Isolated Hepatocytes Research Articles

DIETHYLDITHIOCARBAMATE (DEDC) ENHANCES QUINONE MEDIATED OXIDATIVE STRESS CYTOTOXICITY IN ISOLATED HEPATOCYTES BY FORMING TOXIC QUINONE CONJUGATES

The copper-chelating thiol drug, diethyldithiocarbamate (DEDC) had previously been used to inhibit superoxide dismutase (SOD) and enhance oxidative stress mediated cytotoxicity. Using isolated rat hepatocytes, it was confirmed that DEDC enhances oxidative stress cytotoxicity induced by 1,4-naphthoquinone (1,4-NQ) and 1,4-naphthoquinone-2-sulphonate (1,4-NQ-2S). However, equimolar concentrations of DEDC also enhances cytotoxicity induced by benzoquinone, previously shown to cause cytotoxicity as a result of alkylation and not oxidative stress. Higher DEDC concentrations on the other hand protected against benzoquinone-induced cytotoxicity. Finally, the susceptibility of hepatocytes to quinone mediated oxidative stress cytotoxicity was not enhanced if the DEDC was removed before incubating the hepatocytes with naphthoquinone or benzoquinone. Enhanced oxidative stress cytotoxicity was only observed if the DEDC was present when hepatocytes were treated with quinones. It was concluded that DEDC forms conjugates with quinones which undergo futile redox cycling in the hepatocyte and form H2O2 as well as increase the susceptibility of hepatocytes to H2O2.

Mitochondrial glutathione status during Ca 2+ ionophore-induced injury to isolated hepatocytes

In this study the Ca 2+ ionophore, A23187, was used to determine the effects of disrupted Ca 2+ homeostasis on cellular thiols. Isolated rat hepatocytes were incubated with varying concentrations of extracellular Ca 2+ and A23187 to induce accumulation or loss of cellular Ca 2+. These treatments resulted in loss of mitochondrial and cytosolic glutathione (GSH), loss of protein-thiols, and cell injury. This injury was dependent on the concentrations of ionophore and extracellular Ca 2+. A correlation was found between cell injury and the loss of mitochondrial GSH, while the loss of cytosolic glutathione preceded both these events. The time course of protein-thiol loss appeared secondary to the loss of non-protein thiols. In the absence of extracellular Ca 2+, the antioxidants α-tocopherol and diphenyl- p-phenylenediamine both totally prevented A23187-induced cell injury and loss of mitochondrial GSH, and thus protected the cells from the effects of mobilization of intracellular Ca 2+. In the presence of extracellular Ca 2+, cell injury as well as the loss of mitochondrial GSH were only partially prevented by antioxidant treatment. The mitochondrial Ca 2+ channel blocker, ruthenium red, protected hepatocytes from A23187-induced injury in the absence of extracellular Ca 2+. Leupeptin, an inhibitor of Ca 2+-activated proteases, and dibucaine, a phospholipase inhibitor, did not affect cytotoxicity. Our results indicate that the level of mitochondrial GSH may be important for cell survival during ionophore-induced perturbation of Cellular Ca 2+ homeostasis.

Isolated rat hepatocyte couplets in short-term culture: structural characteristics and plasma membrane reorganization.

Studies of canalicular bile secretion have been limited due to lack of direct access to the canalicular lumen. Isolated rat hepatocyte couplets, consisting of two hepatocytes enclosing a canalicular space, have been proposed as a primary secretory unit that may be useful for direct studies of unmodified canalicular bile secretion. The present study examines their structural characteristics and plasma membrane reorganization. The canalicular space of freshly isolated hepatocyte couplets is freely permeable to ruthenium red, but within 4 hr the junctional borders reseal in most couplets which then exclude ruthenium red from the luminal area. These resealed spaces expand in 61.8 +/- 10% of couplets as secretion is elaborated and after 4 hr in monolayer culture, 12.7 +/- 4.7% of the canalicular spaces are dilated to diameters greater than 3 microns. Normal-appearing microvilli line these canalicular membranes in the majority of dilated spaces as assessed by electron microscopy. Immediately after isolation, Mg++-ATPase, a histochemical marker for canalicular membranes, is located as a discrete band corresponding to the normal in vivo circumferential distribution of the canalicular membrane domain. However, this pattern of Mg++-ATPase staining rapidly diminishes and reorganizes at the remaining canalicular pole within several hours. This membrane reorganization is a microfilament-dependent process, since it is inhibited by cytochalasin D but not by colchicine, an inhibitor of microtubular function or cycloheximide, an inhibitor of new protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the liver P2-purinoceptor involved in the activation of glycogen phosphorylase.

