PGE2 enhanced TNFα-mediated IL-8 induction in monocytic cell lines and PBMC.

BACKGROUND & PURPOSE: Recent studies suggested a role of prostaglandin E2 (PGE2) in the expression of the chemokine IL-8 by monocytes. The function of EP4 receptor for TNFα-induced IL-8 expression was studied in monocytic cell lines. EXPERIMENTAL APPROACH: IL-8 mRNA and protein induction as well as IL-8 promoter activity and transcription factor activation were assessed in monocytic cell lines, primary blood mononuclear cells (PBMC) and transgenic HEK293 cells expressing the EP4 receptor. KEY RESULTS: In monocytic cell lines THP-1, MonoMac and U937 PGE2 had only a marginal impact on IL-8 induction but strongly enhanced TNFα-induced IL-8 mRNA and protein synthesis. Similarly, in PBMC IL-8 mRNA induction was larger by simultaneous stimulation with TNFα and PGE2 than by either stimulus alone. The EP4 receptor subtype was the most abundant EP receptor in all three cell lines and in PBMC. Stimulation of THP-1 cells with an EP4 specific agonist enhanced TNFα-induced IL-8 mRNA and protein formation to the same extent as PGE2. In HEK293 cells expressing EP4, but not in wild type HEK293 cells lacking EP4, PGE2 enhanced TNFα-induced IL-8 protein and mRNA synthesis. In THP-1 cells, the enhancement of TNFα-mediated IL-8 mRNA induction by PGE2 was mimicked by a PKA-activator. Furthermore in these cells PGE2 induced expression of transcription factor C/EBPß, enhanced NF-κB activation by TNFα and inhibited TNFα-mediated AP-1 activation. PGE2 and TNFα synergistically activated transcription factor CREB, induced C/EBPß expression and enhanced the activity of an IL-8 promoter fragment containing -223 bp upstream of the transcription start site. CONCLUSIONS AND IMPLICATIONS: These findings suggest that a combined stimulation of TNFα and PGE2/EP4 signal chains in monocytic cells leads to maximal IL-8 promoter activity, as well as IL-8 mRNA and protein induction, by activating the PKA/CREB/C/EBPß as well as NF-κB signal chains.

Synergistic acceleration of thyroid hormone degradation by phenobarbital and the PPAR alpha agonist WY14643 in rat hepatocytes.

Energy balance is maintained by controlling both energy intake and energy expenditure. Thyroid hormones play a crucial role in regulating energy expenditure. Their levels are adjusted by a tight feedback-controlled regulation of thyroid hormone production/incretion and by their hepatic metabolism. Thyroid hormone degradation has previously been shown to be enhanced by treatment with phenobarbital or other antiepileptic drugs due to a CAR-dependent induction of phase II enzymes of xenobiotic metabolism. We have recently shown, that PPAR alpha agonists synergize with phenobarbital to induce another prototypical CAR target gene, CYP2B1. Therefore, it was tested whether a PPAR alpha agonist could enhance the phenobarbital-dependent acceleration of thyroid hormone elimination. In primary cultures of rat hepatocytes the apparent half-life of T3 was reduced after induction with a combination of phenobarbital and the PPAR alpha agonist WY14643 to a larger extent than after induction with either compound alone. The synergistic reduction of the half-life could be attributed to a synergistic induction of CAR and the CAR target genes that code for enzymes and transporters involved in the hepatic elimination of T3, such as OATP1A1, OATP1A3, UGT1A3 and UGT1A10. The PPAR alpha-dependent CAR induction and the subsequent induction of T3-eliminating enzymes might be of physiological significance for the fasting-induced reduction in energy expenditure by fatty acids as natural PPAR alpha ligands. The synergism of the PPAR alpha agonist WY14643 and phenobarbital in inducing thyroid hormone breakdown might serve as a paradigm for the synergistic disruption of endocrine control by other combinations of xenobiotics.

Inhibition by PGE2 of glucagon-induced increase in phosphoenolpyruvate carboxykinase mRNA and acceleration of mRNA degradation in cultured rat hepatocytes.

