Rat Liver Non-parenchymal Cells Research Articles

Binding of concanavalin A and ricin 120 to parenchymal and non-parenchymal cells of rat liver

Binding of concanavalin A and ricin 120 to parenchymal and non-parenchymal cells of rat liver

A separation chamber to sort cells, nuclei, and chromosomes at moderate g forces. II. Studies on velocity sedimentation and equilibrium density centrifugation of mammalian cells

A separation chamber having a surface of 50 cm 2 and a height of 2 cm is described for the rapid separation of cells and cell organelles at acceleration forces from 10 to 90 g. To eliminate wall sedimentation artifacts, the chamber was positioned 20 cm from the rotor axis in a speed-controlled centrifuge. The chamber has flow deflectors for the undisturbed introduction of the sample layer and the gradient; an antivortex cross prevents swirling upon acceleration and deceleration. To illustrate the use of the separation chamber, examples of velocity sedimentation and of equilibrium density centrifugation are given: (i) human monocytes (70% were 90% pure) are separated from lymphocytes in 10 min at 20 g; (ii) nonparenchymal rat liver cells are separated in 10 min at 16 g in 97% pure endothelial cells and 99% pure Kupffer cells; (iii) equilibrium density centrifugation of human peripheral blood cells at about 90 g permits the separation of erythrocytes, monocytes, lymphocytes, neutrophils, eosinophils, and basophils in one run. B cells are separated from T cells. The movement of swinging buckets is analyzed in mathematical terms and a simple method is offered to determine the position of cells in density gradients with the use of a small programmable calculator.

A comparison of the accumulation of ricin by hepatic parenchymal and non-parenchymal cells and its inhibition of protein synthesis

Rat liver non-parenchymal cells in vivo were found to accumulate 125I-labelled ricin to a much greater extent than parenchymal cells. Similarly, in monolayer cell cultures, the rate of ricin uptake by non-parenchymal Kupffer cells was several times that by parenchymal cells. Evidence is provided also to suggest that ricin is primarily recognized by Kupffer cells via terminal mannose residues in the toxin, whereas ricin uptake by parenchymal cells was consistent with a role of previously postulated galactosyl-containing cell receptors. Protein synthesis in Kupffler cells in vitro, although observed to occur at a lower rate than in parenchymal cells was 100–1000-times more sensitive to inhibition by ricin. The selective damage known to be caused to liver sinusoids by ricin, therefore, may reflect both the relative efficiency with the toxins is taken up by these cells and the extreme sensitivity of protein synthesis in the cells to inhibition by ricin.

Uptake and 25-hydroxylation of vitamin D3 by isolated rat liver cells.

The physiological roles played by hepatocytes and nonparenchymal cells of rat liver in the metabolism of vitamin D3 have been investigated. Tritium-labeled vitamin D3 dissolved in ethanol was administered intravenously to two rats. Isolation of the liver cells 30 and 70 min after the injection showed that vitamin D3 had been taken up both by the hepatocytes and by the nonparenchymal liver cells. The relative proportion of vitamin D3 that accumulated in the nonparenchymal cells increased with time. Perfusion of the isolated rat liver with [3H] vitamin D3 added to the perfusate confirmed the ability of both cell types to efficiently take up vitamin D3 from the circulation. By a method based on high pressure liquid chromatography and isotope dilution-mass fragmentography it was found that isolated liver cells in suspension had a considerable capacity to take up vitamin D3 from the medium. About 2.5 fmol of vitamin D3 were found to be associated with each hepatocyte or nonparenchymal cell after 1 h of incubation. 25-Hydroxylation in vitro was found to be carried out only by the hepatocytes. The rate of hydroxylation was about the same whether the cells were isolated from normal or rachitic rats (3.5 and 4 pmol of 25-hydroxyvitamin D3 formed per h per 10(6) cells, respectively). The possibility that the nonparenchymal cells might serve as a storage site for vitamin D3 in the liver is discussed.

