scholarly journals Tacrine Sinusoidal Uptake and Biliary Excretion in Sandwich-Cultured Primary Rat Hepatocytes

2014 ◽  
Vol 17 (3) ◽  
pp. 427 ◽  
Author(s):  
Amal Kaddoumi ◽  
Loqman A. Mohamed
Keyword(s):  
Biliary Excretion ◽  
Rat Hepatocytes ◽  
Hepatic Uptake ◽  
Hepatic Transport ◽  
Glycoprotein P ◽  
Hepatic Disposition ◽  
Primary Rat Hepatocytes ◽  
Fold Reduction ◽  
Kinetics Of

PURPOSE. The knowledge of hepatic disposition kinetics of tacrine, a first cholinesterase inhibitor was approved by FDA for the treatment of Alzheimer’s disease (AD), would help to understand its hepatotoxicity, its therapeutic effect, and improve the management of patients with AD. The current study aims to characterize tacrine hepatic transport kinetics and study the role of organic cation transporters (OCTs), P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP2) in tacrine sinusoidal uptake and biliary excretion. METHODS. Modulation of tacrine hepatic uptake and efflux, biliary excretion index (BEI%), were performed in sandwich-cultured primary rat hepatocytes (SCHs) using transporters inhibitors. Conformation of the integrity of SCHs model was established by capturing images with light-contrast and fluorescence microscopy. RESULTS. Tacrine uptake in SCHs was carrier-mediated process and saturable with apparent Km of 31.5±9.6 µM and Vmax of 908±72 pmol/min/mg protein. Tetraethyl ammonium (TEA), cimetidine and verapamil significantly reduced tacrine uptake with more pronounced effect observed with verapamil which caused 3-fold reduction in tacrine uptake, indicating role for OCTs. Tacrine has a biliary excretion in SCHs with maximum BEI% value of 22.9±1.9% at 10 min of incubation. Addition of MK571 and valspodar decreased the BEI% of tacrine by 40 and 60% suggesting roles for canalicular MRP2 and P-gp, respectively. CONCLUSIONS. Our results show that in addition to metabolism, tacrine hepatic disposition is carrier-mediated process mediated by sinusoidal OCTs, and canalicular MRP2 and P-gp.This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

Role of bicarbonate in biliary excretion of diisothiocyanostilbene disulfonate

1988 ◽  
Vol 255 (6) ◽  
pp. G713-G722
Author(s):  
M. S. Anwer ◽  
K. Nolan ◽  
W. G. Hardison
Keyword(s):  
Biliary Excretion ◽  
Rat Hepatocytes ◽  
Hepatic Uptake ◽  
Disulfonic Acid ◽  
Base Line ◽  
Hepatic Transport ◽  
Isolated Rat Hepatocytes ◽  
Canalicular Membrane ◽  
Major Mechanism ◽  
Rat Livers

Hepatic transport of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) was studied in isolated perfused rat livers and in isolated rat hepatocytes to determine if DIDS-induced decrease in biliary HCO3- excretion is due to a DIDS-HCO3- exchange and/or due to inhibition of Cl(-)-HCO3- exchange. In isolated perfused rat livers, DIDS reversibly decreased biliary HCO3- concentration and excretion. The changes in biliary HCO3- concentration were inversely related to biliary DIDS concentration. DIDS was concentrated in bile, indicating active hepatic transport. Replacement of perfusate HCO3- with equimolar dimethyloxazolidinedione (DMO) or tricine decreased biliary excretion, but not hepatic uptake, of DIDS. Biliary excretion of DIDS was also associated with a decrease in bile pH, and this decrease in pH was greater in the presence of HCO3-. HCO3-, but not DMO or tricine, stimulated DIDS efflux from preloaded hepatocytes. DIDS efflux was also temperature dependent and increased with increasing extracellular pH. Collectively, these results are consistent with the presence of a DIDS-HCO3- (OH-) exchange mechanism at the canalicular membrane. HCO3(-)-dependent Cl- uptake in hepatocytes was competitively inhibited by DIDS (Ki = 0.24 mM), confirming the presence of DIDS-inhibitable Cl(-)-HCO3- exchange. However, the ability of DIDS to decrease biliary HCO3- excretion persisted when perfusate Cl- was replaced by isethionate. Moreover, biliary HCO3- concentration returned to base line despite the presence of 2-6 mM DIDS in bile. Thus it seems unlikely that the inhibition of Cl(-)-HCO3- exchange by DIDS is a major mechanism of inhibition of HCO3- excretion. We, therefore, conclude that a DIDS-HCO3- (OH-) exchange at the canalicular membrane is the most likely explanation for the observed decrease in biliary HCO3- excretion.

