Bidirectional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles.

We determined the trans effects of extracellular reduced glutathione (GSH) on the rate of efflux of endogenous labeled GSH from freshly isolated rat hepatocytes. The presence of GSH (10 mM) in the medium significantly stimulated the fractional rate of efflux of [35S]GSH from 5.2 to 12.6%/15 min (p < 0.01). This effect was concentration-dependent, had sigmoid type of kinetics (D50 of 0.32 mM), and was reversible upon removal of external GSH. trans-Stimulation (counter-transport) was also observed with 5 mM oxidized glutathione (GSSG) and ophthalmic acid (fractional [35S] GSH efflux: 13.4% +/- 4.1 and 8.8% +/- 2.3 in 15 min, respectively, compared with control: 4.7 +/- 2.5/15 min). Bromosulphthalein-glutathione (BSP-GSH, 5 mM) in Krebs buffer inhibited the fractional [35S]GSH efflux (1.1%/15 min), whereas in Cl(-)-free buffer, GSH efflux was stimulated (14.2%/15 min) compared with control. trans-Stimulation was independent of chloride. BSP-GSH cis-inhibited and trans-stimulated the initial rate of GSH transport in basolateral-enriched membrane vesicles (bLPM) but not in canalicular-enriched membrane vesicles (cLPM). gamma-Glutamyl compounds also cis-inhibited and trans-stimulated GSH transport in bLPM vesicles. GSH-depleted hepatocytes incubated with 10 mM [35S]GSH accumulated more GSH than repleted cells, but the initial rate of uptake of radioactivity was faster in repleted cells. In contrast, repleted hepatocytes incubated with tracer or 50 microM [35S]GSH did not take up GSH. Thus, the sinusoidal membrane GSH transporter exhibits low affinity kinetics with sigmoid features for both GSH uptake and trans-stimulation of efflux, explaining the lack of uptake of GSH at low physiologic extracellular concentrations. Therefore, our findings support and explain the widely held view that GSH transport is unidirectional under physiologic conditions. However, the efflux of GSH may also occur in exchange for the uptake of organic anions and gamma-glutamyl compounds.     

ATP-dependent transport for glucuronides in canalicular plasma membrane vesicles

Using rat liver canalicular plasma membrane vesicles, it has been verified that the transport of p-nitrophenyl glucuronide (NPG) across membranes is an ATP-dependent process; the apparent Km for NPG was 20 microM. S-(2,4-dinitrophenyl)-glutathione (DNP-SG) inhibited NPG uptake dose-dependently, and NPG or testosterone glucuronide did ATP-dependent DNP-SG uptake similarly. These results suggest that transport of glucuronide is mediated by an ATP-dependent glutathione S-conjugate carrier.     

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.     

Mechanisms of active transport in isolated bacterial membrane vesicles. 18. The mechanism of action of carbonylcyanide m-chlorophenylhydrazone

Experiments were performed under several conditions seeking evidence for turnover of protein-bound lipoic acid in Escherichia coli analogous to that described for the 4′-phosphopantetheine moiety of the E. coli acyl carrier protein. Pulse-chase, chase experiments using both low and saturating concentrations of lipoic acid, chase experiments in the presence of chloramphenicol, which prevents incorporation of lipoic acid into the protein-bound form, and chase experiments in cells in which the free pool of lipoic acid was reduced by osmotic shock all failed to demonstrate any turnover of protein-bound lipoic acid.     

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.     

Inhibition of taurocholate efflux from rat hepatic canalicular membrane vesicles by glutathione disulfide

In right-side out rat hepatic canalicular membrane vesicles glutathione disulfide (GSSG) inhibited the efflux of taurocholate approx. 70% in the presence or approx. 55% in the absence of a valinomycin-mediated K+ diffusion potential; maximal inhibition occurred at 5 mM GSSG. The inhibition by GSSG was abolished by dithioerythritol. Neither dithioerythritol alone nor GSH inhibited taurocholate efflux. S-(2,4-Dinitrophenyl)glutathione and N-ethylmaleimide showed intermediate inhibitory effects.     

Active transport of manganese in isolated membrane vesicles of Bacillus subtilis.

Membrane vesicles isolated from cells of bacillus subtilis W23 accumulate manganese in the presence of an energy source. The artificial electron donor system ascorbate and phenazine methosulfate or reduced nicotinamide adenine dinucleotide and phenazine methosulfate can supply the energy for the uptake. D-Lactate in the presence or absence of phenazine methosulfate would not support manganese accumulation. Anaerobiosis, cyanide, m-chlorophenyl carbonylcyanide hydrozone, valinomycin, gramicidin, and p-hydroxy-mercuribenzoate inhibit the uptake. The inhibition by p-hydroxymercuribenzoate is prevented by excess dithiothreitol. Potassium fluoride or sodium arsenate has no effect on the uptake. The manganese transport system in the B. subtilis vesicles exhibits Michaelis-Menten kinetics with a Km of 13 muM and a Vmax of 1.7 nmol/min per mg (dry weight) of membranes. The uptake of manganese is specific and is not inhibited by 0.1 mM CaCL2 or Mgcl2.     

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.     

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.     

The use of isolated membrane vesicles to study epithelial transport processes

Summary Epithelia are multicompartment and multicomponent systems performing transcellular and paracellular transport in a very complex manner. One way to get a deeper understanding of the function of such a complex system is to dissect it into the single components and then, after having defined the components under well-controlled conditions, to try to describe the behavior of the whole system on the basis of the properties of the single components.This article deals with the analysis of isolated plasma membranes derived from the luminal and contraluminal face of epithelial cells, predominantly renal proximal tubular and small intestinal cells. It is aimed to give an overview of methods used to isolate and separate plasma membranes, to study their transport properties as membrane vesicles, and also to address the question of how information gained with the isolated membranes corresponds to observations made in the intact cell using other, notably electrophysiological, measurements. The review also critically evaluates the limitations of the approach and thereby tries to set the work on isolated membranes in the proper perspective within the field of transport physiology.     

Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles

Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity. In contrast to effects induced by taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine. These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity. In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion.     

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.