ABSTRACT
The multispecific transcription factor and tumor suppressor FOXO3 is an important mediator of apoptosis, but the mechanisms that control its proapoptotic function are poorly understood. There has long been evidence that acetylation promotes FOXO3-driven apoptosis and recently a specific JNK (c-Jun N-terminal kinase)-dependent S574 phosphorylated form (p-FOXO3) has been shown to be specifically apoptotic. This study examined whether acetylation and S574 phosphorylation act independently or in concert to regulate the apoptotic function of FOXO3. We observed that both sirtuins 1 and 7 (SIRT1 and SIRT7) are able to deacetylate FOXO3 in vitro and in vivo, and that lipopolysaccharide (LPS) treatment of THP-1 monocytes induced a rapid increase of FOXO3 acetylation, partly by suppression of SIRT1 and SIRT7. Acetylation was required for S574 phosphorylation and cellular apoptosis. Deacetylation of FOXO3 by SIRT activation or SIRT1 or SIRT7 overexpression prevented its S574 phosphorylation and blocked apoptosis in response to LPS. We also found that acetylated FOXO3 preferentially bound JNK1, and a mutant FOXO3 lacking four known acetylation sites (K242, 259, 290 and 569R) abolished JNK1 binding and failed to induce apoptosis. This interplay of acetylation and phosphorylation also regulated cell death in primary human peripheral blood monocytes (PBMs). PBMs isolated from alcoholic hepatitis patients had high expression of SIRT1 and SIRT7 and failed to induce p-FOXO3 and apoptosis in response to LPS. PBMs from healthy controls had lower SIRT1 and SIRT7 and readily formed p-FOXO3 and underwent apoptosis when similarly treated. These results reveal that acetylation is permissive for generation of the apoptotic form of FOXO3 and the activity of SIRT1 and particularly SIRT7 regulate this process in vivo, allowing control of monocyte apoptosis in response to LPS.
Subject(s)
Apoptosis , Forkhead Box Protein O3/metabolism , Serine/metabolism , Acetylation , Adult , Cell Line , Female , Hepatitis, Alcoholic/immunology , Hepatitis, Alcoholic/metabolism , Humans , Lipopolysaccharides/immunology , Male , Middle Aged , Mitogen-Activated Protein Kinase 8/metabolism , Mitogen-Activated Protein Kinases/metabolism , Monocytes/immunology , Monocytes/metabolism , Phosphorylation , Protein Binding , Proteolysis , Signal Transduction , Sirtuins/metabolismABSTRACT
Forkhead box O3 (FOXO3) is a multispecific transcription factor that is responsible for multiple and conflicting transcriptional programs such as cell survival and apoptosis. The protein is heavily post-translationally modified and there is considerable evidence that post-transcriptional modifications (PTMs) regulate protein stability and nuclear-cytosolic translocation. Much less is known about how FOXO3 PTMs determine the specificity of its transcriptional program. In this study we demonstrate that exposure of hepatocytes to ethanol or exposure of macrophages to lipopolysaccharide (LPS) induces the c-Jun N-terminal kinase (JNK)-dependent phosphorylation of FOXO3 at serine-574. Chromatin immunoprecipitation (ChIP), mRNA and protein measurements demonstrate that p-574-FOXO3 selectively binds to promoters of pro-apoptotic genes but not to other well-described FOXO3 targets. Both unphosphorylated and p-574-FOXO3 bound to the B-cell lymphoma 2 (Bcl-2) promoter, but the unphosphorylated form was a transcriptional activator, whereas p-574-FOXO3 was a transcriptional repressor. The combination of increased TRAIL (TNF-related apoptosis-inducing ligand) and decreased Bcl-2 was both necessary and sufficient to induce apoptosis. LPS treatment of a human monocyte cell line (THP-1) induced FOXO3 S-574 phosphorylation and apoptosis. LPS-induced apoptosis was prevented by knockdown of FOXO3. It was restored by overexpressing wild-type FOXO3 but not by overexpressing a nonphosphorylatable S-574A FOXO3. Expression of an S-574D phosphomimetic form of FOXO3 induced apoptosis even in the absence of LPS. A similar result was obtained with mouse peritoneal macrophages where LPS treatment increased TRAIL, decreased Bcl-2 and induced apoptosis in wild-type but not FOXO3(-/-) cells. This work thus demonstrates that S-574 phosphorylation generates a specifically apoptotic form of FOXO3 with decreased transcriptional activity for other well-described FOXO3 functions.