scholarly journals Evidence that Ca2+-release-activated Ca2+ channels in rat hepatocytes are required for the maintenance of hormone-induced Ca2+ oscillations

2003 ◽  
Vol 370 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Roland B. GREGORY ◽  
Gregory J. BARRITT
Keyword(s):  
High Selectivity ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Cation Channels ◽  
Isolated Rat Hepatocytes ◽  
Pharmacological Agents ◽  
Crac Channels ◽  
Kinetics Of ◽  
Ca2 Channels

Store-operated Ca2+ channels in liver cells have been shown previously to exhibit a high selectivity for Ca2+ and to have properties indistinguishable from those of Ca2+-release-activated Ca2+ (CRAC) channels in mast cells and lymphocytes [Rychkov, Brereton, Harland and Barritt (2001) Hepatology 33, 938—947]. The role of CRAC channels in the maintenance of hormone-induced oscillations in the cytoplasmic free Ca2+ concentration ([Ca2+]cyt) in isolated rat hepatocytes was investigated using several inhibitors of CRAC channels. 2-Aminoethyl diphenylborate (2-APB; 75μM), Gd3+ (1μM) and 1-{β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SK&F 96365; 50μM) each inhibited vasopressin- and adrenaline (epinephrine)-induced Ca2+ oscillations (measured using fura-2). The characteristics of this inhibition were similar to those of inhibition caused by decreasing the extracellular Ca2+ concentration to zero by addition of EGTA. The effect of 2-APB was reversible. In contrast, LOE-908 {(R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamidemesylate}(30μM), used commonly to block Ca2+ inflow through intracellular-messenger-activated, non-selective cation channels, did not inhibit the Ca2+ oscillations. In the absence of added extracellular Ca2+, 2-APB, Gd3+ and SK&F 96365 did not alter the kinetics of the increase in [Ca2+]cyt induced by a concentration of adrenaline or vasopressin that induces continuous Ca2+ oscillations at the physiological extracellular Ca2+ concentration. Ca2+ inflow through non-selective cation channels activated by maitotoxin could not restore Ca2+ oscillations in cells treated with 2-APB to block Ca2+ inflow through CRAC channels. Evidence for the specificity of the pharmacological agents for inhibition of CRAC channels under the conditions of the present experiments with hepatocytes is discussed. It is concluded that Ca2+ inflow through CRAC channels is required for the maintenance of hormone-induced Ca2+ oscillations in isolated hepatocytes.

Effects of ion substitution on transport and choleretic effect of ouabain

1987 ◽  
Vol 252 (3) ◽  
pp. G357-G364
Author(s):  
M. S. Anwer
Keyword(s):  
Choline Chloride ◽  
Inorganic Ions ◽  
Hepatic Uptake ◽  
Isolated Perfused Rat Liver ◽  
Electrolyte Excretion ◽  
Hepatic Transport ◽  
Osmotic Effect ◽  
Ion Substitution ◽  
Choleretic Effect

The role of inorganic ions in hepatic transport and choleretic effect of ouabain was studied in isolated perfused rat liver to verify whether Na+-coupled ouabain uptake into hepatocytes is responsible for the choleretic effect. Hepatic uptake and clearance of ouabain were not significantly affected when perfusate Na+ was replaced by Li+ or choline+, chloride by nitrate or isethionate, or bicarbonate by tricine. However, these ion substitutions, with the exception of Li+, significantly reduced ouabain-induced choleresis and biliary electrolyte excretion. When ouabain was infused at different rates followed by perfusion without ouabain, changes in bile flow paralleled biliary excretion of ouabain rather than hepatic uptake. These results indicate that hepatic uptake of ouabain is not Na+ dependent and that the osmotic effect of biliary excreted ouabain is responsible for its choleretic effect. A part of the choleretic effect (30%) must also involve other mechanisms, since a permeable anion-like nitrate failed to substitute for perfusate chloride. Results of infusion studies also showed that ouabain was concentrated in liver (liver/perfusate = 30) and in bile (bile/liver = 15), indicating that ouabain is transported against its concentration gradient across both sinusoidal and canalicular membranes.