In cultured rat hepatocytes the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) is known to be induced by glucagon via an elevation of cAMP. Prostaglandin E2 has been shown to antagonize the glucagon-activated cAMP formation, glycogen phosphorylase activity and glucose output in hepatocytes. It was the purpose of the current investigation to study the potential of PGE2 to inhibit the glucagon-induced expression of PCK on the level of mRNA and enzyme activity. PCK mRNA and enzyme activity were increased by 0.1 nM glucagon to a maximum after 2 h and 4 h, respectively. This increase was completely inhibited if 10 microM PGE2 was added concomitantly with glucagon. This inhibition by PGE2 of glucagon-induced PCK activity was abolished by pertussis toxin treatment. When added at the maximum of PCK mRNA at 2 h, PGE2 accelerated the decay of mRNA and reduced enzyme activity. This effect was not reversed by pertussis toxin treatment. Since in liver PGE2 is derived from Kupffer cells, which play a key role in the local inflammatory response, the present data imply that during inflammation PGE2 may reduce the hepatic gluconeogenic capacity via a Gi-linked signal chain.

Increase of urate formation by stimulation of sympathetic hepatic nerves, circulating noradrenaline and glucagon in the perfused rat liver.

In the isolated rat liver perfused in situ stimulation of the nerve bundles around the portal vein and the hepatic artery caused an increase of urate formation that was inhibited by the alpha 1-blocker prazosine and the xanthine oxidase inhibitor allopurinol. Moreover, nerve stimulation increased glucose and lactate output and decreased perfusion flow. Infusion of noradrenaline had similar effects. Compared to nerve stimulation infusion of glucagon led to a less pronounced increase of urate formation and a twice as large increase in glucose output but a decrease in lactate release without affecting the flow rate. Insulin had no effect on any of the parameters studied.

Exclusive expression of the Gs-linked prostaglandin E2 receptor subtype 4 mRNA in mononuclear Jurkat and KM-3 cells and coexpression of subtype 4 and 2 mRNA in U-937 cells.

Prostaglandin E2 (PGE2) is regarded as a potent regulator of the immune system. It can regulate apoptosis in mononuclear cells and modulate the cytokine secretion pattern from T-helper cell subpopulations via an increase in cyclic AMP (cAMP). Of the 4 PGE2 receptor subtypes (EP1-EP4) that are defined pharmacologically by their affinity to subtype-specific ligands and their coupling to G proteins, EP2 and EP4 receptors couple to Gs. It is as yet unknown which of these two receptor subtypes mediates the immunomodulatory effects. By quantitative RT-PCR, the mRNA for EP4 receptors was demonstrated and quantified in the human mononuclear cell lines Jurkat, KM-3 and U-937. However, EP2 receptor mRNA was only present in U-937 cells and was 100-fold less abundant than EP4 receptor mRNA. PGE2 increased cAMP formation with an ED50 of 50-100 nM in all cell lines. cAMP formation was inhibited by the EP4R-specific antagonist AH23848. Since AH23848 inhibited PGE2-induced cAMP formation in U-937 cells to a similar extent as in Jurkat and KM-3, EP2 receptors seem to play, if any, only a secondary role for the PGE2-mediated cAMP formation in U-937 cells.

The C-terminal domain of the human EP4 receptor confers agonist-induced receptor desensitization in a receptor hybrid with the rat EP3beta receptor.

Prostaglandin E2 receptors (EPR), which belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains, can be classified into four subtypes according to their ligand binding and G protein coupling specificity. Of these, EP3betaR is coupled to Gi, whereas EP4R is coupled to Gs. EP4R, in contrast to EP3betaR, shows agonist-induced desensitization. The C-terminal domain and the third intracellular loop of these receptors have been implicated in G protein coupling specificity and desensitization. Here, receptor hybrids consisting of the main portion of rat EP3betaR and either the C-terminal domain or the third intracellular loop of human EP4R were used to study the contribution of the respective receptor domains to G protein coupling and desensitization. Neither the EP4R C-terminal domain nor the EP4R third intracellular loop alone was sufficient to change the coupling specificity of the rEP3hEP4 receptor hybrids from Gi to Gs or to confer additional Gs coupling. However, the EP4R C-terminal domain but not the third intracellular loop was necessary and sufficient to mediate rapid agonist-induced, second messenger-independent desensitization in the Gi-coupled hybrid receptors.

Stimulation of glycogen phosphorylase in rat hepatocytes via prostanoid release from Kupffer cells by recombinant rat anaphylatoxin C5a but not by native human C5a in hepatocyte/Kupffer cell co-cultures.

