thromboxane-a2 and Endotoxemia

Thromboxane (Tx) A2 has been suggested to be involved in the development of sepsis-induced acute kidney injury (AKI). Therefore, we investigated the impact of cyclooxygenase (COX)-1 and COX-2 activity on lipopolysaccharide (LPS)-induced renal TxA2 formation, and on endotoxemia-induced AKI in mice. Injection of LPS (3 mg/kg ip) decreased glomerular filtration rate (GFR) and the amount of thrombocytes to ∼50% of basal values after 4 h. Plasma and renocortical tissue levels of TxB2 were increased ∼10- and 1.7-fold in response to LPS, respectively. The COX-1 inhibitor SC-560 attenuated the LPS-induced fall in GFR and in platelet count to ∼75% of basal levels. Furthermore, SC-560 abolished the increase in plasma and renocortical tissue levels of TxB2 in response to LPS. The COX-2 inhibitor SC-236 further enhanced the LPS-induced decrease in GFR to ∼40% of basal values. SC-236 did not alter thrombocyte levels nor the LPS-induced increase in plasma and renocortical tissue levels of TxB2. Pretreatment with clopidogrel inhibited the LPS-induced drop in thrombocyte count, but did not attenuate the LPS-induced decrease in GFR and the increase in plasma TxB2 levels. This study demonstrates that endotoxemia-induced TxA2 formation depends on the activity of COX-1. Our study further indicates that the COX-1 inhibitor SC-560 has a protective effect on the decrease in renal function in response to endotoxin. Therefore, our data support a role for TxA2 in the development of AKI in response to LPS.

Topics: Acute Kidney Injury; Animals; Blood Platelets; Cyclooxygenase 1; Cyclooxygenase 2 Inhibitors; Disease Models, Animal; Down-Regulation; Endotoxemia; Endotoxins; Glomerular Filtration Rate; Kidney; Male; Membrane Proteins; Mice, Inbred C57BL; Platelet Aggregation Inhibitors; Pyrazoles; Signal Transduction; Thromboxane A2; Time Factors

The aim of this study was to determine the effects of endotoxin on p38 MAPK activation in equine platelets and leukocytes in vivo and in vitro and its role in thromboxane (Tx) production with reference to equine endotoxaemia.. Six adult Thoroughbred horses were used for in vivo infusion studies and separate in vitro studies. For in vivo studies, following collection of a pre-infusion sample, horses were infused with E. Coli O55:B5 LPS (30 ng/kg; 30 min) during and after which platelets were harvested. For in vitro studies isolated platelets and leukocytes were exposed to LPS (10 pg/ml-1 microg/ml). p38 MAPK activity was assessed by SDS-PAGE followed by immunoblotting. TxA2 release was measured by radioimmunoassay.. LPS infusion caused increased phospho-p38 MAPK in equine platelets and leukocytes (1492 +/- 486 % and 83 +/- 45 above basal, respectively) from 10 min after the start of the infusion, which returned to basal by 60 min. In vitro, platelets were 1,000 times more sensitive to LPS than leukocytes in terms of both TxA2 production (EC50 66 pg/ml versus 110 ng/ml, respectively) and p38 MAPK phosphorylation (EC50 11.1 +/- 2 pg/ml versus 14.8 +/- 4 ng/ml, respectively). p38 MAPK inhibitors SB203580 and PD169316 attenuated LPS-induced TxA2 release in platelets, but not leukocytes.. In vivo, LPS stimulates TxA2 production and p38 MAPK phosphorylation in equine platelets and leukocytes at a concentration within a similar range to those reported in clinical endotoxaemia. These data suggest that LPS-induced eicosanoid production in the early phase of clinical endotoxaemia may involve direct effects of LPS upon platelets, mediated via activation of p38 MAPK.

