Prostaglandin E2 affects differently the release of inflammatory mediators from resident macrophages by LPS and muramyl tripeptides.

LPS and MTP-PE (liposome-encapsulated N-acetyl-muramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-:[1',2'dipalmitoyl -sni-glycero-3-(hydroxy-phosphoryl-oxyl)] etylamide) induce in liver macrophages a synthesis and release of TNF-alpha, nitric oxide and prostanoids. Both agents induce an expression of mRNA's encoding TNF-alpha, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and of corresponding proteins. LPS and MTP-PE induce a rapid activation of the extracellular regulated kinase (ERK) isoenzymes-1 and -2. Inhibition of map kinase isoenzymes leads to a decreased release of TNF-alpha, nitric oxide and prostaglandin (PG) E2 after both agents. The transcription factors NF-kappaB and AP-1 are strongly activated by LPS within 30 minutes. MTP-PE induces a weak activation of both transcription factors only after 5 hours. Inhibition of NF-kappaB inhibits the LPS- but not the MTP-PE-induced release of TNF-alpha, nitric oxide and PGE2. PGE2 release after LPS is higher than after MTP-PE. Exogenously added PGE2 inhibits the activation of map kinase and TNF-alpha release by LPS, but not by MTP-PE. Release of nitric oxide after LPS and MTP-PE is enhanced after prior addition of PGE2. PGD2 is without any effect. MTP-PE, but not LPS, induces a cytotoxicity of Kupffer cells against P815 tumor target cells. The MTP-PE-induced cytotoxicity is reduced by TNF-alpha neutralizing antibodies, indicating the involvement of TNF-alpha. Thus our results suggest that the different potencies of LPS and MTP-PE as immunomodulators probably result from different actions on Kupffer cells, resulting in differences in the amounts and kinetics of released TNF-alpha and PGE2, and that PGE2 plays an important regulatory role in the action of LPS, but not in the actions of MTP-PE.

Differential activation of transcription factors NF-kappa B and AP-1 in rat liver macrophages.

Liver macrophages (Kupffer cells) respond to many stimulations with the production of bioactive substances including cytokines, eicosanoids, and inorganic radicals. In this study the activation of transcription factors by substances inducing cytokine gene expression or superoxide formation in rat Kupffer cells was examined. Using primary cultures of rat Kupffer cells the role of NF-kappa B and activator protein 1 (AP-1) in the expression of the tumor necrosis factor-alpha (TNF-alpha) gene by lipopolysaccharide (LPS) was investigated. Both transcription factors were strongly activated but with different kinetics. Maximal DNA-binding activity was induced with 50 ng of LPS/mL of medium and persisted for at least 24 hours. At that time, NF-kappa B- as well as AP-1-DNA complexes decreased their mobilities in native gels. Among the cytokines tested only TNF-alpha and macrophage colony-stimulating factor (M-CSF) were able to activate NF-kappa B in Kupffer cells. Phorbol ester and zymosan activated AP-1 but not NF-kappa B; the treatment of zymosan yielding a modified form of AP-1. Of all substances found to interfere with TNF-alpha production by Kupffer cells (pyrrolidine dithiocarbamate, dexamethasone, prostaglandin E2, interleukin [IL]-4, IL-10, and transforming growth factor-beta [TGF-beta]) only pyrrolidine dithiocarbamate was able to completely inhibit the activation of NF-kappa B by LPS. Although not abrogating the LPS activation of NF-kappa B, dexamethasone inhibited that of AP-1. The results indicate a direct participation of NF-kappa B in the regulation of TNF-alpha synthesis and a differential effect of LPS on NF-kappa B and AP-1, respectively.

Effects of hypoxia on the oxygen-dependent metabolism of prostaglandins and adenosine in liver cells.

In this study, the capacity of hepatocytes to degrade prostaglandins diminished if the partial oxygen pressure dropped below 5%. This decrease was accompanied by an increased lactate/pyruvate ratio, a decrease in fatty acid oxidation and a drop in the ATP level. The degradation of exogenous adenosine increased with decreasing oxygen tension. At a partial oxygen pressure below 10%, the conversion of uric acid to allantoin, the final catabolite of adenosine in the rat, was strongly inhibited, resulting in the accumulation of uric acid in the medium. A good correlation was observed between the partial oxygen pressure, the oxidation of uric acid to allantoin and the degradation of prostaglandins D2 and E2, suggesting a peroxisomal pathway of hepatic prostaglandin oxidation. Subcellular fractionation of liver homogenates revealed peroxisomes as the site of degradation of prostaglandins D2 and E2 augmented by cytosolic components. The similarity of the degradation products found in the cell-free system, in hepatocytes and in the perfused liver further supports a peroxisomal degradation of prostaglandins in vivo. Stimulated liver macrophages (Kupffer cells) produced the same amount and pattern of eicosanoids at 1% and 21% O2. Even the formation of superoxide remained unaffected down to a partial pressure of 1%. At partial O2 pressures below 1%, the production of prostaglandins and superoxide became strongly inhibited. These results indicate that essential oxygenation reactions in activated Kupffer cells, including prostaglandin synthesis, possess high affinities to oxygen, while the peroxisomal pathway of prostaglandin oxidation in hepatocytes is sensitive to an O2 tension as low as 5%.

