Rate Of Taurocholate Uptake Research Articles

Xenobiotics Inhibit Hepatic Uptake and Biliary Excretion of Taurocholate in Rat Hepatocytes

Reports suggest that troglitazone, and to a lesser extent bosentan, may alter bile acid homeostasis by inhibiting the bile salt export pump. The present studies examined the hypothesis that these xenobiotics may modulate multiple hepatic bile acid transport mechanisms. In suspended rat hepatocytes, troglitazone (10 microM) decreased the initial rate of taurocholate uptake approximately 3-fold; the initial uptake rate of estradiol-17beta-D-glucuronide, a substrate of the organic anion transporting polypeptides, also was decreased approximately 4-fold. Bosentan (100 microM) decreased the initial uptake rate of taurocholate and estradiol-17beta-D-glucuronide by approximately 12- and approximately 7-fold, respectively. In sandwich-cultured rat hepatocytes, 10-min accumulation of taurocholate in cells + bile canaliculi (408 +/- 57 pmol/mg protein) was decreased significantly by troglitazone (157 +/- 17 pmol/mg protein, respectively) only in the presence of Na+, the driving force for the sodium taurocholate cotransporting polypeptide. A similar decrease with 10-fold higher concentrations of bosentan was noted. The biliary excretion index of taurocholate (55 +/- 8%) was decreased in the presence of 10 microM troglitazone (27 +/- 2%) and 100 microM bosentan (10 +/- 6%). In conclusion, xenobiotics may alter hepatic bile acid transport by inhibiting both hepatic uptake and biliary excretion.

Cross-talk between Protein Kinases Cζ and B in Cyclic AMP-mediated Sodium Taurocholate Co-transporting Polypeptide Translocation in Hepatocytes

Cyclic AMP stimulates taurocholate (TC) uptake and sodium taurocholate co-transporting polypeptide (Ntcp) translocation in hepatocytes via the phosphoinositide-3 kinase (PI3K) signaling pathway. The aim of the present study was to determine whether protein kinase (PK) Czeta, one of the downstream mediators of the PI3K signaling pathway, is involved in cAMP-mediated stimulation of TC uptake. Studies were conducted in isolated rat hepatocytes and in HuH-7 cells stably transfected with rat liver Ntcp (HuH-Ntcp cells). Studies in hepatocytes showed that cAMP activates PKCzeta in a PI3K-dependent manner without inducing translocation of PKCzeta to the plasma membrane. Inhibition of cAMP-induced PKCzeta activity by myristoylated PKC (zeta/lambda) pseudosubstrate, a specific inhibitor of PKCzeta, and Gö 6850, a PKC inhibitor, resulted in inhibition of cAMP-induced increases in TC uptake and Ntcp translocation. Studies in HuH-Ntcp cells showed that inhibition of cAMP-induced PKCzeta activation by dominant-negative (DN) PKCzeta resulted in inhibition of cAMP-induced increases in TC uptake and Ntcp translocation. DN PKCzeta also inhibited wild-type PKCzeta-induced increases in PKCzeta activity, TC uptake, and Ntcp translocation. Myristoylated PKC (zeta/lambda) pseudosubstrate and DN PKCzeta also inhibited cAMP-induced activation of PKB in hepatocytes and HuH-Ntcp cells, respectively. Neither DN PKB nor constitutively active PKB affected cAMP-induced activation of PKCzeta, and wild-type PKCzeta did not activate PKB. Taken together, these results suggest that cAMP-induced activation of PKB is dependent on cAMP-induced stimulation of PKCzeta. It is proposed that cAMP-induced Ntcp translocation involves the activation of the PI3K/PKCzeta signaling pathway followed by the activation of the PI3K/PKB signaling pathway.

Ontogenetic development of rat intestinal bile acid transport requires thyroxine but not corticosterone.

