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PubMed1977

Biosynthesis and structure of lipopolysaccharide in an outer membrane-defective mutant of Escherichia coli J5.

Membrane-defective mutants of Escherichia coli J5 were isolated on the basis of supersensitivity to the antibiotic novobiocin. These mutants display an increased sensitivity to a wide range of antibiotics and to several dyes and detergents. In addition, several mutants leak the periplasmic enzymes, alkyline phosphatase and ribonuclease. This evidence indicates an outer membrane defect in these mutants. The inner and outer membranes of one mutant were separated and subjected to compositional analysis. A deficiency in galactose containing lipopolysaccharide in the outer membrane of the mutant was observed. Two possible causes of this deficiency were examined and discounted: defective galactose uptake into the cell, and defective translocation of lipopolysaccharide from the inner membrane. Extraction and chemical analysis of mutant and wild type lipopolysaccharides suggests that the mutant is defective in the enzyme which transfers glucose to the growing lipopolysaccharide core, UDPglucose transferase. Thus, the mutant's deficiency in galactose-containing lipopolysaccharide can be ascribed to the fact that addition of glucose to the lipopolysaccharide core is a prerequisite for galactose addition. The physiological implications of this alteration are discussed.

Biological Transport, Active↗

PubMed1978

Interactions between lipopolysaccharide and phosphatidylethanolamine in molecular monolayers.

Lipopolysaccharide and phosphatidylethanolamine are the two major lipid constituents of the membrane of Salmonella typhimurium. Interactions between the purified lipopolysaccharide and phosphatidylethanolamine were studied in molecular monolayers at air-water interfaces. The equilibrium surface pressures of mixed films of lipopolysaccharide and phosphatidylethanolamine were determined as a function of the film composition. The plot of the equilibrium surface pressrue vs. the area occupied by phosphatidylethanolamine molecules exhibited two distinct regions. Below a phosphatidylethanolamine surface concentration at which 55% of the surface was occupied by phosphatidylethanolamine molecules, the equilibrium pressure was invariant and had the value of a pure lipopolysaccharide monolayer at maximum compression. At phosphatidylethanolamine surface concentrations in excess of 55% surface area occupation (phosphatidylethanolamine/lipopolysaccharide (mol/mol) greater than 16), the equilibrium surface pressure was a function of the surface concentration of phosphatidylethanolamine. The results suggest a simple model in which lipopolysaccharide and phosphatidylethanolamine form a complex in which each lipopolysaccharide molecule is surrounded ("lipidated") by a shell of approx. 16 phosphatidylethanolamine molecules.

Lipopolysaccharides↗

PubMed1986

Fluorescent detection of lipopolysaccharide interactions with model membranes.

We have modeled the initial interaction of bacterial lipopolysaccharide (endotoxin) with mammalian cells as consisting of two steps. The first step, adherence, we have previously shown to be ionic in nature and contains the necessary elements to determine the observed cell specificity of lipopolysaccharide interactions. The second step, coalescence, is the hypothetical insertion of the Lipid A component of lipopolysaccharide into the cell membrane lipid bilayer. Using small, unilamellar vesicles composed of phosphatidylcholine to model the cell membrane lipid bilayer, we found that lipopolysaccharide interacted with these vesicles to change the fluidity of the lipid bilayer, as measured by an increase in the fluorescence anisotropy of diphenylhexatriene in the vesicles. Since this increase in diphenylhexatriene anisotropy could not be attributed to a transfer of diphenylhexatriene between non-interacting lipopolysaccharide aggregates and vesicles, we concluded that the lipopolysaccharide aggregate coalesced with the lipid bilayer.

Cell Membrane↗

PubMed1990

Partitioning of hydrophobic probes into lipopolysaccharide bilayers.

Lipophilic solutes permeate rapidly through lipid bilayer membranes. However, the outer membrane of enteric bacteria, which is composed of a lipopolysaccharide monolayer outer leaflet and the glycerophospholipid inner leaflet, shows extremely low permeability to hydrophobic solutes. In order to examine the cause of this exceptionally low permeability, the lipid/water partition behavior of various lipophilic probes was determined by using lipopolysaccharides of various chemotypes and glycerophospholipids. With all probes, under many different conditions, the lipopolysaccharide/water partition coefficients were generally about an order of magnitude smaller than the phospholipid/water partition coefficients, and this result is consistent with the low permeability of the lipopolysaccharide monolayer, and hence the asymmetric bilayer found in the outer membrane. Furthermore, organic polycations significantly increased the partition of N-phenylnaphthylamine into lipopolysaccharides, a result again consistent with the permeability-increasing effect of such cations on intact outer membrane. Very defective, 'deep rough' lipopolysaccharides of chemotypes Rd2, Rd1 and Re, had only slightly (20-75%) higher partition coefficients in comparison with the more complete lipopolysaccharides, and this difference is probably not enough to explain the approximately 100-fold increase in lipophile permeability seen in deep rough strains.