Production of tumor necrosis factor-alpha, interleukin-1 and interleukin-6 in the perfused rat liver.

The kinetics of the production and release of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) and interleukin-6 (IL-6) were investigated in the perfused rat liver and in primary cultures of Kupffer cells after stimulation with lipopolysaccharide (LPS). A small and transient accumulation of TNF-alpha could be detected immunohistochemically and by cytotoxicity assay in the intracellular space about 1 h after addition of LPS to the cultured cells. TNF-alpha release in the perfused liver followed similar kinetics as those found in the serum of LPS-treated rats and in primary cultures of rat Kupffer cells. The cytotoxic TNF-alpha activity of the perfusate attained its maximum (11.5 +/- 2.6 U/ml) 90 min after LPS stimulation and remained nearly constant for further 150 min. 2 microM dexamethasone reduced the production of TNF-alpha by 10 g of liver during 240 min from 46 to 16 x 10(3) units. The production of IL-1 and IL-6 by 10 g of liver during the initial 240 min was 3 and 530 x 10(3) IU, respectively. The maximal concentrations of IL-1 (1.4 +/- 0.7 IU/ml) and IL-6 (157 +/- 60 IU/ml) were found 240 min after LPS addition. The production of IL-1 was totally suppressed by 2 microM dexamethasone.(ABSTRACT TRUNCATED AT 250 WORDS)

The contraction of hepatic stellate (Ito) cells stimulated with vasoactive substances. Possible involvement of endothelin 1 and nitric oxide in the regulation of the sinusoidal tonus.

We have studied the contractility of liver sinusoidal stellate (Ito) cells stimulated with endothelin 1, nitric-oxide donors and eicosanoids. Contraction and relaxation of stellate cells were detected by the use of a silicone-rubber method that revealed the traction forces exerted by these cells. Endothelin 1 was a strong elicitor for stellate-cell contraction. 78, 55, 59 and 56% of stellate cells were contracted 2.5, 5, 10 and 20 min, respectively, after exposure to 10 nM endothelin 1. The effect of endothelin 1 was dose dependent and still detectable at an endothelin 1 concentration of 100 pM. Concomitantly, an endothelin-dependent formation of inositol phosphates was apparent; values of InsP, InsP2, and InsP3 were 881 +/- 99%, 1965 +/- 368%, and 791 +/- 120% of control, respectively, 20 min after addition of 10 nM endothelin 1. In addition, endothelin 1 caused a transient increase of [Ca2+]i in stellate cells from a basal value of 121 +/- 9 nM to maximal 1015 +/- 86 nM. These endothelin-1 effects were much stronger than those of the thromboxane-A2 analogue U46619 and of prostaglandin F2 alpha. In contrast, Iloprost, prostaglandin E2, and sodium nitroprusside promoted stellate-cell relaxation; for example, 82, 83 and 71% of stellate cells relaxed 5, 10, and 20 min, respectively, after addition of 500 microM sodium nitroprusside to contacted cells. Prostaglandin E2 and Iloprost led to elevation of cAMP levels in stellate cells from a basal value of 9.2 +/- 0.8 pmol/well to 55.1 +/- 8.0 and 122.2 +/- 12.2 pmol/well 10 min after addition of prostaglandin E2 (5 microM) and Iloprost (5 microM), respectively, in the presence of 3-isobutyl-1-methylxanthine (0.5 mM). However, sodium nitroprusside was a trigger for cGMP accumulation. Intracellular cGMP increased from a basal value of 0.9 +/- 0.07 pmol/well to 13.4 +/- 6.7 pmol/well 10 min after addition of 500 microM sodium nitroprusside into the medium. It is interesting that Iloprost and sodium nitroprusside also induced the disappearance of actin stress fibers in contracted cells; F-actin stress fibers became less numerous and de-aggregated; more than 90% of stellate cells were void of stress fibers after 10 microM Iloprost treatment for 30 min. Thus, endothelin 1, eicosanoids and sodium nitroprusside are able to modulate the contractility of stellate cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of endothelin-1 action on the perfused rat liver.

