The isolation of hormone-sensitive rat hepatocytes by a modified enzymatic technique
Abstract
Hepatocytes that are similar to the perfused liver in glucagon sensitivity can be obtained in a high, reproducible yield by modifications of the well-known enzymatic technique for the preparation of isolated liver cells. The major modifications are: (a) a simple, economic, and temperature-controlled apparatus for the recirculating perfusion of the isolated rat liver; (b) the use of substrate-fortified calcium-free Krebs-Henseleit bicarbonate buffer; and (c) high perfusion rates, which lead to the isolation of hepatocytes with normal ultrastructure and metabolic activities.
From 4 × 108 to 5 × 108 cells can be routinely isolated from an 8- to 10-g liver independent of the collagenase preparations applied. The rat liver cells are viable (90–95%) by various criteria including electron microscopy and exclusion of 0.2% trypan blue. When studying various incubation techniques, it was observed that the use of gelatin in the medium is preferred as compared to albumin Fraction V or fatty acid-free albumin which tended to inhibit gluconeogenic rates from various substrates in calcium-free medium. Addition of calcium chloride to the incubation medium strikingly improved gluconeogenesis from lactate. Various procedures for calculating the number of cells corresponding to 1 g wet liver tissue are discussed in detail.
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Response to comment on "Determination of the hepatocellularity number for human, dog, rabbit, rat and mouse livers from protein concentration measurements"
2007, Toxicology in VitroDetermination of the hepatocellularity number for human, dog, rabbit, rat and mouse livers from protein concentration measurements
2006, Toxicology in VitroBiologically based scaling factors have to be used to predict in vivo metabolic clearance of xenobiotics from data obtained in vitro. Although standard values for the hepatocellularity numbers for different species are used in the literature, detailed information on the determination of these values has only been presented for humans and rats, and somewhat different results have been obtained in different studies. The present work was undertaken in order to determine the number of hepatocytes per gram of liver for human, dog, rabbit, rat and mouse livers. Hepatocellularity numbers were calculated from the ratio between the liver protein concentration and the protein concentration in the corresponding hepatocyte suspension. For human, rabbit, rat and mouse livers, the hepatocellular values were in the same range, more precisely 139 ± 25, 114 ± 20, 117 ± 30 and 135 ± 10 million cells per gram of liver, respectively. However, for the dog liver, the corresponding value was as high as 215 ± 45 million cells per gram. These values should be of importance during the scaling process of intrinsic clearance for xenobiotics in hepatocytes to in vivo hepatic clearance.
An integrated approach to model hepatic drug clearance
2006, European Journal of Pharmaceutical SciencesIt has been well accepted that hepatic drug extraction depends on the blood flow, vascular binding, transmembrane barriers, transporters, enzymes and cosubstrate and their zonal heterogeneity. Models of hepatic drug clearances have been appraised with respect to their utility in predicting drug removal by the liver. Among these models, the “well-stirred” model is the simplest since it assumes venous equilibration, with drug emerging from the outflow being in equilibrium with drug within the liver, and the concentration is the same throughout. The “parallel tube” and dispersion models, and distributed model of Goresky and co-workers have been used to account for the observed sinusoidal concentration gradient from the inlet and outlet. Departure from these models exists to include heterogeneity in flow, enzymes, and transporters. This article utilized the physiologically based pharmacokinetic (PBPK) liver model and its extension that include heterogeneity in enzymes and transporters to illustrate how in vitro uptake and metabolic data from zonal hepatocytes on transport and enzymes may be used to predict the kinetics of removal in the intact liver; binding data were also necessary. In doing so, an integrative platform was provided to examine determinants of hepatic drug clearance. We used enalapril and digoxin as examples, and described a simple liver PBPK model that included transmembrane transport and metabolism occurring behind the membrane, and a zonal model in which the PBPK model was expanded three sets of sub-compartments that are arranged sequentially to represent zones 1, 2, and 3 along the flow path. The latter model readily accommodated the heterogeneous distribution of hepatic enzymes and transporters. Transport and metabolic data, piecewise information that served as initial estimates, allowed for the unknown efflux and other intrinsic clearances to be estimated. The simple or zonal PBPK model provides predictive views on the hepatic removal of drugs and metabolites.
Hepatoprotective mechanisms of Yan-gan-wan
2005, Hepatology ResearchA herbal prescription, Yan-gan-wan (YGW), has been known to offer hepatoprotective effects in Asian countries for years. This study investigated its mechanisms of action.
The effects of YGW on CCl4 induced liver damage were tested in mice and cultured hepatocytes. Microarray analysis screened genes affected by YGW. YGWs effects on the expression of cytochrome P450 (CYP) 2E1 and other isozymes were determined. YGWs effects on TNFα expression and NF-κB activation in Kupffer cells (KC), and TNFα promoter activity in RAW264.7 cells, were also assessed.
