Basolateral Liver Plasma Membrane Vesicles Research Articles

Modulation of Na+/Mg2+ exchanger stoichiometry ratio by Cl− ions in basolateral rat liver plasma membrane vesicles

The Na+/Mg²+ exchanger represents the main Mg²+ extrusion mechanism operating in mammalian cells including hepatocytes. We have previously reported that this exchanger, located in the basolateral domain of the hepatocyte, promotes the extrusion of intravesicular trapped Mg²+ for extravesicular Na+ with ratio 1. This electrogenic exchange is supported by the accumulation of tetraphenyl-phosphonium within the vesicles at the time when Mg²+ efflux occurs. In this present study, the role of extra- and intra-vesicular Cl⁻ on the Na+/Mg²+ exchange ratio was investigated. The results reported here suggest that Cl⁻ ions are not required for the Na+ to Mg²+ exchange to occur, but the stoichiometry ratio of the exchanger switches from electrogenic (1Na(in)+ :1Mg(out)²+) in the presence of intravesicular Cl⁻ to electroneutral (2Na(in)+ :1 Mg(out)²+) in their absence. In basolateral liver plasma membrane vesicles loaded with MgCl₂ labeled with ³⁶Cl⁻, a small but significant Cl⁻ efflux (~30 nmol Cl⁻/mg protein/1 min) is observed following addition of NaCl or Na-isethionate to the extravesicular medium. Both Cl⁻ and Mg²+ effluxes are inhibited by imipramine but not by amiloride, DIDS, niflumic acid, bumetanide, or furosemide. In vesicles loaded with Mg-gluconate and stimulated by Na-isethionate, an electroneutral Mg²+ extrusion is observed. Taken together, these results suggest that the Na+/Mg²+ exchanger can operate irrespective of the absence or the presence of Cl⁻ in the extracellular or intracellular environment. Changes in trans-cellular Cl⁻ content, however, can affect the modus operandi of the Na+/Mg²+ exchanger, and consequently impact "cellular" Na+ and Mg²+ homeostasis as well as the hepatocyte membrane potential.

Involvement of ERK1/2 and p38 in Mg2+ accumulation in liver cells

Activation of PKC signaling induces Mg(2+) accumulation in liver cells. To test the hypothesis that PKC induces Mg(2+) accumulation via MAPKs activation, hepatocytes were incubated in the presence of PD98059 and SB202190 as specific inhibitors of ERK1/2 and p38, respectively, and stimulated for Mg(2+) accumulation by addition of PMA or OAG. Accumulation of Mg(2+) within the cells was measured by atomic absorbance spectrophotometry in the acid extract of cell pellet. The presence of either inhibitor completely abolished Mg(2+) accumulation irrespective of the dose of agonist utilized while having no discernible effect on beta -adrenoceptor mediated Mg(2+) extrusion. A partial inhibition on alpha (1)-adrenoceptor mediated Mg(2+) extrusion was observed only in cells treated with PD98059. To confirm the inhibitory effect of PD98509 and SB202190, total and basolateral liver plasma membrane vesicles were purified in the presence of either MAPK inhibitor during the isolation procedure. Consistent with the data obtained in intact cells, liver plasma membrane vesicles purified in the presence of PD98509 or SB202190 lost the ability to accumulate Mg(2+)in exchange for intra-vesicular entrapped Na(+) while retaining the ability to extrude entrapped Mg(2+) in exchange for extra-vesicular Na(+). These data indicate that ERK1/2 and p38 are involved in mediating Mg(2+) accumulation in liver cells following activation of PKC signaling. The absence of a detectable effect of either inhibitor on beta -adrenoceptor induced, Na(+)-dependent Mg(2+) extrusion in intact cells and in purified plasma membrane vesicles further support the hypothesis that Mg(2+) extrusion and accumulation occur through distinct and differently regulated transport mechanisms.

Plasma membrane domain localization and transcytosis of the glucagon-induced hepatic system A carrier.