Human anaphylatoxin C3a had previously been shown to increase glycogenolysis in perfused rat liver and prostanoid formation in rat liver macrophages. Surprisingly, human C5a, which in other systems elicited stronger responses than C3a, did not increase glycogenolysis in perfused rat liver. Species incompatibilities within the experimental system had been supposed to be the reason. The current study supports this hypothesis: (1) In rat liver macrophages that had been maintained in primary culture for 72 h recombinant rat anaphylatoxin C5a in concentrations between 0.1 and 10 micrograms/ml increased the formation of thromboxane A2, prostaglandin D2, E2 and F2 alpha 6- to 12-fold over basal within 10 min. In contrast, human anaphylatoxin C5a did not increase prostanoid formation in rat Kupffer cells. (2) The increase in prostanoid formation by recombinant rat C5a was specific. It was inhibited by a neutralizing monoclonal antibody. (3) In co-cultures of rat hepatocytes and rat Kupffer cells but not in hepatocyte mono-cultures recombinant rat C5a increased glycogen phosphorylase activity 3-fold over basal. This effect was inhibited by incubation of the co-cultures with 500 microM acetylsalicyclic acid. Thus, C5a generated either locally in the liver or systemically e.g. in the course of sepsis, may increase hepatic glycogenolysis by a prostanoid-mediated intercellular communication between Kupffer cells and hepatocytes.

Possible role for ligand binding of histidine 81 in the second transmembrane domain of the rat prostaglandin F2alpha receptor.

For the five principal prostanoids PGD2, PGE2, PGF2alpha, prostacyclin and thromboxane A2 eight receptors have been identified that belong to the family of G-protein-coupled receptors. They display an overall homology of merely 30%. However, single amino acids in the transmembrane domains such as an Arg in the seventh transmembrane domain are highly conserved. This Arg has been identified as part of the ligand binding pocket. It interacts with the carboxyl group of the prostanoid. The aim of the current study was to analyze the potential role in ligand binding of His-81 in the second transmembrane domain of the rat PGF2alpha receptor, which is conserved among all PGF2alpha receptors from different species. Molecular modeling suggested that this residue is located in close proximity to the ligand binding pocket Arg 291 in the 7th transmembrane domain. The His81 (H) was exchanged by site-directed mutagenesis to Gln (Q), Asp (D), Arg (R), Ala (A) and Gly (G). The receptor molecules were N-terminally extended by a Flag epitope for immunological detection. All mutant proteins were expressed at levels between 50% and 80% of the wild type construct. The H81Q and H81D receptor bound PGF2alpha with 2-fold and 25-fold lower affinity, respectively, than the wild type receptor. Membranes of cells expressing the H81R, H81A or H81G mutants did not bind significant amounts of PGF2alpha. Wild type receptor and H81Q showed a shallow pH optimum for PGF2alpha binding around pH 5.5 with almost no reduction of binding at higher pH. In contrast the H81D mutant bound PGF2alpha with a sharp optimum at pH 4.5, a pH at which the Asp side chain is partially undissociated and may serve as a hydrogen bond donor as do His and Gln at higher pH values. The data indicate that the His-81 in the second transmembrane domain of the PGF2alpha receptor in concert with Arg-291 in the seventh transmembrane domain may be involved in ligand binding, most likely not by ionic interaction with the prostaglandin's carboxyl group but rather as a hydrogen bond donor.

Increase of glucose and lactate output and decrease of flow by human anaphylatoxin C3a but not C5a in perfused rat liver.

The complement fragments C3a and C5a were purified from zymosan-activated human serum by column chromatographic procedures after the bulk of the proteins had been removed by acidic polyethylene glycol precipitation. In the isolated in situ perfused rat liver C3a increased glucose and lactate output and reduced flow. Its effects were enhanced in the presence of the carboxypeptidase inhibitor DL-mercaptomethyl-3-guanidinoethylthio-propanoic acid (MERGETPA) and abolished by preincubation of the anaphylatoxin with carboxypeptidase B or with Fab fragments of an anti-C3a monoclonal antibody. The C3a effects were partially inhibited by the thromboxane antagonist BM13505. C5a had no effect. It is concluded that locally but not systemically produced C3a may play an important role in the regulation of local metabolism and hemodynamics during inflammatory processes in the liver.

Molecular cloning and expression of a prostaglandin E2 receptor of the EP3 beta subtype from rat hepatocytes.

