Properties of the canalicular bile acid transport system in rat liver.
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ABSTRACT:
SUBMITTER: Meier PJ
PROVIDER: S-EPMC1147728 | biostudies-other | 1987 Mar
REPOSITORIES: biostudies-other
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Project description:The present study identifies and characterizes a novel ATP-dependent bile-salt transport system in isolated canalicular rat liver plasma-membrane (cLPM) vesicles. ATP (1-5 mM) stimulated taurocholate uptake into cLPM vesicles between 6- and 8-fold above equilibrium uptake values (overshoot) and above values for incubations in the absence of ATP. The ATP-dependent portion of taurocholate uptake was 2-fold higher in the presence of equilibrated KNO3 as compared with potassium gluconate, indicating that the stimulatory effect of ATP was not due to the generation of an intravesicular positive membrane potential. Saturation kinetics revealed a very high affinity (Km approximately 2.1 microM) of the system for taurocholate. The system could only minimally be stimulated by nucleotides other than ATP. Furthermore, it was preferentially inhibited by conjugated univalent bile salts. Further strong inhibitory effects were observed with valinomycin, oligomycin, 4,4'-di-isothiocyano-2,2'-stilbene disulphonate, sulphobromophthalein, leukotriene C4 and N-ethylmaleimide, whereas nigericin, vanadate, GSH, GSSG and daunomycin exerted only weak inhibitory effects or none at all. These results indicate the presence of a high-affinity primary ATP-dependent bile-salt transport system in cLPM vesicles. This transport system might be regulated in vivo by the number of carriers present at the perspective transport site(s), which, in addition to the canalicular membrane, might also include pericanalicular membrane vesicles.
| S-EPMC1132698 | biostudies-other
Project description:BackgroundObese Zucker rats (ZR) have been used as an experimental model for non-alcoholic fatty liver disease and are particularly susceptible to various types of liver injury. Bile secretory function has not been assessed in ZR.AimTo study bile secretion and expression of the main hepatobiliary transporters in ZR.MethodsBile flow and biliary secretion of lipids and glutathione were determined in eight and 14 week old obese ZR and their lean controls. Protein mass and mRNA of the Na(+)/taurocholate cotransporting polypeptide (Ntcp), the bile salt export pump (Bsep), and the multidrug resistant associated protein 2 (Mrp2) were assessed by western and northern blot, respectively. The effects of administration of a tumour necrosis factor alpha inactivator (etanercept) and an insulin sensitiser (rosiglitazone) were assessed in obese ZR while leptin was given to non-obese rats to study its effect on Mrp2 expression.ResultsZR exhibited increased body weight and hyperlipidaemia. Only 14 week old obese ZR has fatty liver. Decreased bile flow and biliary lipid and glutathione secretion as well as reduced hepatic transport of both taurocholate and bromosulphthalein were found in obese ZR. Hepatic Mrp2 protein mass was markedly reduced (-70%) in obese rats while Ntcp and Bsep protein levels were similar to lean rats. Downregulation of Mrp2 seems to involve both transcriptional and post-transcriptional mechanisms probably related to insulin and leptin resistance.ConclusionsObese ZR exhibit an impaired bile secretory function with significant functional and molecular alterations consistent with mild cholestasis. A defective hepatobiliary transport capacity may be a contributory factor in rendering the obese ZR more susceptible to liver injury.
| S-EPMC1774316 | biostudies-literature
Project description:Beta-catenin plays important roles in liver physiology and hepatocarcinogenesis. While studying the role of ?-catenin in diet-induced steatohepatitis, we recently found that liver-specific ?-catenin knockout (KO) mice exhibit intrahepatic cholestasis. This study was undertaken to further characterize the role of ?-catenin in biliary physiology. KO mice and wild-type (WT) littermates were fed standard chow or a diet supplemented with 0.5% cholic acid for 2 weeks. Chow-fed KO mice had higher serum and hepatic total bile acid levels and lower bile flow rate than WT mice. Expression levels of bile acid biosynthetic genes were lower and levels of major bile acid exporters were similar, which therefore could not explain the KO phenotype. Despite loss of the tight junction protein claudin-2, KO mice had preserved functional integrity of tight junctions. KO mice had bile canalicular morphologic abnormalities as evidenced by staining for F-actin and zona occludens 1. Electron microscopy revealed dilated and tortuous bile canaliculi in KO livers along with decreased canalicular and sinusoidal microvilli. KO mice on a cholic acid diet had higher hepatic and serum bile acid levels, bile ductular reaction, increased pericellular fibrosis, and dilated, misshapen bile canaliculi. Compensatory changes in expression levels of several bile acid transporters and regulatory genes were found in KO livers.Liver-specific loss of ?-catenin leads to defective bile canalicular morphology, bile secretory defect, and intrahepatic cholestasis. Thus, our results establish a critical role for ?-catenin in biliary physiology.