Topics: Animals; Blood Platelets; Dose-Response Relationship, Drug; Endotoxemia; Endotoxins; Enzyme Inhibitors; Female; Horse Diseases; Horses; Imidazoles; Leukocytes; Male; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Platelet Activation; Pyridines; Signal Transduction; Thromboxane A2

We examined the role of thromboxane A2 (TXA2) in LPS-induced hyperresponsiveness of hepatic portal circulation to endothelins (ETs) and whether Kupffer cells are the primary source of TXA2 release in response to ET-1 in endotoxemia. After 6 h of LPS (1 mg/kg body wt ip) or saline (control), liver was isolated and perfused with recirculating Krebs-Henseleit bicarbonate buffer at a constant flow rate (100 ml.min(-1).kg body wt(-1)). ET-1 (10 pmol/min) was infused for 10 min. Portal pressure (PP) was continuously monitored during perfusion. Perfusate was sampled for enzyme immunoassay of thromboxane B2 (TXB2; the stable metabolite of TXA2) and lactate dehydrogenase (LDH) assay. ET-1 infusion resulted in a significantly greater increase of PP in the LPS group than in controls. Both TXA2 synthase inhibitor furegrelate (Fureg) and TXA2 receptor antagonist SQ-29548 (SQ) substantially blocked enhanced increase of PP in the LPS group (4.9 +/- 0.4 vs. 3.6 +/- 0.5 vs. 2.6 +/- 0.6 mmHg for LPS alone, LPS + Fureg, and LPS + SQ, respectively; P < 0.05) while having no significant effect on controls. GdCl3 for inhibition of Kupffer cells had similar effects (4.9 +/- 0.4 mmHg vs. 2.9 +/- 0.4 mmHg for LPS alone and GdCl3 + LPS, respectively; P < 0.05). In addition, the attenuated PP after ET-1 was found concomitantly with significantly decreased releases of TXB2 and LDH in LPS rats treated with Fureg, SQ, and GdCl3 (886.6 +/- 73.4 vs. 110.8 +/- 0.8 vs. 114.8 +/- 54.7 vs. 135.2 +/- 45.2 pg/ml, respectively; P < 0.05). After 6 h of LPS, Kupffer cells in isolated cell preparations released a significant amount of TXA2 in response to ET-1. These results clearly indicate that hyperresponsiveness of hepatic portal circulation to ET-1 in endotoxemia is mediated at least in part by TXA2-induced receptor activation, and Kupffer cells are likely the primary source of increased TXA2 release.

Topics: Animals; Benzofurans; Bridged Bicyclo Compounds, Heterocyclic; Endothelin-1; Endotoxemia; Fatty Acids, Unsaturated; Gadolinium; Hydrazines; Kupffer Cells; Lipopolysaccharides; Liver; Male; Portal Pressure; Portal System; Rats; Rats, Sprague-Dawley; Receptors, Thromboxane; Thromboxane A2; Thromboxane-A Synthase

Although thromboxanes (TXs), whose synthesis is regulated by cyclooxygenase (COX), have been suggested to promote inflammation in the liver, little is known about the role of TXA(2) in leukocyte endothelial interaction during endotoxemia. The present study was conducted to investigate the role of TXA(2) as well as that of COX in lipopolysaccharide (LPS)-induced hepatic microcirculatory dysfunction in male C57Bl/6 mice. We observed during in vivo fluorescence microscopic study that LPS caused significant accumulation of leukocytes adhering to the hepatic microvessels and non-perfused sinusoids. Levels of serum alanine transaminase (ALT) and tumor necrosis factor alpha (TNF alpha) also increased. LPS raised the TXB(2) level in the perfusate from isolated perfused liver. A TXA(2) synthase inhibitor, OKY-046, and a TXA(2) receptor antagonist, S-1452, reduced LPS-induced hepatic microcirculatory dysfunction by inhibiting TNF alpha production. OKY-046 suppressed the expression of an intercellular adhesion molecule (ICAM)-1 in an LPS-treated liver. In thromboxane prostanoid receptor-knockout mice, hepatic responses to LPS were minimized in comparison with those in their wild-type counterparts. In addition, a selective COX-1 inhibitor, SC-560, a selective COX-2 inhibitor, NS-398, and indomethacin significantly attenuated hepatic responses to LPS including microcirculatory dysfunction and release of ALT and TNF alpha. The effects of the COX inhibitors on hepatic responses to LPS exhibited results similar to those obtained with TXA(2) synthase inhibitor, and TXA(2) receptor antagonist. In conclusion, these results suggest that TXA(2) is involved in LPS-induced hepatic microcirculatory dysfunction partly through the release of TNF alpha, and that TXA(2) derived from COX-1 and COX-2 could be responsible for the microcirculatory dysfunction during endotoxemia.