We have utilized canalicular (cLPM) and basolateral (blLPM) liver plasma membrane vesicles to investigate the domain localization of several Na(+)-dependent amino acid transporters. Neutral amino acid transport by systems N and ASC was measurable in both domains but was greater in blLPM vesicles. Sodium-dependent glutamate uptake (system X-) was preferentially localized to cLPM. The absolute activity and domain distribution of these three carriers remained unchanged after treatment of rats with a combination of glucagon and dexamethasone. A low level of basal system A activity was present in both the blLPM and cLPM. Glucagon-induced system A activity was first observed in blLPM vesicles approximately 60 min posthormone treatment and, in cLPM vesicles, approximately 30 min later. In situ perfusion of glucagon-treated rat liver with the membrane-impermeant protein modification reagent glutathione maleimide specifically inactivated blLPM system A activity and abolished the delayed arrival of hormone-induced activity to the cLPM. Transient perfusion of the liver with glutathione maleimide followed by a recovery period in vivo showed that the reagent did not irreversibly inactivate the transcytotic process and also provided an independent demonstration of the time delay between arrival of the carrier activity at the two membrane surfaces. These results support the concept of a transcytotic process in which the blLPM is the sorting site for the hormone-induced system A carrier proteins that eventually reach the cLPM.

Shift from Biliary to Urinary Elimination of Acetaminophen-Glucuronide in Acetaminophen-Pretreated Rats

Despite its toxicity, acetaminophen (APAP) is used increasingly as an analgesic, antipyretic, and anti-inflammatory agent. We examined the effect of prior exposure to APAP on its biliary and urinary elimination. The biliary and urinary elimination of a test dose of APAP (150 mg/kg i.v.) was determined in male Wistar rats 24 h after pretreatment with vehicle, a single dose (1.0 g/kg i.p.), or increasing daily doses (0.2, 0.3, 0.6, and 1.0 g/kg/day i.p.) of APAP. Although elimination of the parent APAP was minimally affected, biliary excretion of APAP glucuronide was significantly decreased 70 and 80%, whereas urinary excretion was significantly increased 90 and 100% in the groups pretreated with single and repeated doses of APAP, respectively, relative to vehicle controls. Western analysis and confocal immunofluorescent microscopy indicated a marked increase in hepatic expression of multidrug resistance-associated protein 3 (Mrp3) in both groups pretreated with APAP, relative to expression of Mrp2. ATP-dependent transport of [3H]taurocholate, an Mrp3 substrate, was significantly increased in basolateral liver plasma membrane vesicles from rats pretreated with repeated doses of APAP relative to controls. Enterohepatic recirculation of APAP glucuronide after administration of the same test dose of the drug was significantly decreased in rats pretreated with repeated doses of APAP. These data indicate that APAP pretreatment induced a shift from biliary to urinary elimination of APAP glucuronide, consistent with the increased expression of Mrp3 in the basolateral domain of the hepatocyte. We postulate that decreased enterohepatic recirculation contributes to decreased APAP hepatotoxicity by reducing liver exposure.

ATP modulates sulfobromophthalein uptake in rat liver plasma membrane vesicles.

The hepatic uptake of the bilirubin-bilirubin-sulfobromophthalein (BSP) group of organic anions is a carrier-mediated process and is accounted for by at least four distinct plasma membrane proteins (bilitranslocase, BSP/bilirubin-binding protein, organic anion-binding protein and the organic anion transport protein). In order to investigate the regulation of basolateral organic anion uptake, BSP transport was measured in rat basolateral liver plasma membrane vesicles in the presence of ATP. ATP significantly stimulated the electroneutral uptake of BSP with an increment in Vmax compared with control (1.57 +/- 0.14 vs 0.73 +/- 0.06 nmol BSP/mg protein per 15 s, respectively; P < 0.001) while the apparent K(m) was not changed significantly (12 +/- 1 vs 12 +/- 2 mumol/L). The stimulatory effect was dose-dependent for ATP (K(m) 1.01 +/- 0.37 mmol/L). ATP had no detectable effect on the electrogenic component of BSP transport. Other nucleotides (ADP, AMP, GTP) and non-hydrolysable ATP did not enhance BSP uptake, suggesting that ATP hydrolysis was necessary for the effect. This was supported by the lack of effect on BSP uptake when ATP was added in the presence of vanadate. The addition of phorbol-12-myristate 13-acetate, an activator of protein kinase C (PKC), increased BSP uptake in a dose-dependent manner in the presence, but not in the absence, of ATP. Incubation of vesicles with staurosporine, an inhibitor of PKC activity, resulted in a dose-dependent inhibition of ATP-sensitive BSP transport. These data indicate that electroneutral BSP hepatic uptake is modulated by ATP. The effect is related to ATP hydrolysis and involves the activity of PKC.

