D-galactosamine-induced cell damage enhances specific binding of prostaglandin E1 to rat hepatocytes.

Specific bindings of [3H]prostaglandin E1 ([3H]PGE1), 125I-glucagon and [3H]norepinephrine to D-galactosamine (GalN)-treated rat hepatocytes in primary culture were investigated. After a two hour-treatment with GalN (1 and 10mg/ml), hepatocytes showed an enhanced specific binding to [3H]PGE1, whereas 125I-glucagon binding was little affected and [3H]norepinephrine binding was strongly diminished. Scatchard plot analysis indicated an increase of binding sites of [3H]PGE1. This unusual manner of [3H]PGE1 binding is suggested to indicate a special property of PGE1 receptor and may be associated with the cytoprotective effect of prostaglandins.     

Apoptosis vs. necrosis: glutathione-mediated cell death during rewarming of rat hepatocytes

Hypothermia induces injury in its own right, but the mechanisms involved in the cell damage are still unclear. The aim of this study was to test the effects that glutathione (GSH) depletion induces on cell death in isolated rat hepatocytes, kept at 4 degrees C for 20 h, by modulating intracellular GSH concentration with diethylmaleate and buthionine sulfoximine (DEM and BSO). Untreated hepatocytes showed Annexin V stained cells (AnxV(+)), scarce propidium iodide stained cells (PI(+)) and presented a low level of lactate dehydrogenase (LDH) leakage after 20 h at 4 degrees C and rewarming at 37 degrees C. When DEM and BSO were added before cold storage, we observed a few AnXV(+) cells and an increase in PI(+) cells associated with LDH release in the incubation medium. Conversely, the addition of DEM and BSO only during rewarming caused a marked increase in cell death by apoptosis. Production of reactive oxygen species (ROS) and thiobarbituric acid species (TBARS), associated with a decrease in GSH concentrations, was higher when DEM and BSO were added before cold storage. Cells treated with DEM and BSO before cold storage showed lower ATP energy stores than hepatocytes treated with DEM and BSO only during rewarming. Pretreatment of hepatocytes with deferoxamine protected against apoptotic and necrotic morphology in conditions of GSH depletion. These results suggest that pretreatment of hepatocytes with DEM and BSO before cold storage induces necrosis, while the treatment of hepatocytes only during rewarming increases apoptosis. In both conditions, iron represents a crucial mediator of cell death.     

氟对大鼠肝组织细胞凋亡与DNA损伤的影响

目的研究大鼠染氟后肝组织细胞凋亡及DNA损伤情况。方法 SD大鼠随机分为对照组、低氟组、中氟组、高氟组,每组12只,分别饮用含氟化钠为0、50、100、200 mg/L的去离子水,均饲标准营养大鼠饲料,染氟120 d。肉眼观察牙齿的变化,采用氟离子选择电极法测定大鼠尿氟,HE染色观察组织病理学变化,彗星实验检测细胞DNA损伤,流式细胞术检测肝脏组织细胞凋亡率。结果低氟组、中氟组、高氟组大鼠尿氟分别为（23.52±2.91）、（30.16±4.78）、（61.23±3.98）mg/L,均显著高于对照组（0.07±0.02）mg/L,差异有统计学意义（P〈0.01）。不同剂量染氟大鼠肝组织细胞呈现不同程度肿胀,肝组织内出现多种灶状病变。各染氟组大鼠肝细胞拖尾率及拖尾长度与相应的对照组相比,差异均有统计学意义,并且肝细胞拖尾率及拖尾长度随染氟剂量的加大而增大。不同剂量染氟组细胞凋亡率与对照组相比,均明显增高,而且高、中氟组肝细胞凋亡率显著高于低氟组（P〈0.01）。结论氟化物可导致大鼠肝细胞DNA损伤,诱导细胞凋亡,一定浓度的氟化物诱导大鼠肝细胞凋亡与DNA损伤之间存在着相关性。     

Responsiveness of RNA degradation to amino acids in cultured rat hepatocytes: comparison with isolated rat hepatocytes.

The role of amino acids in the regulation of RNA degradation was investigated in cultured hepatocytes from fed rats previously labeled in vivo with [6-14C]orotic acid. Rates of RNA degradation were determined between 42 and 48 h of culture from the release of radioactive cytidine in the presence of 0.5 mM unlabeled cytidine. The fractional rate was about 4.4 +/- 0.4%/h in the absence of amino acids (0x). The catabolism of RNA was decreased to basal level (1.5 +/- 0.3%/h) by the addition of amino acids at 10 times normal plasma concentration (10x). The inhibition of RNA degradation, expressed as percentage of maximal deprivation-induced response (0x minus 10x), averaged 60% at normal plasma levels of amino acids. The degree of responsiveness was greatly improved as compared to freshly isolated hepatocytes (20%) and was similar to the sensitivity previously observed with perfused livers. In cultured hepatocytes, the sensitivity of RNA degradation to amino acids was not affected by varying the volume of medium from 1 to 4 ml per dish. In freshly isolated hepatocytes, the inhibitory effect of amino acids was not modified by changing the cell density from 0.5 to 5 x 10(6) cells per ml. In the range of normal plasma concentration of amino acids, the low sensitivity of RNA degradation in isolated hepatocytes persisted with inhibition ranging from 10 to 20%. These findings suggest that the control of RNA degradation in both cultured and isolated hepatocytes is not affected by the total quantity of amino acids available in the medium, but their concentration is crucial. Electron microscopy observations and the inhibitory effect of 3-methyl-adenine in cultured rat hepatocytes partially confirmed the role of the lysosomal system in the increase of RNA degradation and its regulation by amino acids.     