1-Naphthylamine↗

PubMed1994

Role of platelet activating factor in acute pancreatitis induced by lipopolysaccharides in rabbits.

In the present study we demonstrated that a single injection of endotoxin (lipopolysaccharides, E. Coli 0111-B4) into the superior pancreaticoduodenal artery of rabbits induced a dose-dependent acute necrotizing pancreatitis. The lesions observed by light microscopy were significant for 10 micrograms lipopolysaccharides and were maximal for 20 micrograms. After 24 h the main findings were edema, acinar cell vacuolisation, polymorphonuclear neutrophil infiltration and tissue necrosis. The pancreatic lesions developed strictly in the area supplied by the artery injected with lipopolysaccharides, without significant intestinal involvement. Since platelet-activating factor (1-O-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine, PAF; 50-500 ng), a phospholipid mediator of endotoxin-induced inflammation and shock, was previously shown to cause an acute necrotizing pancreatitis in rabbits, the role of PAF in the development of acute pancreatitis induced by lipopolysaccharides was studied by evaluating: (1) the synergism between doses of lipopolysaccharides (5-10 micrograms), which produced a mild tissue injury, and doses of PAF (10 ng) not producing, per se, any significant injury, and (2) the effect of three structurally unrelated PAF receptor antagonists. The results obtained demonstrated that 10 ng of PAF significantly potentiated pancreatic tissue damage induced by 10 micrograms of lipopolysaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗

PubMed1995

Effect of lipopolysaccharide on the physical conformation of the erythrocyte cytoskeletal proteins.

Red blood cell deformability is important for effective circulation in the capillaries. It is known that red cell deformability is significantly reduced during septic shock. Surface to volume ratio, physical effects of the cytoskeletal proteins and the fluidity of lipid bilayer are some of the important intrinsic factors that regulate this mechanical function. Alterations in the physical conformation of cytoskeletal proteins in septic conditions could significantly alter their function. In this study, erythrocytes in whole blood were treated with lipopolysaccharide, the outer covering of Gram-negative bacteria released during Gram-negative sepsis. Electron paramagnetic resonance spectroscopy in conjunction with a protein-specific maleimide nitroxide spin label covalently bound to cytoskeletal proteins was used to investigate the resulting changes occurring in the physical state of cytoskeletal proteins in isolated membranes. Treatment of red blood cells with a lipopolysaccharide concentration as low as 40 micrograms/mL of blood solution for 90 minutes showed a significant decrease in the relevant EPR parameter (p < 0.01) of the spin label bound to subsequently isolated membranes, suggestive of a decreased segmental motion of the spin label and an increase in cytoskeletal protein-protein interactions. These results suggest a marked conformational alteration in the cytoskeletal proteins induced by the lipopolysaccharide and may explain, in part, the marked reduction in red blood cell deformability during septic shock. Bacterial lipopolysaccharide does not exert most of its effects on the host directly, but rather elicits the production of host factors that leads to complex septic shock. Leukocytes, endothelial tissue and many other cells release these mediators. Leukocytes are thought to be a particularly important source of such mediators, including cytokines (tumor necrosis factor, interleukins, etc.), oxygen free radicals, proteases, and hydrolyses. In order to characterize the possible mechanism by which the lipopolysaccharide acts on the physical state of the erythrocyte cytoskeleton, erythrocytes void of leukocytes and plasma were treated with lipopolysaccharide. The relevant EPR parameter showed no significant change over the control value. These results indicate that the leukocytes and their factors are responsible for the rearrangements seen in the cytoskeletal proteins of the erythrocyte membrane.

Animals↗

PubMed1996

Effects of chronic intrahippocampal infusion of lipopolysaccharide in the rat.