Evidence that interference with binding to hepatic cytosol binders can inhibit bile acid excretion in rats

We previously identified that Y' bile acid binders (3alpha- hydroxysteroid dehydrogenases) interact with bile acids in intact rat hepatocytes using [3beta-3H, C24-14C]bile acids and that indomethacin, a competitive inhibitor of 3alpha-hydroxysteroid dehydrogenase, inhibits 3H-loss from the C3-position of bile acids as well as inhibits hepatic bile acid removal and excretion. To study the kinetics of these inhibitory effects, glycocholate transport was studied in the absence and presence of indomethacin in the single-pass perfused rat liver. Indomethacin decreased net hepatic glycocholate uptake in the perfused liver, which was confirmed in isolated hepatocytes and basolateral liver plasma membrane vesicles. However, indomethacin markedly increased the sinusoidal efflux and decreased the biliary excretion of glycocholate in the perfused liver. These observations indicate that the effect of indomethacin to delay biliary glycocholate excretion is related to either intracellular or canalicular glycocholate transport. The latter possibility seemed unlikely because indomethacin did not inhibit electrogenic or adenosine triphosphate (ATP)-dependent glycocholate uptake by canalicular liver plasma membrane vesicles. Thus, the current data support an important role for binding of bile acids to cytosolic proteins in overall hepatic transport and suggest that specific interference with cytosolic binding can interfere with the excretion of bile acids. (Hepatology 1996 Jun;23(6):1642-9)

Evidence That Interference With Binding to Hepatic Cytosol Binders Can Inhibit Bile Acid Excretion in Rats

We previously identified that Y' bile acid binders (3alpha-hydroxysteroid dehydrogenases) interact with bile acids in intact rat hepatocytes using [3beta-3H, C24-14C]bile acids and that indomethacin, a competitive inhibitor of 3alpha-hydroxysteroid dehydrogenase, inhibits 3H-loss from the C3-position of bile acids as well as inhibits hepatic bile acid removal and excretion. To study the kinetics of these inhibitory effects, glycocholate transport was studied in the absence and presence of indomethacin in the single-pass perfused rat liver. Indomethacin decreased net hepatic glycocholate uptake in the perfused liver, which was confirmed in isolated hepatocytes and basolateral liver plasma membrane vesicles. However, indomethacin markedly increased the sinusoidal efflux and decreased the biliary excretion of glycocholate in the perfused liver. These observations indicate that the effect of indomethacin to delay biliary glycocholate excretion is related to either intracellular or canalicular glycocholate transport. The latter possibility seemed unlikely because indomethacin did not inhibit electrogenic or adenosine triphosphate (ATP)-dependent glycocholate uptake by canalicular liver plasma membrane vesicles. Thus, the current data support an important role for binding of bile acids to cytosolic proteins in overall hepatic transport and suggest that specific interference with cytosolic binding can interfere with the excretion of bile acids.

High affinity transporter of reduced glutathione plays a main role in glutathione transport in rat basolateral liver plasma membrane vesicles (bLPM): [formula omitted] Dept. of GI and Liver Diseases, Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan

High affinity transporter of reduced glutathione plays a main role in glutathione transport in rat basolateral liver plasma membrane vesicles (bLPM): [formula omitted] Dept. of GI and Liver Diseases, Institute for Adult Diseases, Asahi Life Foundation, Tokyo, Japan

P-221 A kinetic analysis of reduced glutathione (GSH) transport in rat basolateral liver plasma membrane vesicles (bLPM)

P-221 A kinetic analysis of reduced glutathione (GSH) transport in rat basolateral liver plasma membrane vesicles (bLPM)

Role of BSP/Bilirubin Binding Protein and Bilitranslocase in Glutathione Uptake in Rat Basolateral Liver Plasma Membrane Vesicles

Glutathione (GSH) efflux in the liver is mediated by carrier proteins sensitive to membrane potential (electrogenic), and it is inhibited by organic anions. The hepatic uptake of tetrabromosulfophthalein (BSP) is also a carrier-mediated function accomplished at least by two different transport mechanisms, bilitranslocase (BTL) and BSP/Bilirubin Binding Protein (BBBP). The two proteins operate in parallel, the former operating electro-genically, the latter being insensitive to membrane potential (electroneutral). To investigate the relationship between transport mechanisms for GSH and organic anions, the initial uptake rate of 35S-labelled BSP into basolateral rat liver plasma membranes was measured in the presence of external or intravesicular GSH. Electrogenic and electroneutral BSP uptake were neither cis-inhibited nor trans-stimulated by GSH. 3H glycine-GSH uptake in LPMV was not modified by pre-incubating LPMV with anti-BTL or anti-BBBP antibodies. These data indicate that GSH hepatic uptake is mediated neither by BBBP nor BTL.