Role of extracellular Ca2+ in hepatic bile formation and taurocholate transport

1985 ◽  
Vol 249 (6) ◽  
pp. G711-G718 ◽  
Author(s):  
M. S. Anwer ◽  
L. M. Clayton
Keyword(s):  
Control Period ◽  
Rat Hepatocytes ◽  
Experimental Period ◽  
Isolated Hepatocytes ◽  
Hepatic Uptake ◽  
Hepatic Bile ◽  
Bile Formation ◽  
Isolated Rat Hepatocytes ◽  
Diffusional Permeability

The role of extracellular Ca2+ in hepatic bile formation, biliary membrane permeability, and taurocholate (TC) transport was studied in isolated perfused rat livers and in isolated rat hepatocytes to determine the functional importance of paracellular permeability in biliary bile acid excretion. Each liver was perfused for 1 h with perfusate containing 1.3 mM Ca2+ (control period) followed by another hour with 1.3, 0.5, 0.1, 0.05, 0.03, or 0.01 mM Ca2+ (experimental period). Basal bile flow and biliary excretion of added TC declined significantly only at and below 0.05 mM perfusate Ca2+ and was associated with an increase in bile-to-perfusate concentration ratio of [3H]inulin (B/P inulin ratio). A twofold increase in the diffusional permeability coefficient at 0.05 mM and a sixfold increase at 0.03 and 0.01 mM perfusate Ca2+ could explain the increased in B/P inulin ratios. Time-dependent increases in cell-to-medium concentration ratios of inulin were less in the absence than in the presence of Ca2+. Hepatic uptake rates of TC determined in isolated hepatocytes and from perfusate disappearance of added TC and efflux rates of TC from preloaded hepatocytes were not significantly affected by Ca2+ removal. It is possible that the observed decline in biliary TC excretion at low perfusate Ca2+ is due to regurgitation of secreted TC back into the perfusate followed by reuptake. This was supported by an accumulation of perfusate radioactivity when TC uptake inhibitors (furosemide and bumetanide) were added to the perfusate (0.03 mM Ca2+) 60 min after the addition of [14C]TC.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The role of conjugation reactions in enhancing biliary secretion of bile acids

1983 ◽  
Vol 214 (3) ◽  
pp. 923-927 ◽  
Author(s):  
D A Vessey ◽  
J Whitney ◽  
J L Gollan
Keyword(s):  
Bile Acid ◽  
Bile Acids ◽  
Hepatic Uptake ◽  
Biliary Secretion ◽  
Side Chain ◽  
Isolated Perfused Rat Liver ◽  
Rat Livers ◽  
Kinetics Of ◽  
Methylene Group

Shortening the five-carbon carboxylic acid side chain of cholic acid by one methylene group gave rise to a bile acid (norcholate) that was not a substrate for the bile acid-conjugating enzymes. The metabolism and biliary secretion of norcholate in intact liver was examined in the isolated perfused rat liver system. When rat livers were perfused with 14-20 microM solutions of norcholate for 10 min, norcholate was found in the unconjugated form in liver, venous effluent and bile. Neither tauronorcholate nor glyconorcholate was detectable by high-pressure liquid chromatography or fast-atom-bombardment mass spectrometry. The kinetics of hepatic uptake and biliary secretion of norcholate was compared with that for cholate, taurocholate and chemically synthesized tauronorcholate. The latter three bile acids were completely cleared from the perfusate and efficiently secreted into the bile. However, norcholate was incompletely extracted from the perfusate, and this was shown to be at least partially due to its relatively lower rate of hepatic uptake. Furthermore, the rate of norcholate secretion into bile was greatly reduced relative to the secretion of cholate or chemically synthesized tauronorcholate, even though the concentration of norcholate in the liver was comparatively high. These data demonstrate that the conjugation of bile acids greatly facilitates their secretion into bile.

scholarly journals Activation of glucokinase gene expression by hepatic nuclear factor 4α in primary hepatocytes

2002 ◽  
Vol 365 (1) ◽  
pp. 223-228 ◽  
Author(s):  
Ulrike ROTH ◽  
Kurt JUNGERMANN ◽  
Thomas KIETZMANN
Keyword(s):  
Gene Expression ◽  
Glucose Utilization ◽  
Rat Hepatocytes ◽  
Nuclear Factor ◽  
Primary Hepatocytes ◽  
Glucokinase Gene ◽  
Primary Rat Hepatocytes ◽  
Key Enzyme

Glucokinase (GK) is a key enzyme for glucose utilization in liver and shows a higher expression in the perivenous zone. In primary rat hepatocytes, the GK gene expression was activated by HNF (hepatic nuclear factor)-4α via the sequence −52/−39 of the GK promoter. Venous pO2 enhanced HNF-4 levels and HNF-4 binding to the GK—HNF-4 element. Thus, HNF-4α could play the role of a regulator for zonated GK expression.

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