scholarly journals ATP-dependent transport of unconjugated bilirubin by rat liver canalicular plasma membrane vesicles

1998 ◽  
Vol 331 (1) ◽  
pp. 99-103 ◽  
Author(s):  
Lorella PASCOLO ◽  
Enrique J. BAYON ◽  
Felicia CUPELLI ◽  
J. Donald OSTROW ◽  
Claudio TIRIBELLI
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Atp Hydrolysis ◽  
Organic Anion ◽  
Membrane Vesicles ◽  
Organic Anion Transporter ◽  
Unconjugated Bilirubin ◽  
Plasma Membrane Vesicles ◽  
Anion Transporter ◽  
Dependent Transport

The transport of highly purified 3H-labelled unconjugated bilirubin (UCB) was investigated in rat liver plasma membrane vesicles enriched in the canalicular domain and found to be stimulated (more than 5-fold) by the addition of ATP. Other nucleotides, such as AMP, ADP, GTP and a non-hydrolysable ATP analogue (adenosine 5´-[α,β-methylene] triphosphate), did not stimulate [3H]UCB transport, indicating that ATP hydrolysis was necessary for the stimulatory effect. [3H]UCB uptake occurred into an osmotically sensitive space. At an unbound bilirubin concentration ([Bf]) below saturation of the aqueous phase (no more than 70 nM UCB), the ATP-dependent transport followed saturation kinetics with respect to [Bf], with a Km of 26±8 nM and a Vmax of 117±11 pmol per 15 s per mg of protein. Unlabelled UCB inhibited the uptake of [3H]UCB, indicating that UCB was the transported species. Inhibitors of ATPase activity such as vanadate or diethyl pyrocarbonate decreased the ATP effect (59±11% and 100% respectively). Daunomycin, a known substrate for multidrug resistance protein-1, and taurocholate did not inhibit the ATP-dependent [3H]UCB transport, suggesting that neither mdr-1 nor the canalicular bile acid transporter is involved in the canalicular transport of UCB. [3H]UCB uptake (both with and without ATP) in canalicular vesicles obtained from TR- rats was comparable to that in vesicles obtained from Wistar rats, indicating that the canalicular multispecific organic anion transporter, cMOAT, does not account for UCB transport. These results indicate that UCB is transported across the canalicular membrane of the liver cell by an ATP-dependent mechanism involving an as yet unidentified transporter.

scholarly journals ATP-dependent transport of glutathione conjugate of 7β,8α-dihydroxy-9α,10α-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene in murine hepatic canalicular plasma membrane vesicles

1998 ◽  
Vol 332 (3) ◽  
pp. 799-805 ◽  
Author(s):  
Sanjay K. SRIVASTAVA ◽  
Xun HU ◽  
Hong XIA ◽  
Richard J. BLEICHER ◽  
Howard A. ZAREN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Atp Hydrolysis ◽  
Membrane Vesicles ◽  
Competitive Inhibitor ◽  
Dependent Manner ◽  
Plasma Membrane Vesicles ◽  
High Affinity ◽  
Dependent Transport ◽  
Detoxification Pathway ◽  
Stimulation Of

Glutathione (GSH) S-transferases (GSTs) have an important role in the detoxification of (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE], which is the ultimate carcinogen of benzo[a]pyrene. However, the fate and/or biological activity of the GSH conjugate of (+)-anti-BPDE [(-)-anti-BPD-SG] is not known. We now report that (-)-anti-BPD-SG is a competitive inhibitor (Ki 19 µM) of Pi-class isoenzyme mGSTP1-1, which among murine hepatic GSTs is most efficient in the GSH conjugation of (+)-anti-BPDE. Thus the inhibition of mGSTP1-1 activity by (-)-anti-BPD-SG might interfere with the GST-catalysed GSH conjugation of (+)-anti-BPDE unless one or more mechanisms exist for the removal of the conjugate. The results of the present study indicate that (-)-anti-BPD-SG is transported across canalicular liver plasma membrane (cLPM) in an ATP-dependent manner. The ATP-dependent transport of (-)-anti-[3H]BPD-SG followed Michaelis–Menten kinetics (Km 46 µM). The ATP dependence of the (-)-anti-BPD-SG transport was confirmed by measuring the stimulation of ATP hydrolysis (ATPase activity) by the conjugate in the presence of cLPM protein, which also followed Michaelis–Menten kinetics. In contrast, a kinetic analysis of ATP-dependent uptake of the model conjugate S-[3H](2,4-dinitrophenyl)-glutathione ([3H]DNP-SG) revealed the presence of a high-affinity and a low-affinity transport system in mouse cLPM, with apparent Km values of 18 and 500 µM respectively. The ATP-dependent transport of (-)-anti-BPD-SG was inhibited competitively by DNP-SG (Ki 1.65 µM). Likewise, (-)-anti-BPD-SG was found to be a potent competitive inhibitor of the high-affinity component of DNP-SG transport (Ki 6.3 µM). Our results suggest that GST-catalysed conjugation of (+)-anti-BPDE with GSH, coupled with ATP-dependent transport of the resultant conjugate across cLPM, might be the ultimate detoxification pathway for this carcinogen.

scholarly journals Characterization of a sodium-dependent transport system for butyrobetaine into rat liver plasma membrane vesicles

Hepatology ◽  
1998 ◽  
Vol 28 (2) ◽  
pp. 521-525 ◽  
Author(s):  
Simona Berardi ◽  
Bruno Stieger ◽  
Sandra Wächter ◽  
Brigitte O'Neill ◽  
Stephan Krähenbühl
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Transport System ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Liver Plasma Membrane ◽  
Sodium Dependent ◽  
Dependent Transport

scholarly journals The MRP4/ABCC4 Gene Encodes a Novel Apical Organic Anion Transporter in Human Kidney Proximal Tubules: Putative Efflux Pump for Urinary cAMP and cGMP