Hepatic Na(+)-dicarboxylate cotransport: identification, characterization, and acinar localization.

Rat liver perfusion studies suggest that the transport of alpha-ketoglutarate (KG) and related dicarboxylates exhibits acinar heterogeneity, in that the uptake and subsequent metabolism of these organic anions appears to occur predominantly in the perivenous region. However, the isolated perfused liver as an experimental model cannot distinguish intra-acinar differences in either the rate of solute uptake and/or efflux or intracellular binding and/or metabolism. Therefore, the driving forces and acinar localization of KG transport were examined using rat basolateral liver plasma membrane vesicles (blLPMV) isolated from control animals and animals treated 24 h before with selective perivenous and periportal toxins [carbon tetrachloride (CCl4) and allyl alcohol (AA), respectively]. In control blLPMV, [14C]KG uptake into an osmotically sensitive space was markedly stimulated by an inwardly directed Na+ gradient but not by inwardly directed gradients of other monovalent cations. The Na+ ionophore, gramicidin, had a small but significant inhibitory effect on Na(+)-dependent KG uptake, demonstrating that KG uptake was not the result of an intravesicular positive Na+ diffusion potential. The protonophore, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, had no effect on Na+ gradient-driven KG uptake, indicating that KG uptake was not the indirect result of coordinated activities of Na-H and KG-OH exchange. Na+ gradient-driven KG uptake was electrogenic (occurring with the net transfer of positive charge), and cis-inhibited by other tricarboxylic acid cycle intermediates, including succinate, fumarate, and malate and by citrate, but not by the dicarboxylates oxalate and malonate nor by glutamate and taurocholate (TC).(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma membrane domain localization and transcytosis of the glucagon-induced hepatic system A carrier

We have utilized canalicular (cLPM) and basolateral (blLPM) liver plasma membrane vesicles to investigate the domain localization of several Na(+)-dependent amino acid transporters. Neutral amino acid transport by systems N and ASC was measurable in both domains but was greater in blLPM vesicles. Sodium-dependent glutamate uptake (system X-) was preferentially localized to cLPM. The absolute activity and domain distribution of these three carriers remained unchanged after treatment of rats with a combination of glucagon and dexamethasone. A low level of basal system A activity was present in both the blLPM and cLPM. Glucagon-induced system A activity was first observed in blLPM vesicles approximately 60 min posthormone treatment and, in cLPM vesicles, approximately 30 min later. In situ perfusion of glucagon-treated rat liver with the membrane-impermeant protein modification reagent glutathione maleimide specifically inactivated blLPM system A activity and abolished the delayed arrival of hormone-induced activity to the cLPM. Transient perfusion of the liver with glutathione maleimide followed by a recovery period in vivo showed that the reagent did not irreversibly inactivate the transcytotic process and also provided an independent demonstration of the time delay between arrival of the carrier activity at the two membrane surfaces. These results support the concept of a transcytotic process in which the blLPM is the sorting site for the hormone-induced system A carrier proteins that eventually reach the cLPM.

Organic cation transport by rat liver plasma membrane vesicles: studies with tetraethylammonium.

