Rat Liver Non-parenchymal Cells Research Articles

Nanotechnology for therapy and imaging of liver diseases

Nanotechnology has been considered for the improved delivery of various therapeutic agents, including drugs and genes. Indeed, liposomes and nanoparticles equipped with homing devices for the targeting of receptors over-expressed on the hepatic tissue have improved the treatment of various liver diseases. In this review, various nanotechnology approaches employed for the treatment/imaging of liver disease, either in preclinical or in clinic are discussed.

Expression of MMPs and TIMPs in liver fibrosis – a systematic review with special emphasis on anti-fibrotic strategies

In liver tissue matrix metalloproteinases (MMPs) and their specific inhibitors (tissue inhibitors of metalloproteinases, TIMPs) play a pivotal role in both, fibrogenesis and fibrolysis. The current knowledge of the pathophysiology of liver fibrogenesis with special emphasis on MMPs and TIMPs is presented. A systematic literature search was conducted. All experimental models of liver fibrosis that evaluated a defined anti-fibrotic intervention in vivo or in vitro considering MMPs and TIMPs were selected. The methodological quality of all these publications has been critically appraised using an objective scoring system and the content has been summarized in a table.

β 2 -Glycoprotein I (Apolipoprotein H) Modulates Uptake and Endocytosis Associated Chemiluminescence in Rat Kupffer Cells

g 2-Glycoprotein I (g 2GPI) is known to influence macrophage uptake of particles with phosphatidylserine containing surfaces, as apoptotic thymocytes and unilamellar vesicles in vitro. Nevertheless, effects upon macrophage activation induced by this interaction are still unknown. g 2GPI influence upon the reactive species production by Kupffer cells was evaluted in order to investigate whether g 2GPI modulates the macrophage response to negatively charged surfaces. Chemiluminescence of isolated non-parenchymal rat liver cells was measured after phagocytosis of opsonized zymosan or phorbolmyristate acetate (PMA) stimulation, in the presence and absence of large unilamellar vesicles (LUVs) containing 25mol% phosphatidylserine (PS) or 50mol% cardiolipin (CL) and complementary molar ratio of phosphatidylcholine (PC). g 2GPI decreased by 50% the chemiluminescence response induced by opsonized zymosan, with a 66% reduction of the initial light emission rate. PMA stimulated Kupffer cell chemiluminescence was insensitive to human or rat g 2GPI. Albumin (500 w g/ml) showed no effect upon chemiluminescence. g 2GPI increased PS/PC LUV uptake and degradation by Kupffer cells in a concentration-dependent manner, without leakage of the internal contents of the LUVs, as shown by fluorescence intensity enhancement. LUVs opsonized with antiphospholipid antibodies (aPL) from syphilitic patients increased light emission by Kupffer cells. Addition of g 2GPI to the assay reduced chemiluminescence due to opsonization with purified IgG antibodies from systemic lupus erythematosus (SLE or syphilis (Sy) patient sera. A marked net increase in chemiluminescence is observed in the presence of Sy aPL antibodies, whereas a decrease was found when SLE aPL were added to the assay, in the presence or absence of g 2GPI. At a concentration of 125 w g/ml, g 2 GPI significantly reduced Kupffer cell Candida albicans phagocytosis index and killing score by 50 and 10%, respectively. The present data strongly suggest that particle uptake in the presence of g 2GPI is coupled to an inhibition of reactive species production by liver macrophages during the respiratory burst, supporting the role of g 2GPI as a mediator of senescent cell removal.

Immunolocalization of a novel cholesteryl ester hydrolase in the endoplasmic reticulum of murine and human hepatocytes.