Effect of cadmium acetate on the uptake and degradation of formaldehyde-treated 125I-labelIed human serum albumin in rat liver non-parenchymal cells in vitro

Effect of cadmium acetate on the uptake and degradation of formaldehyde-treated 125I-labelIed human serum albumin in rat liver non-parenchymal cells in vitro

Uptake and degradation of rat and human very low density (remnant) apolipoprotein by parenchymal and non-parenchymal rat liver cells

1. 1. The relative contribution of parenchymal and non-parenchymal cells to the in vivo hepatic uptake of serum apolipoproteins was measured 30 min after intravenous injection of radioiodinated rat very low density lipoprotein (VLDL) remnants, rat and human VLDL, low density lipoprotein (LDL) and high density lipoprotein (HDL). Using rat VLDL, VLDL-remnants, LDL and HDL, respectively, the non-parenchymal cells contain 4.7, 4.9, 6.1 and 5.3 times the amount of trichloroacetic acid-precipitable radioactivity per mg cell protein as compared to parenchymal cells. For human VLDL, LDL and HDL these values are 5.1, 12.0 and 5.9, respectively. 2. 2. The abilities of homogenates of human liver, rat liver parenchymal cells and rat liver non-parenchymal cells to hydrolyze human and rat iodinated VLDL apoprotein were determined by measuring the amount of trichloroacetic acid-soluble (non-iodide) radioactivity liberated upon incubation at the optimal pH of 4.2. Non-parenchymal cells possess a 8–21-fold higher maximal capacity to degrade VLDL apoprotein per mg of cell protein than parenchymal cells. This factor is 5–6 for VLDL-remnant apoprotein degradation measured at low (suboptimal) apolipoprotein concentrations. 3. 3. It is concluded that, in addition to parenchymal cells, the non-parenchymal cells play an important role in the hepatic uptake and possibly degradation of VLDL-(remnant) apoprotein.

Properties of binding of lipases to non-parenchymal rat liver cells

The binding of a heparin-releasable acylglycerol hydrolase from rat liver (liver lipase) to non-parenchymal liver cells was studied in vitro. The binding of the partially purified liver lipase to the cells was found to be rapid. Within l min 80% of the maximal binding occurred at either 4 or 25°C. The binding capacity of the cells was saturable, with a maximal binding of 46 mU lipase activity per mg cell protein. Albumin did not prevent the binding and neither did glucose, galactose or methyl- α- mannoside. Mg 2+, but not Ca 2+, promoted the binding of the lipase to the cells. The in vitro bound enzyme activity was releasable from the cells by heparin. Both the soluble and bound liver lipase could be completely inhibited by an antibody against liver lipase. Lipoprotein lipase, derived from rat adipose tissue, was also found to bind preferentially to non-parenchymal liver cells. The presented results show that non-parenchymal liver cells contain 10 5 binding sites per cell for heparin-releasable lipases. Lipase secreted from parenchymal cells in vitro was also bound to the non-parenchymal cells. The data suggest that in vivo the liver lipase bound to endothelial liver cells can be derived from parenchymal cells.

Characteristics of acid lipase and acid cholesteryl esterase activity in parenchymal and non-parenchymal rat liver cells

1.(1) Parenchymal and non-parenchymal cells were isolated from rat liver. The characteristics of acid lipase activity with 4-methylumbelliferyl oleate as substrate and acid cholesteryl esterase activity with cholesteryl[1-14C]oleate as substrate were investigated. The substrates were incorporated in egg yolk lecithin vesicles and assays for total cell homogenates were developed, which were linear with the amount of protein and time. With 4-methylumbelliferyl oleate as substrate, both parenchymal and non-parechymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 2.5 times higher than for parenchymal cells. It is concluded that 4-methylumbelliferyl oleate hydrolysis is catalyzed by similar enzyme(s) in both cell types.2.(2) With cholesteryl[1-14C] oleate as substrate both parenchymal and nonparenchymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 11.4 times higher than for parenchymal cells. It is further shown that the cholesteryl ester hydrolysis in both cell types show different properties.3.(3) The high activity and high affinity of acid cholesteryl esterase from non-parenchymal cells for cholesterol oleate hydrolysis as compared to parenchymal cells indicate a relative specialization of non-parenchymal cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells possess the enzymic equipment to hydrolyze very efficiently internalized cholesterol esters, which supports the suggestion that these cell types are an important site for lipoprotein catabolism in liver.

Isolation of a lipocyte-rich fraction from rat liver nonparenchymal cells.

A modified pronase digestion procedure is described for isolating nonparenchymal liver cells from vitamin A treated rats, which yielded a 15-23% population of lipocytes in the total cell suspension. The criteria for defining a lipocyte was the appearance of vitamin A containing cells as determined by fluorescent microscopy. Measurement of alcohol dehydrogenase and retinol dehydrogenase activities indicated that these enzyme activities were not present in the isolated nonparenchymal cells. A lipocyte-rich fraction of nonparenchymal cells was obtained by centrifugation of purified nonparenchymal cells in a linear Metrizamide gradient. Vitamin A fluorescence and chemical assay of vitamin A in the cell fractions indicated a four-fold enrichment of lipocytes in the cell fraction with d = 1.043 g/ml. Fractions high in vitamin A also had numerous cells with fat droplets as shown by transmission electron microscopy.