Evidence has been presented for the existence in rat liver of P2-purinoceptors which are involved in the control of glycogenolysis. Isolated rat hepatocytes and purified liver plasma membranes have been used to study the binding of the ATP analogue adenosine 5'-[alpha- [35S]thio]triphosphate (ATP alpha [35S]) to these postulated P2-purinoceptors. The nucleotide analogue behaves as a full agonist for the activation of glycogen phosphorylase in isolated hepatocytes, 0.3 microM being required for half-maximal activation. Specific binding of ATP alpha [35S] to hepatocytes and plasma membranes occurs within 1 min and is essentially reversible. The analysis of the dose-dependency at equilibrium indicates the presence of binding sites with Kd of 0.23 microM with hepatocytes and Kd of 0.11 microM with plasma membranes. The relative affinities of 10 nucleotide analogues were deduced from competition experiments for ATP alpha [35S] binding to hepatocytes, and these correlated highly with their biological activity (activation of glycogen phosphorylase in hepatocytes). For all the agonists, binding occurs in the same concentration range as the biological effect. These data clearly suggest that the detected binding sites correspond to the physiological P2-purinoceptors involved in the regulation of liver glycogenolysis. The rank order of potency of some ATP analogues suggests that liver possesses the P2Y-subclass of P2-purinoceptors.

Tert.-Butyl hydroperoxide metabolism and stimulation of the pentose phosphate pathway in isolated rat hepatocytes

The metabolism of tert.-butyl hydroperoxide (TBHP) by the glutathione peroxidase/reductase system in isolated hepatocytes results in the rapid depletion of reduced glutathione and NADPH. The regeneration of NADPH can occur through the pentose phosphate pathway, but only when the pathway is stimulated, for example, by NADP + and possibly oxidized glutathione, both of which can be elevated in hepatocytes exposed to TBHP. TBHP is a cytotoxicant and the role of NADPH and the pentose phosphate pathway in protecting hepatocytes from TBHP-induced injury is unknown. Isolated rat hepatocytes exposed to TBHP (0.5 m m) for 30 min metabolized more [1- 14C]glucose to 14CO 2 than control (638.2 ± 96.2 vs 306.9 ± 69.5 dpm/10 6 cells) whereas 14CO 2 evolution from [6- 14C]glucose was unchanged, indicating that TBHP increases the activity of the pentose phosphate pathway and not glycolysis. TBHP (0.25 m m) metabolism also resulted in a rapid oxidation of hepatocyte NADPH from 2.85 ± 0.32 to 0.55 ± 0.24 nmol/10 6 cells which rapidly returned to 3.58 ± 0.27 nmol NADPH/10 6 cells. Inhibition of the pentose phosphate pathway with 6-aminonicotinamide (70 mg/kg; 5 hr prior to hepatocyte isolation) inhibited TBHP-stimulated 14CO 2 evolution from [1- 14C]glucose and decreased the rate of NADP + reduction. Hepatocytes isolated from 6-aminonicotinamide-treated animals were more susceptible to TBHP-induced cell injury than were control hepatocytes. These data demonstrate the following: (1) The metabolism of TBHP by isolated hepatocytes stimulated the activity of the pentose phosphate pathway; and (2) inhibition of the pentose phosphate pathway with 6-aminonicotinamide potentiated the toxicity of TBHP to isolated rat hepatocytes. These results suggest that the regeneration of NADPH by the pentose phosphate pathway may play a significant role in protecting hepatocytes from TBHP-induced damage.

Incorporation of the 1-pro-R and 1-pro-S hydrogen atoms of ethanol in the reduction of acids in the liver of intact rats and in isolated hepatocytes

Ethanol oxidation causes redox effects. The coupling of this oxidation via NADH to intermediary metabolism was studied in order to reveal the underlying mechanisms. Isolated rat hepatocytes were incubated with [1,1-2H2]-, (1R)-[1-2H]- and (1S)-[1-2H]-ethanol and the 2H incorporation was measured in lactate, beta-hydroxybutyrate, fumarate, malate, succinate, alpha-oxoglutarate and citrate. The results differed in the following ways from results obtained in intact rats. Lactate became labelled to an increasing extent, and in more than one position, indicating labelling of pyruvate. A small and constant fraction of malate and fumarate was formed without access to [2H]coenzyme. Addition of aspartate increased this fraction, which was concluded to be formed in the mitochondria. Citrate was essentially unlabelled. The 2H from (1R)-[1-2H]ethanol contributed to malate to a larger extent and to beta-hydroxybutyrate to a smaller extent, and 2H from (1S)-[1-2H]ethanol contributed to lactate to a smaller extent. These results indicate that the exchange via shuttle system was less efficient in isolated hepatocytes than in intact rats. The 2H incorporation was independent of concentration of [1,1-2H2]ethanol when this was above 5mM. Additions known to increase ethanol elimination, and cyanamide, which decreases it, had no marked effect on the 2H incorporation. This indicates equilibration of the NADH bound to alcohol dehydrogenase with free NADH. Disulfiram and cyanamide caused a decrease in the relative incorporation from (1S)-[1-2H]ethanol into malate in liver of intact rats. Addition of cyanamide to incubations with hepatocytes resulted in a decrease of the contribution of 2H from (1S)-[1-2H]ethanol in lactate, beta-hydroxybutyrate and malate. This indicates that acetaldehyde was only oxidized in the mitochondrial compartment.

