Basolateral Plasma Membrane Vesicles Research Articles

Inhibition by trichloroethylene and 1,1,2-trichloro-1,2,2-trifluoroethane of taurocholate uptake into basolateral rat liver plasma membrane vesicles

It has previously been shown that trichloroethylene (TRI) and 1,1,2-trichloro-1,2,2-tri-fluoroethane (FC 113) interfere with the transport of bile acids by isolated human and rat hepatocytes in a dose-dependent and reversible manner. This finding may explain the rise in serum bile acids (SBA) following exposure to these chemicals. However, the effect of these compounds on the transport of bile acids across the cellular membrane in the absence of confounding variables, such as interference by intracellular metabolism, binding to cytosolic proteins and intracellular conjugation, has not been investigated. Accordingly, in vitro effects of TRI and FC 113 on uptake of [3H]taurocholate (TC) into purified basolateral (blLPM) and canalicular (cLPM) rat liver plasma membrane vesicles were examined by a rapid filtration technique. Both TRI and FC 113 caused a dose-dependent inhibition of TC uptake into blLPM vesicles at an approximate concentration of 3 mm and 72 μm, respectively. Initial rates of TC uptake in the presence of TRI and FC 113 were significantly inhibited by 69 and 61%, respectively (P < 0.05). In contrast, these chemicals had no effect on TC uptake into cLPM vesicles. This confirms studies in intact cells where these solvents were found to inhibit the uptake of bile acids by hepatocytes rather than interfere with the process of efflux. In conclusion, and consistent with the previous findings, the data suggest that the mechanism of TRI and FC 113-induced elevation of SBA may, in part, be due to selective inhibition of bile acid transport by the parent compounds at the basolateral domain of the hepatocyte plasma membrane.

Magnesium Transport Across the Basolateral Plasma Membrane of the Fish Enterocyte

In tilapia (Oreochromis mossambicus) intestine, Mg2+ transport across the epithelium involves a transcellular, Na+- and Na+/K+-ATPase dependent pathway. In our search for the Mg2+ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na+/Mg2+ antiport or ATP-driven transport. Evidence is provided, however, that Mg2+ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl- gradient stimulated Mg2+ uptake (as followed with the radionuclide 27Mg) twofold. As Cl--stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg2+ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg2+ uptake by the stilbene derivative, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid. Kinetic analyses of the Cl--stimulated Mg2+ uptake yielded a Km (Mg2+) of 6.08 +/- 1.29 mmol . l-1 and a Km (Cl-) of 26.5 +/- 6.5 mmol . l-1, compatible with transport activity at intracellular Mg2+- and Cl--levels. We propose that Mg2+ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism.

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.

Isolation and characterization of luminal and basolateral plasma membrane vesicles from the medullary thick ascending loop of Henle

Transport processes in the medullary thick ascending limb of Henle (MTAL) have previously been investigated predominantly using tubule suspensions, perfusion of the loop of Henle in vivo, or individual perfused tubules [1, 2]. One important approach to investigate mechanisms of transport and luminal-basolateral sidedness of ion movement directly has been to isolate highly purified vesicle preparations of both types of plasma membranes of epithelia, such as the renal proximal tubule and the small intestine [reviewed in 3—5]. Regarding the MTAL, however, isolation of luminal (LMV) and basolateral (BLMV) plasma membrane vesicles has posed considerable difficulties, and has generally yielded mixed plasma membrane fractions consisting of both LMV and BLMV [5]. It is also important to remember that most vesicle studies aimed at characterizing transport processes in the MTAL have been performed on plasma membrane vesicles derived either from the entire outer medulla or from outer medullary homogenates [5], and the results then extrapolated to the MTAL. Clearly, for direct investigation into the basic mechanisms of transtubular solute transport in the MTAL, it would be valuable to isolate both types of plasma membranes free of contamination with intracellular as well as with plasma membranes from other nephron segments. In the present paper, we describe a new and simple approach that allowed the simultaneous isolation of LMV and BLMV with high yields from a single homogenate sample of purified isolated rat MTALs. Because this is the first report of the isolation of both types of MTAL plasma membrane vesicles, we wanted to examine whether these membrane preparations may serve as a useful model to characterize the transport properties of the two surfaces of this nephron segment. This report describes our evidence for the expression of different Na/H antiporters on the LMV and BLMV isolated from rat MTAL.