Metabolism of ethanol and acetaldehyde in parenchymal and non-parenchymal rat liver cells

Suspensions of isolated parenchymal (P) and non-parenchymal (NP) cells were prepared by collagenase perfusion followed by centrifugation of the primary cell suspension. Suspensions of P cells were able to metabolize ethanol (8–16 nmoles/min/10 6 viable cells) while NP cells did not metabolize ethanol at all. Acetaldehyde was metabolized in P-cell suspensions at rates ranging from 14 to 20 nmoles/min/10 6 viable cells. Some acetaldehyde metabolism also occurred in NP-cell suspensions (0.18–0.33 nmoles/min/10 6 viable cells). In accordance with these studies on ethanol and acetaldehyde metabolism we found alcohol dehydrogenase activity only in homogenates of P cells, and aldehyde dehydrogenase activity in homogenates of P cells was 20 times higher per cell than in homogenates of NP cells. It was concluded that the P cells of rat liver are responsible for ethanol metabolism and probably also responsible for most, if not all metabolism of acetaldehyde arising from ethanol oxidation. Biochemical effects which are consequences of ethanol metabolism are probably not found in NP cells.

Role of parenchymal and non-parenchymal rat liver cells in the uptake of cholesterolester-labeled serum lipoproteins

Very low density lipoprotein (VLDL)-remnants, prepared by extrahepatic circulation of VLDL, labeled biosynthetically in the cholesterol (ester) moiety, were injected intravenously into rats in order to determine the relative contribution of parenchymal and non-parenchymal liver cells to the hepatic uptake of VLDL-remnant cholesterol (esters). 82.7% of the injected radioactivity is present in liver, measured 30 min after injection. The non-parenchymal liver cells contain 3.1±0.1 times the amount of radioactivity per mg cell protein as compared to parenchymal cells. The hepatic uptake of biosynthetically labeled (screened) low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterolesters amounts to 26.8% and 24.4% of the injected dose, measured 6 h after injection. The non-parenchymal cells contain 4.3±0.8 and 4.1±0.7 times the amount of radioactivity per mg cell protein as compared to parenchymal cells for LDL and HDL, respectively. It is concluded that in addition to parenchymal cells, the non-parenchymal cells play an important role in the hepatic uptake of cholesterolesters from VLDL-remnants, LDL and HDL.

Endocytosis of beta-N-acetylglucosaminidase from sections of mucolipidosis-II and-III fibroblasts by non-parenchymal rat liver cells.

beta-N-Acetylglucosaminidase isolated from the secretions of fibroblasts of mucolipidosis-II and -III patients is internalized by cultured non-parenchymal rat liver cells. The rate of endocytosis compared with that of beta-N-acetylglucosaminidase from control fibroblasts was 11 and 19% for the enzyme from mucolipidosis-II and -III patients respectively. The inhibition of endocytosis by mannan indicates that the beta-N-acetylglucosaminidase from mucolipidosis-II and -III patients is recognized by cell-surface receptors specific for mannose.

Incorporation of labelled amino acids into proteins of isolated parenchymal and nonparenchymal rat liver cells in the absence and presence of ethanol

Parenchymal and nonparenchymal cells were isolated from perfused rat livers and incubated at 37°C in the absence and presence of ethanol (50 mM). 1. 1. Nonparenchymal cells prepared by means of centrifugation showed a higher rate of incorporation of l-[U- 14C]valine into protein than nonparenchymal cells prepared by means of pronase. Cells prepared by the former method were used for further studies. 2. 2. Protein degradation was present in suspensions of both parenchymal and nonparenchymal cells evidenced by increasing levels of branched amino acids in the intracellular and extracellular compartment during cell incubation. 3. 3. The rate of cellular protein synthesis (corrected for precursor pool specific radioactivity) was of the same order of magnitude in nonparenchymal and parenchymal cells when expressed as nmol valine incorporated per mg protein. This rate was also close to the value found in intact liver by other workers. 4. 4. Approximately 25% of the total radioactivity incorporated during incubation for 2 h was found in proteins released to the medium from parenchymal cells, while the corresponding figure for nonparenchymal cells was 3.5%. 5. 5. Ethanol inhibited incorporation of labelled valine into stationary and medium proteins of parenchymal cells. No such effects were found in nonparenchymal cells. 6. 6. Nonparenchymal cells did not metabolize ethanol while parenchymal cells did, shown by changes in lactate pyruvate ratio and medium pH. It was concluded that nonparenchymal cells are capable of synthesizing proteins at a rate comparable to that found in parenchymal cells. Protein synthesis in parenchymal cells was inhibited by ethanol, but nonparenchymal protein synthesis was unaffected. This difference may be linked to the ability of the former cell type to metabolize ethanol.