Endothelin-1 (ET-1) was found to be a very potent stimulus for contraction and glycogenolysis in the perfused rat liver. At 1 nM it caused a dramatic increase in portal pressure of 22.1 +/- 2.7 cm water and enhanced the glucose output up to 3-fold. Extracellular Ca2+ and protein kinase C were involved in the signal transduction of ET-1. ET-1 action does not seem to be mediated by endogenous eicosanoids. The effects of ET-1 were significantly reduced in the presence of 1 microM Iloprost, a prostaglandin I2 analogue, or by 100 microM sin-1, a nitric oxide donor. In cultured hepatocytes, glycogenolysis was also stimulated by ET-1 although to an extent too small to explain the high glucose output found in the perfused liver.

Fate of exogenous and endogenous prostaglandins D2 and E2 in the perfused rat liver.

The degradation of radiolabelled exogenous PGD2 and PGE2 was compared to that of endogenous labelled prostaglandins synthesized after stimulation with PMA in the perfused rat liver. With exogenous PGD2 and PGE2 the same degradation products were found in the perfused liver as in hepatocyte primary cultures. The major metabolite of PGD2 was dinor-PGD2 while tetranor-PGE1 was the main degradation product of PGE2. Some polar metabolites and tritiated water were also formed. The metabolites detected with endogenous prostaglandins were similar to those obtained with exogenous PGD2. Over 99% of the labelled prostaglandins were degraded in the recirculating perfusion within 40 min. In the open perfusion system, 95% of endogenous PGD2 was calculated to be degraded after a single passage through the liver, which suggests that hepatocytes play an important role in the removal of biologically active prostaglandins.

Ganglioside biosynthesis in rat liver: different distribution of ganglioside synthases in hepatocytes, Kupffer cells, and sinusoidal endothelial cells.

The activities of five glycolipid-glycosyltransferases, GL2, GM3, GM2, GM1, and GD1a 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 GM3 synthase, but only 14% of GM2, 19% of GM1, and 67% of GD1a synthase activity. Sinusoidal endothelial cells expressed a pattern of enzyme activities quite similar to that of Kupffer cells with the exception of higher GM2 synthase activity. Activity of GL2 synthase was distributed unifromly 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 GL2 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 GM3.

Output and effects of thromboxane produced by the liver perfused with phorbol myristate acetate.

The capacity of the perfused rat liver to produce thromboxane after stimulation by phorbol myristate acetate was examined. A total of 109 +/- 20 and 155 +/- 28 pmol/g liver were found in the perfusate and in the bile, respectively, after 40 min. The amount of thromboxane recovered in the perfusate and in the bile accounted for 12.6% of the production calculated from the same number of Kupffer cells in primary cultures, indicating that a major part of thromboxane was taken up and inactivated by hepatocytes. The effect of endogenously synthesized thromboxane on the liver was assessed by using CGS 13080, a thromboxane synthase inhibitor, or BM 13.177, a thromboxane receptor antagonist. 20 nM CGS 13080 in the perfusate inhibited the synthesis of thromboxane and at the same time the elevation of portal pressure and glycogenolysis following administration of phorbol 12-myristate 13-acetate (PMA). The thromboxane receptor antagonist BM 13.177 did not inhibit the synthesis of thromboxane, but reduced the PMA-related elevation of portal pressure and glycogenolysis to the same extent (greater than 60%) as CGS 13080. Sodium nitroprusside, a vasodilator, inhibited the rise in portal pressure caused by PMA to the same extent as CGS 13080 or BM 13.177 but reduced the increase in glycogenolysis only by 25%. These results indicate that thromboxane released by stimulated Kupffer cells of the liver elevates portal pressure and glycogenolysis in the perfused rat liver, although by different mechanisms.

Remodeling of a rat hepatocyte plasma membrane glycoprotein. De- and reglycosylation of dipeptidyl peptidase IV.