Choline transport in rat liver basolateral plasma membrane vesicles

Previous studies have demonstrated several pathways for the sinusoidal uptake of endogenous and exogenous organic cations, including two distinct organic cation:H+ exchanges and a separate carrier-mediated system driven by the inside-negative membrane potential. In this study, the driving force(s) for the uptake of the endogenous quaternary amine, choline, were determined in rat liver basolateral plasma membrane (blLPM) vesicles. Choline uptake into an osmotically sensitive space was not stimulated by an outwardly directed H+ or inwardly directed Na+ gradient. Instead, an inside-negative K+ diffusion potential stimulated choline uptake, suggesting the presence of a conductive pathway for choline uptake in bILPM vesicles. Conductive choline uptake was confirmed by inducing variable changes in the transmembrane potential with anions of different membrane permeability. Choline uptake in blLPM vesicles exhibitied 1) temperature dependency; 2) trans-stimulation; and 3) saturability, with an approximate Michaelis constant (Km) of 0.34 mmol/L and maximum velocity (Vmax) of 0.45 nmol/mg protein/15 s. Choline uptake in blLPM vesicles was cis-inhibited by the structurally similar derivative, hemicholinium-3 and acetylcholine, but not by substrates for other organic cation transport processes identified in blLPM vesicles, including thiamine, tetraethylammonium (TEA), tri-n-butyl-methylammonium (TBuMA), and N'-methylnicotinamide (NMN). These findings demonstrate an electrogenic pathway on the sinusoidal membrane for the uptake of this essential nutrient and support the existence of multiple pathways for the sinusoidal uptake of endogenous and exogenous organic cations. (Hepatology 1996 Jul;24(1):192-7)

Choline transport in rat liver basolateral plasma membrane vesicles.

Previous studies have demonstrated several pathways for the sinusoidal uptake of endogenous and exogenous organic cations, including two distinct organic cation:H+ exchanges and a separate carrier-mediated system driven by the inside-negative membrane potential. In this study, the driving force(s) for the uptake of the endogenous quaternary amine, choline, were determined in rat liver basolateral plasma membrane (blLPM) vesicles. Choline uptake into an osmotically sensitive space was not stimulated by an outwardly directed H+ or inwardly directed Na+ gradient. Instead, an inside-negative K+ diffusion potential stimulated choline uptake, suggesting the presence of a conductive pathway for choline uptake in bILPM vesicles. Conductive choline uptake was confirmed by inducing variable changes in the transmembrane potential with anions of different membrane permeability. Choline uptake in blLPM vesicles exhibitied 1) temperature dependency; 2) trans-stimulation; and 3) saturability, with an approximate Michaelis constant (Km) of 0.34 mmol/L and maximum velocity (Vmax) of 0.45 nmol/mg protein/15 s. Choline uptake in blLPM vesicles was cis-inhibited by the structurally similar derivative, hemicholinium-3 and acetylcholine, but not by substrates for other organic cation transport processes identified in blLPM vesicles, including thiamine, tetraethylammonium (TEA), tri-n-butyl-methylammonium (TBuMA), and N'-methylnicotinamide (NMN). These findings demonstrate an electrogenic pathway on the sinusoidal membrane for the uptake of this essential nutrient and support the existence of multiple pathways for the sinusoidal uptake of endogenous and exogenous organic cations.