We have recently purified a cholesteryl ester hydrolase (CEH) from rat liver microsomes. Antibodies raised against the purified protein specifically reacted with a 106-kd protein and neutralized 90% of the CEH activity of rat liver microsomes (J Lipid Res 1999;40:715-725). In this work we have used the anti-CEH antibody to study both the subcellular distribution of the protein in hepatocytes as well as its tissue-specific expression in rat. Western blotting of subcellular fractions obtained from isolated rat hepatocytes revealed that the immunoreactive 106-kd CEH was exclusively localized in microsomes. The antibody also recognized a 106-kd protein in microsomes from mouse and human liver but not from rat nonparenchymal liver cells. Confocal microscopy of HepG2 cells revealed that CEH immunoreactive material colocalized with calnexin, a marker of the endoplasmic reticulum. Furthermore, high-resolution immunoelectron microscopy of rat liver thin sections exclusively localized the CEH immunoreactivity to the endoplasmic reticulum of the hepatocyte. No CEH immunoreactivity was observed in microsomes derived from adrenal glands, ovaries, testis, pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, brain, aorta, and macrophages. We report a CEH localized to the endoplasmic reticulum, erCEH, in the mammalian hepatocyte. The subcellular localization and tissue-restricted pattern of expression of erCEH suggests that it might have unique functions in liver cholesterol metabolism.

Dexamethasone coupled to albumin is selectively taken up by rat nonparenchymal liver cells and attenuates LPS-induced activation of hepatic cells

Background/Aims: The human serum albumin (HSA) conjugate Dexa 10-HSA was designed to specifically deliver the anti-inflammatory drug dexamethasone (Dexa) to nonparenchymal cells (NPC) in the rat liver. NPC play an important role in the pathogenesis of acute and chronic inflammatory liver diseases like fibrosis. Targeting Dexa to these cells might reduce its adverse effects and increase the efficacy. Methods: Tissue and intrahepatic distributions of Dexa 10-HSA were assessed in normal and fibrotic rats with 125I-labelled conjugate and by immunohistochemistry. The effect of the conjugate on lipopolysaccharide (LPS)-induced inflammation and cell activation was studied in vitro with precision-cut liver slices and in vivo. Results: Ten minutes after i.v. injection 72±13% and 65±12% of a tracer dose of Dexa 10-HSA had been taken up in normal and fibrotic livers, respectively. Unconjugated Dexa also accumulated in livers, but cellular distribution studies revealed an accumulation in parenchymal cells (NPC vs. parenchymal cell (PC) ratio=0.29±11, p<0.005) whereas Dexa 10-HSA accumulated in nonparenchymal cells (NPC/PC ratio=7.9±3.1). Both coupled and uncoupled Dexa showed effective inhibition of LPS-induced NO x and TNFα production in precision-cut liver slices. At low concentrations (0.02 μM), however, Dexa 10-HSA was more efficient at inhibiting TNFα production than uncoupled Dexa. In fibrotic rats Dexa 10-HSA (3 mg/kg) and an equimolar amount of uncoupled Dexa (0.22 mg/kg) both significantly promoted survival after LPS-induced acute inflammation. Conclusion: Dexa 10-HSA was at least as effective as uncoupled Dexa at inhibiting LPS-induced effects, which indicates that HSA-bound Dexa is pharmacologically active. Coupling Dexa to HSA shifted the accumulation of Dexa from the PC to the NPC of livers. Since mediator release from NPC is crucial in the initiation and propagation of the fibrotic process, selective delivery of Dexa in NPC may improve the efficacy and safety of corticosteroid treatment of liver fibrosis.

Expression patterns of matrix metalloproteinases and their inhibitors in parenchymal and non-parenchymal cells of rat liver: regulation by TNF-α and TGF-β1