Topics: Alanine Transaminase; Animals; Bridged Bicyclo Compounds; Cyclooxygenase 1; Cyclooxygenase 2; Endotoxemia; Enzyme Inhibitors; Fatty Acids, Monounsaturated; Isoenzymes; Lipopolysaccharides; Liver Circulation; Male; Membrane Proteins; Methacrylates; Mice; Mice, Inbred C57BL; Mice, Knockout; Microcirculation; Prostaglandin Antagonists; Prostaglandin-Endoperoxide Synthases; Receptors, Thromboxane; Thromboxane A2

Circulatory failure in sepsis arises from vascular hyporesponsiveness, in which nitric oxide (NO) derived from inducible NO synthase (iNOS) plays a major role. Details of the cross talk between thromboxane (TX) A2 and the iNOS-NO system, however, remain unknown. We intended to clarify the role of TXA2, via the cross talk, in vascular hyporesponsiveness.. We examined cytokine-induced iNOS expression and NO production in cultured vascular smooth muscle cells (VSMCs) and cytokine-induced hyporesponsiveness of the aorta from mice lacking the TXA2 receptor (TP-/- mice). The cytokine-induced iNOS expression and NO production observed in wild-type VSMCs were significantly augmented in TP-/- VSMCs, indicating an inhibitory effect of endogenous TXA2 on iNOS expression. Furthermore, in indomethacin-treated wild-type VSMCs, U-46619, a TP agonist, inhibited cytokine-induced iNOS expression and NO production in a concentration-dependent manner, effects absent from TP-/- VSMCs. In an ex vivo system, the cytokine-induced hyporesponsiveness of aortas to phenylephrine was significantly augmented in TP-/- aorta but was almost completely canceled by aminoguanidine, an iNOS inhibitor. Accordingly, cytokine-induced NO production was significantly higher in TP-/- aorta than in wild-type aorta. Moreover, U-46619 significantly suppressed lipopolysaccharide-induced NO production in vivo only in wild-type mice.. These results suggest that TXA2 has a protective role against the development of vascular hyporesponsiveness via its inhibitory action on the iNOS-NO system under pathological conditions such as sepsis.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta; Cells, Cultured; Cytokines; Endotoxemia; Enzyme Induction; Humans; Indomethacin; Male; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phenylephrine; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2; Vasoconstrictor Agents

Topics: Endothelins; Endotoxemia; Epoprostenol; Humans; Hypertension, Portal; Liver Cirrhosis; Portasystemic Shunt, Surgical; Radioimmunoassay; Thromboxane A2; Vasoconstriction; Vasodilation

In this study, gram-positive Staphylococcus aureus lipoteichoic acid (LTA) dose-dependently (0.1-1.0 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human platelets stimulated by agonists. LTA also dose-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. LTA (0.5 and 1.0 microg/ml) also significantly inhibited thromboxane A2 formation stimulated by collagen in human platelets. Moreover, LTA (0.1-1.0 microg/ml) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatrience. Rapid phosphorylation of a platelet protein of Mr. 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (30 nM). This phosphorylation was markedly inhibited by LTA (0.5 and 1.0 microg/ml) within a 10-min incubation period. These results indicate that the antiplatelet activity of LTA may be involved in the following pathways: LTA's effects may initially be due to induction of conformational changes in the platelet membrane, leading to a change in the activity of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca+2 mobilization and phosphorylation of P47 protein. Therefore, LTA-mediated alteration of platelet function may contribute to bleeding diathesis in gram-positive septicemic and endotoxemic patients.