Rat hepatocytes have previously been reported to possess prostaglandin E2 receptors of the EP3-type (EP3-receptors) that inhibit glucagon-stimulated glycogenolysis by decreasing cAMP. Here, the isolation of a functional EP3 beta receptor cDNA clone from a rat hepatocyte cDNA library is reported. This clone can be translated into a 362-amino-acid protein, that displays over 95% homology to the EP3 beta receptor from mouse mastocytoma. The amino- and carboxy-terminal region of the protein are least conserved. Transiently transfected HEK 293 cells expressed a single binding site for PGE2 with an apparent Kd of 15 nM. PGE2 > PGF2 alpha > PGD2 competed for [3H]PGE2 binding sites as did the EP3 receptor agonists M&B 28767 = sulprostone > misoprostol but not the EP1 receptor antagonist SC 19220. In stably transfected CHO cells M&B 28767 > sulprostone = PGE2 > misoprostol > PGF2 alpha inhibited the forskolin-elicited cAMP formation. Thus, the characteristics of the EP3 beta receptor of rat hepatocytes closely resemble those of the EP3 beta receptor of mouse mastocytoma.

The C-terminal domain of the Gs-coupled EP4 receptor confers agonist-dependent coupling control to Gi but no coupling to Gs in a receptor hybrid with the Gi-coupled EP3 receptor.

Prostaglandin E2 receptors (EPR) belong to the family of G-protein-coupled receptors with 7 transmembrane domains. They form a family of four subtypes, which are linked to different G-proteins. EP1R are coupled to Gq, EP2 and EP4R to Gs and EP3R to Gi. Different C-terminal splice variants of the bovine EP3R are coupled to different G-proteins. A mouse EP3R whose C-terminal domain had been partially truncated no longer showed agonist-induced Gi-protein activation and was constitutively active. In order to test the hypothesis that the C-terminal domain confers coupling specificity of the receptors on the respective G-proteins, a cDNA for a hybrid rEP3hEP4R, containing the N-terminal main portion of the Gi-coupled rat EP(3beta)R including the 7th transmembrane domain and the intracellular C-terminal domain of the Gs-coupled human EP4R, was generated by PCR. HEK293 cells transiently transfected with the chimeric rEP3hEP4R cDNA expressed a plasma membrane PGE2 binding site with a slightly lower Kd value for PGE2 but an identical binding profile for receptor-specific ligands as cells transfected with the native rat EP(3beta)R. In HepG2 cells stably transfected with the chimeric rEP3hEP4R cDNA PGE2 did not increase cAMP formation characteristic of Gs coupling but attenuated the forskolin-stimulated cAMP synthesis characteristic of Gi coupling. This effect was inhibited by pre-treatment of the cells with pertussis toxin. Thus, the hybrid receptor behaved both in binding and in functional coupling characteristics as the native rat EP(3beta)R. Apparently, the intracellular C-terminal domain did not confer coupling specificity but coupling control, i.e. allowed a signalling state of the receptor only with agonist binding.

Stimulation by anaphylatoxin C5a of glycogen phosphorylase in rat hepatocytes via prostanoid release from hepatic stellate cells but not sinusoidal endothelial cells.

In the perfused rat liver, the anaphylatoxin C5a has been shown to enhance glucose output. Since hepatocytes lack C5a receptor mRNA, the metabolic effect of C5a must be elicited indirectly via C5a receptor expressing non-parenchymal liver cells. Kupffer cells were found to be able to mediate the C5a action via release of prostanoids. However, elimination of the Kupffer cells by pretreatment of the animals with gadolinium chloride reduced the metabolic effect of C5a to only about 40%. Therefore, it was investigated whether not only Kupffer cells but in addition also hepatic stellate cells or sinusoidal endothelial cells released prostanoids in response to C5a. In isolated hepatic stellate cells but not in sinusoidal endothelial cells, recombinant rat C5a induced a time- and dose-dependent release of thromboxane B2 and prostaglandins D2, E2 and F2alpha. The rate of prostanoid release was maximal within the first two minutes and then declined again. C5a-induced prostanoid release from hepatic stellate cells was smaller than that from Kupffer cells and it differed in the prostanoid ratios (PGE2/PGD2/PGF2alpha/TXB2 = 1:1:0.1:0.6 and 1:4:1:3, respectively). RrC5a activated hepatocellular glycogen phosphorylase via prostanoid release in cocultures of hepatocytes with hepatic stellate cells but not with sinusoidal endothelial cells. Thus, the part of the rrC5a-induced glucose output in the perfused rat liver, which was not abrogated by elimination of the Kupffer cells with gadolinium chloride, most likely was mediated by prostanoids released from hepatic stellate cells.