Effect of endotoxin on bile acid transport in rat liver: a potential model for sepsis-associated cholestasis

Intrahepatic cholestasis in the setting of extrahepatic bacterial infection has been attributed to the effects of endotoxin and cytokines such as tumor necrosis factor-alpha (TNF-alpha) on bile acid transport. To define the mechanism of sepsis-associated cholestasis, taurocholate transport was examined in basolateral (bLPM) and canalicular (cLPM) rat liver plasma membrane vesicles derived from control and endotoxin [lipopolysaccharide (LPS)]-treated animals and in plasma membrane vesicles prepared after TNF-alpha treatment. Na(+)-dependent [3H]taurocholate uptake and both membrane-potential-dependent and ATP-dependent [3H]taurocholate transport were reduced in bLPM and cLPM vesicles, respectively, after LPS treatment. In membrane vesicles from TNF-alpha-treated animals, Na(+)-dependent [3H]taurocholate uptake was also reduced. Northern blot hybridization, using cDNA probes for the putative sinusoidal bile acid transporter (Ntcp) and canalicular ecto-adenosinetriphosphatase, demonstrated decreased mRNA levels after LPS and TNF-alpha treatment. Immunoblot analysis of membrane extracts from LPS-treated animals revealed decreased levels of these putative bile acid transporters. Impaired bile acid transport at the sinusoidal and canalicular membrane domains by these and other mediators of the inflammatory response may account for sepsis-associated cholestasis.

Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB-HSA affinity constants (K'f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 microM, K'f (litre/mol) was inversely related to [HSA], irrespective of the [Bf]/[HSA] ratio. K'f was 2.066 x 10(6) + (3.258 x 10(8)/[HSA]). When 50 mM KC1 was isoosmotically substituted for sucrose, the K'f value was significantly lower {2.077 x 10(6) + (1.099 x 10(8)/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] < or = 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with K(m) values of 28 +/- 7 and 57 +/- 8 nM respectively (mean +/- S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112 +/- 12 versus 45 +/- 4 pmol of UCB. mg-1 of protein. 15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K'f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with K(m) values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K'f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

The effects of cyanobacteria and the cyanobacterial toxin microcystin-LR on Ca2+ transport and Na+/K+-ATPase in tilapia gills

The effects of cytotoxic substances from cyanobacteria on ionic transport processes in tilapia (Oreochromis mossambicus) were examined. Inhibitory effects on ionic transport including whole-body Ca2+ fluxes and P-type ATPases of the gill were found. The compounds tested were (1) purified microcystin-LR (MC-LR), a heptapeptide hepatotoxin produced by the cyanobacterium Microcystis aeruginosa, (2) extracts from M. aeruginosa strain PCC 7820, a strain producing MC-LR and other microcystin variants, and (3) extracts of M. aeruginosa CYA 43, a strain producing toxins including small quantities of MC-LR. Whole-body Ca2+ influx was inhibited by a 24 h exposure to extracts of M. aeruginosa CYA 43 and 7820, but not by exposure to an equivalent amount (90 mg l-1) of purified MC-LR. Shorter exposure times (4 h) were ineffective. Fish exposed to extracts from M. aeruginosa CYA 43 showed significant plasma hypocalcaemia. Both strains of M. aeruginosa inhibited Ca2+ uptake by basolateral plasma membrane vesicles (BLMVs), endoplasmic reticulum (ER) and mitochondria, as well as BLMV K+-dependent p-nitrophenol phosphatase (pNPPase) activity. The hydrophobic fractions of the cyanobacterial extracts were the most potent, inhibiting BLMV, ER and mitochondrial Ca2+ uptake by up to 99 %, but they were less inhibitory of BLMV K+-dependent pNPPase activity. Purified MC-LR was without effect on these preparations. In conclusion, cytotoxic substances from cyanobacteria have the potential to disrupt normal physiological processes dependent upon Ca2+ transport processes in tilapia gills.

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.

Reconstitution of a passive Ca(2+)-transport pathway from the basolateral plasma membrane of rat parotid gland acinar cells.