Background/Aims: Although matrix metalloproteinases (MMPs) and their specific inhibitors (TIMPs) play an essential role in liver injury associated with tissue remodeling, the cellular origin of MMPs/TMPs within the liver remains to be clarified. Methods: Different liver cell populations were analysed with respect to their expression by reverse transcription-polymerase chain reaction, Northern blot analysis and zymography. Results: MMP and TIMP coding transcripts were detectable in all liver cell types by reverse transcription-polymerase chain reaction; however, the cellular expression levels were markedly different as assessed by Northern blot analysis. Gelatinase-B was predominantly expressed in Kupffer cells, gelatinase-A in hepatic stellate cells and rat liver myofibroblasts and stromelysins-1, -2 as well as collagenase in hepatic stellate cells. Membrane type-1 MMP (MMP-14) was found in significant amounts in all liver cells. TIMP-1 coding m-RNAs were present mainly in hepatic stellate cells and rat liver myofibroblasts, TIMP-2 additionally in Kupffer cells, while TIMP-3 expression was detectable only in hepatocytes. During in vitro activation of hepatic stellate cells, MMP expression was mostly downregulated, while TIMP expression was enhanced, thereby providing an explanation for matrix accumulation co-localised with these cells during chronic liver injury. In general, TNF-α stimulated both MMP and TIMP expression of hepatic stellate cells, while TGF-β1 induced TIMP expression only. Conclusions: Collectively these data demonstrate that all resident liver cells are involved in matrix degradation to some extent and that hepatic stellate cells play an important role in matrix breakdown in addition to matrix synthesis. The cytokine-specific regulation of MMP/TIMP expression in hepatic stellate cells suggests that the initial matrix breakdown following liver injury might be enhanced by TNF-α, while diminished matrix degradation during chronic tissue injury might be due to the action of TGF-β1 through TIMP induction.

Differential expression of prostanoid receptors in hepatocytes, Kupffer cells, sinusoidal endothelial cells and stellate cells of rat liver

Background/Aims: Prostanoids produced by nonparenchymal cells modulate the function of parenchymal and nonparenchymal liver cells during homeostasis and inflammation via eight classes of prostanoid receptors coupled to different G-proteins. Prostanoid receptor expression in parenchymal and nonparenchymal cells was studied in order to get a better insight into the complex prostanoid-mediated intrahepatic signaling network. Methods: RNA was isolated from freshly purified parenchymal and nonparenchymal rat liver cells and the mRNA level of all eight prostanoid receptor classes was determined by newly developed semiquantitative reverse transcription-polymerase chain reaction protocols. Results: The mRNAs for the prostanoid receptors were differentially expressed. Hepatocytes were the only cell type which contained the mRNA of the G q-linked prostaglandin F 2α receptor; they were devoid of any mRNA for the G s-linked prostanoid receptors. Kupffer cells possessed the largest amount of mRNA for the G s-linked prostaglandin E 2 receptor subtype 2. Endothelial cells expressed high levels of mRNA for the G q-linked thromboxane receptor and medium levels of mRNA for the G s-linked prostacyclin receptor, while stellate cells had the highest levels of mRNA for the prostacyclin receptor. The mRNAs for the G q-linked prostaglandin E 2 receptor subtype 1 and the G i-linked prostaglandin E 2 receptor subtype 3 were expressed in hepatocytes and all nonparenchymal cell types atsimilar high levels, whereas the mRNA of the G s-linked prostaglandin D 2 receptor was expressed in all nonparenchymal cells at very low levels. Conclusions: In hepatocytes the prostaglandin F 2α receptor can mediate an increase in glucose output via an increase of intracellular InsP 3 while cAMP-dependent glucose output can be inhibited via the subtype 3 prostaglandin E 2 receptor. The subtype 2 prostaglandin E 2 receptor can restrain the inflammatory response of Kupffer cells via an increase in intracellular cAMP. The thromboxane receptor and the prostacyclin receptor in sinusoidal endothelial and the prostacyclin receptor in stellate cells may be involved in the regulation of sinusoidal blood flow and filtration.

Differential expression of transforming growth factor-β and its receptors in hepatocytes and nonparenchymal cells of rat liver after CCl 4 administration