2002 ◽  
Vol 13 (3) ◽  
pp. 595-603 ◽  
Author(s):  
Rémon A. M. H. van Aubel ◽  
Pascal H. E. Smeets ◽  
Janny G. P. Peters ◽  
René J. M. Bindels ◽  
Frans G. M. Russel
Keyword(s):  
Efflux Pump ◽  
Organic Anion ◽  
Membrane Vesicles ◽  
Human Kidney ◽  
Proximal Tubules ◽  
Organic Anion Transporter ◽  
Anion Transporter ◽  
Dependent Transport ◽  
Camp And Cgmp ◽  
Kidney Proximal Tubules

ABSTRACT. The cyclic nucleotides cAMP and cGMP play key roles in cellular signaling and the extracellular regulation of fluid balance. In the kidney, cAMP is excreted across the apical proximal tubular membrane into urine, where it reduces phosphate reabsorption through a dipyridamole-sensitive mechanism that is not fully understood. It has long been known that this cAMP efflux pathway is dependent on ATP and is inhibited by probenecid. However, its identity and whether cGMP shares the same transporter have not been established. Here the expression, localization, and functional properties of human multidrug resistance protein 4 (MRP4) are reported. MRP4 is localized to the proximal tubule apical membrane of human kidney, and membrane vesicles from Sf9 cells expressing human MRP4 exhibit ATP-dependent transport of [3H]cAMP and [3H]cGMP. Both probenecid and dipyridamole are potent MRP4 inhibitors. ATP-dependent [3H]methotrexate and [3H]estradiol-17β-d-glucuronide transport by MRP4 and interactions with the anionic conjugates S-(2,4-dinitrophenyl)-glutathione, N-acetyl-(2,4-dinitrophenyl)-cysteine, α-naphthyl-β-d-glucuronide, and p-nitrophenyl-β-d-glucuronide are also demonstrated. In kidneys of rats deficient in the apical anionic conjugate efflux pump Mrp2, Mrp4 expression is maintained at the same level. It is concluded that MRP4 is a novel apical organic anion transporter and the putative efflux pump for cAMP and cGMP in human kidney proximal tubules.

scholarly journals ATP-dependent transport of bilirubin glucuronides by the multidrug resistance protein MRP1 and its hepatocyte canalicular isoform MRP2

1997 ◽  
Vol 327 (1) ◽  
pp. 305-310 ◽  
Author(s):  
Gabriele JEDLITSCHKY ◽  
Inka LEIER ◽  
Ulrike BUCHHOLZ ◽  
Johanna HUMMEL-EISENBEISS ◽  
Brian BURCHELL ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Membrane Vesicle ◽  
Organic Anion ◽  
Membrane Vesicles ◽  
Organic Anion Transporter ◽  
Multidrug Resistance Protein ◽  
Leukotriene C4 ◽  
Anion Transporter ◽  
Resistance Protein ◽  
Dependent Transport

Bilirubin is secreted from the liver into bile mainly as monoglucuronosyl and bisglucuronosyl conjugates. We demonstrate for the first time that ATP-dependent transport of both bilirubin glucuronides is mediated by the multidrug resistance protein (MRP1) as well as by the distinct canalicular (apical) isoform MRP2, also termed cMRP or cMOAT (canalicular multispecific organic anion transporter). In membrane vesicles from MRP1-transfected HeLa cells mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin (each at 0.5 μM) were transported with rates of 5.3 and 3.1 pmol/min per mg of protein respectively. Rat hepatocyte canalicular membrane vesicles, which contain Mrp2 (the rat equivalent of MRP2), transported mono[3H]glucuronosylbilirubin and bis[3H]glucuronosylbilirubin at rates of 8.9 and 8.5 pmol/min per mg of protein, whereas membrane vesicles from mutant liver lacking Mrp2 showed no transport of the conjugates. In membrane vesicles from human hepatoma Hep G2 cells, which predominantly expressed MRP2, transport rates were 8.3 and 4.4 pmol/min per mg of protein for monoglucuronosylbilirubin and bisglucuronosylbilirubin respectively. ATP-dependent transport of the glutathione S-conjugate [3H]leukotriene C4, an established high-affinity substrate for MRP1 and MRP2, was inhibited by both bilirubin glucuronides with IC50 values between 0.10 and 0.75 μM. The ratios of leukotriene C4 transport and bilirubin glucuronide transport, determined in the same membrane vesicle preparation, indicated substrate specificity differences between MRP1 and MRP2 with a preference of MRP2 for the glucuronides.

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

1996 ◽  
Vol 316 (3) ◽  
pp. 999-1004 ◽  
Author(s):  
Lorella PASCOLO ◽  
Savino DEL VECCHIO ◽  
Ronald K. KOEHLER ◽  
J. Enrique BAYON ◽  
Cecile C. WEBSTER ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Experimental Conditions ◽  
Saturation Kinetics ◽  
Basolateral Plasma Membrane ◽  
Component C ◽  
Uptake Studies

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 μM, K´f (litre/mol) was inversely related to [HSA], irrespective of the [Bt]/[HSA] ratio. K´f was 2.066×106+(3.258×108/[HSA]). When 50 mM KCl was iso-osmotically substituted for sucrose, the K´f value was significantly lower {2.077×106+(1.099×108/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] ⩽ 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with Km 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 Km 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.

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