Subject(s)
Apoptosis , Forkhead Box Protein O3/metabolism , Gene Expression Regulation , Transcription, Genetic , Animals , Cell Line, Tumor , Forkhead Box Protein O3/genetics , Humans , Mice , Mice, Knockout , Phosphorylation , SerineABSTRACT
Hepatitis C virus (HCV) infection results in several changes in mitochondrial function including increased reactive oxygen species (ROS) production and greater sensitivity to oxidant, Ca(2+) and cytokine-induced cell death. Prior studies in protein over-expression systems have shown that this effect can be induced by the core protein, but other viral proteins and replication events may contribute as well. To evaluate the specific role of core protein in the context of viral replication and infection, we compared mitochondrial sensitivity in Huh7-derived HCV replicon bearing cells with or without core protein expression with that of cells infected with the JFH1 virus strain. JFH1 infection increased hydrogen peroxide production and sensitized cells to oxidant-induced loss of mitochondrial membrane potential and cell death. An identical phenomenon occurred in genome-length replicons-bearing cells but not in cells bearing the subgenomic replicons lacking core protein. Both cell death and mitochondrial depolarization were Ca(2+) dependent and could be prevented by Ca(2+) chelation. The difference in the mitochondrial response of the two replicon systems could be demonstrated even in isolated mitochondria derived from the two cell lines with the 'genome-length' mitochondria displaying greater sensitivity to Ca(2+) -induced cytochrome c release. In vitro incubation of 'subgenomic' mitochondria with core protein increased oxidant sensitivity to a level similar to that of mitochondria derived from cells bearing genome-length replicons. These results indicate that increased mitochondrial ROS production and a reduced threshold for Ca(2+) and ROS-induced permeability transition is a characteristic of HCV infection. This phenomenon is a direct consequence of core protein interactions with mitochondria and is present whenever core is expressed, either in infection, full-length replicon-bearing cells, or in over-expression systems.
Subject(s)
Hepacivirus/pathogenicity , Mitochondrial Membranes/physiology , Viral Core Proteins/toxicity , Virulence Factors/toxicity , Calcium/metabolism , Cations, Divalent/metabolism , Cell Death , Cell Line , Hepatocytes/virology , Humans , Hydrogen Peroxide/toxicity , Membrane Potential, Mitochondrial , Oxidants/toxicityABSTRACT
BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) frequently results from synergism between chemical and infectious liver carcinogens. Worldwide, the highest incidence of HCC is in regions endemic for the foodborne contaminant aflatoxin B1 (AFB1) and hepatitis B virus (HBV) infection. Recently, gut microbes have been implicated in multisystemic diseases including obesity and diabetes. Here, the hypothesis that specific intestinal bacteria promote liver cancer was tested in chemical and viral transgenic mouse models. METHODS: Helicobacter-free C3H/HeN mice were inoculated with AFB1 and/or Helicobacter hepaticus. The incidence, multiplicity and surface area of liver tumours were quantitated at 40 weeks. Molecular pathways involved in tumourigenesis were analysed by microarray, quantitative real-time PCR, liquid chromatography/mass spectrometry, ELISA, western blot and immunohistochemistry. In a separate experiment, C57BL/6 FL-N/35 mice harbouring a full-length hepatitis C virus (HCV) transgene were crossed with C3H/HeN mice and cancer rates compared between offspring with and without H hepaticus. RESULTS: Intestinal colonisation by H hepaticus was sufficient to promote aflatoxin- and HCV transgene-induced HCC. Neither bacterial translocation to the liver nor induction of hepatitis was necessary. From its preferred niche in the intestinal mucus layer, H hepaticus activated nuclear factor-kappaB (NF-kappaB)-regulated networks associated with innate and T helper 1 (Th1)-type adaptive immunity both in the lower bowel and liver. Biomarkers indicative of tumour progression included hepatocyte turnover, Wnt/beta-catenin activation and oxidative injury with decreased phagocytic clearance of damaged cells. CONCLUSIONS: Enteric microbiota define HCC risk in mice exposed to carcinogenic chemicals or hepatitis virus transgenes. These results have implications for human liver cancer risk assessment and prevention.