Background/Aims: Transforming growth factor-β (TGF-β) is a family of multifunctional proteins that regulate hepatocyte proliferation, and biosynthesis of the extracellular matrix. In this study we examined whether modulation of TGF-β receptor expression contributes to the liver diseases. Methods: The mRNA expression of TGF-β1, TGF-β type I receptor (TGFβRI), TGF-β II receptor (TGFβRII) and TGF-β type III receptor (TGFβRIII) in rat livers injured by CCl 4 administration was studied by Northern blotting. The mRNA expression patterns were confirmed by in situ hybridization. Result: The peak of TGF-β1 mRNA expression was observed 48 h after acute intoxication with CCl 4 in nonparenchymal cells. However, the levels of TGFβRI and TGFβRII mRNA expression decreased from 24 h to 48 h and from 12 h to 48 h, respectively, and returned to the normal level by 72 h. TGFβRII mRNA expression was depressed more and for longer than that or TGFβRI mRNA. Analysis in separated hepatocytes and nonparenchymal cells from the injured livers indicated that the mRNA changes occurred in hepatocytes. Nonparenchymal cells expressed TGFβRI and TGFβRII mRNAs at constant levels during liver regeneration. TGFβRIII mRNA, which also decreased after 12 h, was not apparent in hepatocytes but only in nonparenchymal cells. Conclusions: These observations suggest that: (i) whenever TGF-β1 is increased in CCl 4-treated livers, it may induce liver fibrogenesis via nonparenchymal cells; (ii) mitoinhibitory effect of TGF-β1 on hepatocytes is transiently relieved by down-regulation of TGF-β receptors for 72 h post-damage; and (iii) the resistance to TGF-β growth inhibition between24 to 48 h may be predominantly due to down-regulation of the expression of TGFβRII.

Regulation of CD95 (APO-1/Fas) receptor and ligand expression by lipopolysaccharide and dexamethasone in parenchymal and nonparenchymal rat liver cells.

The effect of lipopolysaccharide (LPS) on the expression of CD95 (APO-1/Fas) receptor and ligand (CD95L) was studied in primary cultures of rat liver Kupffer cells (KCs), sinusoidal endothelial cells (SECs), and parenchymal cells (PCs) at the messenger RNA (mRNA) level and by means of immunocytochemistry. LPS treatment of KCs and SECs led to a three- to five-fold increase in CD95L mRNA levels within 6 hours, which declined thereafter. Within 24 hours, the number of KCs and SECs staining positive for CD95L strongly increased. After a lag phase of 12 hours after LPS addition, in both cell types the mRNA levels for the soluble CD95 isoform increased approximately 10-fold; however, the number of KCs and SECs staining positive for transmembrane CD95 remained low and did not significantly increase. Compared with nonparenchymal cells, CD95L mRNA levels in primary hepatocyte cultures were low in the absence and presence of LPS. On the other hand, functionally active CD95 expression markedly increased in response to LPS in these cells. Dexamethasone diminished the LPS-induced stimulation of CD95L expression in nonparenchymal cells but markedly stimulated CD95L expression in PCs. Apoptosis of PCs and thymic lymphocytes was stimulated by the addition of supernatants derived from LPS-treated KC or SEC cultures and was apparently mediated by CD95L as assessed by its sensitivity to inhibitors of the CD95-dependent apoptotic pathway in PCs. The data suggest a complex and timely coordinated interplay between the various liver cell populations with respect to LPS-induced activation of the apoptotic machinery with potential relevance for immunoregulation.

Gene expression of N -acetylglucosaminyltransferases III and V: A possible implication for liver regeneration

N-acetylglucosaminyltransferases III and V (GnT-III and -V) are key enzymes in the synthesis of the branches of asparagine-linked oligosaccharides. Although their messenger RNAs (mRNAs) are expressed in various rat tissues, they are not detected in normal rat liver. Expression of the GnT-III and -V mRNAs, however, increased in regenerating liver after two-thirds partial hepatectomy compared with sham-operated rats. The enzymatic activities of GnT-III and -V increased in proportion to mRNA expression. To determine which type of cells in the liver have high activities of these glycosyltransferases, hepatocytes and nonparenchymal cells were separated by means of a two-step collagenase perfusion technique. GnT-III activity was only detected in nonparenchymal cells of normal rat liver. However, during liver regeneration, GnT-III activity increased and thus was also detectable in hepatocytes. GnT-V activity was detected in both types of cells. These data were supported by reverse transcription-polymerase chain reaction results. Although the mechanism underlying the induction of these glycosyltransferases is unknown, lectin blot analysis showed that oligosaccharides in many glycoproteins, including hepatocyte growth factor, a major growth factor associated with liver regeneration, were newly synthesized during liver regeneration. This is the first report on the expression of glycosyltransferases during liver regeneration and suggests that there are different mechanisms involved in regulation of the genes of GnT-III and -V during liver regeneration.