Absorption of bile acids by the distal ileum is an essential component of the enterohepatic circulation. In neonatal rats, the appearance of the apical sodium-dependent bile acid transporter (ASBT) at 17 d of age coincides with increases in serum corticosterone and thyroxine. We tested the hypothesis that these hormones modulate ASBT expression during ileal development. Taurocholate uptake into the isolated ileum of normal 20-d-old pups exhibited saturable (K(m) = 0.52 mM, J(max) = 0.34 pmol mg/min) and nonsaturable (K(diff) = 0.015 min(-1)) components and was two to five times greater than uptake in the proximal intestine. Hypothyroid or euthyroid pups received daily thyroxine injections starting at 6 d of age. At 12 d of age, serum concentrations of thyroxine, ileal abundance of ASBT mRNA, and ileal rates of taurocholate uptake were low in hypothyroid pups that received an injection of vehicle (HT-) or thyroxine (HT+) and in euthyroid pups that received an injection of vehicle (ET-) or thyroxine (ET+). At 20 and 26 d, ileal ASBT mRNA abundance and taurocholate uptake rate remained low in HT- pups but increased dramatically in ET- and ET+ pups, paralleling the increase in serum thyroxine. Restoration of normal plasma thyroxine in HT- pups by thyroxine injections (HT+) restored normal ASBT development. Sodium-glucose co-transporter activity and mRNA expression were independent of serum thyroxine levels. Corticosterone levels were significantly lower in pups that were adrenalectomized at 10 d of age. ASBT mRNA abundance and taurocholate uptake rate increased markedly with age but were the same in adrenalectomized, sham-operated, and nonoperated pups. Hence, endogenous thyroxine but not corticosterone regulates the developmentally timed appearance of ASBT.

Partial maintenance of taurocholate uptake by adult rat hepatocytes cultured in a collagen sandwich configuration.

This study was designed to characterize taurocholate uptake properties in primary cultures of rat hepatocytes maintained under different matrix conditions. Hepatocytes isolated from male Wistar rats (230-280 g) were cultured on a simple collagen film, on a substratum of gelled collagen or between two layers of gelled collagen (sandwich configuration). Hepatocyte morphology, taurocholate uptake properties, and expression of the sinusoidal transport protein. Na+/taurocholate-cotransporting polypeptide (Ntcp) were examined in these cultures at day 0 and day 5. By day 5, monolayer integrity had deteriorated in simple collagen cultures. In contrast, cell morphology was preserved in hepatocytes maintained in a sandwich configuration. At day 5, taurocholate accumulation at 5 min in hepatocytes cultured on a simple collagen film, on a substratum of gelled collagen, and in a sandwich configuration was approximately 13%, 20% and 35% of day-0 levels, respectively, and occurred predominately by a Na+-dependent mechanism. The initial taurocholate uptake rate vs. concentration (1-200 microM) profile was best described by a combined Michaelis-Menten and first-order function. In all cases, the estimated apparent Km values were comparable for day-0 and day-5 hepatocytes (3241 microM). In contrast, the Vmax values of hepatocytes cultured on a simple collagen film, on gelled collagen and in a sandwich configuration were approximately 5, 6 and 14% of the values at day 0, respectively; values for the first-order rate constant were 5-, 3- and 2-fold lower, respectively. Immunoblot analysis indicated that at day 5 Ntcp expression in hepatocytes cultured in a sandwich configuration was greater than in hepatocytes cultured on a simple collagen film. A collagen sandwich configuration reestablishes normal morphology and partially restores bile acid uptake properties in primary cultures of rat hepatocytes.

Vasopressin and phorbol-12,13-dibutyrate inhibit glucagon- or cyclic AMP-stimulated taurocholate uptake in isolated rat hepatocytes

Bile salt uptake by hepatocytes is modulated in part by changes in intracellular cyclic AMP. We studied the effect of activation of protein kinase C on cyclic AMP-mediated taurocholate uptake in isolated rat hepatocytes. Both dibutyryl cyclic AMP (2 x 10(-6) mol/L) and glucagon (10(-6) mol/L), which increase intracellular cyclic AMP, enhanced the initial uptake rate of taurocholate into hepatocytes, with maximal increases of 45% to 50% over the basal uptake rate. Vasopressin (10(-9) mol/L), a hormone known to activate protein kinase C, and phorbol-12,13-dibutyrate (10(-5) mol/L) significantly inhibited the glucagon-stimulated increase in taurocholate uptake rate (72% +/- 10% and 105% +/- 13% inhibition, respectively). Basal (unstimulated) taurocholate uptake rate was not affected by vasopressin or phorbol-12,13-dibutyrate. Down-regulation of the glucagon-stimulated transport was rapid and persisted during the 20-min experimental period. Angiotensin II had a similar but more transient inhibitory effect. Vasopressin and phorbol-12,13-dibutyrate suppression of glucagon-stimulated taurocholate uptake rate was not accompanied by diminished cyclic AMP levels. Moreover, vasopressin and phorbol-12,13-dibutyrate inhibited dibutyryl cyclic AMP-stimulated taurocholate uptake rate can be dissociated from alterations in the cyclic AMP levels.