Recently, an organic cation:H+ antiport was selectively identified on the sinusoidal domain of rat liver with the use of the endogenous organic cation N1-methylnicotinamide (NMN). Absence of NMN+:H+ exchange on canalicular membrane suggested that this transport process was primarily involved in organic cation uptake, leaving the mechanism(s) for organic cation secretion into bile unknown. To further define hepatic organic cation transport, we examined the characteristics of tetraethylammonium (TEA) transport in basolateral (blLPM) and canalicular (cLPM) rat liver plasma membrane vesicles. In cLPM vesicles, under voltage-clamped conditions, an outwardly directed H+ gradient stimulated [14C]TEA uptake compared with [14C]TEA uptake under pH-equilibrated conditions, consistent with electroneutral TEA:H+ exchange. The proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone had no effect on [14C]TEA uptake, demonstrating that pH-dependent [14C]TEA uptake was not the result of a H+ diffusion potential. In the absence of a pH gradient, the intravesicular presence of TEA trans-stimulated uptake of [14C]TEA. Procainamide ethobromide (PAEB), vecuronium, and tributylmethylammonium (TBuMA), organic cations selectively excreted in bile, cis-inhibited pH-dependent TEA uptake. In contrast, in blLPM vesicles, no pH gradient-dependent [14C]TEA uptake was demonstrated. Instead, basolateral [14C]TEA uptake was significantly stimulated by a valinomycin-induced intravesicular-negative K+ diffusion potential. Basolateral [14C]TEA uptake was also cis-inhibited by PAEB, vecuronium, and TBuMA, but not by NMN. Conversely, PAEB, vecuronium, and TBuMA had no effect on basolateral pH-dependent [3H]NMN uptake. These findings suggest that organic cation transport, with TEA as a model quaternary amine, across the canalicular membrane is driven by an electroneutral organic cation:H+ exchange and that the transport of certain organic cations across the basolateral membrane is via a carrier-mediated system stimulated by an inside-negative membrane potential.

Identification of sodium-dependent and sodium-independent dicarboxylate transport systems in rat liver basolateral membrane vesicles.

The mechanisms involved in the hepatocellular uptake of Krebs-cycle intermediates were investigated in isolated basolateral (sinusoidal and lateral) rat liver plasma membrane (blLPM) vesicles. An inwardly directed Na+ gradient markedly stimulated uptake of 2-oxoglutarate and succinate into voltage- and pH-clamped blLPM vesicles. This Na(+)-dependent portion of the dicarboxylate uptake was characterized by (a) saturability with increasing substrate concentrations (Km = 6.4-10 mM; Vmax approximately 0.2 nmol min-1 mg protein-1), (b) cis-inhibition by lithium (10 mM), other Krebs-cycle dicarboxylates (1 mM) and DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; 1 mM) but not by sulphate, monocarboxylates, oxalate, acidic amino acids, bile salts and probenecid, (c) stimulation by an intravesicular negative K(+)-diffusion potential indicating electrogenic [(Na+)n greater than 2-succinate] cotransport, and (d) a pH optimum for transport between 7.0 and 7.5. In the absence of Na+, an inside alkaline pH gradient also markedly stimulated 2-oxoglutarate uptake. This pH-gradient-driven 2-oxoglutarate uptake was insensitive to lithium, but could also be inhibited by DIDS and succinate. Furthermore, saturation kinetics demonstrated Km (approximately 34 mM) and Vmax (approximately 0.8 nmol min-1 mg protein-1) values that were clearly different from those of the Na(+)-dependent uptake system. These results indicate the occurrence of two separate dicarboxylate transport systems along the sinusoidal border of hepatocytes, one being a Na(+)-dicarboxylate symporter and the other representing an anion-exchange system.

Impaired alanine uptake by basolateral liver plasma membrane vesicles from rats consuming ethanol

Chronic ethanol consumption reduces alanine transport by rat basolateral liver plasma membrane (blLPM) vesicles; however, the mechanism for this effect remains uncertain. It may be related to the ethanol-induced changes in blLPM fluidity and lipid composition; alternatively ethanol might reduce the number of transporters in the blLPM. To investigate the effect of blLPM fluidity and lipid composition on Na(+)-dependent alanine uptake these parameters were altered in vitro. Increasing the blLPM fluidity had no effect on Na(+)-dependent alanine uptake by blLPM vesicles or the activity of amino acid transport systems, A and ASC. Because ethanol is known to reduce the blLPM cholesterol content, the influence of altering blLPM cholesterol on alanine transport by these membranes was investigated next. Neither an increase nor a decrease of the cholesterol content of the blLPM altered Na(+)-dependent alanine uptake or the activity of system A or ASC. Finally, the influence of chronic ethanol consumption on the specific binding of [3H]alanine to blLPM was studied. The dissociation constant for alanine binding to blLPM from ethanol-fed rats and their pair-fed controls was similar (1.9 +/- 0.2 vs. 2.0 +/- 0.3 mM); however, the maximal binding capacity for alanine was significantly (P less than 0.05) lower in the blLPM from ethanol-fed rats (316 +/- 53 pmol/mg protein) compared with their pair-fed controls (527 +/- 79 pmol/mg protein). These studies do not support the hypothesis that ethanol-induced changes in blLPM fluidity are responsible for the impaired alanine transport; they do suggest that ethanol may reduce the