Topics: Calcium Signaling; Cell Membrane; Collagen; Cytosol; Dose-Response Relationship, Drug; Endotoxemia; Enzyme Activation; Gram-Negative Bacterial Infections; Gram-Positive Bacterial Infections; Hemorrhagic Disorders; Humans; L-Lactate Dehydrogenase; Lipopolysaccharides; Membrane Fluidity; Membrane Lipids; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peptides; Phorbol 12,13-Dibutyrate; Phosphatidylinositols; Phosphorylation; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Kinase C; Protein Processing, Post-Translational; Sepsis; Shock, Septic; Staphylococcus aureus; Teichoic Acids; Thromboxane A2; Thromboxane B2

The involvement of PAF, TXA2 and NO in LPS-induced pulmonary neutrophil sequestration an hyperlactataemia was studied in conscious rats. As pharmacological tools WEB 2170 (PAF receptor antagonist, 20 mg/kg), camongarel (inhibitor of TXA2 synthase, 30 mg/kg), N(G)-nitro L-arginine methyl ester (L-NAME -- non-selective nitric oxide synthase inhibitor, 30 mg/kg) were used.. Plasma lactate and NO2-/NO3- levels as well as myeloperoxidase (MPO) activity in lung tissue were measured one and five hours after administration of LPS (4 mg/kg(-1)).. LPS induced a twofold increase in plasma lactate levels and nearly 10-fold increase in plasma NO2-/NO3- levels five but not one hour after LPS administration. However, LPS-induced increase in pulmonary MPO activity was seen at both time intervals. Neither WEB 2170 nor camonagrel changed one or five hours responses to LPS (lactate, NO2-/NO3-, MPO). L-NAME potentiated LPS-induced rise in MPO activity in the lung and this potentiation was not affected by WEB 2170 or camonagrel. L-NAME supressed plasma NO2-/NO3- response and substantially potentiated plasma lactate response to LPS and both effects were partially reversed by WEB 2170 or camonagrel.. In summary, we demonstrated that PAF and TXA 2 play a role in overproduction of lactate during endotoxaemia in NO-deficient rats. However, these lipids do not mediate endotoxin-induced sequestration of neutrophils in the lung.

Topics: Animals; Azepines; Cell Movement; Endotoxemia; Indans; Lactic Acid; Lipopolysaccharides; Lung; Male; Neutrophils; NG-Nitroarginine Methyl Ester; Nitric Oxide; Platelet Activating Factor; Platelet Membrane Glycoproteins; Rats; Rats, Wistar; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Thromboxane A2; Triazoles

The effect of endotoxin treatment in vivo on platelet activating factor (PAF)-induced glycogenolysis was studied in the perfused rat liver. The addition of PAF (20 nM) to the perfusate increased glucose production concomitant with suppression of oxygen consumption in control rats without endotoxin treatment. At 6 h after endotoxin administration, PAF caused severe suppression of oxygen consumption, but glucose production was greatly inhibited. At 24 h after endotoxin treatment, PAF caused less suppression of oxygen consumption than the control, and glucose production was partially restored. The metabolic responses in the control rat were abolished by the simultaneous presence of cyclooxygenase- and lipoxygenase-inhibitors. Combined use of leukotriene (LT) D4- and thromboxane (Tx) A2-receptor antagonists inhibited the metabolic responses in the rat given endotoxin 6 h before. The efflux of Tx B2 during PAF-infusion decreased 24 h after endotoxin treatment, and Tx A2 receptor antagonist, but not LT D4 receptor antagonist, prevented the suppression of oxygen consumption. These results suggest that different eicosanoids are involved in PAF-induced glycogenolysis in different stages of endotoxemia, and that LT D1 may also play a role in PAF-induced glycogenolysis.

Topics: Animals; Eicosanoids; Endotoxemia; Glycogen; Leukotriene Antagonists; Leukotriene D4; Lipopolysaccharides; Liver; Male; Membrane Proteins; Oxygen Consumption; Perfusion; Platelet Activating Factor; Prostaglandin D2; Rats; Rats, Sprague-Dawley; Receptors, Immunologic; Receptors, Leukotriene; Receptors, Prostaglandin; Receptors, Thromboxane; Thromboxane A2