The present paper demonstrates the terminal de- and reglycosylation of a rat hepatocyte plasma membrane glycoprotein, dipeptidyl peptidase IV (DPP IV). Cultured hepatocytes were used in pulse-chase experiments with [3H]L-fucose and [14C]N-acetyl-D-mannosamine as markers for terminal carbohydrates, [3H]D-mannose as marker of a core-sugar, and [35S]L-methionine for labeling the protein backbone. Membrane DPP IV was immunoprecipitated with a polyclonal antibody which bound selectively at 4 degrees C to the cell-surface glycoprotein. The times of maximal labeling of hepatocyte plasma membrane DPP IV were 6-9 min for [3H]L-fucose, 20 min for [3H]D-mannose, and 25 min for [35S]L-methionine. When antibodies were bound to cell-surface DPP IV at 4 degrees C, the immune complex remained stable for more than 1 h after rewarming to 37 degrees C, despite ongoing metabolic and membrane transport processes. This was shown by pulse labeling with [35S]L-methionine at 37 degrees C, followed by cooling to 4 degrees C, and addition of antibody against plasma membrane DPP IV. During rewarming, the radioactivity in the complex remained constant. In a similar experiment with [3H]L-fucose, the radioactivity in the immune complex declined rapidly, indicating a defucosylation of the plasma membrane glycoprotein. Using the same experimental design with [3H]D-mannose, the radioactivity in the immune complex remained constant, showing that the core-sugar D-mannose is not cleaved from the membrane glycoprotein. Terminal reglycosylation (refucosylation and resialylation) was demonstrated as follows. Hepatocytes were maintained at 37 degrees C in a medium supplemented with tunicamycin in order to block the de novo synthesis of N-glycosidically bound carbohydrate chains. At 4 degrees C the antibody against DPP IV bound only to cell surface glycoprotein. During the rewarming period at 37 degrees C, radioactivity from [3H]L-fucose and [14C]N-acetyl-D-mannosamine became incorporated into the immune complex. This indicates a fucosylation and sialylation of the glycoprotein originally present at the cell surface. The mechanisms whereby terminal de- and reglycosylation of plasma membrane glycoproteins may occur during membrane recycling are discussed.

Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins.

The initial plasma clearance and organ distribution of alpha 1-acid glycoprotein and alpha 2-macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1-deoxymannojirimycin (high-mannose type). Deglycosylated glycoproteins (Asn-GlcNAc) were obtained by endoglucosaminidase H treatment of high-mannose-type glycoproteins. Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high-mannose type. 64% of unglycosylated and 80% of deglycosylated alpha 1-acid glycoprotein disappeared from the plasma. The respective values for alpha 2-macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low-molecular-mass polypeptides. Whereas complex-type alpha 1-acid glycoprotein and alpha 2-macroglobulin showed no accumulation in various organs, hybrid-type alpha 1-acid glycoprotein and alpha 2-macroglobulin were present in spleen and liver. High-mannose-type alpha 1-acid glycoprotein and alpha 2-macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high-mannose-type glycoproteins. Competition experiments with mannan and GlcNAc-bovine-serum-albumin showed a mannose/GlcNAc receptor-mediated removal. Whereas unglycosylated alpha 1-acid glycoprotein was taken up by the kidney, unglycosylated alpha 2-macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn-GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor-mediated uptake mainly into the spleen. We conclude that N-linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.

Net prostaglandin release by perfused rat liver after stimulation with phorbol 12-myristate 13-acetate.

Phorbol myristate acetate, which was shown previously to elicit eicosanoid synthesis in primary cultures of Kupffer cells, led to a net release of prostaglandins (PG) D2 and E2 from the perfused rat liver. While a substantial amount of PGD2 (the major prostaglandin of Kupffer cells) left the liver, very little PGE2 was found in the effluent. Considerable amounts of immunologically reactive PGD2 and E2 were secreted with the bile. PGE2 rather than PGD2 was able to stimulate glycogenolysis and to increase perfusion pressure. These effects were, however, strongly dependent on the direction of the flow. If the liver was perfused in a retrograde fashion, i.e., from the vena cava to the portal vein, phorbol myristate acetate or PGE2 exerted only minor effects. These observations suggest a topological heterogeneity of producer and responder cells, respectively, in the liver sinusoid.

Regulation of synthesis and secretion of major rat acute-phase proteins by recombinant human interleukin-6 (BSF-2/IL-6) in hepatocyte primary cultures.

