Digitonin-collagenase perfusion for efficient separation of periportal or perivenous hepatocytes.
Ontology highlight
ABSTRACT:
SUBMITTER: Lindros KO
PROVIDER: S-EPMC1145048 | biostudies-other | 1985 Jun
REPOSITORIES: biostudies-other
Similar Datasets
Project description:A technique is described which allows preparations of hepatocytes, enriched in either periportal or perivenous hepatocytes ('PP-cells' and 'PV-cells' respectively), in a yield of about 30-50% compared with control cell preparations. The liver is first perfused for 40-60s with digitonin (4 mg/ml) to destroy selectively either the periportal or the perivenous part of the microcirculatory unit, and then the remaining hepatocytes are isolated by the ordinary collagenase perfusion technique. In periportal cells the activities of alanine aminotransferase and pyruvate kinase were 29.4 and 18.7 mumol/min per mg of DNA respectively. The rate of gluconeogenesis was 0.402 mumol/min per mg of DNA. In perivenous cells the corresponding values were 9.55, 22.1 and 0.244 mumol/min per mg of DNA respectively. These data support the concept of a zonation of glucose metabolism within the microcirculatory unit of the liver, with the afferent part (periportal zone) having a 2-fold, more active gluconeogenesis than the efferent part (perivenous zone).
| S-EPMC1145170 | biostudies-other
Project description:The expression of albumin and alpha 1-inhibitor 3 genes was investigated in rat cell suspensions enriched in periportal (n = 10) and perivenous (n = 10) hepatocytes obtained by the digitonin-collagenase technique. The degree of enrichment of the cell suspensions was assessed: (1) by enzymic assays for the periportal marker alanine aminotransferase and for the perivenous marker glutamine synthetase; and (2) by their content of mRNAs for the periportal marker hepatic glutaminase and for glutamine synthetase. The existence of an antegrade intra-lobular gradient for albumin and alpha 1-inhibitor 3 mRNAs was demonstrated, with periportal:perivenous ratios of 2.33 and 3.80, respectively. However, no gradient was demonstrated for the respective protein contents with corresponding ratios of 0.98 and 1.21. A certain degree of overlap existed between periportal and perivenous suspensions for their content in albumin and alpha 1-inhibitor 3 mRNAs. A morphometrical analysis of the surface of digitonin-permeabilized hepatic tissue revealed that this overlap could be explained by a variable extent of permeabilization of the mediolobular zone from one rat to another and from one lobule to another in a given animal. These results suggest that while the digitonin-collagenase technique is well suited for studies in vitro of proteins expressed in sharp intra-lobular gradients or restricted to an intra-lobular compartment, it is not completely reliable for proteins distributed in continuous moderate intra-lobular gradients, such as albumin and alpha 1-inhibitor 3.
| S-EPMC1136458 | biostudies-other
Project description:A previously described digitonin-perfusion technique [Quistorff, Grunnet & Cornell (1985) Biochem. J. 226, 289-297], by which intracellular material of rat liver could be liberated, has been refined, now allowing release of cytosol of high purity from both periportal and perivenous parts of the same liver. The cytosolic fractions are obtained by perfusing the liver for short intervals (10-20 s) with digitonin (4-5 mg/ml), first in the normal perfusion direction and then, after an interval of 1-2 min, in the retrograde direction, the eluate being collected during and after both intervals. The technique is termed 'dual-digitonin-pulse perfusion'. The eluate fractions showed a peak specific activity of the cytosolic enzymes alanine aminotransferase (ALAT), lactate dehydrogenase (LDH) and pyruvate kinase (PK) of 3-5-fold higher than obtained in a biopsy from the same liver. For glutamine synthetase (GS) a 10-fold higher specific activity was obtained. Zonation, defined as the ratio of the specific activities in periportal and perivenous eluates, of ALAT, LDH and PK was 10, 1.7 and 0.70 respectively. Zonation of GS was less than 0.01. These factors may be modified by a slight zonation of cytosolic protein of 1.2-1.3. Peak concentrations in the eluate of ATP, ADP, Pi, NAD+ and glycerol 3-phosphate were 32.5 +/- 11.4, 19.9 +/- 4.3, 71.9 +/- 25.4, 2.41 +/- 0.83 and 6.84 +/- 2.74 nmol/mg of protein for periportal eluates. There was no difference between periportal and perivenous eluates except for glycerol 3-phosphate, which was significantly higher in perivenous eluates, 12.8 +/- 4.5 nmol/mg of protein.
