Fluid endocytosis in isolated rat parenchymal and non-parenchymal liver cells

Fluid endocytosis in rat liver parenchymal (hepatocytes) and non-parenchymal cells was studied by measuring uptake of [¹²⁵I]polyvinylpyrrolidone (PVP). Radioactive sucrose preparations were also tested but turned out to be unsuitable because of impurities of radioactive glucose and fructose. Fluid endocytosis was temperature dependent without any transition temperature. The rate of endocytosis was inhibited by inhibitors of the glycolytic and the respiratory pathway. Colchicine, but not cytochalasin B, inhibited the uptake of [¹²⁵I]PVP in hepatocytes. Therefore, intact microtubuli, but not microfilaments may be required for normal rate of fluid endocytosis in hepatocytes. Colchicine reduced the rate of fluid endocytosis in the non-parenchymal liver cells. Subcellular fractionation by isopycnic centrifugation in sucrose gradients indicated that [¹²⁵I]PVP taken up by the hepatocytes accumulated in the lysosomes. The rate of uptake expressed as volume of fluid internalized per unit time (endocytic index) was calculated to 0.08 μl/h/10⁶ cells for hepatocytes and 0.07 μl/h/10⁶ cells for non-parenchymal liver cells.     

Hormonal control of glycogenolysis in parenchymal liver cells by Kupffer and endothelial liver cells

Conditioned media of isolated Kupffer and endothelial liver cells were added to incubations of parenchymal liver cells, in order to test whether secretory products of Kupffer and endothelial liver cells could influence parenchymal liver cell metabolism. With Kupffer cell medium an average stimulation of glucose production by parenchymal liver cells of 140% was obtained, while endothelial liver cell medium stimulated with an average of 127%. The separation of the secretory products of Kupffer and endothelial liver cells in a low and a high molecular weight fraction indicated that the active factor(s) had a low molecular weight. Media, obtained from aspirin-pretreated Kupffer and endothelial liver cells, had no effect on the glucose production by parenchymal liver cells. Because aspirin blocks prostaglandin synthesis, it was tested if prostaglandins could be responsible for the effect of media on parenchymal liver cells. It was found that prostaglandin (PG) E1, E2, and D2 all stimulated the glucose production by parenchymal liver cells, PGD2 being the most potent. Kupffer and endothelial liver cell media as well as prostaglandins E1, E2, and D2 stimulated the activity of phosphorylase, the regulatory enzyme in glycogenolysis. The data indicate that prostaglandins, present in media from Kupffer and endothelial liver cells, may stimulate glycogenolysis in parenchymal liver cells. This implies that products of Kupffer and endothelial liver cells may play a role in the regulation of glucose homeostasis by the liver.     

Changes in intracellular calcium induced by acute hypothermia in parenchymal, endothelial, and Kupffer cells of the rat liver.

Disturbances in intracellular calcium have been implicated in liver graft damage after cold preservation and warm reperfusion. Despite improvements noted with the use of calcium channel blockers, such as nisoldipine, the exact nature and cellular basis of the presumed changes in intracellular calcium as well as the actual target of these blockers remain unclear. Isolated rat parenchymal, endothelial, and Kupffer cells were cultured and changes in intracellular calcium measured in vitro after acute hypothermia (5-8 degrees C) by fluorescence imaging using FURA-2. Between 50 and 80% of parenchymal, endothelial, and Kupffer cells exhibited significant increases in baseline calcium that were gradual and sustained for the duration of acute hypothermia. Removal of extracellular calcium completely abolished the positive response of hepatocytes and diminished the proportion of responding endothelial and Kupffer cells. The calcium channel blocker nisoldipine (1 microM) slightly diminished the proportion of positive responders in parenchymal but not in endothelial or Kupffer cells. However, nisoldipine did not modify the amplitude of the calcium rise in responding cells of all types. Acute hypothermia causes calcium influx into a majority of parenchymal, endothelial, and Kupffer cells. Nisoldipine does not effectively prevent these changes in intracellular calcium. Pathways of calcium entry resistant to the drug or other than voltage-dependent calcium channels may thus be involved.     