Molecular cloning and characterization of the four rat prostaglandin E2 prostanoid receptor subtypes.

We have characterized the rat prostanoid EP1, EP2, EP3alpha and EP4 receptor subtypes cloned from spleen, hepatocyte and/or kidney cDNA libraries. Comparison of the deduced amino acid sequences of the rat EP receptors with their respective homologues from mouse and human showed 91% to 98% and 82% to 89% identity, respectively. Radioreceptor binding assays and functional assays were performed on EP receptor expressing human embryonic kidney (HEK) 293 cells. The KD values obtained with prostaglandin E2 for the prostanoid receptor subtypes EP1, EP2, EP3alpha and EP4 were approximately 24, 5, 1 and 1 nM, respectively. The rank order of affinities for various prostanoids at the prostanoid receptor subtypes EP2, EP3alpha and EP4 receptor subtypes was prostaglandin E2 = prostaglandin E1 > iloprost > prostaglandin F2alpha > prostaglandin D2 > U46619. The rank order at the prostanoid EP1 receptor was essentially the same except that iloprost had the highest affinity of the prostanoids tested. Of the selective ligands, butaprost was selective for prostanoid EP2, M&B28767 and sulprostone were selective for EP3alpha and enprostil displayed dual selectivity, interacting with both prostanoid receptor subtypes EP1 and EP3alpha. All four receptors coupled to their predominant signal transduction pathways in HEK 293 cells. Notably, using a novel aequorin luminescence assay to monitor prostanoid EP1 mediated increases in intracellular calcium, both iloprost and sulprostone were identified as partial agonists. Finally, by Northern blot analysis EP3 transcripts were most abundant in liver and kidney whereas prostanoid EP2 receptor mRNA was expressed in spleen, lung and testis and prostanoid EP1 receptor mRNA transcripts were predominantly expressed in the kidney. The rat prostanoid EP1 probes also detected additional and abundant transcripts present in all the tissues examined. These were found to be related to the expression of a novel protein kinase gene and not the prostanoid EP1 gene [Batshake, B., Sundelin, J., 1996. The mouse genes for the EP1 prostanoid receptor and the novel protein kinase overlap. Biochem. Biophys. Res. Commun. 227. 1329-1333].

NF-κB-dependent IL-8 induction by prostaglandin E(2) receptors EP(1) and EP(4).

BACKGROUND AND PURPOSE: Recent studies suggested a role for PGE(2) in the expression of the chemokine IL-8. PGE(2) signals via four different GPCRs, EP(1) -EP(4) . The role of EP(1) and EP(4) receptors for IL-8 induction was studied in HEK293 cells, overexpressing EP(1) (HEK-EP(1) ), EP(4) (HEK-EP(4) ) or both receptors (HEK-EP(1) + EP(4) ). EXPERIMENTAL APPROACH: IL-8 mRNA and protein induction and IL-8 promoter and NF-κB activation were assessed in EP expressing HEK cells. KEY RESULTS: In HEK-EP(1) and HEK-EP(1) + EP(4) but not HEK or HEK-EP(4) cells, PGE(2) activated the IL-8 promoter and induced IL-8 mRNA and protein synthesis. Stimulation of HEK-EP(1) + EP(4) cells with an EP(1) -specific agonist activated IL-8 promoter and induced IL-8 mRNA and protein, whereas a specific EP(4) agonist neither activated the IL-8 promoter nor induced IL-8 mRNA and protein synthesis. Simultaneous stimulation of HEK- EP(1) + EP(4) cells with both agonists activated IL-8 promoter and induced IL-8 mRNA to the same extent as PGE(2) . In HEK-EP(1) + EP(4) cells, PGE(2) -mediated IL-8 promoter activation and IL-8 mRNA induction were blunted by inhibition of IκB kinase. PGE(2) activated NF-κB in HEK-EP(1) , HEK-EP(4) and HEK-EP(1) + EP(4) cells. In HEK-EP(1) + EP(4) cells, simultaneous activation of both receptors was needed for maximal PGE(2) -induced NF-κB activation. PGE(2) -stimulated NF-κB activation by EP(1) was blocked by inhibitors of PLC, calcium-signalling and Src-kinase, whereas that induced by EP(4) was only blunted by Src-kinase inhibition. CONCLUSIONS AND IMPLICATIONS: These findings suggest that PGE(2) -mediated NF-κB activation by simultaneous stimulation of EP(1) and EP(4) receptors induces maximal IL-8 promoter activation and IL-8 mRNA and protein induction.

