Urea synthesis in freshly isolated and in cultured periportal and perivenous hepatocytes.
Ontology highlight
ABSTRACT:
SUBMITTER: Poso AR
PROVIDER: S-EPMC1147276 | biostudies-other | 1986 Oct
REPOSITORIES: biostudies-other
Similar Datasets
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: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:Intact rat liver cells from the perivenous region were isolated by collagenase perfusion after first destroying the periportal region by a brief portal infusion of digitonin. Periportal cells were isolated after retrograde digitonin infusion. Significantly higher alanine aminotransferase, gamma-glutamyltransferase and lactate dehydrogenase activities and lower glutamate dehydrogenase and pyruvate kinase activities in periportal than in perivenous cells demonstrate marked separation. The high yield allows further characterization in vitro of the cell populations.
| S-EPMC1145048 | 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: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: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:Metabolites are involved in a diverse range of intracellular processes, including a cell's response to a changing extracellular environment. Using single-cell capillary electrophoresis coupled to electrospray ionization mass spectrometry, we investigated how placing individual identified neurons in culture affects their metabolic profile. First, glycerol-based cell stabilization was evaluated using metacerebral neurons from Aplysia californica; the measurement error was reduced from ∼24% relative standard deviation to ∼6% for glycerol-stabilized cells compared to those isolated without glycerol stabilization. In order to determine the changes induced by culturing, 14 freshly isolated and 11 overnight-cultured neurons of two metabolically distinct cell types from A. californica, the B1 and B2 buccal neurons, were characterized. Of the more than 300 distinctive cell-related signals detected, 35 compounds were selected for their known biological roles and compared among each measured cell. Unsupervised multivariate and statistical analysis revealed robust metabolic differences between these two identified neuron types. We then compared the changes induced by overnight culturing; metabolite concentrations were distinct for 26 compounds in the cultured B1 cells. In contrast, culturing had less influence on the metabolic profile of the B2 neurons, with only five compounds changing significantly. As a result of these culturing-induced changes, the metabolic composition of the B1 neurons became indistinguishable from the cultured B2 cells. This observation suggests that the two cell types differentially regulate their in vivo or in vitro metabolomes in response to a changing environment.
| S-EPMC3474288 | biostudies-literature
Project description:1. In hepatocytes isolated from 24 h-starved rats, urea production from ammonia was stimulated by addition of lactate, in both the presence and the absence of ornithine. The relationship of lactate concentration to the rate of urea synthesis was hyperbolic. 2. Other glucose precursors also stimulated urea production to varying degrees, but none more than lactate. Added oleate and butyrate did not stimulate urea synthesis. 3. Citrulline accumulation was largely dependent on ornithine concentration. As ornithine was increased from 0 to 40 mM, the rate of citrulline accumulation increased hyperbolically, and was half-maximal when ornithine was 8-12 mM. 4. The rate of citrulline accumulation was independent of the presence of lactate, but with pyruvate the rate increased. 5. The rate of urea production continued to increase as ornithine was varied from 0 to 40 mM. 6. It was concluded that intermediates provided by both ornithine and lactate are limiting for urea production from ammonia in isolated liver cells. It was suggested that the stimulatory effect of lactate lies in increased availability of cytosolic aspartate for condensation with citrulline.
| S-EPMC1164223 | 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:Bovine mammary function at molecular level is often studied using mammary tissue or primary bovine mammary epithelial cells (pbMECs). However, bulk tissue and primary cells are heterogeneous with respect to cell populations, adding further transcriptional variation in addition to genetic background. Thus, understanding of the variation in gene expression profiles of cell populations and their effect on function are limited. To investigate the mononuclear cell composition in bovine milk, we analyzed a single-cell suspension from a milk sample. Additionally, we harvested cultured pbMECs to characterize gene expression in a homogeneous cell population. Using the Drop-seq technology, we generated single-cell RNA datasets of somatic milk cells and pbMECs. The final datasets after quality control filtering contained 7,119 and 10,549 cells, respectively. The pbMECs formed 14 indefinite clusters displaying intrapopulation heterogeneity, whereas the milk cells formed 14 more distinct clusters. Our datasets constitute a molecular cell atlas that provides a basis for future studies of milk cell composition and gene expression, and could serve as reference datasets for milk cell analysis.
| S-EPMC8282601 | biostudies-literature