a t1/2 of approximately 30 s. A vasopressin antagonist blocked the activation of transport, if added prior to vasopressin. However, experiments designed to determine the effect of hormone occupancy per se on transport activity indicated that continued hormone occupancy was not required. When the external medium was nominally Ca2+ free and an antagonist was added 1 min after vasopressin, Ca2+ entry, even 8 min after antagonist addition, was rapid. Conversely, preincubation with vasopressin antagonist in medium containing 0.5 mM Ca2+ dramatically lowered plasma membrane Ca2+ permeability. The ER Ca2+ pool emptied by vasopressin was refilled in the presence of vasopressin antagonist plus 0.5 mM Ca2+, but did not refill when the medium contained no added Ca2+. Under the conditions of these experiments, the Ca2+ levels of the ER hormone-sensitive Ca2+ pool were estimated as well as intracellular concentrations of inositol-1,4,5-trisphosphate. The Ca2+ levels of the endoplasmic reticulum correlated inversely with plasma membrane Ca2+ permeability, whereas cellular concentrations of inositol-1,4,5-trisphosphate did not correlate with Ca2+ permeability.(ABSTRACT TRUNCATED AT 400 WORDS)