Adenosine transport in rat liver plasma membrane vesicles

Liver plasma membrane ecto-ATPase activity is largely restricted to the bile canalicular membrane. To determine whether a transport process is also selectively present on this membrane surface to reclaim adenosine derived from the intracanalicular degradation of ATP, the characteristics of hepatic nucleoside transport were examined in canalicular (cLPM) and basolateral (blLPM) rat liver plasma membrane vesicles. In the presence of the adenosine deaminase inhibitor, deoxycoformycin, an inwardly directed Na+ gradient markedly stimulated [3H]adenosine uptake in cLPM vesicles. Canalicular Na(+)-dependent [3H]adenosine uptake was enhanced by an intravesicular-negative membrane potential and inhibited by dissipation of the Na+ gradient with gramicidin D. Both purine and pyrimidine nucleosides inhibited canalicular adenosine transport. 6-[(4-Nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, an inhibitor of nucleoside transport in erythrocytes and nonepithelial cells, had no effect on canalicular adenosine transport. Canalicular Na(+)-dependent [3H]adenosine uptake exhibited saturability with a Michaelis-Menten constant of 8.3 microM and a maximum transport rate of 7.6 pmol.5 s-1.mg protein-1. In contrast, [3H]adenosine uptake in blLPM vesicles was not stimulated by an inwardly directed Na+ gradient. These findings demonstrate asymmetric distribution of hepatic Na(+)-dependent nucleoside transport. Reclamation of intracanalicular adenosine resulting from ecto-ATPase activity may explain the presence of this transport process selectively on the bile canalicular membrane.

Transport of N1-methylnicotinamide by organic cation-proton exchange in rat liver membrane vesicles

The characteristics of hepatic organic cation transport were examined in basolateral (blLPM) and canalicular (cLPM) rat liver plasma membrane vesicles, using the naturally occurring organic cation, N1-methylnicotinamide (NMN). In blLPM vesicles, an outwardly directed H+ gradient (pHin 5.9/pHout 7.9) stimulated [3H]NMN uptake compared with [3H]NMN uptake under pH-equilibrated conditions. The time course of [3H]NMN uptake exhibited a transient "over-shoot" phenomenon, consistent with active transport. The proton ionophore, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, had no effect on [3H]NMN uptake, demonstrating that pH-dependent [3H]NMN uptake was not the result of a H+ diffusion potential. An outwardly directed H+ gradient also stimulated [3H]NMN uptake under voltage-clamped conditions, consistent with electroneutral NMN-H+ exchange. Under conditions that effectively dissipated the H+ gradient, no active transport of [3H]NMN was observed. In the absence of a pH gradient, the intravesicular presence of NMN trans-stimulated the uptake of [3H]NMN. NMN-H+ exchange was differentiated from sinusoidal Na(+)-H+ exchange by determining sensitivity to amiloride. The substrate specificity of NMN-H+ exchange in blLPM vesicles was examined by determining the cis-inhibitory effects of typical endogenous and exogenous substrates of other epithelial organic cation-H+ exchangers. Kinetic analysis of initial rates of carrier-mediated [3H]NMN uptake over a NMN concentration range of 0.05-15 mM demonstrated that uptake occurred via two saturable transport systems, one a high-affinity low-capacity process and the other a low-affinity high-capacity type. In contrast, in cLPM vesicles, no pH gradient-dependent [3H]NMN uptake was demonstrated. These findings are consistent with the presence of an organic cation-H+ antiport on the sinusoidal membrane, with features distinct from the renal antiport, such as substrate specificity and membrane localization, that may account for differences in drug disposition by these two organs.