| S-EPMC1147818 | biostudies-other
Project description:Periportal hepatocytes isolated by digitonin/collagenase perfusion produced urea faster than did similarly prepared perivenous hepatocytes, in both the presence and the absence of amino acids and various urea precursors. There was no difference between the two cell types in rates of intracellular proteolysis. The initial difference in urea synthesis persisted for 5 days during primary culture, but then gradually disappeared. Our results demonstrate that the periportal dominance of urea formation is unrelated to the currently existing acinar microenvironment in the intact liver, but probably reflects differences in acinar key enzyme activities only slowly converging during culture.
| S-EPMC1147276 | biostudies-other
Project description:The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.
| S-EPMC1135093 | biostudies-other
Project description:Perivenous and periportal hepatocytes were isolated by the digitonin/collagenase perfusion technique. The specific activity of phosphate-activated glutaminase was 2.33-fold higher in periportal cells than in perivenous cells. Similarly, the relative abundance of glutaminase mRNA was 2.6-fold higher in samples from periportal cells. The distribution of glutaminase activity and mRNA was compared with those for glutamine synthetase (predominantly perivenous) and phosphoenolpyruvate carboxykinase (predominantly periportal). The results suggest that phosphate-activated glutaminase is predominantly expressed in the periportal zone of the liver acinus.
| S-EPMC1131276 | biostudies-other
Project description:The uptake and metabolism of 35S-labelled sulphur amino acids were compared in periportal (PP) and perivenous (PV) rat hepatocytes, isolated by digitonin/collagenase perfusion, to identify the factors underlying the previously observed [Kera, Penttilä & Lindros, Biochem. J. (1988) 254, 411-417] higher rate of GSH replenishment in PP cells. The buthionine sulphoximine-inhibitable synthesis of GSH was faster in PP than in PV hepatocytes with both cysteine (6.1 versus 5.0 mumol/h per g of cells) and methionine (4.5 versus 3.3 mumol/h per g) as well as with endogenous precursors and L-2-oxo-4-thiazolidinecarboxylate as substrates. However, the uptake of cysteine by PP cells was slower than by PV cells (8.6 versus 10.3 mumol/h per g of cells), whereas methionine was taken up at similar rates. The activity of gamma-glutamylcysteine synthetase (GCS) was slightly higher in digitonin lysates from the PP than from the PV zone. Production of sulphate, the major catabolite of [35S]cysteine sulphur, as well as incorporation of the label into protein occurred at similar rates in PP and PV cells. Taurine, on the other hand, was produced from [35S]cysteine much faster by PV than by PP cells (0.7 versus 0.1 mumol/h per g of cells). Accordingly, the taurine content of PV hepatocytes tended to be higher and to increase faster during incubation with methionine. These results imply that metabolism of taurine is highly zonated within the acinus. They also suggest that both the slightly lower GCS activity and the fast metabolism of cysteine to taurine limit the capacity of PV hepatocytes to synthesize GSH.