Specific gene expression of ATP-binding cassette transporters and nuclear hormone receptors in rat liver parenchymal,endothelial, and Kupffer cells

Hepatic cholesterol(ester) uptake from serum coupled to intracellular processing and biliary excretion are important features in the removal of excess cholesterol from the body. ATP-binding cassette (ABC) transporters play an important role in hepatic cholesterol transport. The liver consists of different cell types, and ABC transporters may exert different physiological functions dependent on the individual cell type. Therefore, in the current study, using real time PCR we compared the mRNA expression of ABC transporters and genes involved in the regulation of cholesterol metabolism in liver parenchymal, endothelial, and Kupffer cells. It appears that liver parenchymal cells contain high expression levels compared with endothelial and Kupffer cells of scavenger receptor class BI ( approximately 3-fold), peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma (8-20-fold), cholesterol 7alpha-hydroxylase A1 (>100-fold), and ABCG5/G8 ( approximately 5-fold). Liver endothelial cells show a high expression of cholesterol 27-hydroxylase, liver X receptor (LXR)beta, PPARdelta, and ABCG1, suggesting a novel specific role for these genes in endothelial cells. In Kupffer cells, the expression level of LXRalpha, ABCA1, and in particular ABCG1 is high, leading to an ABCG1 mRNA expression level that is 70-fold higher than in parenchymal cells. It can be calculated that 51% of the total liver ABCG1 expression resides in Kupffer cells and 24% in endothelial cells, suggesting an intrahepatic-specific role for ABCG1 in Kupffer and endothelial cells. Because of a specific stimulation of ABCG1 in parenchymal cells by a high cholesterol diet, the contribution of parenchymal cells to the total liver increased from 25 to 60%. Our data indicate that for studies of the role of ABC transporters and their regulation in liver, their cellular localization should be taken into account, allowing proper interpretation of metabolic changes, which are directly related to their (intra)cellular expression level.     

Modes of endocytosis of latex particles in sinusoidal endothelial and Kupffer cells of normal and perfused rat liver

The endocytosis of latex particles (0.33, 0.46 and 0.80 micron in diameter) in the sinusoidal endothelial and Kupffer cells of the rat liver was studied electron microscopically. When the liver was perfused with serum-free oxygenated Krebs Ringer bicarbonate, latex particles of all three sizes were taken up by the endothelial cells. After a 10-min perfusion, particles were incorporated by the luminal cell surface of the perikarya or of the thick portion of the endothelial cells. A large patch of bristle coat was surrounding the ingested particle. The number of ingested particles in the endothelial cells, however, was much less than in the Kupffer cells. In in vivo experiments, no endocytosis of the latex particles was observed in the endothelial cells. In the Kupffer cells, particles were engulfed by the ruffled membranes or sank into the cytoplasm without a large patch of the bristle coat both in the perfusion system and in vivo. These observations show that at least 0.80 micron latex particles are taken up by the bristle-coated membranes in the sinusoidal endothelial cells of the perfused liver. The endocytic mechanism for latex particles in the endothelial cells is different from that of the Kupffer cells.     

Interaction in vivo and in vitro of apolipoprotein E-free high-density lipoprotein with parenchymal, endothelial and Kupffer cells from rat liver.