Biosynthesis of methylguanidine in isolated rat hepatocytes and in vivo.

To clarify the organ in which methylguanidine is synthesized, high doses of creatinine, which is known to stimulate the synthesis of methylguanidine, were administered to male Wistar rats intraperitoneally. Various tissues of the rats were frozen by a freeze clamp method before and 1, 2 and 3 h after injection, and methylguanidine was determined by high-pressure liquid chromatography using 9,10-phenanthrenequinone for fluorometric determination. We found evidence that the liver, kidney, lung, muscle, red blood cells and gut flora synthesize methylguanidine. In addition, we measured the synthesis of methylguanidine in isolated hepatocytes prepared from normal rats following the addition of creatinine, arginine and guanidinoacetic acid to the incubation medium. Synthesis of methylguanidine was observed only in those incubations which contained creatinine, and was dependent on the concentration of creatinine in the media and on the incubation period. Isolated rat hepatocytes also synthesized guanidine in the presence of guanidinoacetic acid. These results indicate that the liver is one of the organs which synthesize methylguanidine and also that creatinine is the precursor.

Isolated rat hepatocyte couplets: a primary secretory unit for electrophysiologic studies of bile secretory function.

Hepatocyte couplets were isolated by collagenase perfusion from rat liver. Between adjacent cells, the bile canaliculus forms a closed space into which secretion occurs. As in intact liver, Mg2+-ATPase is localized at the canalicular lumen, the organic anion fluorescein is excreted, and secretion is modified by osmotic gradients. By passing a microelectrode through one cell into the canalicular vacuole, a transepithelial potential profile was obtained. In 27 cell couplets the steady-state intracellular (-26.3 +/- 5.3 mV) and intracanalicular (-5.9 +/- 3.3 mV) potentials were recorded at 37 degrees C with reference to the external medium. Input resistances were determined within the cell (86 +/- 23 M omega) and in the bile canalicular lumen (32 +/- 17 M omega) by passing current pulses through the microelectrode. These data define electrical driving forces for ion transport across the sinusoidal, canalicular, and paracellular barriers and indicate ion permeation across a leaky paracellular junctional pathway. These findings indicate that the isolated hepatocyte couplet is an effective model for electrophysiologic studies of bile secretory function.

Metabolic labeling of dolichol and dolichyl phosphate in isolate hepatocytes.

Isolated hepatocytes from rat liver were incubated with [3H]mevalonate, and the labeling of polyprenols in the microsomal fraction was followed. After a 1-min incubation the alpha-unsaturated forms of polyprenyl-P2, -P, and polyprenol were mainly labeled and at this time point only 2, 8, and 17%, respectively, of the label was associated with the saturated forms. In the case of the free alcohol 2 h of incubation was required before all the labeling was recovered in the saturated form. After 1 min polyprenols and polyprenyl-P with 20 and 21 isoprene residues demonstrated much higher specific labeling than the shorter compounds, but after 5 min these differences were greatly reduced. In experiments utilizing short incubation times and chasing no evidence has been obtained that the phosphorylated form is a precursor of the free alcohol or vice versa, that the free alcohol is a precursor of the phosphorylated form. In human liver about 1% of the dolichol is present in the alpha-unsaturated form. These experiments suggest that: 1) the alpha-unsaturated form is the precursor of the alpha-saturated free alcohol, 2) dolichol does not necessarily arise directly from dephosphorylation of the phosphorylated form, and 3) the free alcohol is for the most part not phosphorylated under in vivo conditions in rat liver.

Receptor binding properties of monoiodotyrosyl insulin isomers purified by high performance liquid chromatography.