Gene expression of N-acetylglucosaminyltransferases III and V: A possible implication for liver regeneration

N-acetylglucosaminyltransferases III and V (GnT-III and -V) are key enzymes in the synthesis of the branches of asparagine-linked oligosaccharides. Although their messenger RNAs (mRNAs) are expressed in various rat tissues, they are not detected in normal rat liver. Expression of the GnT-III and -V mRNAs, however, increased in regenerating liver after two-thirds partial hepatectomy compared with sham-operated rats. The enzymatic activities of GnT-III and -V increased in proportion to mRNA expression. To determine which type of cells in the liver have high activities of these glycosyltransferases, hepatocytes and nonparenchymal cells were separated by means of a two-step collagenase perfusion technique. GnT-III activity was only detected in nonparenchymal cells of normal rat liver. However, during liver regeneration, GnT-III activity increased and thus was also detectable in hepatocytes. GnT-V activity was detected in both types of cells. These data were supported by reverse transcription-polymerase chain reaction results. Although the mechanism underlying the induction of these glycosyltransferases is unknown, lectin blot analysis showed that oligosaccharides in many glycoproteins, including hepatocyte growth factor, a major growth factor associated with liver regeneration, were newly synthesized during liver regeneration. This is the first report on the expression of glycosyltransferases during liver regeneration and suggests that there are different mechanisms involved in regulation of the genes of GnT-III and -V during liver regeneration.

Polymerized albumin receptor on rat liver cells.

The polymerized albumin hypothesis was proposed for the mechanism of a hepatitis B virus (HBV) infection of human liver parenchymal cells on the basis that a receptor for polymerized albumin treated with glutaraldehyde was detected on isolated human liver parenchymal cells. However, some controversy exists regarding this hypothesis, because a receptor for formaldehyde-treated bovine serum albumin (f-BSA) has been found on liver non-parenchymal cells. Therefore, we characterized the uptake of polymerized rat serum albumin (p-RSA) and f-BSA by rat liver in vivo, and their bindings to liver cells in vitro. Most p-RSA and f-BSA was taken up by the liver after intravenous administration, and the uptake of p-RSA was inhibited by a 1,000-fold excess of f-BSA. In addition, more than 80% of p-RSA taken up by the liver was found in the non-parenchymal cells, and the remainder was found in the parenchymal cells. P-RSA as well as f-BSA could bind to isolated rat liver parenchymal and non-parenchymal cells. Furthermore, p-RSA and f-BSA could bind to isolated rat liver cell plasma membranes, and these bindings were completely inhibited by 1,000-fold excess of either f-BSA or p-RSA. These results indicate that there is a receptor, which can recognize both p-RSA and f-BSA, on not only rat liver non-parenchymal cells but also the parenchymal cells. It is also indicated that the receptor on the parenchymal cells as well as the non-parenchymal cells is involved in the in vivo uptake of p-RSA.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo formation of aflatoxin B1-DNA adducts in parenchymal and non-parenchymal cells of rat liver.

The induction of hepatocellular carcinoma from liver parenchymal cells in laboratory animals by aflatoxin B1 (AFB1) is well documented. In contrast no tumours arising from the sinusoidal cell population have been reported after exposure to AFB1. The apparent resistance of the latter cell type was investigated at the level of DNA adduct formation in vivo in male Sprague-Dawley rats. Liver parenchymal and non-parenchymal cell populations were isolated from rats at 20 min and 1, 24 and 72 h after administration of 240 microCi (0.6 mg) [G-3H]AFB1/kg. AFB1-DNA binding was observed in both liver cell subpopulations and was 3- to 5-fold higher in parenchymal cells than in non-parenchymal cells. The major DNA adduct found in parenchymal cells at 1 h after AFB1 administration was 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-gua), whereas at later time points the persistent secondary adduct, AFB1-formamidopyrimidine, predominated. In contrast, AFB1-gua was not observed at any time in DNA from non-parenchymal cells and the secondary adducts predominated throughout. These observations are discussed with reference to the susceptibility of different liver cell types to AFB1-carcinogenesis and the possible roles of the major AFB1-DNA adduct species.