The interaction of apolipoprotein (apo) E-free high-density lipoprotein (HDL) with parenchymal, endothelial and Kupffer cells from liver was characterized. At 10 min after injection of radiolabelled HDL into rats, 1.0 +/- 0.1% of the radioactivity was associated with the liver. Subfractionation of the liver into parenchymal, endothelial and Kupffer cells, by a low-temperature cell-isolation procedure, indicated that 77.8 +/- 2.4% of the total liver-associated radioactivity was recovered with parenchymal cells, 10.8 +/- 0.8% with endothelial cells and 11.3 +/- 1.7% with Kupffer cells. It can be concluded that inside the liver a substantial part of HDL becomes associated with endothelial and Kupffer cells in addition to parenchymal cells. With freshly isolated parenchymal, endothelial and Kupffer cells the binding properties for apo E-free HDL were determined. For parenchymal, endothelial and Kupffer cells, evidence was obtained for a saturable, specific, high-affinity binding site with Kd and Bmax. values respectively in the ranges 10-20 micrograms of HDL/ml and 25-50 ng of HDL/mg of cell protein. In all three cell types nitrosylated HDL and low-density lipoproteins did not compete for the binding of native HDL, indicating that lipids and apo B are not involved in specific apo E-free HDL binding. Very-low-density lipoproteins (VLDL), however, did compete for HDL binding. The competition of VLDL with apo E-free HDL could not be explained by label exchange or by transfer of radioactive lipids or apolipoproteins between HDL and VLDL, and it is therefore suggested that competition is exerted by the presence of apo Cs in VLDL. The results presented here provide evidence for a high-affinity recognition site for HDL on parenchymal, liver endothelial and Kupffer cells, with identical recognition properties on the three cell types. HDL is expected to deliver cholesterol from peripheral cells, including endothelial and Kupffer cells, to the liver hepatocytes, where cholesterol can be converted into bile acids and thereby irreversibly removed from the circulation. The observed identical recognition properties of the HDL high-affinity site on liver parenchymal, endothelial and Kupffer cells suggest that one receptor may mediate both cholesterol efflux and cholesterol influx, and that the regulation of this bidirectional cholesterol (ester) flux lies beyond the initial binding of HDL to the receptor.     

In vivo and in vitro uptake and degradation of acetylated low density lipoprotein by rat liver endothelial, Kupffer, and parenchymal cells

Isolation and separation of rat liver cells into endothelial, Kupffer, and parenchymal cell fractions were performed at different times after injection of human 125I-acetyl low density lipoproteins (LDL). In order to minimize degradation and redistribution of the injected lipoprotein during cell isolation, a low temperature (8 degrees C) procedure was applied. Ten min after injection, isolated endothelial cells contained 5 times more acetyl-LDL apoprotein per mg of cell protein than the Kupffer cells and 31 times more than the hepatocytes. A similar relative importance of the different cell types in the uptake of acetyl-LDL was observed 30 min after injection. For studies on the in vitro interaction of endothelial and Kupffer cells with acetyl-LDL, the cells were isolated with a collagenase perfusion at 37 degrees C. Pure endothelial (greater than 95%) and purified Kupffer cells (greater than 70%) were obtained by a two-step elutriation method. It is demonstrated that the rat liver endothelial cell possesses a high affinity receptor specific for the acetyl-LDL because a 35-fold excess of unlabeled acetyl-LDL inhibits association of the labeled compound for 70%, whereas unlabeled native human LDL is ineffective. Binding to the acetyl-LDL receptor is coupled to rapid uptake and degradation of the apolipoprotein. Addition of the lysosomotropic agents chloroquine (50 microM) or NH4Cl (10 mM) resulted in more than 90% inhibition of the high affinity degradation, indicating that this occurs in the lysosomes. With the purified Kupffer cell fraction, the cell association and degradation of acetyl-LDL was at least 4 times less per mg of cell protein than with the pure endothelial cells. Although cells isolated with the cold pronase technique are also still able to bind and degrade acetyl-LDL, it appeared that 40-60% of the receptors are destroyed or inactivated during the isolation procedure. It is concluded that the rat liver endothelial cell is the main cell type responsible for acetyl-LDL uptake.     

Cadmium metabolism by rat liver endothelial and Kupffer cells.