| S-EPMC1131638 | biostudies-other
Project description:Glycogen synthesis in hepatocyte cultures is dependent on: (1) the nutritional state of the donor rat, (2) the acinar origin of the hepatocytes, (3) the concentrations of glucose and gluconeogenic precursors, and (4) insulin. High concentrations of glucose (15-25 mM) and gluconeogenic precursors (10 mM-lactate and 1 mM-pyruvate) had a synergistic effect on glycogen deposition in both periportal and perivenous hepatocytes. When hepatocytes were challenged with glucose, lactate and pyruvate in the absence of insulin, glycogen was deposited at a linear rate for 2 h and then reached a plateau. However, in the presence of insulin, the initial rate of glycogen deposition was increased (20-40%) and glycogen deposition continued for more than 4 h. Consequently, insulin had a more marked effect on the glycogen accumulated in the cell after 4 h (100-200% increase) than on the initial rate of glycogen deposition. Glycogen accumulation in hepatocyte cultures prepared from rats that were fasted for 24 h and then re-fed for 3 h before liver perfusion was 2-fold higher than in hepatocytes from rats fed ad libitum and 4-fold higher than in hepatocytes from fasted rats. The incorporation of [14C]lactate into glycogen was 2-4-fold higher in periportal than in perivenous hepatocytes in both the absence and the presence of insulin, whereas the incorporation of [14C]glucose into glycogen was similar in periportal and perivenous hepatocytes in the absence of insulin, but higher in perivenous hepatocytes in the presence of insulin. Rates of glycogen deposition in the combined presence of glucose and gluconeogenic precursors were similar in periportal and perivenous hepatocytes, whereas in the presence of glucose alone, rates of glycogen deposition paralleled the incorporation of [14C]glucose into glycogen and were higher in perivenous hepatocytes in the presence of insulin. It is concluded that periportal and perivenous hepatocytes utilize different substrates for glycogen synthesis, but differences between the two cell populations in the relative utilization of glucose and gluconeogenic precursors are dependent on the presence of insulin and on the nutritional state of the rat.
| S-EPMC1131100 | biostudies-other
Project description:Hydrodynamic tail vein injection (HTV) is the "gold standard" for delivering naked DNA vectors to mouse liver, thereby transfecting predominately perivenous hepatocytes. While HTV corrects metabolic liver defects such as phenylketonuria or cystathionine β-synthase deficiency, correction of spf ash mice with ornithine transcarbamylase (OTC) deficiency was not possible despite overexpression in the liver, as the OTC enzyme is primarily expressed in periportal hepatocytes. To target periportal hepatocytes, we established hydrodynamic retrograde intrabiliary injection (HRII) in mice and optimized minicircle (MC) vector delivery using luciferase as a marker gene. HRII resulted in a transfection efficiency below 1%, 100-fold lower than HTV. While HRII induced minimal liver toxicity compared with HTV, overexpression of luciferase by both methods, but not of a natural liver-specific enzyme, elicited an immune response that led to the elimination of luciferase expression. Further testing of MC vectors delivered via HRII in spf ash mice did not result in sufficient therapeutic efficacy and needs further optimization and/or selection of the corrected cells. This study reveals that luciferase expression is toxic for the liver. Furthermore, physical delivery of MC vectors via the bile duct has the potential to treat defects restricted to periportal hepatocytes, which opens new doors for non-viral liver-directed gene therapy.
| S-EPMC9630613 | biostudies-literature
Project description:Compensatory proliferation triggered by hepatocyte loss is required for liver regeneration and maintenance but also promotes development of hepatocellular carcinoma (HCC). Despite extensive investigation, the cells responsible for hepatocyte restoration or HCC development remain poorly characterized. We used genetic lineage tracing to identify cells responsible for hepatocyte replenishment following chronic liver injury and queried their roles in three distinct HCC models. We found that a pre-existing population of periportal hepatocytes, located in the portal triads of healthy livers and expressing low amounts of Sox9 and other bile-duct-enriched genes, undergo extensive proliferation and replenish liver mass after chronic hepatocyte-depleting injuries. Despite their high regenerative potential, these so-called hybrid hepatocytes do not give rise to HCC in chronically injured livers and thus represent a unique way to restore tissue function and avoid tumorigenesis. This specialized set of pre-existing differentiated cells may be highly suitable for cell-based therapy of chronic hepatocyte-depleting disorders.
| S-EPMC4545590 | biostudies-literature