Subject(s)
Aflatoxin B1/toxicity , Hepatitis B/complications , Intestines/microbiology , Liver Neoplasms, Experimental/etiology , Adaptive Immunity , Animals , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Chemokines/blood , Cocarcinogenesis , Female , Helicobacter Infections/complications , Helicobacter hepaticus , Hepatitis B/immunology , Immunity, Innate , Interleukin-12 Subunit p40/blood , Liver Neoplasms, Experimental/microbiology , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Oxidative Stress/physiology , Sex Factors , Signal Transduction/physiology , Th1 Cells/immunologyABSTRACT
ClC-3 is a highly conserved voltage-gated chloride channel, which together with ClC-4 and ClC-5 belongs to one subfamily of the larger group of ClC chloride channels. Whereas ClC-5 is localized intracellularly, ClC-3 has been reported to be a swelling-activated plasma membrane channel. However, recent studies have shown that native ClC-3 in hepatocytes is primarily intracellular. Therefore, we reexamined the properties of ClC-3 in a mammalian cell expression system and compared them with the properties of endogenous swelling-activated channels. Chinese hamster ovary (CHO)-K1 cells were transiently transfected with rat ClC-3. The resulting chloride currents were Cl(-) > I(-) selective, showed extreme outward rectification, and lacked inactivation at positive voltages. In addition, they were insensitive to the chloride channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and were not inhibited by phorbol esters or activated by osmotic swelling. These properties are identical to those of ClC-5 but differ from those previously attributed to ClC-3. In contrast, nontransfected CHO-K1 cells displayed an endogenous swelling-activated chloride current, which was weakly outward rectifying, inactivated at positive voltages, sensitive to NPPB and DIDS, and inhibited by phorbol esters. These properties are identical to those previously attributed to ClC-3. Therefore, we conclude that when expressed in CHO-K1 cells, ClC-3 is an extremely outward rectifying channel with similar properties to ClC-5 and is neither activated by cell swelling nor identical to the endogenous swelling-activated channel. These data suggest that ClC-3 cannot be responsible for the swelling-activated chloride channel under all circumstances.
Subject(s)
Chloride Channels/metabolism , Animals , CHO Cells , Cell Size , Chloride Channels/genetics , Cricetinae , Electric Conductivity , Hepatocytes/cytology , Hepatocytes/metabolism , Hypotonic Solutions , Ion Channel Gating , Isotonic Solutions , Kinetics , Patch-Clamp Techniques , Rats , Recombinant Fusion Proteins/metabolism , TransfectionABSTRACT
The molecular identities of functional chloride channels in hepatocytes are largely unknown. We examined the ClC-3 chloride channel in rat hepatocytes and found that mRNA for two different isoforms is present. A short form is identical to the previously reported sequence for rat ClC-3, and a long form contains a 176-bp insertion immediately upstream of the translation initiation site. This predicts a 58-amino acid NH(2) terminal insertion. Both long and short form mRNA was expressed in diverse tissues of the rat. Transient transfection of the long form in CHO-K1 cells resulted in currents with an I(-) > B(-) > Cl(-) selectivity sequence, outward rectification, and inactivation at positive voltages. Short form currents had identical ionic selectivity but displayed a more extreme outward rectification and showed no voltage-dependent inactivation. Immunofluorescence and immunoblots localized native ClC-3 preferentially but not exclusively to the canalicular membrane. We have therefore identified a new isoform of rat ClC-3 and shown that expression of both isoforms produces functional channels. In hepatocytes, ClC-3 is located in association with the canalicular membrane.