Astrogliosis and microglial activation are associated with many neurodegenerative disorders including multiple sclerosis, its animal model experimental allergic encephalomyelitis, and Alzheimer's disease. To address the hypothesis that chronic astroglial or microglial activation could be contributing factors to neuronal death or injury, the immunostimulant lipopolysaccharide was infused into the hippocampus for 16 days using Alzet mini-osmotic pumps attached to a cannula. Placement of the cannula and infusion of vehicle for 16 days caused a hippocampal lesion with a volume of 0.5 +/- 0.1 mm3. Infusion of lipopolysaccharide at the dose of 2.0 micrograms/day produced a lesion of 4.9 +/- 1.3 mm3 (P < 0.01, Newman-Keuls), whereas, a lower dose of 0.2 microgram/day caused a lesion of 1.3 +/- 0.3 mm3 (P < 0.05). The lesion was defined as a focal necrotic reaction with fibrin deposits outlining an area at an early stage of encapsulation. No apparent neuronal loss was observed by Cresyl Violet staining outside the encapsulated necrotic area. There was a pronounced astrogliosis and an increase in activated macrophages throughout the lipopolysaccharide-infused hippocampus as determined by glial fibrillary acidic protein and ED-1 immunohistochemistry, respectively. Choline acetyltransferase and glutamic acid decarboxylase enzyme activities, used as functional measures of neuronal viability for cholinergic and GABAergic neurons, respectively, were unaffected in the hippocampus following a 16 day infusion of lipopolysaccharide at the doses of 0.2, 0.6 and 2.0 micrograms/day. In addition, unilateral infusion of lipopolysaccharide into the hippocampus did not affect 24 h locomotion when tested on day 13, body temperature or weight gain. Under the experimental conditions employed in the present study, chronic infusion of lipopolysaccharide into the hippocampus resulted in a dose-dependent focal necrotic lesion at the site of infusion. In tissue surrounding the encapsulated lesion, neurons were present among the reactive astrocytes and increased number of macrophages suggesting that astrocytes and macrophages can be activated without causing neuronal loss.

Animals↗

PubMed1996

Indomethacin and thromboxane A2/prostaglandin H2 antagonist SQ29,548 impair in vitro contractions of aortic rings of ex vivo-treated lipopolysaccharide rats.

Lipopolysaccharide treated rats (25 mg/kg i.v.) were killed after 60 min and rat aortic rings were mounted in an isolated organ bath for measurement of isometric contractions in response to phenylephrine (0.01-10 microM) or potassium chloride (10 mM). Aortic rings from lipopolysaccharide-treated rats showed reduced contractility to phenylephrine and potassium chloride when compared to those from saline-treated rats. Indomethacin 10 microM, added in vitro further impaired phenylephrine-induced contraction of aortic rings from lipopolysaccharide ex vivo treated rats but was ineffective on aortic rings from saline treated rats. A similar pattern was observed when potassium chloride was used. Administration in vitro of thromboxane A2 receptor antagonist SQ29,548 gave a similar effect to indomethacin. Aortic rings collected from rat treated in vivo with dexamethasone (10 mg/kg) showed a reduction in phenylephrine induced contractions that was not further reduced by in vitro treatment with indomethacin (10 microM). Similarly, when rat aortic rings were incubated in vitro (60 min) with lipopolysaccharide (0.4 mg/ml) a reduction of phenylephrine- and potassium chloride-induced contraction was observed, but addition of either indomethacin or SQ29,548 did not further reduce contraction. Our results suggest that under these experimental conditions, in the early phase of endotoxin shock, synthesis of cyclooxygenase products (such as endoperoxides or thromboxane A2) occurs probably as a compensatory mechanism to lipopolysaccharide induced hypocontractility from the interaction, in vivo, between lipopolysaccharide, endothelium, circulating cells and vascular smooth muscles.

6-Ketoprostaglandin F1 alpha↗

PubMed2004

Effects of lipopolysaccharide on epithelium-dependent relaxation in coaxial bioassay.