| S-EPMC2947599 | biostudies-literature
Project description:Transport processes in the canalicular membrane are key elements in bile formation and are the driving force of the enterohepatic circulation of bile salts. The canalicular membrane is constantly exposed to the detergent action of bile salts. One potential element protecting the canalicular membrane from the high canalicular bile salt concentrations may be bile salt resistant microdomains, however additional factors are likely to play a role. To obtain more insights into the molecular composition of the canalicular membrane, the proteome of highly purified rat canalicular membrane vesicles was determined. Isolated rat canalicular membrane vesicles were stripped from adhering proteins, deglycosylated and protease digested before subjecting the samples to shot gun proteomic analysis. The expression of individual candidates was studied by PCR, Western blotting and immunohistochemistry. A total of 2449 proteins were identified, of which 1282 were predicted to be membrane proteins. About 50% of the proteins identified here were absent from previously published liver proteomes. In addition to ATP8B1, four more P4-ATPases were identified. ATP8A1 and ATP9A showed expression specific to the canalicular membrane, ATP11C at the bLPM and ATP11A in an intracellular vesicular compartment partially colocalizing with RAB7A and EEA1 as markers of the endosomal compartment. This study helped to identify additional P4-ATPases from rat liver particularly in the canalicular membrane, previously not known to be expressed in liver. These P4-ATPases might be contributing for maintaining transmembrane lipid homeostasis in hepatocytes.
| S-EPMC4922570 | biostudies-literature
Project description:Farnesoid X receptor (FXR) and Takeda G-protein coupled receptor 5 (TGR5) are the two known bile acid (BA) sensitive receptors and are expressed in the intestine and liver as well as in extra-enterohepatic tissues. The physiological effects of extra-enterohepatic FXR/TRG5 remain unclear. Further, the extent BAs escape liver reabsorption and how they interact with extra-enterohepatic FXR/TGR5 is understudied. We investigated if hepatic BA reuptake differed between BAs agonistic for FXR and TGR5 compared to non-agonists in the rat. Blood was collected from the portal vein and inferior caval vein from anesthetized rats before and 5, 20, 30, and 40 min post stimulation with sulfated cholecystokinin-8. Plasma concentrations of 20 different BAs were assessed by liquid chromatography coupled to mass spectrometry. Total portal vein BA AUC was 3-4 times greater than in the vena cava inferior (2.7 ± 0.6 vs. 0.7 ± 0.2 mM x min, p < 0.01, n = 8) with total unconjugated BAs being 2-3-fold higher than total conjugated BAs (AUC 8-10 higher p < 0.05 for both). However, in both cases, absolute ratios varied greatly among different BAs. The average hepatic reuptake of BAs agonistic for FXR/TGR5 was similar to non-agonists. However, as the sum of non-agonist BAs in vena portae was 2-3-fold higher than the sum agonist (p < 0.05), the peripheral BA pool was composed mostly of non-agonist BAs. We conclude that hepatic BA reuptake varies substantially by type and does not favor FXR/TGR5 BAs agonists.
| S-EPMC7288213 | biostudies-literature
Project description:Solute transport through the bone lacunar-canalicular system is believed to be essential for osteocyte survival and function but has proved difficult to measure. We report an approach that permits direct measurement of real-time solute movement in intact bones. By using fluorescence recovery after photobleaching, the movement of a vitally injected fluorescent dye (sodium fluorescein) among individual osteocytic lacunae was visualized in situ beneath the periosteal surface of mouse cortical bone at depths up to 50 microm with laser scanning confocal microscopy. Transport was analyzed by using a two-compartment mathematical model of solute diffusion that accounted for the characteristic anatomical features of the lacunar-canalicular system. The diffusion coefficient of fluorescein (376 Da) was determined to be 3.3 +/- 0.6 x 10(-6) cm2/sec, which is 62% of its diffusion coefficient in water and is similar to diffusion coefficients measured for comparably sized molecules in cartilage. The diffusion of fluorescein in bone is also consistent with the presence of an osteocyte pericellular matrix whose structure resembles that proposed for the endothelial glycocalyx [Squire, J. M., Chew, M., Nneji, G., Neal, C., Barry, J. & Michel, C. (2001) J. Struct. Biol. 136, 239-255]. To our knowledge, this is the first instance where the dynamics of molecular movement has been measured directly in the bone lacunar-canalicular system. This in situ imaging approach should also facilitate the analysis of convection-based transport mechanisms in bones of living animals.