Subject(s)
Chloride Channels , Liver/chemistry , Animals , Base Sequence , CHO Cells , Chloride Channels/analysis , Chloride Channels/chemistry , Chloride Channels/genetics , Cricetinae , Electrophysiology , Fluorescent Antibody Technique , Gene Expression/physiology , Ion Channel Gating/physiology , Isomerism , Liver/cytology , Male , Membrane Potentials/physiology , Membrane Proteins/analysis , Membrane Proteins/chemistry , Membrane Proteins/genetics , Molecular Sequence Data , Molecular Weight , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Sequence Homology, Amino Acid , TransfectionSubject(s)
Fatty Liver/etiology , Hepatitis C, Chronic/complications , Liver Neoplasms, Experimental/etiology , Animals , Disease Models, Animal , Fatty Liver/pathology , Female , Gene Expression , Genes, Viral , Hepacivirus/genetics , Hepatitis C, Chronic/pathology , Humans , Liver Neoplasms, Experimental/pathology , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Transgenic , Open Reading Frames , Phenotype , Viral Proteins/geneticsABSTRACT
Intestinal absorption of bile acids depends on a sodium-bile acid cotransport protein in the apical membrane of the ileal epithelial cell. Transport is Na+-dependent, but the Na+-bile acid stoichiometry and electrogenicity of transport are not known. Studies in whole intestine, isolated cells, and ileal membrane vesicles have been unable to resolve this issue because transport currents are small and can be obscured by other ionic conductances and transport proteins present in these membranes. In this study, the human apical sodium-bile acid transporter was expressed in stably transfected Chinese hamster ovary cells that lack other bile acid transporters. The Na+-dependent transport of a fluorescent bile acid analog, chenodeoxycholyl-Nepsilon-nitrobenzoxadiazol-lysine, was monitored by fluorescence microscopy in single, voltage-clamped cells. Bile acid movement was bidirectional and voltage-dependent with negative intracellular voltage-stimulating influx. A 3-fold reduction in extracellular Na+ produced a negative 52 mV shift of the flux-voltage relationship, consistent with a 2:1 Na+:bile acid coupling stoichiometry. No Na+- or voltage-dependent uptake was observed in nontransfected Chinese hamster ovary cells. These results indicate that the cotransport of bile acids and Na+ by human apical sodium-bile acid transporter is electrogenic and bidirectional and is best explained by a 2:1 Na+:bile acid coupling stoichiometry. These results suggest that membrane potential may regulate bile acid transport rates under physiological and pathophysiological conditions.
Subject(s)
Bile Acids and Salts/metabolism , Carrier Proteins/metabolism , Organic Anion Transporters, Sodium-Dependent , Symporters , Animals , Biological Transport , CHO Cells , Carrier Proteins/genetics , Cell Polarity , Cholic Acids/metabolism , Cricetinae , Electric Conductivity , Humans , Lysine/analogs & derivatives , Lysine/metabolism , Patch-Clamp Techniques , Recombinant Proteins/metabolism , Sodium/pharmacology , Taurocholic Acid/metabolismSubject(s)
Antidotes/therapeutic use , Ethylene Glycol/poisoning , Pyrazoles/therapeutic use , Emergency Nursing , Fomepizole , Humans , Male , Middle AgedABSTRACT
The liver plays a major role in metabolism and elimination of leukotrienes (LT). It produces cysteinyl leukotrienes (cLT), and cLT have been implicated in hepatocellular toxicity in several models of lipopolysaccharide (LPS)-associated liver injury. However, the liver cell types responsible for cLT production are poorly defined, and the expression of the LT-synthesis enzymes, 5-lipoxygenase (5-LO) and LTC4 synthase (LTC4-S), in liver cells has never been demonstrated. The aim of the present study was to examine the ability of rat liver cells to produce cLT by determining whether hepatocytes, Kupffer cells, and sinusoidal endothelial cells express mRNA and enzyme activities of the LT-synthesis enzymes and whether expression is altered by LPS. 5-LO mRNA was expressed in whole liver, and expression was enhanced by LPS. Cell fractionation studies demonstrated that expression was present in Kupffer cells and sinusoidal endothelial cells, but not in hepatocytes. LTC4-S mRNA was detected in whole liver, hepatocytes, and sinusoidal endothelial cells, but not in Kupffer cells. Semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) showed that LPS increased LTC4-S expression in hepatocytes by a factor of 3 (n = 3; P < .03). LTC4-S enzyme activity in the microsomal fraction of hepatocytes was also increased from 0.52 +/- 0.13 to 1.90 +/- 0.66 nmol . mg protein-1 . 5 min-1 (n = 6; P < .015) after LPS treatment. These results indicate that hepatocytes do not possess the ability for de novo synthesis of cLT from arachidonic acid, but they may actively participate in cLT production by conjugation of LTA4 with glutathione to produce LTC4. LPS enhances LTC4-S expression in hepatocytes. This intrinsic cLT production may contribute to hepatocellular injury during inflammation.