Expression of insulin-like growth factor II (IGF-II) and IGF-II, IGF-I and insulin receptors mRNAs in isolated non-parenchymal rat liver cells.

The insulin-like growth factor II (IGF-II) is involved in embryonic growth. Modifications of its expression might play a role in the development of primary liver cancer in humans and woodchucks. In the liver, little information is available on the cell types involved in its synthesis. We have investigated the expression of IGF-II as well as IGF-II, IGF-I and insulin receptor mRNAs in non parenchymal liver cell preparations in rats of various ages. The results indicate that Kupffer cells, endothelial cells and fat-storing cells express both IGF-II and the three different receptor mRNAs. Furthermore, a switch from a fetal to an adult IGF-II mRNA profile was obtained in the different cell preparations. Therefore, our results indicate that regulation of IGF-II gene expression can be analyzed through these isolated liver cell preparations. These results might also be important in investigating the potential role of IGF-II in liver carcinogenesis.

Presence of Liver CGRP/Amylin Receptors in Only Nonparenchymal Cells and Absence of Direct Regulation of Rat Liver Glucose Metabolism by CGRP/Amylin

Human calcitonin gene-related peptide (hCGRP-1) and human amylin (hA) have been reported to increase hepatic glucose output in vivo and to bind with high affinity to rat liver plasma membranes, resulting in increased cAMP production. These observations have led to the hypothesis that CGRP or amylin may be physiological regulators of liver glucose metabolism. Liver plasma membranes are derived from several cell types, including parenchymal (hepatocyte), Kupffer, endothelial, lipid storage, and smooth muscle cells. Because the parenchymal cell is responsible for the contribution of the liver to whole-body glucose homeostasis, it is important to verify the location and activity of the CGRP/amylin receptor to this cell. These studies separate liver cells prepared by collagenase digestion into parenchymal and nonparenchymal fractions by metrizamide gradient and differential centrifugation. 125I-labeled [Tyr-0]hCGRP-1 bound with high affinity to nonparenchymal cell fraction and was displaced by both hCGRP-1 and hA. hCGRP-1 bound with greater affinity than hA (Kd = 2.1 +/- 1.6 x 10(-11) vs. 2.6 +/- 1.2 x 10(-8) M) in a manner similar to the binding reported for liver plasma membrane fraction. Linear regression of receptor concentration against nonparenchymal cell count per milliliter was significant (r = 0.999, P = 0.026), leading to an estimate of 3000 receptors/cell. The parenchymal cell fraction bound very little 125I-[Tyr-0]hCGRP-1, and regression of receptor concentration against parenchymal cell count per milliliter was not significant (r = -0.708, P = 0.29), suggesting that binding was not due to parenchymal cells but instead to contamination by nonparenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatocyte growth factor/hepatopoietin A stimulates the growth of rat kidney proximal tubule epithelial cells (RPTE), rat nonparenchymal liver cells, human melanoma cells, mouse keratinocytes and stimulates anchorage-independent growth of SV-40 transformed RPTE

Hepatocyte growth factor/hepatopoietin A is a mitogen for primary hepatocytes and may mediate regeneration after liver damage. To date, the activity of this novel factor has been restricted to hepatocytes. We now show that the factor is also a mitogen for a number of primary epithelial cells but is inactive with human foreskin fibroblasts, human endothelial cells and HEP3B cells. The factor also substitutes for HBGF-2 (basic FGF) in stimulating the anchorage-independent growth of SV-40 transformed rat kidney epithelial cells. Therefore, hepatocyte growth factor/hepatopoietin A appears to act on a variety of epithelial, but not mesenchymal, cells which respond to HBGFs.