Vasopressin and Phorbol–12, 13–Dibutyrate Inhibit Glucagon– or Cyclic Amp–Stimulated Taurocholate Uptake in Isolated Rat Hepatocytes

ABSTRACT Bile salt uptake by hepatocytes is modulated in part by changes in intracellular cyclic AMP. We studied the effect of activation of protein kinase C on cyclic AMP–mediated taurocholate uptake in isolated rat hepatocytes. Both dibutyryl cyclic AMP (2 × 10-6 mol/L) and glucagon (10-6 mol/L), which increase intracellular cyclic AMP, enhanced the initial uptake rate of taurocholate into hepatocytes, with maximal increases of 45% to 50% over the basal uptake rate. Vasopressin (10-9 mol/L), a hormone known to activate protein kinase C, and phorbol–12, 13–dibutyrate (10-5 mol/L) significantly inhibited the glucagon–stimulated increase in taurocholate uptake rate (72% ± 10% and 105% ± 13% inhibition, respectively). Basal (unstimulated) taurocholate uptake rate was not affected by vasopressin or phorbol–12, 13–dibutyrate. Down–regulation of the glucagon–stimulated transport was rapid and persisted during the 20–min experimental period. Angiotensin II had a similar but more transient inhibitory effect. Vasopressin and phorbol–12, 13–dibutyrate suppression of glucagon–stimulated taurocholate uptake rate was not accompanied by diminished cyclic AMP levels. Moreover, vasopressin and phorbol–12, 13–dibutyrate inhibited dibutyryl cyclic AMP–stimulated taurocholate uptake rate can be dissociated from alterations in the cyclic AMP levels. (Hepatology 1994;20:159-165.)

Bile acid transport by basal membrane vesicles of human term placental trophoblast

The aim of this work was to investigate the first step in the vectorial translocation of bile acids from the fetus to the mother, which is the transfer across the basal (i.e., fetal-facing) plasma membrane of the trophoblast. Thus, the uptake of [14C]taurocholate by basal plasma membrane vesicles obtained from normal human term placentas was studied. Taurocholate retention into vesicles was studied using a rapid filtration technique that was modified to reduce the taurocholate binding to the filters and to the external surface of the vesicles. Using 100 μmol/L substrate, the membrane vesicles showed a temperature-dependent, Na+-independent transport of taurocholate into an osmotically reactive intravesicular space. The initial rate of taurocholate influx in the presence of 100 mmol/L KNO3 followed saturation kinetics (apparent Km for taurocholate = 670 ± 128 μmol/L; Vmax = 1.86 ± 0.28 nmol/mg protein · 60 s at 37 °C). Over the 6.9–7.9 pH range neither internal nor external pH nor inward nor outward proton gradients affected the uptake of taurocholate. When the electrical potential difference across the basal membrane was manipulated by external anion replacement (Cl−, SCN−, SO42−, or NO3−) or by valinomycin-induced K+-diffusion potential (vesicle inside negative), taurocholate uptake was not significantly modified. Taurocholate uptake was cis-inhibited in the presence of 1 mmol/L glycocholate, 0.5 mmol/L 4,4′-diisothiocyanostilbene-2,2′-disulfonate and 0.5 mmol/L sulfobromophthalein. However, 1 mmol/L probenecid or 0.5 mmol/L p-aminohippurate had no effect. Moreover, preloading the vesicles with 100 mmol/L HCO3− (but not with 100 mmol/L Cl− or 50 mmol/L SO42−) induced a significant enhancement in the initial rate of taurocholate uptake. In summary, these findings provide strong evidence for the presence of an electroneutral transport system for taurocholate in the basal plasma membrane of human chorionic trophoblast. They also suggest that this is likely to be an anion-exchange system.