Subject(s)
Arachidonate 5-Lipoxygenase/genetics , Gene Expression , Glutathione Transferase/genetics , Leukotrienes/biosynthesis , Liver/enzymology , Animals , Endothelium, Vascular/enzymology , Kupffer Cells/enzymology , Lipopolysaccharides/pharmacology , Male , Microsomes, Liver/enzymology , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
The Na(+)-bile acid cotransporters NTCP and ASBT are largely responsible for the Na(+)-dependent bile acid uptake in hepatocytes and intestinal epithelial cells, respectively. This review discusses the experimental methods available for demonstrating electrogenicity and examines the accumulating evidence that coupled transport by each of these bile acid transporters is electrogenic. The evidence includes measurements of transport-associated currents by patch clamp electrophysiological techniques, as well as direct measurement of fluorescent bile acid transport rates in whole cell patch clamped, voltage clamped cells. The results support a Na+:bile acid coupling stoichiometry of 2:1.
Subject(s)
Bile Acids and Salts/metabolism , Carrier Proteins/metabolism , Organic Anion Transporters, Sodium-Dependent , Sodium/metabolism , Symporters , Animals , Biological Transport, Active , Electrophysiology , Humans , Intestinal Mucosa/metabolism , Kinetics , Liver/metabolism , Membrane Potentials , Patch-Clamp TechniquesABSTRACT
Endotoxin (LPS) can cause hepatocellular injury under several circumstances, and leukotrienes have been implicated as a contributing factor. Since ion channel activation has been associated with cytotoxicity, the aim of this study was to determine the circumstances under which LPS and/or leukotrienes activate ionic conductances in hepatocytes. LPS treatment of rats increased Cl- conductance in hepatocytes from 232+/-42 to 1236+/-134 pS/pF. Voltage dependence and inhibitor specificity of this conductance were similar to that of a swelling-activated Cl- conductance, and internal dialysis with nucleoside analogues suggested control by an inhibitory G protein. The lipoxygenase inhibitor nordihydroguaiaretic acid, the specific leukotriene D4 (LTD4) receptor antagonist MK-571, and the 5-lipoxygenase activating protein inhibitor MK-886 all significantly inhibited the conductance. Intracellular dialysis with LTD4 (1.5 microM) elevated intracellular Ca2+ from 143+/-6.5 to 388+/-114 nM within 6 min and stimulated an outwardly rectifying conductance from 642+/-159 to 1669+/-224 pS/pF (n = 9, P < 0.001). In hepatocytes prepared from untreated rats, this concentration of intracellular LTD4 neither raised intracellular Ca2+ nor activated the conductance. The LTD4 response could be induced in normal hepatocytes by culture with either conditioned medium from LPS-treated macrophages or purified TNF-alpha. In conclusion, intracellular LTD4 activates a chloride conductance in hepatocytes isolated from rats treated with LPS or primed in vitro with TNF-alpha. Changes in the hepatocellular accumulation of leukotrienes therefore mediate channel activation and may contribute to liver injury during sepsis and other inflammatory conditions.
Subject(s)
Chloride Channels/physiology , Leukotriene D4/pharmacology , Lipopolysaccharides/pharmacology , Liver/physiology , Membrane Proteins , Receptors, Leukotriene , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Animals , Calcium/metabolism , Calcium/pharmacology , Cells, Cultured , Chloride Channels/antagonists & inhibitors , Culture Media, Conditioned , Electric Conductivity , Escherichia coli , Leukotriene Antagonists , Lipoxygenase Inhibitors/pharmacology , Male , Patch-Clamp Techniques , Rats , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
BACKGROUND/AIMS: Photodynamic therapy using porphyrins or related compounds and laser light is an investigational treatment for neoplasms. The aim of this study was to establish whether this might be applicable for hepatocellular carcinoma using protoporphyrin synthesized in the tissue from administered delta-aminolevulinic acid. METHODS: We measured porphyrin accumulation in normal rat hepatocytes and Morris hepatoma cells in culture, and in subcutaneously implanted hepatomas and other tissues of the rat after administration of delta-aminolevulinic acid, and assessed cell and tissue damage after application of laser light. RESULTS: Porphyrin accumulation after delta-aminolevulinic acid was added to the medium was greater and continued to increase for a longer period of time in hepatoma cells than in hepatocytes (1337+/-42 vs 513+/-31 fluorescence units/cell at 8 h, means+/-SE, p<0.001). After intraperitoneal injection of delta-aminolevulinic acid to rats with subcutaneously growing hepatomas, porphyrin content in tumor and liver was similar at 4 h but was higher in tumor at 6 h. Laser light caused necrosis of normal and malignant liver cells in culture and subcutaneous hepatomas in vivo. CONCLUSIONS: We conclude from these in vitro and in vivo studies that porphyrin accumulation after administration of delta-aminolevulinic acid in this hepatoma is substantial and time dependent, and delivery of laser light locally can cause tumor photosensitization and necrosis.