Influence of the culture time of DNA damage and repair in isolated rat hepatocytes exposed to nitochlorobenzene derivatives

The induction of DNA damage on 1.5 and 24-h cultured hepatocytes was tested after a 3-h exposure to 5 and 50 μM mono-, and trinitrochlorobenzene (100-00-5; 97-00-7; 88-88-0). DNA-repair synthesis, elicited by nitrochlorobenzene treatment, was also estimated 24 and 48 h after the withdrawal of the nitro-aryl halides. DNA damage and repair were evaluated by determining the DNA elution rate in alkali. A dose-related rate of DNA damage was obtained by exposure of 1.5-h-cultured hepatocytes to 5 and 50 μM nitrochlorobenzenes. DNA of 24-h-cultured cells was not affected by nitrochlorobenzene treatment. The data obtained by exposure to 5 μM methyl methanesulfate (66-27-3) and nitrosodimethylamine (62-75-9), direct and indirect methylating agents, suggest that 24-h-cultured liver cells are still able to transform nitrosodimethylamine but not nitrochlorobenzenes.Isolated hepatocytes maintain their capability of repairing the induced DNA damage when cultured for 24 and 48 h in fresh medium. The system offers an interesting model to investigate the perturbations related to the metabolism of xenobiotics.     

Calcium metabolism in rat hepatocytes.

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.     

Metabolic activity in isolated hepatocytes from cold exposed rats subjected to 24-hour fasting.

The aim of this work was to study the metabolic activity in isolated hepatocytes from control rats and rats exposed for 15 or 30 days to cold, all subjected to 24-h fasting. Hepatocyte oxygen consumption was used as an index of metabolic activity. The results show that 24-h fasting induces a decrease in energy expenditure at the level of the liver in cold-exposed rats but not in control animals.     

Influence of the culture time on DNA damage and repair in isolated rat hepatocytes exposed to nitrochlorobenzene derivatives

The induction of DNA damage on 1.5- and 24-h cultured hepatocytes was tested after a 3-h exposure to 5 and 50 microM mono-, di-, and trinitrochlorobenzene (100-00-5; 97-00-7; 88-88-0). DNA-repair synthesis, elicited by nitrochlorobenzene treatment, was also estimated 24 and 48 h after the withdrawal of the nitro-aryl halides. DNA damage and repair were evaluated by determining the DNA elution rate in alkali. A dose-related rate of DNA damage was obtained by exposure of 1.5-h-cultured hepatocytes to 5 and 50 microM nitrochlorobenzenes . DNA of 24-h-cultured cells was not affected by nitrochlorobenzene treatment. The data obtained by exposure to 5 microM methyl methanesulfonate (66-27-3) and nitrosodimethylamine (62-75-9), direct and indirect methylating agents, suggest that 24-h-cultured liver cells are still able to transform nitrosodimethylamine but not nitrochlorobenzenes . Isolated hepatocytes maintain their capability of repairing the induced DNA damage when cultured for 24 and 48 h in fresh medium. The system offers an interesting model to investigate the perturbations related to the metabolism of xenobiotics.     

Purpurogallin protects both ventricular myocytes and aortic endothelial cells of rats against oxyradical damage.

Rat ventricular myocytes have been isolated and cultured by two separate procedures. Using phase-contrast and electron microscopies, we illustrate that (a) definitive cell damage is produced when myocytes are exposed to xanthine oxidase--hypoxanthine and (b) purpurogallin between 0.25 and 1.0 mM prolongs survival of both myocyte preparations in a dose-dependent manner. The cytoprotection produced by 1 mM purpurogallin exceeds that given by 2 mM each of ascorbate, Trolox, and mannitol, or 24,200 IU superoxide dismutase/L and (or) 92,000 IU catalase/L. Furthermore, we noted, for the first time, that purpurogallin markedly protects rat aortic endothelial cells, a key target of free radical generation and attack. In contrast, Trolox has a negligible effect here. Mechanistically, we showed that purpurogallin inhibits urate formation by xanthine oxidase more potently than allopurinol. Also, the compound diminishes formation of superoxide-reduced cytochrome c. Therefore, purpurogallin is a potent protector of ventricular myocytes and aortic endothelial cells, both of which are important cells in the cardiovascular system.     

