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Background & purpose: Recent studies suggested a role of prostaglandin E-2 (PGE(2)) in the expression of the chemokine IL-8 by monocytes. The function of EP4 receptor for TNF alpha-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 PGE(2) had only a marginal impact on IL-8 induction but strongly enhanced TNFa-induced IL-8 mRNA and protein synthesis. Similarly, in PBMC IL-8 mRNA induction was larger by simultaneous stimulation with TNF alpha and PGE(2) 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 TNF alpha-induced IL-8 mRNA and protein formation to the same extent as PGE(2). In HEK293 cells expressing EP4, but not in wild type HEK293 cells lacking EP4, PGE(2) enhanced TNFainduced IL-8 protein and mRNA synthesis. In THP-1 cells, the enhancement of TNF alpha-mediated IL-8 mRNA induction by PGE(2) was mimicked by a PICA-activator. Furthermore in these cells PGE(2) induced expression of transcription factor C/EBPS, enhanced NF-KB activation by TNFa and inhibited TNF alpha-mediated AP-1 activation. PGE(2) and TNF alpha synergistically activated transcription factor CREB, induced C/EBPS 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 TNF alpha and PGE(2)/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 PICA/CREB/C/EB1313 as well as NF-kappa B signal chains.
In a subset of patients, non-alcoholic fatty liver disease (NAFLD) is complicated by cell death and inflammation resulting in non-alcoholic steatohepatitis (NASH), which may progress to fibrosis and subsequent organ failure. Apart from cytokines, prostaglandins, in particular prostaglandin E-2 (PGE(2)), play a pivotal role during inflammatory processes. Expression of the key enzymes of PGE(2) synthesis, cyclooxygenase 2 and microsomal PGE synthase 1 (mPGES-1), was increased in human NASH livers in comparison to controls and correlated with the NASH activity score. Both enzymes were also induced in NASH-diet-fed wild-type mice, resulting in an increase in hepatic PGE(2) concentration that was completely abrogated in mPGES-1-deficient mice. PGE(2) is known to inhibit TNF-alpha synthesis in macrophages. A strong infiltration of monocyte-derived macrophages was observed in NASH-diet-fed mice, which was accompanied with an increase in hepatic TNF-alpha expression. Due to the impaired PGE(2) production, TNF-alpha expression increased much more in livers of mPGES-1-deficient mice or in the peritoneal macrophages of these mice. The increased levels of TNF-alpha resulted in an enhanced IL-1 beta production, primarily in hepatocytes, and augmented hepatocyte apoptosis. In conclusion, attenuation of PGE(2) production by mPGES-1 ablation enhanced the TNF-alpha-triggered inflammatory response and hepatocyte apoptosis in diet-induced NASH.
The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.
The suitability of a newly developed cell-based functional assay was tested for the detection of the activity of a range of neurotoxins and neuroactive pharmaceuticals which act by stimulation or inhibition of calcium-dependent neurotransmitter release. In this functional assay, a reporter enzyme is released concomitantly with the neurotransmitter from neurosecretory vesicles. The current study showed that the release of a luciferase from a differentiated human neuroblastoma-based reporter cell line (SIMA-hPOMC1-26-GLuc cells) can be stimulated by a carbachol-mediated activation of the Gq-coupled muscarinic-acetylcholine receptor and by the Ca2+-channel forming spider toxin α-latrotoxin. Carbachol-stimulated luciferase release was completely inhibited by the muscarinic acetylcholine receptor antagonist atropine and α-latrotoxin-mediated release by the Ca2+-chelator EGTA, demonstrating the specificity of luciferase-release stimulation. SIMA-hPOMC1-26-GLuc cells express mainly L- and N-type and to a lesser extent T-type VGCC on the mRNA and protein level. In accordance with the expression profile a depolarization-stimulated luciferase release by a high K+-buffer was effectively and dose-dependently inhibited by L-type VGCC inhibitors and to a lesser extent by N-type and T-type inhibitors. P/Q- and R-type inhibitors did not affect the K+-stimulated luciferase release. In summary, the newly established cell-based assay may represent a versatile tool to analyze the biological efficiency of a range of neurotoxins and neuroactive pharmaceuticals which mediate their activity by the modulation of calcium-dependent neurotransmitter release.