The metabolism of cadmium was investigated in Wistar-rat liver non-parenchymal cells. Kupffer and endothelial cells, the major cell populations lining the sinusoidal tracts, were isolated by collagenase dispersion and purified by centrifugal elutriation. At 20 h after subcutaneous injection of the metal salt (1.5 mg of Cd/kg body weight), endothelial cells accumulated 2-fold higher concentrations of Cd than did Kupffer or parenchymal cells. Most of the Cd in non-parenchymal cells was associated with cytosolic metallothionein (MT), the low-Mr heavy-metal-binding protein(s). When MT was quantified in cytosols from cells isolated from control rats by a 203Hg competitive-binding assay, low levels were found to be present in Kupffer, endothelial and parenchymal cells. Cd injection significantly increased MT levels in all three cell types. The induction of MT synthesis was investigated in vitro by using primary monolayer cultures. The incorporation of [35S]cysteine into MT increased 47% over constitutive levels in endothelial-cell cultures after the addition of 0.8 microM-Cd2+ to the medium for 10 h. MT synthesis in Kupffer cells was not observed. The lack of MT synthesis by monolayer cultures of Kupffer cells in vitro was associated with a decreased capacity of these cells to accumulate heavy metals from the extracellular medium. This apparent decreased ability to transport metals did not reflect a general defect in either cellular function or metabolic activity, since isolated Kupffer cells incorporated [3H]leucine into protein at rates comparable with those shown by liver parenchymal cells and readily phagocytosed particles.     

Autoradiographic localizatio of [125I]-C-ANP compared to [125I]-ANP in rat tissue

Summary Whole-body autoradiography demonstrated the different distribution of [¹²⁵I]-C-ANP and [¹²⁵I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [¹²⁵I]-ANP and [¹²⁵I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [¹²⁵I]-ANP administration were detected, no or just few radioactivity was found after administration of [¹²⁵I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [¹²⁵I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.This work is part of the doctoral thesis of Frank Heidemann to be presented at the Ludwig-Maximilians-Universität München, FRG     

Conditioned media of Kupffer and endothelial liver cells influence protein phosphorylation in parenchymal liver cells. Involvement of prostaglandins.

The possible role of Kupffer and endothelial liver cells in the regulation of parenchymal-liver-cell function was assessed by studying the influence of conditioned media of isolated Kupffer and endothelial cells on protein phosphorylation in isolated parenchymal cells. The phosphorylation state of three proteins was selectively influenced by the conditioned media. The phosphorylation state of an Mr-63,000 protein was decreased and the phosphorylation state of an Mr-47,000 and an Mr-97,000 protein was enhanced by these media. These effects could be mimicked by adding either prostaglandin E1, E2 or D2. Both conditioned media and prostaglandins stimulated the phosphorylase activity in parenchymal liver cells, suggesting that the Mr-97,000 phosphoprotein might be phosphorylase. Parenchymal liver cells secrete a phosphoprotein of Mr-63,000 and pI 5.0-5.5. The phosphorylation of this protein is inhibited by Kupffer- and endothelial-liver-cell media, and prostaglandins E1, E2 and D2 had a similar effect. The data indicate that Kupffer and endothelial liver cells secrete factors which influence the protein phosphorylation in parenchymal liver cells. This forms further evidence that products from non-parenchymal liver cells, in particular prostaglandin D2, might regulate glucose homoeostasis and/or other specific metabolic processes inside parenchymal cells. This stresses the concept of cellular communication inside the liver as a way by which the liver can rapidly respond to extrahepatic signals.     

Multiple forms of acid phosphatase in rat liver parenchymal,endothelial, and kupffer cells

Purified suspensions of highly viable parenchymal, endothelial, and Kupffer cells were prepared from rat liver. In the liver cell classes, total activities of acid phosphatase were determined with 4-methylumbelliferylphosphate, 1-naphthylphosphate, and p-nitrophenylphosphate. The specific enzyme activities were different for each type of cell and, even within one cell class, the enzymes showed different conversion rates for the three substrates. These results indicate the presence of multiple forms of acid phosphatase enzymes in each cell class. The inhibiting effects of tartrate, fluoride, and alloxan on the acid phosphatase activities were investigated. Depending on the substrate used, the inhibitors inactivated the enzymes at different rates, which also indicates the presence of multiple forms of acid phosphatase enzymes in the liver cell classes. By means of an isoelectric focusing technique, acid phosphatase enzymes could be separated on the basis of their differences in isoelectric points. One form with an isoelectric point around 4 is found in Kupffer cells, whereas another form with an isoelectric point of about 7 is found in parenchymal cells. Endothelial cells possess both forms. These findings suggest a specificity in the function of this lysosomal enzyme in each cell class.     