Collagenase perfusion of rat liver induces DNA damage and DNA repair in hepatocytes

Evidence is presented that the collagenase perfusion of adult rat liver results in significant damage to nuclear DNA as evaluated by the alkaline elution technique. The extent of the damage is related to the perfusion time as well as to the clostridial enzyme preparation used. The DNA structure of isolated cells is almost completely repaired within 12 h of their culture in chemically defined medium.     

Adhesion of rat hepatocytes to collagen

Rat hepatocytes, freshly isolated with a collagenase perfusion technique, were found to attach within 1 h on collagen substrates and on culture dishes coated with cold insoluble globulin (CIG) or asialoceruloplasmin (AC). Spreading was observed on collagen and CIG but not on AC. Both attachment and spreading occurred in a simple balanced salt solution in the absence of serum. In the absence of serum no attachment was observed on plain plastic dishes or on dishes coated with serum albumin or other plasma proteins, unless divalent manganese ions were present. In the presence of manganese the hepatocytes attached to all surfaces tested, but no spreading occurred. Attachment to collagen occurred equally well to collagens type I or type III both in the native, fibrillar state and in the denatured state. Collagen attachment required magnesium ions but did not appear to involve the collagen-linked carbohydrates. Different mechanisms were found to operate in hepatocyte attachment to collagen and to AC; the latter is most likely mediated by the hepatocyte surface receptor involved in recognition and uptake of asialoglycoproteins. The role of CIG in hepatocyte attachment to collagen was investigated. Data are presented suggesting that this glycoprotein, which mediates the adhesion of fibroblasts to collagen, is not required for hepatocyte attachment to collagen.     

Metabolism of hypoxanthine in isolated rat hepatocytes.

The hepatic metabolism of hypoxanthine was investigated by studying both the fate of labelled hypoxanthine, added at micromolar concentrations to isolated rat hepatocyte suspensions, and the kinetic properties of purified hypoxanthine/guanine phosphoribosyltransferase from rat liver. More than 80% of hypoxanthine was oxidized towards allantoin; less than 5% of the label was incorporated into the purine mononucleotides, and a similar proportion appeared transiently in inosine. The maximal velocity of oxidation (approx. 750nmol/min per g of cells) was in close agreement with the known activity of xanthine oxidase in liver extracts. In contrast, the maximal velocity of the incorporation of labelled hypoxanthine into mononucleotides reached only 30nmol/min per g of cells, compared with an activity of hypoxanthine/guanine phosphoribosyltransferase, measured at substrate concentrations analogous to those prevailing intracellularly, of 500nmol/min per g of cells. Hypoxanthine incorporation into the mononucleotides was decreased by allopurinol, anoxia and ethanol, despite inhibition of its oxidation under these conditions; it was increased by incubation of the cells in supraphysiological concentrations of Pi. Allopurinol and anoxia decreased the concentration of phosphoribosyl pyrophosphate inside the cells by respectively 40 and 60%, ethanol had no effect on the concentration of this metabolite and Pi increased its concentration up to 10-fold. The kinetic study of purified hypoxanthine/guanine phosphoribosyltransferase showed that a mixture of ATP, IMP, GMP and GTP, at the concentrations prevailing in the liver cell, decreased the V max. of the enzyme 6-fold, increased its Km for hypoxanthine from 1 to 4 microM and its Km for phosphoribosyl pyrophosphate from 2.5 to 25 microM. In the presence of 5 microM-hypoxanthine and 2.5 microM-phosphoribosyl pyrophosphate, the mixture of nucleotides inhibited the activity of purified hypoxanthine/guanine phosphoribosyltransferase by 95%. It is concluded that this inhibition results in a limited participation of hypoxanthine/guanine phosphoribosyltransferase in the control of the production of allantoin by the liver.     

Effects of tumor necrosis factor-alpha on insulin stimulated amino acid transport in cultured rat hepatocytes

It has been suggested that tumor necrosis factor alpha (TNF-alpha) plays a pivotal role in the pathogenesis of insulin resistance. It could act directly or indirectly in liver. The aim of this study was to determine direct short time (4 h) and long time (24 h) action of TNF-alpha on amino acid transport in cultured rat hepatocytes and possible role of protein kinase C (PKC) in insulin signal pathway and insulin resistance. Hepatocytes were isolated by a modified collagenase perfusion technique and cultured for 24 h in M 199 medium. In the presence of insulin basal alpha-amino isobutyric acid (AIB) uptake was increased 55%. TNF-alpha in short time action did not change basal AIB transport, but significantly (25%) increased insulin stimulated uptake. Short time action of TNF-alpha was ameliorated by phorbol ester treatment. These results indicated that PKC activation is important in insulin signaling and TNF-alpha action. TNF-alpha acting directly did not cause insulin resistance in cultured hepatocytes.