The regulation of the three major acute-phase proteins alpha 2-macroglobulin, cysteine proteinase inhibitor and alpha 1-antitrypsin by recombinant human interleukin-1 beta, recombinant human interleukin-6 and recombinant human tumor necrosis factor alpha was studied in rat hepatocyte primary cultures. Synthesis and secretion of the acute-phase proteins was measured after labeling with [35S]methionine and immunoprecipitation. Incubation of hepatocytes with interleukin-6 led to dose-dependent and time-dependent changes in the synthesis of the three major acute-phase proteins and albumin, similar to those occurring in vivo during experimental inflammation. alpha 2-Macroglobulin and cysteine proteinase inhibitor synthesis was induced 54-fold and 8-fold, respectively, 24 h after the addition of 100 units/ml interleukin-6. At the same time synthesis of the negative acute-phase protein albumin was reduced to 30% of controls. Half-maximal effects were achieved with 4 units interleukin-6/ml. Interleukin-1 beta had only a partial effect on the regulation of the four patients studied: only a twofold stimulation of alpha 2-macroglobulin and a 60% reduction of albumin synthesis were observed. Tumor necrosis factor alpha did not alter the synthesis of acute-phase proteins. The stimulation of alpha 2-macroglobulin and cysteine proteinase inhibitor synthesis by interleukin-6 was inhibited by interleukin-1 beta in a dose-dependent manner. In pulse-chase experiments the effect of interleukin-1 beta, interleukin-6 and tumor necrosis factor alpha on the secretion of acute-phase proteins was examined. Interleukin-6 markedly accelerated the secretion of total proteins and alpha 2-macroglobulin, whereas the secretion of cysteine proteinase inhibitor, alpha 1-antitrypsin and albumin was not affected. The inhibition of N-glycosylation by tunicamycin abolished the effect of interleukin-6 on the secretion of alpha 2-macroglobulin, indicating a possible role of interleukin-6 on N-glycosylation.

Stimulation of prostaglandin release by Ca2+-mobilizing agents from the perfused rat liver. A comparative study on the action of ATP, UTP, phenylephrine, vasopressin and nerve stimulation.

Several Ca2+-mobilizing agents were tested for their potential to elicit the net release of prostaglandins from the isolated perfused rat liver. Among these ATP and UTP only led to an efficient stimulation of PGD2 and PGE2 synthesis. 20 microM ATP or 20 microM UTP increased the release of PGD2 8-fold and that of PGE2 2 to 3-fold. In total, at least 40 times more PGD2 than PGE2 left the liver after stimulation. The time course of prostaglandin release was similar for both nucleotides. Vasopressin had almost no effect on the release of both prostaglandins and on portal vein pressure. But phenylephrine and nerve stimulation while raising the PGD2 efflux only slightly caused an elevation of PGE2 outflow and portal pressure.

Hepatocyte heterogeneity in response to extracellular ATP.

1. The metabolic and hemodynamic effects of extracellular ATP in perfused rat liver were compared during physiologically antegrade (portal to hepatic vein) and retrograde (hepatic to portal vein) perfusion. ATP in concentrations up to 100 microM was completely hydrolyzed during a single liver passage regardless of the perfusion direction. 2. The ATP(20 microM)-induced increases of glucose output, perfusion pressure and ammonium ion release seen during antegrade perfusions were diminished by 85-95% when the perfusion was in the retrograde direction, whereas the amount of Ca2+ mobilized from the liver was decreased by only 60%. The maximal rate of initial K+ uptake following ATP was dependent on the amount of Ca2+ mobilized regardless of the direction of perfusion. In the presence of UMP (1 mM), an inhibitor of ATP hydrolysis by membrane-bound nucleotide pyrophosphatase, the effect of the direction of perfusion on the glycogenolytic response to ATP (20 microM) was largely diminished. 3. For a maximal response of glucose output, Ca2+ release and perfusion pressure to extracellular ATP, concentrations of about 20 microM, 50 microM and 100 microM were required during antegrade perfusion, respectively. These maximal responses could also be obtained during retrograde perfusion, but higher ATP concentrations were required (120 microM, 80 microM, above 200 microM, respectively). 4. 14CO2 production from [1-14C]glutamate which occurs predominantly in the perivenous hepatocytes capable of glutamine synthesis was stimulated by extracellular ATP (20 microM); it was only slightly affected by the direction of perfusion. In antegrade perfusions, ATP (20 microM) increased 14CO2 production from 88 to 162 nmol g-1 min-1, compared to an increase from 91 to 148 nmol g-1 min-1 in retrograde perfusion. 5. The data are interpreted to suggest that (a) extracellular ATP is predominantly hydrolyzed by a small hepatocyte population located at the perivenous outflow of the acinus; (b) glycogenolysis to glucose is predominantly localized in the periportal area; (c) contractile elements (sphincters) exist near the inflow of the sinusoidal bed; (d) a considerable portion of the Ca2+ mobilized by ATP is derived from liver cells that do not contribute to hepatic glucose output.