Mechanisms of transport of nontransferrin-bound iron in basolateral and canalicular rat liver plasma membrane vesicles

Although most iron in plasma is bound to transferrin, recent evidence suggests that the nontransferrin-bound fraction contributes to hepatic iron loading and toxicity seen in iron-overload disorders. Our studies of isolated perfused rat liver previously demonstrated saturable uptake of nontransferrin-bound iron that continues despite hepatic iron overload. To further characterize the mechanism of transport of this form of iron, we measured binding of 55Fe-labeled ferrous ascorbate to rat liver plasma membrane vesicles under varying conditions. Binding of 5 mumol/L iron by both basolateral and canalicular membranes was time-dependent and linear for the first 5 sec. Initial rate of binding of ferrous ascorbate to basolateral membrane vesicles was temperature dependent and increased by calcium but, in contrast to the perfused rat liver, was not inhibited by other divalent cations. Binding velocities by basolateral membrane vesicles were saturable at increasing iron concentration (Km = 33 mumol/L, Vmax = 16 pmol/mg protein/sec). Ferrous iron binding by canalicular membrane vesicles was also temperature dependent, but initial association rates were not saturable over the concentration range studied (2 to 20 mumol/L). We conclude that nontransferrin-bound iron associates with basolateral liver plasma membrane vesicles by a saturable mechanism sensitive to temperature and calcium and consistent with a membrane carrier. Other divalent cations do not inhibit membrane association but may compete for a subsequent cytosolic binding site.

Ethanol consumption decreases alanine uptake by rat basolateral liver plasma membrane vesicles

Alterations of amino acid metabolism may play an important role in the pathogenesis of ethanol-induced liver disease. Previous studies indicate that ethanol added in vitro inhibits amino acid uptake by cultured hepatocytes and liver plasma membrane vesicles; however, the effect of chronic ethanol consumption on amino acid uptake by the liver remains unknown. Therefore, the present studies were performed to determine if chronic ethanol consumption impairs alanine uptake by rat basolateral liver plasma membrane vesicles. Male Sprague-Dawley rats were pair-fed for 6 weeks a diet containing 36% of calories as ethanol or a control diet in which ethanol was isocalorically replaced with carbohydrate. Chronic ethanol consumption reduced basolateral liver plasma membrane sodium-dependent alanine transport activity by 36.3% ± 15.9% (p < 0.01). This reduction was caused primarily by impaired activity of amino acid transport system A. The response of system A to glucagon was reduced in the ethanol-fed rats, suggesting that impaired hormonal regulation is at least partially responsible for the lower system-A activity. Kinetic analysis shows that ethanol consumption reduces the Vmax of sodium-dependent alanine transport without affecting the Km. These studies indicate that chronic ethanol consumption reduces alanine uptake by the rat liver. They further show that the reduced uptake is at least partially caused by an intrinsic defect in membrane-transport processes rather than another regulatory mechanism.

Taurocholate transport by basolateral plasma membrane vesicles isolated from human liver.

Transport of taurocholate into the hepatocyte against unfavorable chemical and electrical gradients occurs via a sodium-dependent, carrier-mediated transport system. Although this cotransporter has been characterized in the rodent, it has not been demonstrated in man. Therefore, we utilized human liver, obtained via multiorgan donation but not used for transplantation, to prepare basolateral (sinusoidal) liver plasma membrane vesicles by a Percoll gradient method. Na+,K+-ATPase, a marker enzyme for the basolateral domain, was enriched 28.9-fold in the final membrane fraction compared with homogenate, whereas the bile canalicular membrane enzymes Mg++-ATPase and alkaline phosphatase were enriched only 3.4- and 6.4-fold, respectively. Marker enzyme activities for endoplasmic reticulum, lysosomes and mitochondria were not enriched compared with homogenate. Integrity of the membrane vesicles was confirmed by the demonstration of Na+-dependent concentrative uptake of the amino acid L-alanine (estimated intravesicular volume of 0.59 microliter per mg protein). An inwardly directed 100 mM Na+ gradient stimulated the initial rate of 2.5 microM taurocholate uptake and energized a transient 2-fold accumulation of the bile acid above equilibrium ("overshoot"). In contrast, uptake was slower and no overshoot occurred with a K+ gradient. A negative intravesicular potential, created by altering accompanying anions or by valinomycin-induced K+ diffusion potentials, did not enhance taurocholate uptake, suggesting an electroneutral cotransport mechanism. Chloride as the accompanying anion stimulated the initial rate of uptake compared with anions of lesser or greater lipid permeability. Na+-dependent taurocholate (4 microM) uptake was significantly inhibited by 250 microM cholate, taurocholate, glycocholate, taurochenodeoxycholate and bromsulfophthalein.(ABSTRACT TRUNCATED AT 250 WORDS)