Binding of liver lipase to parenchymal and non-parenchymal rat liver cells

Summary The present paper describes the in vitro binding of liver lipase partially purified from postheparin rat serum to isolated liver cells. The binding of liver lipase to non-parenchymal liver cells was 100 times more, per mg cell protein, than to parenchymal cells. The in vitro bound lipase to non-parenchymal cells was largely (80%) releasable by heparin. The presented data suggest that in vivo the heparin-releasable liver lipase is mainly (85–92%) located at the surface of non-parenchymal cells.

Biosynthesis of prostaglandins in parenchymal and nonparenchymal rat liver cells

Rat hepatocyte suspensions were separated into parenchymal and nonparenchymal (endothelial-rich) cells and the conversion of arachidonate into prostaglandin E was studied. Most of the intracellular labeled arachidonate was incorporated into phospholipids. Fractionation of parenchymal cell phospholipids by thin-layer chromatography showed the highest specific activity in the phosphatidylserine plus phosphatidylinositol zone followed by phosphatidylcholine and phosphatidylethanolamine; sphingomyelin was not labeled. The conversion of arachidonate into prostaglandins was 1% and 4.3% in parenchymal and nonparenchymal cells respectively. Nonparenchymal cells synthesized 5-fold more prostaglandin E-like material than parenchymal cells. Quantitation of prostaglandin E biosynthesis by radioimmunoassay revealed that in the presence of cold arachidonate sinusoidal cells synthesized 1200 pg of immunogenic prostaglandin E per mg protein, in parenchymal cells the figure was 175 pg per mg protein. Parenchymal cells had little effect on platelet aggregation, whereas nonparenchymal cells inhibited aggregation in a dose-dependent fashion. This suggests that sinusoidal liver cells synthesize a prostacyclin-like compound in addition to prostaglandin E 2.

In vivo uptake of human and rat low density and high density lipoprotein by parenchymal and nonparenchymal cells from rat liver

The relative contribution of the parenchymal and nonparenchymal rat liver cells to the hepatic uptake of human and rat high density lipoprotein (HDL) and low density lipoprotein (LDL) was determined in vivo. Non-parenchymal cells, isolated 6 h after intravenous injection of iodinated human HDL and LDL, contained respectively 4.2 and 6.3 times the amount of trichloroacetic acid-precipitable radioactivity per mg cell protein as compared to parenchymal cells. For rat iodinated HDL and LDL these factors were 3.4 and 4.1, respectively. These results indicate that nonparenchymal liver cells play a substantial role in the hepatic uptake of human and rat HDL and LDL in vivo.

The effect of α-mannose-terminal oligosaccharides on the survival of glycoproteins in the circulation: Rapid uptake and catabolism of bovine pancreatic ribonuclease B by nonparenchymal cells of rat liver

125 I-Labeled ribonuclease B (RNase B), injected intravenously, is rapidly cleared from the rat circulation ( t 12 ∼ 15 min). Clearance of this glycoprotein depends on specific recognition of the α-mannosyl residues of the attached oligosaccharide and is inhibited by the coinjection of an excess of unlabeled RNase B or yeast mannan, but not by RNase A, dextrans, galactan, or asialofetuin. Following 125 I-labeled RNase B injection into nephrectomized rats, radioactivity accumulated primarily in the lysosomal subcellular fraction in the liver and was rapidly degraded to low molecular weight iodinated products. Free iodide began to accumulate in the stomach and intestines after 30 min. Liver, spleen, and bone marrow were most active, on a weight basis, in the clearance of RNase B. At 15 min after the injection of 125 I-labeled RNase B, parenchymal and nonparenchymal cells were isolated from liver following perfusion with dilute formalin and collagenase. More than 90% of the radioactivity was localized in the nonparenchymal cell fraction. The evidence indicates that mannose-dependent glycoprotein clearance from the circulation occurs primarily in tissues characteristic of the reticuloendothelial system.