Ganglioside biosynthesis in rat liver: Different distribution of ganglioside synthases in hepatocytes, kupffer cells, and sinusoidal endothelial cells

The activities of five glycolipid-glycosyltransferases, GL 2, GM 3, GM 2, GM 1, and GD 1a synthase, were determined in a cell-free system with homogenate protein of total rat liver, isolated hepatocytes, Kupffer cells, and sinusoidal endothelial cells. In rat liver parenchymal and nonparenchymal cells ganglioside synthases were distributed differently. Compared to hepatocytes, Kupffer cells expressed a nearly sevenfold greater activity of GM 3 synthase, but only 14% of GM 2, 19% of GM 1, and 67% of GD 1a synthase activity. Sinusoidal endothelial cells expressed a pattern of enzyme activities quite similar to that of Kupffer cells with the exception of higher GM 2 synthase activity. Activity of GL 2 synthase was distributed uniformly in parenchymal and nonparenchymal cells of rat liver, but differed by sex. It was 1 to 2 orders of magnitude below that of all the other ganglioside synthases investigated. The results indicate GL 2 synthase regulates the total hepatic ganglioside content, and hepatocytes but not nonparenchymal liver cells have high enzymatic capacities to form a-series gangliosides more complex than GM 3.

Procollagen expression by nonparenchymal rat liver cells in experimental biliary fibrosis

To localize the cellular sources of the collagens excessively deposited in the liver in the course of secondary biliary fibrosis, we have analyzed by in situ hybridization the distribution of α2(I), α1(III), and α1(IV) procollagen and albumin RNA transcripts in rat livers up to 6 wk following common bile duct ligation and scission. In normal liver, moderate amounts of α2(I) and α1(III) procollagen RNA were found in nonparenchymal cells, while α1(IV) procollagen gene expression was at the threshold of detection. Following bile duct obstruction, increasing amounts of α2(I), α1(III), and α1(IV) procollagen gene transcripts were observed in cells of the expanding portal tracts and in perisinusoidal cells in areas of excessive collagen deposition. Procollagen gene expressing perisinusoidal cells were colocalized with desmin-immunoreactive cells, suggesting that Ito cells and transitional cells were among the collagen-expressing cell types. Only α1(IV) procollagen transcripts were found in epithelial cells of newly formed bile ducts. Neither normal nor fibrotic liver showed any hybridization signal above background over hepatocytes, indicating that hepatocytes are unlikely to be a major source of hepatic collagen.

Cytochrome P450 in highly purified suspension of nonparenchymal liver cells.

Rat liver nonparenchymal cells (NPC) were prepared by pronase digestion and purified on discontinuous gradients on Nycodenz. Morphological and biochemical characterization of cell suspensions showed that they were free of contamination by hepatocytes. We have confirmed the usefulness of pyruvate kinase activity in monitoring the degree of hepatocyte contamination of NPC and we have derived an equation which allows this carry-over to be calculated. Using highly purified suspensions of NPC we have shown that they contain glucose-6-phosphatase in low but detectable levels. Spectrophotometric studies showed that they contain cytochrome P450, with a specific content of 24 +/- 5 pmole mg-1 cell protein. A potential source of error in previous studies was recognized; namely that peroxidase, present in NPC in high concentration, is able to mask the absorption due to cytochrome P450. Both the presence and inducibility of this enzyme in NPC prepared from rats pretreated with phenobarbital or 3-methylcholanthrene have been confirmed using Western blot analysis.

In vivo studies on the binding sites for lipoprotein (a) on parenchymal and non-parenchymal rat liver cells

The direct correlation between lipoprotein (a) (Lp(a)) concentrations and atherosclerosis stimulated us to investigate the in vivo interaction of Lp(a) with the liver and the various liver cell types. In untreated rats the serum decay of Lp(a) is comparable to that of LDL. By estrogen treatment the interaction of LDL with parenchymal liver cells is increased 17-fold whereas only a 2-fold effect on Lp(a) is found. The decay of Lp(a) in estrogen-treated rats is slower than for LDL. The data indicate that Lp(a) in vivo shows a less efficient interaction than LDL with the estrogen-induced apo-B,E receptor on parenchymal liver cells. It is suggested that the inability of Lp(a) to interact efficiently with the LDL removal system of the liver might be related to its atherogenic action.