| S-EPMC1187997 | biostudies-literature
Project description:ContextAlterations in bile acid (BA) synthesis and transport have the potential to affect multiple metabolic pathways in the pathophysiology of obesity.ObjectiveThe objective of the study was to investigate the effects of obesity on serum fluctuations of BAs and markers of BA synthesis.DesignWe measured BA fluctuations in 11 nonobese and 32 obese subjects and BA transporter expression in liver specimens from 42 individuals and specimens of duodenum, jejunum, ileum, colon, and pancreas from nine individuals.Main outcome measuresWe analyzed serum BAs and markers of BA synthesis after overnight fasting, during a hyperinsulinemic-euglycemic clamp, or a mixed-meal tolerance test and the association of BA transporter expression with body mass index.ResultsBA synthesis markers were 2-fold higher (P < .01) and preferentially 12α-hydroxylated (P < .05) in obese subjects, and both measures were correlated with clamp-derived insulin sensitivity (r = -0.62, P < .0001, and r = -0.39, P = .01, respectively). Insulin infusion acutely reduced serum BAs in nonobese subjects, but this effect was blunted in obese subjects (δBAs -44.2% vs -4.2%, P < .05). The rise in serum BAs postprandially was also relatively blunted in obese subjects (δBAs +402% vs +133%, P < .01). Liver expression of the Na+-taurocholate cotransporting polypeptide and the bile salt export pump were negatively correlated with body mass index (r = -0.37, P = .02, and r = -0.48, P = .001, respectively).ConclusionsObesity is associated with increased BA synthesis, preferential 12α-hydroxylation, and impaired serum BA fluctuations. The findings reveal new pathophysiological aspects of BA action in obesity that may lend themselves to therapeutic targeting in metabolic disease.
| S-EPMC4870845 | biostudies-literature
Project description: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'-[alpha, beta-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.
| S-EPMC1219325 | biostudies-other
Project description:Osteocytes, the most abundant cells in bone, are essential in maintaining tissue homeostasis and orchestrating bone's mechanical adaptation. Osteocytes depend upon load-induced convection within the lacunar-canalicular system (LCS) to maintain viability and to sense their mechanical environment. Using the fluorescence recovery after photobleaching (FRAP) imaging approach, we previously quantified the convection of a small tracer (sodium fluorescein, 376 Da) in the murine tibial LCS under intermittent cyclic loading. In the present study, we first expanded the investigation of solute transport using a larger tracer (parvalbumin, 12.3 kDa), which is comparable in size to some signaling proteins secreted by osteocytes. Murine tibiae were subjected to sequential FRAP tests under rest-inserted cyclic loading while the loading magnitude (0, 2.8, or 4.8 N) and frequency (0.5, 1, or 2 Hz) were varied. The characteristic transport rate k and the transport enhancement relative to diffusion (k/k0) were measured under each loading condition, from which the peak solute velocity in the LCS was derived using our LCS transport model. Both the transport enhancement and solute velocity increased with loading magnitude and decreased with loading frequency. Furthermore, the solute-matrix interaction, quantified in terms of the reflection coefficient through the osteocytic pericellular matrix (PCM), was measured and theoretically modeled. The reflection coefficient of parvalbumin (? = 0.084) was derived from the differential fluid and solute velocities within loaded bone. Using a newly developed PCM sieving model, the PCM's fiber configurations accounting for the measured interactions were obtained for the first time. The present study provided not only new data on the micro-fluidic environment experienced by osteocytes in situ but also a powerful quantitative tool for future study of the PCM, the critical interface that controls both outside-in and inside-out signaling in osteocytes during normal bone adaptation and in pathological conditions.
| S-EPMC3593787 | biostudies-literature
Project description:The pericellular matrix (PCM), a thin coating surrounding nearly all mammalian cells, plays a critical role in many cell-surface phenomena. In osteocytes, the PCM is believed to control both "outside-in" (mechanosensing) and "inside-out" (signaling molecule transport) processes. However, the osteocytic PCM is challenging to study in situ because it is thin (?100?nm) and enclosed in mineralized matrix. To this end, we recently developed a novel tracer velocimetry approach that combined fluorescence recovery after photobleaching (FRAP) imaging with hydrodynamic modeling to quantify the osteocytic PCM in young murine bone. In this study, we applied the technique to older mice expressing or deficient for perlecan/HSPG2, a large heparan-sulfate proteoglycan normally secreted in osteocytic PCM. The objectives were (1) to characterize transport within an altered PCM; (2) to test the sensitivity of our approach in detecting the PCM alterations; and (3) to dissect the roles of the PCM in osteocyte mechanosensing. We found that: (1) solute transport increases in the perlecan-deficient (hypomorphic [Hypo]) mice compared with control mice; (2) PCM fiber density decreases with aging and perlecan deficiency; (3) osteocytes in the Hypo bones are predicted to experience higher shear stress (+34%), but decreased fluid drag force (-35%) under 3-N peak tibial loading; and (4) when subjected to tibial loading in a preliminary in vivo experiment, the Hypo mice did not respond to the anabolic stimuli as the CTL mice did. These findings support the hypothesis that the PCM fibers act as osteocyte's sensing antennae, regulating load-induced cellular stimulations and thus bone's sensitivity and in vivo bone adaptation. If this hypothesis is further confirmed, osteocytic PCM could be new targets to develop osteoporosis treatments by modulating bone's intrinsic sensitivity to mechanical loading and be used to design patient-specific exercise regimens to promote bone formation.
| S-EPMC3962519 | biostudies-literature