Lack of linear correlation between hepatic ligand uptake rate and unbound ligand concentration does not necessarily imply receptor-mediated uptake

Previous studies of the hepatic uptake of several albumin-bound ligands, using constant and variable albumin concentrations, were interpreted as being inconsistent with the traditional mechanism of uptake, defined as an uptake rate directly proportional to unbound ligand concentration, and led to the formulation of the albumin receptor theory of hepatic uptake. Because other experimental designs have failed to confirm the albumin receptor theory, we reexamined, using the isolated perfused rat liver preparation, the traditional uptake mechanism under the conditions used in the original studies, of constant and variable albumin concentration. Livers (n = 6) were perfused in randomized sequence with 10 different solutions containing 24-14C-taurocholate in a single-pass design. Five solutions contained fixed albumin (0.1 mM) and variable taurocholate (3-48 microM) concentrations, and five maintained the taurocholate-albumin ratio fixed at 0.06; absolute concentrations of taurocholate varied from 3-48 microM, and of albumin from 0.05-0.08 mM. At constant albumin concentration in hepatic inflow, elimination rate of taurocholate was linearly related to both total (Cin) and unbound (Cin,u) taurocholate concentration in hepatic inflow, indicating first-order elimination kinetics. When taurocholate and albumin were increased in hepatic inflow in a fixed molar ratio, taurocholate uptake rate was not linearly related to Cin,u but was still consistent with the traditional uptake mechanism. Moreover, the apparent saturation of taurocholate uptake by added albumin was consistent with the reduction in unbound fraction (fu) in accordance with the traditional uptake mechanism. This study shows that although the traditional uptake mechanism dictates that ligand uptake rate is linearly related to unbound ligand concentration within the liver, uptake rate need not necessarily be linearly related to Cin,u. Therefore, experiments in which lack of a linear relationship between uptake rate and Cin,u is found do not necessarily imply receptor-mediated uptake.

Taurocholate uptake by isolated skate hepatocytes: effect of albumin.

Taurocholate transport was analyzed in a well characterized, polarized hepatocyte preparation from the small skate (Raja erinacea), an analbuminemic species that does not synthesize bile acids. In addition, the effect of sodium omission, bovine albumin, ovalbumin, and bovine gamma-globulin on the uptake of taurocholate was studied. Uptake could be divided into nonsaturable (0.48 pmol X min-1 X mg protein-1. microM-1) and saturable components (Km, 32.5 microM and Vmax 110 pmol X min-1 X mg protein-1). No evidence for sodium dependence could be obtained. Glycocholate competitively inhibited taurocholate uptake. The initial rate of taurocholate uptake was also inhibited by sulfobromophthalein, N-(4-azido-2-nitrophenyl)-2-aminoethyl sulfonate, and 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid but not lactate or alanine. Hepatic uptake of taurocholate in albumin solutions (2.5%) was twice as great as expected from estimates of the free taurocholate concentration. In contrast, equimolar concentrations of ovalbumin or bovine gamma-globulin had no effect on measured rates of taurocholate uptake and no evidence for a specific albumin receptor could be found on these hepatocytes by 125I-albumin binding. These studies indicate that a carrier-mediated, sodium-independent transport system for taurocholate uptake is present in skate hepatocytes that is not driven solely by the free concentration of taurocholate.

Initial rate of sodium taurocholate uptake in isolated elutriated hepatocytes from untreated and phenobarbital-treated rats

The initial rate of sodium taurocholate uptake was measured in rat hepatocytes separated by centrifugal elutriation into five cell fractions whose difference in size was verified by flow cytometry. The hepatocytes were prepared from untreated and phenobarbital-treated rats. For untreated animals. the initial rate of taurocholate uptake at concentrations of 5 or 50 μM was the same for hepatocytes prior to fractionation and for each of the five elutriated fractions. Treatment of the animals with phenobarbital was associated with a significant increase in hepatocyte size in all fractions and caused a significant increase in the initial uptake rate. The extent of the rate increase in hepatocytes prior to fractionation was similar to that observed for each of the five hepatocyte subpopulations. Our observation indicates that phenobarbital causes a significant increase in the initial rate of sodium taurocholate uptake and suggests that large and small hepatocytes possess no inherent differences controlling the initial uptake process.