We have previously reported that rat parotid gland basolateral plasma membrane vesicles (BLMV) have a relatively high affinity Ca2+ transport pathway and an unsaturable Ca2+ flux component (Lockwich et al., 1994. J. Membrane Biol. 141:289-296). In this study, we have solubilized BLMV with octylglucoside (1.5%) and have reconstituted the solubilized proteins into proteoliposomes (PrL) composed of E. coli bulk phospholipids, by using a detergent dilution method. PrL exhibited 3-5-fold higher 45Ca2+ influx than control liposomes (without protein). Ca2+ uptake into PrL was dependent on the [protein] in PrL and steady state [Ca2+] in PrL was in equilibrium with external [Ca2+]. These data demonstrate that a passive, protein-mediated Ca2+ transport has been reconstituted from BLMV into PrL. 45Ca2+ influx into liposomes did not saturate with increasing [Ca2+] in the assay medium. In contrast, PrL displayed saturable 45Ca2+ influx and exhibited a single Ca2+ flux component with an apparent KCa = 242 +/- 50.9 microM and Vmax = 13.5 +/- 1.14 nmoles Ca2+/mg protein/ minute. The KCa of Ca(2+)-transport in PrL was similar to that of the high affinity Ca2+ influx component in BLMV while the Vmax was about 4-fold higher. The unsaturable Ca2+ flux component was not detected in PrL. 45Ca2+ influx in PrL was inhibited by divalent cations in the order of efficacy, Zn2+ > Mn2+ > Co2+ = Ni2+, and appeared to be more sensitive to lower concentrations of Zn2+ than in BLMV. Consistent with our observations with BLMV, the carboxyl group reagent N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the reconstituted Ca2+ transport in PrL. Importantly, in both BLMV and PrL, DCCD induced a 40-50% decrease in Vmax of Ca2+ transport without an alteration in KCa. These data strongly suggest that the high affinity, passive Ca2+ transport pathway present in BLMV has been functionally reconstituted into PrL. We suggest that this approach provides a useful experimental system towards isolation of the protein(s) involved in mediating Ca2+ influx in the rat parotid gland basolateral plasma membrane.

In vitro modeling of liver membrane copper transport

The process of hepatobiliary copper (Cu) secretion is still poorly understood Cu secretion as a complex with glutathione and transport via a lysosomal pathway have been proposed. The recent cloning and sequencing of the gene for Wilson disease indicates that Cu transport in liver cells may be mediated by a Cu transporting Ptype ATPase. Biochemical evidence for ATP-dependent Cu transport in mammalian systems, however, has not been reported so far. We have investigated Cu transport in rat liver plasma membrane vesicles enriched in canalicular or basolateral membranes in the presence and absence of ATP (4 mM) and an ATP-regenerating system. The presence of ATP clearly stimulated uptake of radiolabeled Cu (64Cu, 10 μM) into canalicular plasma membrane vesicles and, to a lesser extent, also into basolateral plasma membrane vesicles. ATP-dependent Cu transport was dose-dependently inhibited by the P-type ATPase inhibitor vanadate, and showed saturation kinetics with an estimated K m of 8.6 μM and a V max of 6.9 nmol/min/mg protein. ATP-stimulated Cu uptake was similar in canalicular membrane vesicles of normal Wistar rats and those of mutant GY rats, expressing a congenital defect in the activity of the ATP-dependent canalicular glutathione-conjugate transporter (cMOAT). These studies demonstrate the presence of an ATP-dependent Cu transporting system in isolated plasma membrane fractions of rat liver distinct from cMOAT. (J. Clin. Invest. 1995.95:412-416.) Key words: copper · bile secretion · P-type ATPase · vesicle transport · Wilson disease. (Reproduced from The Journal of Clinical Investigation, 1995, vol. 95, pp. 412-416 by copyright permission of The Society for Clinical Investigation.).

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)

Identification and characterization of a basolateral dicarboxylate/cholate antiport system in rat hepatocytes.