The four monoiodinated forms of pork insulin were prepared by lactoperoxidase catalyzed iodination followed by separation on reverse-phase high-performance liquid chromatography. The procedure was rapid and extremely reproducible, resulting in total separation of the B16, B26, and A14 monoiodinated insulins from one another and from unreacted insulin. The A19 derivative eluted closely to the unreacted insulin, requiring an additional step on ion exchange chromatography for complete purification of this derivative. The receptor binding properties of the derivatives were examined in isolated rat adipocytes and hepatocytes and in cultured human IM-9 lymphocytes. The relative binding affinities of the derivatives varied in the different cell types. At tracer concentrations, the B26 derivative had the highest apparent affinity in IM-9 lymphocytes and in adipocytes, but the A14 had the greatest affinity in hepatocytes. At equivalent concentration of each tracer, IM-9 lymphocytes bound the B26 isomer 144 ± 9% (P < 0.05) and the B16 isomer 118 ± 3% (P < 0.05) as well as the 125I (A14) insulin. Isolated rat adipocytes also bound the B26 to a greater extent at tracer concentrations (117 ± 6%; P < 0.01) compared with A14, but bound B16 less than A14 (91 ± 2%; P < 0.01). Isolated rat hepatocytes bound more of the A14 derivative than either of the B-chain labels. The B26 bound 72 ± 2% (P < 0.01) and the B16 90 ± 1% (P < 0.01) as well as the A14 derivative to isolated hepatocytes. In contrast to the results obtained at tracer concentrations of the isomers, higher concentrations of native insulin were required for 50% competitive displacement (ED50) of the B26 derivative as compared with the other two derivatives in all three cell types examined. These results indicate a higher average affinity for the B26 isomer in the three tissues, and suggest that results obtained from tracer binding alone may not provide an accurate reflection of receptor affinity for the monoiodoinsulin isomers. Competitive displacement of the 125I-labeled derivatives by 127I (nonradioactive) labeled homologues in the IM-9 lymphocytes also showed the B26 derivative to have the highest affinity. The present study demonstrates that monoiodoinsulin selectively labeled on each of the tyrosines can be prepared and separated on HPLC and used to examine insulin receptor binding in different cell types. Furthermore, the data demonstrate different apparent affinities of the selectively-labeled monoiodoinsulin isomers in different cell types, suggesting variability in insulin binding properties in different tissues.

Formation and disposition of N-hydroxylated metabolites of aniline and nitrobenzene by isolated rat hepatocytes.

The formation and secretion of phenylhydroxylamine plus nitrosobenzene was studied in cultures of isolated rat hepatocytes after addition of aniline or nitrobenzene. With aniline concn. up to 10 mM, N-oxygenated metabolites were secreted linearly with time over 2 h. Phenobarbitone pretreatment in vivo for c. 5 d increased aniline N-hydroxylation by a factor of 2.8. Nitrobenzene reduction by isolated rat hepatocytes, yielding phenylhydroxylamine plus nitrosobenzene in the medium, was stimulated 1.9-fold and 4.3-fold after phenobarbitone pretreatment in vivo for 5 and 10 d, respectively. After reduction of nitrobenzene by isolated hepatocytes, the secretion of N-oxygenated products into the medium was non-linear with time for substrate concn. higher than 2.5 mM, probably due to the formation of cytotoxic concn. of nitrosobenzene. Isolated rat hepatocytes reduced phenylhydroxylamine to aniline. Results indicate that isolated rat hepatocytes are a reliable and sensitive system to demonstrate N-oxygenated metabolites of aromatic amino- and reduction of nitrocompounds.

Bioactivation and covalent binding of halothane to liver macromolecules.

In this manuscript we report our attempts to determine if 14C-halothane or its metabolites interact with DNA. Three bioactivation systems were used: in vitro microsomal incubations, isolated hepatocytes, and in vivo administration. Even though we used optimal conditions for bioactivation, no significant covalent binding of 14C to DNA was observed. Slight 14C activity above background (6 dpm/0.1 mg DNA) was observed in the microsomal incubations but is considered insignificant because it was not reduced when NADPH was omitted from the incubations. We are able to demonstrate covalent binding to nuclear lipids and proteins when rats were pretreated with phenobarbital and maintained in a hypoxic environment (14% O2). Similarly, these conditions markedly increased covalent binding of 14C from 14C-halothane to microsomal proteins and lipids. Isolated rat hepatocytes proved to be a viable system for studying the bioactivation of halothane. In this system it was also possible to demonstrate increased binding under N2 and/or phenobarbital pretreatment.

Studies on gluconeogenesis and protein synthesis in isolated hepatocytes.

Glucose production was studied in isolated hepatocytes using various substrates and with increasing substrate concentrations (0-10 mM). Fructose was the best gluconeogenic substrate while other substrates studied stimulated net glucose production in the following decreasing order: lactate, pyruvate, glycerol, galactose, alanine, and succinate. Studies on oxygen consumption showed that endogenous respiration was linear for 60 min and was not altered by extracellular calcium. Studies on the incorporation of 14C-leucine into protein was linear for only 3-4 hr in cells containing low glycogen. However, cells containing high glycogen incorporated 14C-leucine into protein linearly for 8-10 hr. About 3 mg of protein per g per hr was synthesized by isolated cells when incubated for 4 hr with amino acids mixture, glucose, lactate, and insulin.