Subject(s)
Aminolevulinic Acid/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Liver Neoplasms/drug therapy , Photochemotherapy , Photosensitizing Agents/metabolism , Photosensitizing Agents/therapeutic use , Protoporphyrins/biosynthesis , Protoporphyrins/therapeutic use , Animals , Carcinoma, Hepatocellular/pathology , In Vitro Techniques , Laser Therapy , Liver/cytology , Liver/drug effects , Liver/radiation effects , Liver Neoplasms/pathology , Male , Necrosis , Neoplasm Transplantation , Photolysis , Rats , Rats, Sprague-Dawley , Reference ValuesABSTRACT
An outwardly rectifying Cl- conductance was identified in primary isolated rat hepatocytes, and the whole cell patch-clamp technique was used to characterize its properties and mechanisms of activation. With symmetrical Cl(-)-containing solutions on both sides and adenosine 3',5'-cyclic monophosphate (cAMP; 100 microM) in the pipette solution, a large outwardly rectifying conductance (1,014 +/- 153 pS/pF, n = 20) developed in all cells within 3 min. This cAMP-activated conductance was highly anion selective and slowly inactivated at voltages > 80 mV. It was completely inhibited by the anion channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (200 microM, n = 6) and partially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (150 microM, n = 7). It displayed a halide selectivity of I- > Br- > Cl-. In the absence of cAMP, a functionally similar conductance was activated by cell swelling. Reduction of bath osmolality from 300 to 250 mosmol/kg increased membrane conductance from 64 +/- 16.4 to 487 +/- 23 pS/pF (n = 4). This swelling-activated conductance was also highly anion selective and had identical halide selectivity and blocker sensitivity as the cAMP-activated conductance. Although cell swelling was not necessary for cAMP activation, cell shrinkage with hyperosmotic bath (350 mosmol/kg), either before or after exposure to cAMP, inhibited the cAMP-activated conductance. By the determination of conductance as a function of bath osmolality in the presence and absence of cAMP, it was observed that cAMP shifted the osmotic set point for conductance activation without changing either the maximum or minimum conductance. In conclusion, both cAMP and cell swelling activate a large outwardly rectifying Cl- conductance in rat hepatocytes. Its ionic selectivity and sensitivity to channel blockers are identical to those seen for swelling-activated Cl- conductances in many cell types. The conductive properties are not those of cystic fibrosis transmembrane conductance regulator-mediated Cl- conductance. cAMP appears to activate this conductance by altering the volume set point of a swelling-activated channel.
Subject(s)
Chlorides/physiology , Cyclic AMP/pharmacology , Hypotonic Solutions/pharmacology , Liver/drug effects , Liver/physiology , Animals , Cell Separation , Electric Conductivity , Liver/cytology , Osmolar Concentration , Patch-Clamp Techniques , RatsABSTRACT
Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) hyperpolarizes hepatocytes and increases the uptake rate of bile acids. The purpose of this study was to determine to what extent these two phenomena are linked. Fluorescent bile acid analogues (FBA) were used to probe bile acid transport into whole cell patch-clamped hepatocytes. Na(+)-dependent uptake of cholyl-nitrobenz-2-oxa-1,3-diazol-4-yl-lysine (C-NBD-L), an FBA with a net charge of -1, was shown to be electrogenic, whereas uptake of cholylglycylamidofluorescein (CGamF), an FBA with a net charge of -2, was neutral. Incubation of hepatocytes with 8-bromo-cAMP (8-BrcAMP; 100 microM) increased the uptake rate of the electrogenically transported FBA by 25% (P = 0.002), but had no effect on the uptake rate of the electroneutrally transported FBA. Microelectrode impalements revealed that 8-BrcAMP or forskolin hyperpolarized hepatocytes by 6-8 mV. To determine if hyperpolarization is responsible for the cAMP-induced increase in uptake rate, cAMP was directly introduced into hepatocytes during whole cell patch clamp under voltage-clamp conditions. As long as voltage clamp was maintained at -30 mV there was no stimulation of C-NBD-L uptake. However, when voltage clamp was terminated by either pipette removal or current clamp, cAMP increased the uptake rate by 25-34% (P < 0.002). In both of these protocols, cAMP had no effect on uptake of the electroneutrally transported FBA, CGamF. Finally, in voltage-clamped hepatocytes in the absence of cAMP, a 10-mV hyperpolarization increased the uptake rate of C-NBD-L by 23%. We therefore conclude that short-term cAMP-induced stimulation of fluorescent bile acid uptake in hepatocytes is a direct consequence of membrane hyperpolarization.