The mechanisms and driving forces for the uptake of the unconjugated bile acid cholate were investigated both in cultured rat hepatocytes and in rat liver basolateral (sinusoidal) plasma membrane (BLPM) vesicles. Determination of initial uptake rates of [3H]cholate (0.1 microM) into cultured hepatocytes confirmed that the majority (75%) of the transmembrane transport was mediated by Na(+)-independent mechanisms. This portion of cholate uptake consisted of a pH-sensitive moiety representing nonionic diffusion, which may become quantitatively important at low pH and high cholate concentrations, as well as of a saturable (Michaelis constant 7.4 microM), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive transport moiety, suggesting the involvement of a carrier. This latter transport system was functionally characterized by 1) inhibition of cellular cholate uptake in the absence of extracellular sodium by the dicarboxylic acid alpha-ketoglutarate (alpha-KG; 1 mM) and by the organic anion p-aminohippurate (PAH; 1 mM); 2) stimulation of cellular cholate uptake by alpha-KG (10 microM) or PAH (1 mM) in the presence of an inwardly directed sodium gradient; 3) lack of sensitivity toward lithium in BLPM vesicles; 4) trans-stimulation of vesicular cholate uptake by alpha-KG or PAH, but not by benzoate; and 5) cis-inhibition of alpha-KG/alpha-KG self-exchange by extravesicular cholate (400 microM), PAH (5 mM), probenecid, or DIDS. Collectively, these data indicate the presence of a Na(+)-dicarboxylate cotransport-coupled organic anion exchanger in the hepatocyte basolateral plasma membrane that may be involved in cholate uptake in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine triphosphate-dependent copper transport in isolated rat liver plasma membranes.

The process of hepatobiliary copper (Cu) secretion is still poorly understood: Cu secretion as a complex with glutathione and transport via a lysosomal pathway have been proposed. The recent cloning and sequencing of the gene for Wilson disease indicates that Cu transport in liver cells may be mediated by a Cu transporting P-type ATPase. Biochemical evidence for ATP-dependent Cu transport in mammalian systems, however, has not been reported so far. We have investigated Cu transport in rat liver plasma membrane vesicles enriched in canalicular or basolateral membranes in the presence and absence of ATP (4 mM) and an ATP-regenerating system. The presence of ATP clearly stimulated uptake of radiolabeled Cu (64Cu, 10 microM) into canalicular plasma membrane vesicles and, to a lesser extent, also into basolateral plasma membrane vesicles. ATP-dependent Cu transport was dose-dependently inhibited by the P-type ATPase inhibitor vanadate, and showed saturation kinetics with an estimated Km of 8.6 microM and a Vmax of 6.9 nmol/min/mg protein. ATP-stimulated Cu uptake was similar in canalicular membrane vesicles of normal Wistar rats and those of mutant GY rats, expressing a congenital defect in the activity of the ATP-dependent canalicular glutathione-conjugate transporter (cMOAT). These studies demonstrate the presence of an ATP-dependent Cu transporting system in isolated plasma membrane fractions of rat liver distinct from cMOAT.

Prolactin increases mRNA encoding Na(+)-TC cotransport polypeptide and hepatic Na(+)-TC cotransport.

We have shown that prolactin (Prl) increases the transhepatic transport of taurocholate (TC) in postpartum rats and following treatment of ovariectomized (Ovx) rats with ovine Prl (oPrl). The present studies were designed to determine if treatment of Ovx rats with oPrl (100, 300, or 600 micrograms/day, 7 days iv) 1) increases Na(+)-TC cotransport in basolateral plasma membrane vesicles (bLPM), 2) induces a corresponding increase in the steady-state levels of Na(+)-TC cotransport polypeptide (Ntcp mRNA), and 3) if the oPrl-mediated increase in Na(+)-TC cotransport activity is blocked by cycloheximide, an inhibitor of protein synthesis. oPrl (300 micrograms/day) induced a twofold increase in the maximal velocity for Na(+)-TC cotransport in both hepatocytes and bLPM vesicles with little change in the Michaelis constant. Infusion of oPrl at a dose of 100, 300, or 600 micrograms/day increased steady-state Ntcp mRNA four-, ten-, and twofold, respectively. Finally, cycloheximide blocked the oPrl-mediated increase in Na(+)-TC cotransport but did not affect basal activity. These data support the hypothesis that an increase in Ntcp mRNA followed by increased synthesis and incorporation of Ntcp in the plasma membrane is responsible for the oPrl-mediated increase in Na(+)-TC cotransport in the basolateral plasma membrane domain of the hepatocyte.