Activation of inositol phosphate formation by circulating noradrenaline but not by sympathetic nerve stimulation with a similar increase of glucose release in perfused rat liver.

In the isolated rat liver perfused in situ, stimulation of the nerve bundles around the hepatic artery and portal vein caused an increase of glucose and lactate output and a reduction of perfusion flow. These changes could be inhibited completely by alpha-receptor blockers. The possible involvement of inositol phosphates in the intracellular signal transmission was studied. 1. In cell-suspension experiments, which were performed as a positive control, noradrenaline caused an increase in glucose output and, in the presence of 10 mM LiCl, a dose-dependent and time-dependent increase of inositol mono, bis and trisphosphate. 2. In the perfused rat liver 1 microM noradrenaline caused an increase of glucose and lactate output and in the presence of 10 mM LiCl a time-dependent increase of inositol mono, bis and trisphosphate that was comparable to that observed in cell suspensions. 3. In the perfused rat liver stimulation of the nerve bundles around the portal vein and hepatic artery caused a similar increase in glucose and lactate output to that produced by noradrenaline, but in the presence of 10 mM LiCl there was a smaller increase of inositol monophosphate and no increase of inositol bis and trisphosphate. These findings are in line with the proposal that circulating noradrenaline reaches every hepatocyte, causing a clear overall increase of inositol phosphate formation and thus calcium release from the endoplasmic reticulum, while the hepatic nerves reach only a few cells causing there a small local change of inositol phosphate metabolism and thence a propagation of the signal via gap junctions.

Structure of the 5'-flanking region of the rat prostaglandin F(2alpha) receptor gene and its transcriptional control functions in hepatocytes.

Prostaglandin F(2alpha) (PGF(2alpha)), modulates hepatocyte functions via a heptahelical G(q)-coupled PGF(2alpha)-receptor (FP-R) which in liver is expressed exclusively in hepatocytes. The aim of the present study was to isolate the 5'-flanking region of the rat FP-R gene and to elucidate its basal and IL-6-modulated transcription control function in rat hepatocytes. The 5'-non-translated region of the rat hepatocyte FP-R mRNA differed from the corresponding region in rat fetal astrocyte or corpus luteum. It was encoded by exons 1a and 2 which were separated by a 1. 4 kb intron containing the exons 1b and 1c coding for the 5'-untranslated region of rat fetal astrocyte and corpus luteum FP-R mRNA, respectively. The transcription initiation site in hepatocytes was localized 263 bp upstream of the start ATG by 5'-RACE. A DNA-fragment covering the 5'-flanking region of the rFP-R gene from -1 of the transcription initiation site to -2590 bp was cloned and sequenced. Its 3'-two thirds had a 65% sequence identity to the mouse FP-R promoter however no homology to the bovine FP-R promoter. In the overlapping sequence most of the putative transcription factor binding sites were conserved between mouse and rat. The rat promoter contained no classical TATA- or CAAT-boxes but putative binding sites for the transcription factors C/EBP, GATA-1, HNF-1, HNF-3beta, SP-1, and USF. Luciferase reporter gene constructs containing portions of the 5'-flanking region were transfected into rat hepatocytes. Luciferase expression ranked -181 >/= -608 < -1418 > -1821 >/= -2590. The strongest transcriptional activity was conferred by the region between -608 and -1418 containing a cluster of potential HNF-1 and HNF-3beta binding sites that might allow the exclusive expression of FP-R mRNA in hepatocytes. The amount of FP-R mRNA and the luciferase expression under control of the -2590 promoter fragment were reduced by IL-6 in hepatocytes.

Glycogenolytic and antiglycogenolytic prostaglandin E2 actions in rat hepatocytes are mediated via different signalling pathways.