Subject(s)
Cholic Acids/pharmacokinetics , Cyclic AMP/pharmacology , Liver/physiology , Lysine/analogs & derivatives , Animals , Cell Membrane/physiology , Electrophysiology , Fluorescent Dyes , Liver/cytology , Lysine/pharmacokinetics , Microscopy, Fluorescence , Patch-Clamp Techniques , RatsABSTRACT
The transport properties of three different synthetically prepared fluorescent conjugated bile acid analogs (FBA), all with the fluorophore on the side chain, were determined using isolated rat hepatocytes and hepatocyte couplets. The compounds studied were cholylglycylamidofluorescein (CGamF), cholyl(N epsilon-nitrobenzoxadiazolyl [NBD])-lysine (C-NBD-L), and chenodeoxycholyl-(N epsilon-NBD)-lysine (CDC-NBD-L). When hepatocytes were incubated at 37 degrees C with 0.3 mumol/L of FBA and 0.15 mol/L of Na+, cell fluorescence increased linearly with time at a rate (U/min) of 7.8 +/- 0.5 for CGamF, 7.2 +/- 0.3 for C-NBD-L, and 13.7 +/- 1.0 for CDC-NBD-L (mean, +/- SE; n = 40 to 90). Uptake was concentration dependent for concentrations less than 20 mumol/L and was saturable. The Michaelis constant (Km) value (mumol/L) for CGamF was 10.8, for C-NBD-L was 3.8, and for CDC-NBD-L was 3.0. In the absence of Na+, the uptake rate was decreased by 50% for CGamF and by 38% for C-NBD-L; but uptake of CDC-NBD-L was unchanged and thus Na+ independent. Cellular uptake of all three derivatives was specific to hepatocytes and was absent in several nonhepatocyte cell lines. For CGamF and C-NBD-L, both Na(+)-dependent and Na(+)-independent uptake was inhibited by 200-fold excess concentrations of cholyltaurine, dehydrocholyltaurine, and cholate, but for CDC-NBD-L, these nonfluorescent bile acids did not inhibit initial uptake. The intracellular fluorescence of CGamF was strongly pH dependent at an excitation wavelength of 495 nm, but pH independent at 440 nm excitation. In contrast, intracellular fluorescence of C-NBD-L and CDC-NBD-L was pH independent. All three FBA were secreted into the canalicular space of approximately 50% to 60% of couplets. Cellular adenosine triphosphate (ATP) depletion with either CN- or atractyloside inhibited secretion of all three FBA. The multispecific organic anion transporter (MOAT) inhibitor, chlorodinitrobenzene, blocked secretion of fluorescent MOAT substrates at a concentration of 1 mumol/L. At this concentration it did not affect secretion of the three FBA. At higher concentrations, chlorodinitrobenzene partially inhibited the canalicular secretion of CGamF but not the other two FBA. In conclusion, all three FBA are secreted by canalicular membrane bile acid transporters, but the sinusoidal uptake characteristics of CGamF and C-NBD-L are more similar than those of CDC-NBD-L to the transport properties of cholyltaurine. Therefore, C-NBD-L appears to be the best of the three for studies of conjugated trihydroxy-bile acid transport in hepatocytes.
Subject(s)
Bile Acids and Salts/metabolism , Fluorescent Dyes , Liver/metabolism , Animals , Anion Transport Proteins , Bile Canaliculi/metabolism , Biological Transport , Carrier Proteins/metabolism , Chenodeoxycholic Acid/analogs & derivatives , Chenodeoxycholic Acid/metabolism , Cholic Acids/metabolism , Fluoresceins/metabolism , Hydrogen-Ion Concentration , Kinetics , Lysine/analogs & derivatives , Lysine/metabolism , Microscopy, Fluorescence , Rats , Rats, Sprague-Dawley , Sodium/pharmacology , Spectrometry, Fluorescence , ThermodynamicsABSTRACT
Because of the growing number of automobiles equipped with air bags, it is virtually certain that patients will seek treatment in the emergency department for air bag-mediated injury. The emergency nurse must be aware of air bag-mediated injury patterns and prepared for triage and management of any of the potential concurrent injuries caused by air-bag deployment.