ATP-dependent transport of the linear renin-inhibiting peptide EMD 51921 by canalicular plasma membrane vesicles of rat liver: evidence of drug-stimulatable ATP-hydrolysis

Certain peptide drugs, such as the linear hydrophobic renin-inhibitor EMD 51921, are rapidly eliminated via the bile. At the sinosoidal membrane of liver cells EMD 51921 is taken up via a sodium-independent carrier-mediated mechanism, competing for the uptake of bile acids. Until now, the mechanisms of biliary excretion of EMD 51921 were unknown. In this study we describe an ATP-dependent transport system for the enzymatically and metabolically stable hydrophobic linear renin-inhibiting peptide EMD 51921. The ATP-dependent uptake into the osmotic reactive intravesicular space is saturable (Km 12 μM, Vmax 663 pmol/min per mg protein), temperature dependent and specifically requires ATP. Transport is inhibited by vanadate but not by ouabain, EGTA or NaN3, and does not function in basolateral plasma membrane vesicles. Transport is not altered in canalicular membrane vesicles isolated from Tr− rats lacking the canalicular ATP-dependent transport of cysteinyl leukotrienes and related anions. Transport is inhibited by taurocholate, a typical substrate of the canalicular ATP-dependent bile acid transporter, but also by vincristine and daunomycin, substrates of P-glycoproteins. EMD 51921, however, only inhibits the uptake of taurocholate, whereas the transport of daunomycin is not influenced. Taurocholate and EMD 51921 are mutually non- or un-competitive transport inhibitors. Incubation of rat liver canalicular membranes with micromolar concentrations of EMD 51921 resulted in a 1.8–2.5-fold increase in the rate of ATP-hydrolysis. In contrast, ATP-hydrolysis was not affected by fragments of the peptide that are not transported in an ATP-dependent manner. The apparent Km value (EMD) for ATP-hydrolysis is 68 μM. Vmax is 0.032 U/mg protein. ATPase activity is pH dependent. Stimulation of ATP-hydrolysis is inhibited by vanadate, NEM, hydroxymercuribenzoate and ascorbate, but is not affected by ouabain, EGTA or NaN3. EMD 51921 does not stimulate the ATPase activity of the Na+/K+-ATPase isolated from kidney medulla. The EMD-stimulatable ATPase seems to be distinct from the glutathione-S-conjugate stimulatable ATPase and the mdr 1a/b gene products and differs in its characteristics from that of the canalicular ecto-ATPase.

Competition of bile acids on the sulfobromophthalein uptake in basolateral rat liver plasma membrane vesicles

The effect of different bile acids (BA) on the hepatic uptake of sulfobromophthalein (BSP) was investigated in liver plasma membrane vesicles enriched in basolateral fraction. BSP uptake was measured either in the absence (electroneutral component) or in the presence of a membrane potential (electrogenic component) induced by the addition of valinomycin in the presence of an inwardlydirected potassium gradient. BSP uptake was also measured in the presence of different BA [cholate (C), taurocholate (TC), ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC)]. Electrogenic BSP uptake was not affected by BA. Conversely, the electroneutral portion of the BSP uptake was inhibited with an inhibition constant ( K i , μM) of 230 ± 40 for C, 103 ± 33 for TC, 99 ± 34 for UDC and 120 ± 39 for TUDC, respectively (means ± SD, N = 4). The Dixon and Cornish-Bowden plot of the data revealed an uncompetitive type of inhibition for each BA. These data indicate that the electroneutral, but not the electrogenic, BSP transport system is modulated by BA.