Prostaglandin E2 has been reported both to stimulate glycogen-phosphorylase activity (glycogenolytic effect) and to inhibit the glucagon-stimulated glycogen-phosphorylase activity (antiglycogenolytic effect) in rat hepatocytes. It was the purpose of this study to resolve this apparent contradiction and to characterize the signalling pathways and receptor subtypes involved in the opposing prostaglandin E2 actions. Prostaglandin E2 (10 microM) increased glucose output, glycogen-phosphorylase activity and inositol trisphosphate formation in hepatocyte cell culture and/or suspension. In the same systems, prostaglandin E2 decreased the glucagon-stimulated (1 nM) glycogen-phosphorylase activity and cAMP formation. The signalling pathway leading to the glycogenolytic effect of PGE2 was interrupted by incubation of the hepatocytes with 4 beta-phorbol 12-myristate 13-acetate (100 nM) for 10 min, while the antiglycogenolytic effect of prostaglandin E2 was not attenuated. The signalling pathway leading to the antiglycogenolytic effect of prostaglandin E2 was interrupted by an incubation of cultured hepatocytes with pertussis toxin (100 ng/ml) for 18 h, whereas the glycogenolytic effect of prostaglandin E2 was enhanced. The EP1/EP3 prostaglandin-E2-receptor-specific prostaglandin E2 analogue Sulproston had a stronger glycogenolytic potency than the EP3 prostaglandin-E2-receptor-specific prostaglandin E2 analogue Misoprostol. The antiglycogenolytic potency of both agonists was equal. It is concluded that the glycogenolytic and the antiglycogenolytic effects of prostaglandin E2 are mediated via different signalling pathways in hepatocytes possibly involving EP1 and EP3 prostaglandin E2 receptors, respectively.

Eicosanoid-mediated increase in glucose and lactate output as well as decrease and redistribution of flow by complement-activated rat serum in perfused rat liver.

Rat serum, in which the complement system had been activated by incubation with zymosan, increased the glucose and lactate output, and reduced and redistributed the flow in isolated perfused rat liver clearly more than the control serum. Heat inactivation of the rat serum prior to zymosan incubation abolished this difference. Metabolic and hemodynamic alterations caused by the activated serum were dose dependent. They were almost completely inhibited by the cyclooxygenase inhibitor indomethacin and by the thromboxane antagonist 4-[2-(4-chlorobenzesulfonamide)-ethyl]-benzene-acetic acid (BM 13505), but clearly less efficiently by the 5'-lipoxygenase inhibitor nordihydroguaiaretic acid and the leukotriene antagonist N-(3-[3-(4-acetyl-3-hydroxy-2-propyl-phenoxy)-propoxy]-4-chlorine-6-meth yl- phenyl)-1H-tetrazole-5-carboxamide sodium salt (CGP 35949 B). Control serum and to a much larger extent complement-activated serum, caused an overflow of thromboxane B2 and prostaglandin F2 alpha into the hepatic vein. It is concluded that the activated complement system of rat serum can influence liver metabolism and hemodynamics via release from nonparenchymal liver cells of thromboxane and prostaglandins, the latter of which can in turn act on the parenchymal cells.

Increase in prostanoid formation in rat liver macrophages (Kupffer cells) by human anaphylatoxin C3a.

Human anaphylatoxin C3a increases glycogenolysis in perfused rat liver. This action is inhibited by prostanoid synthesis inhibitors and prostanoid antagonists. Because prostanoids but not anaphylatoxin C3a can increase glycogenolysis in hepatocytes, it has been proposed that prostanoid formation in nonparenchymal cells represents an important step in the C3a-dependent increase in hepatic glycogenolysis. This study shows that (a) human anaphylatoxin C3a (0.1 to 10 micrograms/ml) dose-dependently increased prostaglandin D2, thromboxane B2 and prostaglandin F2 alpha formation in rat liver macrophages (Kupffer cells); (b) the C3a-mediated increase in prostanoid formation was maximal after 2 min and showed tachyphylaxis; and (c) the C3a-elicited prostanoid formation could be inhibited specifically by preincubation of C3a with carboxypeptidase B to remove the essential C-terminal arginine or by preincubation of C3a with Fab fragments of a neutralizing monoclonal antibody. These data support the hypothesis that the C3a-dependent activation of hepatic glycogenolysis is mediated by way of a C3a-induced prostanoid production in Kupffer cells.