Administration of YGW reduced the plasma ALT, centrilobular necrosis, neutrophilic infiltration, and TNFα mRNA in the livers of mice acutely given CCl4. The in vivo herb treatment reduced ALT release and necrosis of isolated hepatocytes directly exposed to CCl4. Microarray analysis demonstrated marked reductions in CYP4A10 and 4A14 by YGW but no changes in other CYP isozymes as confirmed by immunoblot analysis. The herb treatment suppressed LPS-stimulated TNFα release in vivo and by cultured KC. Direct addition of the aqueous herb extract suppressed NF-κB activation by KC and TNFα promoter activity in RAW cells under LPS stimulation. This activity to suppress TNFα expression was largely separated into gel filtration fractions with the molecular size of 102–107 Da. YGW also attenuated liver fibrosis induced by chronic treatment of CCl4 or porcine serum.
The protective effects of YGW on CCl4 hepatotoxicity are due in part to inhibition of KC NF-κB activation and TNFα expression by small water soluble molecules, and may also be related to suppressed hepatic expression of CYP4A10 and 4A14 that are considered as alternative prooxidant cytochromes.
Thyroid hormone and dehydroepiandrosterone permit gluconeogenic hormone responses in hepatocytes
2000, Archives of Biochemistry and BiophysicsThe importance of the sn-glycerol- 3-phosphate (G-3-P) electron transfer shuttle in hormonal regulation of gluconeogenesis was examined in hepatocytes from rats with decreased mitochondrial G-3-P dehydrogenase activity (thyroidectomized) or increased G-3-P dehydrogenase activity [triiodothyronine (T3) or dehydroepiandrosterone (DHEA) treated]. Rates of glucose formation from 10 mM lactate, 10 mM pyruvate, or 2.5 mM dihydroxyacetone were somewhat less in hypothyroid cells than in cells from normal rats but gluconeogenic responses to calcium addition and to norepinephrine (NE), glucagon (G), or vasopressin (VP) were similar to the responses observed in cells from normal rats. However, with 2.5 mM glycerol or 2.5 mM sorbitol, substrates that must be oxidized in the cytosol before conversion to glucose, basal gluconeogenesis was not appreciably altered by hypothyroidism but responses to calcium and to the calcium-mobilizing hormones were abolished. Injecting thyroidectomized rats with T3 2 days before preparing the hepatocytes greatly enhanced gluconeogenesis from glyc erol and restored the response to Ca2+ and gluconeogenic hormones. Feeding dehydroepiandrosterone for 6 days depressed gluconeogenesis from lactate or pyruvate but substantially increased glucose production from glycerol in euthyroid cells and restored responses to Ca2+ in hypothyroid cells metabolizing glycerol. Euthyroid cells metabolizing glycerol or sorbitol use the G-3-P and malate/aspartate shuttles to oxidize excess NADH generated in the cytosol. The transaminase inhibitor aminooxyacetate (AOA) decreased gluconeogenesis from glycerol 40%, but had little effect on responses to Ca2+ and NE. However, in hypothyroid cells, with minimal G-3-P dehydrogenase, AOA decreased gluconeogenesis from glycerol more than 90%. Thus, the basal rate of gluconeogenesis from glycerol in the euthyroid cells is only partly dependent on electron transport from cytosol to mitochondria via the malate/aspartate shuttle and almost completely dependent in the hypothyroid state, and the hormone enhancement of the rate in euthyroid cells involves primarily the G-3-P cycle. These data are consistent with Ca2+ being mobilized by gluconeogenic hormones and G-3-P dehydrogenase being activated by Ca2+ so as to permit it to transfer reducing equivalents from the cytosol to the mitochondria.
Effects of 2',3'-dideoxycytidine and 2',3'-dideoxycytidine 5'-triphosphate on phospholipid metabolism in permeabilized rat hepatocytes
1997, Biochemical PharmacologyBoth 2′,3′-dideoxycytidine (ddC) and 2′,3′-dideoxycytidine 5′-triphosphate (ddCTP) inhibit the synthesis of the major phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in permeabilized rat hepatocytes. For PC, this appears to be based on competitive inhibition of cholinephosphotransferase (CDPcholine:l,2-diacylglycerol cholinephosphotransferase; EC 2.7.8.2). The study was based on short-term incubations (6–12 min) of the nucleoside/nucleotide analogs with α-toxin permeabilized rat hepatocytes. At a concentration of 1 mM, ddC and ddCTP decreased the incorporation of radiolabelled glycerol-3-phosphate into PC by approximately 50% as compared with control. This was accompanied by a significant increase in diacylglycerol labelling. In the presence of 1 mM CDP-ethanolamine and increasing concentrations of ddC(TP) (0.01–1 mM), the incorporation of radiolabelled glycerol-3-phosphate into PE was decreased to approximately 60% of the control value. When both PC and PE synthesis were operative, the inhibition by ddC(TP) was restricted to PC synthesis. ddC and ddCTP were found to have inhibition constants (Ki) of 496 μM and 452 μM, respectively, for the inhibition of PC synthesis from CDP-choline. Although the inhibitory concentrations of the nucleoside analog and its triphosphate ester are much higher than the in vivo plasma concentrations, the possibility is raised that the peripheral neuropathy, seen as a dose-dependent adverse effect of ddC treatment in acquired immunodeficiency syndrome therapy is, at least partly, caused by a perturbation of the phospholipid constitution of neuronal membranes.