Autoradiographic localization of [125I]-C-ANP compared to [125I]-ANP in rat tissue.

Whole-body autoradiography demonstrated the different distribution of [125I]-C-ANP and [125I]-ANP to rat tissues. Highest enrichment of radioactivity of both labelled peptides was found in the kidney. In some organs we found remarkable differences between [125I]-ANP and [125I]-C-ANP. In the kidney cortex, especially in the glomeruli, as well as in the endocardium, the zona glomerulosa and the medulla of the adrenal gland, where high levels of radioactivity after [125I]-ANP administration were detected, no or just few radioactivity was found after administration of [125I]-C-ANP. On the other hand in the kidney papilla and the outer subcortical medulla, characteristic blackening was found after [125I]-C-ANP administration. Those differences might be important for the understanding of pharmacological actions of ANP analogues.     

The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells

Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions (Simons, T. J. B. (1979) Nature 281, 337-338). Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C (Weigel, P. H., and Oka, J. A. (1983) J. Biol. Chem. 258, 5089-5094); vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content.     

Isolation and characterization of Kupffer and endothelial cells from the rat liver

Non-parenchymal cell suspensions were prepared from rat livers by three different methods based on a collagenase, a pronase and a combined collagenase-pronase treatment. The highest yield of Kupffer and endothelial cells was obtained with the pronase treatment. Attempts were made for a further purification of these cells by Metrizamide density gradient centrifugation after preferentially loading lysosomal structures in Kupffer cells with Triton WR 1339, Jectofer^®, Neosilvol^®, Zymosan or colloidal carbon. After loading with Triton WR 1339 or Jectofer^®, highly purified endothelial cell suspensions were obtained, but the final Kupffer cell preparations were contaminated with about 20% of endothelial cells. Kupffer and endothelial cells purified in this way showed an altered ultrastructure and contained increased activities of the lysosomal enzymes acid phosphatase, arylsulphatase B and cathepsin D. As an alternative procedure for the purification of Kupffer and endothelial cells, a method based on centrifugal elutriation was employed. With this procedure, highly purified preparations of Kupffer or endothelial cells with a well preserved ultrastructure were obtained. Compared with endothelial cells, purified Kupffer cells had a three times higher cathepsin D activity, whereas the arylsulphatase B activity was three times higher in endothelial cells. The high cathepsin D activity in Kupffer cells could be nearly completely inhibited by the specific cathepsin D inhibitor pepstatin, which excludes a possible contribution to this activity by proteases endocytosed during the isolation of the cells.     

Effects of thiol agents on the accumulation of cadmium by rat liver parenchymal and Kupffer cells

The rate of Cd accumulation by adult rat liver parenchymal cells in serum free primary culture in the presence of 100 μM CdCl₂ was 10 times greater than that by non-parenchymal Kupffer cells. Addition of the monothiol chelating agents, cysteine and penicillamine, decreased Cd uptake in both cell types, the effect becoming more pronounced as the monothiol concentration was increased from 0.1 to 1.0 mM. These monothiols thus appear to reduce the availability of Cd for transport across the cell membrane. In contrast 1–10 molar excesses of the dithiol agents 2,3-dimercaptopropanol (BAL) or dithiothreitol (DTT) stimulated to variable extents the rate of Cd accumulation 2–10-fold in parenchymal cells and by over 100-fold in Kupffer cells. Supplementation of the media with 3% serum had little effect on the Cd accumulation in the presence of monothiols but substantially depressed Cd uptake in the presence of dithiols. Intravenous injection of Cd (0.05 mg/kg CdCl₂) with up to a 10-fold molar excess of cysteine or penicillamine had little effect on the hepatocellular Cd distribution. However Cd uptake by non-parenchymal cells was increased markedly by the simultaneous administration of BAL or DTT in 2 or 10 molar excess. Evidence is provided that these results may be partially explained by the endocytosis, particularly in Kupffer cells, of colloidal complexes of Cd which are formed with the dithiols but not the monothiols. These observations demonstrate that the physicochemical form of Cd determines its hepatocellular distribution which may be an important factor in the manifestation of Cd toxicity after thiol treatment.