Iodine labeled human and rat low-density and high-density lipoprotein degradation by human liver and parenchymal and non-parenchymal cells from rat liver

1. 1. The abilities of homogenates of human liver, rat liver parenchymal cells, rat liver non-parenchymal cells and total rat liver to catabolize human and rat iodinated high-density lipoprotein (HDL) and low-density lipoprotein (LDL) were determined by measuring the amount of trichloroacetic acid-soluble (noniodide) radioactivity liberated upon incubation at the optimum pH of 4.2. 2. 2. A comparison of the capacities of human liver, rat liver and parenchymal and non-parenchymal cells from rat liver indicated that these different preparations are all able to degrade rat iodine-labeled LDL and HDL, with a 5–6-fold higher capacity for HDL as compared to LDL. 3. 3. Iodine-labeled human HDL can be degraded by rat liver, rat parenchymal and rat non-parenchymal cells with a 5–7-fold higher rate than human iodinelabeled LDL. Human liver homogenate was more active in the degradation of both human and rat iodine-labeled LDL and rat HDL as compared to rat liver. 4. 4. A comparison of the capacities of parenchymal and non-parenchymal cells for the degradation of iodine-labeled human and rat LDL and HDL indicates that non-parenchymal cells possess a considerable higher capacity to degrade these lipoproteins per mg of cell protein. 5. 5. The results indicate that a high proportion of the total rat liver capacity for lipoprotein degradation is localized in the non-parenchymal liver cells and this, together with the active endocytic activity, suggests an important role of these liver cells in hepatic lipoprotein catabolism.

Distribution and some properties of NADPH and NADH oxidase in parenchymal and nonparenchymal liver cells

The activities of NADPH and NADH oxidase were determined in homogenates of isolated pure parenchymal and nonparenchymal rat liver cells at neutral (7.4) and acid (5.5) pH. The NADPH oxidase at pH 7.4 is about equally active in parenchymal and nonparenchymal cells and in both cell types is rather insensitive to KCN (1 m m) inhibition. By lowering the pH to 5.5, the NADPH oxidase of the nonparenchymal cells is stimulated (twofold) while the activity in parenchymal cells is decreased. The NADH consumption at neutral pH in parenchymal cells is 75% inhibited by KCN, while this activity in nonparenchymal cells is relatively insensitive to KCN. The NADH oxidase in both parenchymal and nonparenchymal liver cells is less active when the pH is lowered from 7.4 to 5.5. The distribution of NAD(P)H oxidases between parenchymal and nonparenchymal liver cells and the effect of pH on their activities suggest that in the nonparenchymal cells, the NADPH oxidase might play a role in the synthesis of H 2O 2 within the phagocytic vacuole. A scheme is proposed which describes the metabolic events involved in H 2O 2 formation and catabolism of endo(phago)cytosed particles in nonparenchymal liver cells.

Different Types of Mitochondria in Parenchymal and Non-Parenchymal Rat-Liver Cells

1. Intact and pure parenchymal and non-parenchymal cells were isolated from rat liver. The specific activities of several mitochondrial enzymes were determined in both parenchymal and non-parenchymal cell homogenates to characterize the mitochondria in these liver cell types. 2. In general the activities of mitochondrial enzymes were lower in non-parenchymal liver cells than in parenchymal cells. The specific activity of pyruvate carboxylase in non-parenchymal cells expressed as the percentage of that in parenchymal cells was onlu 2% for glutamate dehydrogenase 4.3% and for cytochrome c oxidase 79.4%. Monoamine oxidase, as an exception, has an equal specific activity in both cell types. 3. The activity ratio of pyruvate carboxylase at 10 mM pyruvate over 0.1 mM pyruvate is 3.35 for parenchymal cells and 1.50 for non-parenchymal cells. This indicates that non-parenchymal liver cells only contain the high affinity form of pyruvate carboxylase in contrast to parenchymal cells. 4. The ratio of glycerol-3-phosphate cytochrome c reductase over succinate cytochrome c reductase activity differs from parenchymal (0.01) and non-parenchymal cells (0.10). This might indicate that the glycerol-3-phosphate shuttle, which is important for the transport of reduction equivalents for cytosol to mitochondria is relatively more active in non-parenchymal cells than in parenchymal cells. 5. The activity pattern of mitochondrial enzymes in parenchymal and non-parenchymal cell homogenates indicates that these cell types contain different types of mitochondria. The presence of these different cell types in liver will therefore contribute to the heterogeneity of isolated rat liver mitochondria in which the mitochondria from non-parenchymal cells might be considered as "non-gluconeogenic".