Taurocholate transport by rat liver canalicular membrane vesicles. Evidence for the presence of an Na+-independent transport system.

To elucidate the mechanism of vectorial translocation of bile acids in the liver, taurocholate transport was studied in isolated liver canalicular membrane vesicles by a rapid filtration method. The membrane vesicles revealed temperature-dependent, Na+-independent transport of taurocholate into an osmotically reactive intravesicular space. In the absence of sodium, taurocholate uptake followed saturation kinetics (apparent Km for taurocholate = 43 microM and Vmax = 0.22 nmol/mg protein X 20 s at 37 degrees C) and was inhibited by cholate and probenecid. Transstimulation by unlabeled taurocholate was also demonstrated. When the electrical potential difference across the membranes was altered by anion replacement, a more positive intravesicular potential stimulated, and a more negative potential inhibited, transport of taurocholate by the vesicles. Valinomycin-induced K+-diffusion potential (vesicle inside-positive) enhanced the rate of taurocholate uptake that was not altered by imposed pH gradients. These results indicate that rat liver canalicular plasma membrane contains a sodium-independent taurocholate transport system that translocates the bile acid as an anion across the membrane. In intact hepatocytes, the electrical potential difference across the canalicular membrane probably provides the driving force for taurocholate secretion. The contribution of nonionic diffusion to taurocholate secretion appears to be minimal.

Taurocholate transport by rat liver sinusoidal membrane vesicles: evidence of sodium cotransport.

To elucidate the first step in the vectorial transport of bile acids by the liver, plasma membrane vesicles were isolated from rat liver by differential and sucrose–Ficoll density gradient centrifugation. The membranes were selectively enriched 20–fold in Na+,K+-ATPase activity, a marker of sinusoidal plasma membranes. Electron microscopy of pellets from sinusoidal membrane fraction did not reveal other organelles. The initial rate of taurocholate uptake by the membrane vesicles was stimulated by a sodium gradient but not by a potassium gradient (Naoutside> Nainside). Sodium–dependent uptake of taurocholate was inhibited at low temperature (0oC), reduced by decreasing intravesicular space, and showed transstimulation in tracer exchange experiments indicating that uptake into vesicles, rather than binding to membranes, was occurring. Sodium–dependent taurocholate transport by the vesicles exhibited saturation kinetics with respect to taurocholate (apparent Km = 56 μM, Vmax = 0.65 nmoles per mg × 15 sec at 100 mM NaNO3 and 25oC) and sodium (apparent Km = 48 mM at 50 μM taurocholate). Other cations, such as lithium and choline, did not replace sodium in its stimulatory action. Sodium–dependent taurocholate uptake was selectively inhibited by cholic acid and probenecid. When the electrical potential difference across the vesicle membrane was altered by anion replacement, a more negative intravesicular potential inside the vesicles stimulated and a more positive potential inhibited sodium–dependent taurocholate transport. These data indicate the presence of a taurocholate–sodium cotransport system in the sinusoidal membranes of rat hepatocytes. This sodium–cotransport system probably participates in sodium–dependent uptake of bile acids into the hepatocyte. Bile acid secretion in the liver can be classified as secondary active transport.

Cholestatic steroid hormones inhibit taurocholate uptake into isolated rat hepatocytes

The effect of three cholestatic steroids (norethandrolone, 17-β-estradiol and progesterone) on hepatocellular uptake and secretion of taurocholate was studied in isolated rat liver cells. The steroids decreased the rate of taurocholate uptake. Norethandrolone inhibited uptake noncompetitively with a K i of 18 μM, but had no effect on the activation energy of uptake. 17-β-estradiol and progesterone reduced taurocholate uptake by 50 per cent at concentrations between 40 μM and 50 μM. The secretion of taurocholate from taurocholate-loaded cells was slightly increased by all three steroids at concentrations below 100 μM. A 60 per cent inhibition of secretion was observed in the presence of 500μM norethandrolone. Interference of cholestatic steroids with hepatocellular bile acid uptake may be an important step in the pathogenesis of intrahepatic cholestasis.