Increase in prostanoid formation in rat liver macrophages (Kupffer cells) by human anaphylatoxin C3a
(1993)
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 mug/ml) dose-dependently increased prostaglandin D2, thromboxane B, and prostaglandin F2alpha 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.
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 α1-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.
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 μM 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.
In perfused rat livers, infusion of prostaglandin F₂α (PGF₂α) or noradrenaline increased glucose and lactate output and reduced flow. Glucagon increased glucose output and decreased lactate output without influence on flow. Infusion of phorbol 13-myristate 14-acetate (PMA) for 20 min prior to these stimuli strongly inhibited the metabolic and hemodynamic effects of noradrenaline, reduced the metabolic actions of PGF₂α but did not alter the effects of glucagon. In isolated rat hepatocytes PGF₂α, noradrenaline and glucagon activated glycogen phosphorylase but only PGF₂α and noradrenaline increased intracellular inositol 1,4,5-1risphosphalc (InsP₃). The noradrenaline- or PGF₂α-elicited activation of glycogen phosphorylase and increase in InsP₃ were largely reduced after preincubation of the cells for 10 min with PMA, whereas the glucagon-mediated enzyme activation was not affected. In contra\t to PMA, the phorbol ester 4a-phorbol 13,14-didecanoate. which does not activate protein kinase C, did not attenuate the PGF₂α- and noradrenaline-elicited stimulation of glucose output, glycogen phosphorylase and InsP, formation. Stimulation of InsP₃ formation by AlF₄⁻, which activates phospholipase C independently of the receptor, was not attenuated by prior incubation with PMA. Plasma membranes purified from isolated hepatocytes had both a high-capacity, low-affinity and a low-capacity, high-affinity binding site for PGF₂α. The Kd of the high-capacity, low-affinity binding site was close to the concentration of PGF₂α that increased glycogen phosphorylase activity halfmaximally. Binding to the high-capacity, low-affinity binding site was enhanced by guanosine 5'- 0-(3-thio)triphosphate (GTP[S]). This high-capacity, low-affinity site might thus represent the receptor. The Bmax and Kd of the high-capacity site, as well as the enhancement by GTP[S] of PGF₂α binding to this site, remained unaffected by PMA pretreatment. It is concluded that, in hepatocytes, activation of protein kinase C by PMA interrupted the InsP₃-mediated signal pathway from PGF₂α via a PGF₂α receptor and phospholipase C to glycogen phosphorylase at a point distal of the receptor prior to phospholipase C.
Prostaglandin (PG)F₂α has previously been shown to increase glucose output from perfused livers and isolated hepatocytes, where it stimulated glycogen phosphorylase via an inositol-trisphosphatedependent signal pathway. In this study, PGF₂α binding sites on hepatocyte plasma membranes, that might represent the putative receptor, were characterized. Binding studies could not be performed with intact hepatocytes, because PGF₂α accumulated within the cells even at 4°C. The intracellular accumulation was an order of magnitude higher than binding to plasma membranes. Purified hepatocyte plasma membranes had a high-affinity/low-capacity and a low-affinity/highcapacity binding'site for PGF₂α. The respective binding constants for the high-affinity site were Kd = 3 nM and Bmax = 6 fmol/mg membrane protein, and for the low-affinity site Kd = 426 nM and Bmax = 245 fmol/mg membrane protein. Specific PGF₂α binding to the low-affinity site, but not to the high-affinity site, could be enhanced most potently by GTP[γS] followed by GDP[ϐS] and GTP, but not by ATP[γS] or GMP. PGF₂α competed most potently with [³H]PGF₂α for specific binding to hepatocyte plasma membranes, followed by PGD₂ and PGE₂. Since the low-affinity PGF₂α-binding site had a Kd in the concentration range in which PG had previously been shown to be half-maximally active, and since this binding site showed a sensitivity to GTP, it is concluded that it might represent the receptor involved in the PGF₂α signal chain in hepatocytes. A biological function of the high-affinity site is currently not known.
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 μM) increased glucose output, glycogen-phosphorylase activity and inositol trisphosphate formation in hepatocyte cell culture andor 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 4P-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.