This study investigated the effects of airway inflammation elicited by intraperitoneal and intratracheal lipopolysaccharide administration to guinea pigs on the activity of tracheal epithelium-derived relaxant factor (EpDRF). Acetylcholine induced epithelium-dependent relaxation in precontracted rat anococygeus muscle placed in guinea pig trachea (coaxial bioassay). Indomethacin, N-nitro-l-arginine methyl ester, aminoguanidine and l-canavanine did not alter this relaxation excluding the role of prostaglandins and nitric oxide (NO). Intraperitoneal lipopolysaccharide potentiated the acetylcholine response, which was reversed by aminoguanidine and l-canavanine while intratracheal lipopolysaccharide inhibited acetylcholine-induced relaxation. Lipopolysaccharide pretreatments did not cause epithelial damage but induced inflammatory cell infiltration. These results suggested that systemic lipopolysaccharide administration did not alter the EpDRF response but resulted in NO synthase induction, thus NO participated in relaxation to acetylcholine in coaxial bioassay system. On the other hand, airway inflammation induced by intratracheal lipopolysaccharide attenuated the synthesis/release of EpDRF without altering the epithelium morphology.

Animals↗

PubMed2005

Hyperbaric oxygen protects against lipopolysaccharide-stimulated oxidative stress and mortality in rats.

Free radicals and proinflammatory mediators have been implicated in the pathogenesis of endotoxic shock, a disease with high mortality caused by Gram-negative bacterial endotoxin. Hyperbaric oxygen is used as an adjuvant therapy for various inflammatory diseases and shows beneficial effects in lipopolysaccharide-induced shock syndrome. However, the underlying mechanisms for these effects are still to be defined. In this study, we investigated the effect of hyperbaric oxygen on inflammatory mediators, free radicals, and mortality in endotoxic rats. Wistar-Kyoto rats were injected with lipopolysaccharide (10 mg/kg) and then exposed to aminoguanidine, an inhibitor of inducible nitric oxide (NO) synthase (bolus injection 2 h after lipopolysaccharide), or hyperbaric oxygen (2 ATA for 60 min 1, 4, 9, and 24 h after lipopolysaccharide). Plasma tumor necrosis factor alpha (TNF-alpha), NO, and superoxide anion were detected and the vasorelaxation response and survival rate were assessed. The results demonstrated that increases in plasma TNF-alpha and NO, and the vasohyporeactivity induced by lipopolysaccharide treatment were significantly inhibited by hyperbaric oxygen and aminoguanidine. Mortality and vascular superoxide anion production of lipopolysaccharide treatment were also markedly reduced by hyperbaric oxygen treatment, but were not restored by aminoguanidine. None of the parameters was changed by hyperbaric oxygen treatment alone. Thus, repeated hyperbaric oxygen exposure significantly attenuated the inflammatory mediators, free radicals, and mortality in endotoxic rats.

Acetylcholine↗

PubMed2006

Lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate induction of migration and invasion of glioma cells in vitro and in vivo: Differential inhibitory effects of flavonoids.

In an earlier study, we reported that nitric oxide is involved in lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate-induced malignant transformation via increases in metalloproteinase 9 enzyme activity and inducible nitric oxide synthase gene expression in rat glioma C6 cells, however the mechanism has remained undefined. Lipopolysaccharide plus 12-o-tetradecanoylphorbol 13-acetate, but not lipopolysaccharide or 12-o-tetradecanoylphorbol 13-acetate alone, induced transformation in glioma C6 cells (but not in human glioblastoma cells GBM-8401 cells) without affecting their viability. An increase in inducible nitric oxide synthase protein expression, nitric oxide production, and metalloproteinase 9 enzyme activity is identified lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-treated C6 cells, however lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate and 12-o-tetradecanoylphorbol 13-acetate (but not lipopolysaccharide) addition shows the similar inductive pattern on metalloproteinase 9 enzyme activity without affecting inducible nitric oxide synthase protein expression and nitric oxide production in GBM-8401 cells. Treatment of C6 cells with lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate increases the expression of phosphorylated extracellular regulated protein kinases and Jun N-terminal kinases, but not p38, proteins, and an addition of the extracellular regulated protein kinases inhibitor PD98059 or Jun N-terminal kinases inhibitors SP600125, but not the p38 inhibitor SB203580, significantly blocked lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced inducible nitric oxide synthase protein expression and metalloproteinase 9 enzyme activity accompanied by blocking morphological transformation in C6 cells. Among 19 structurally related flavonoids, kaempferol and wogonin exhibit significant inhibitory effects on lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced morphological transformation and colony formation, and attenuation of inducible nitric oxide synthase, phosphorylated extracellular regulated protein kinases protein expression, and metalloproteinase 9 enzyme activity was observed. 2'-OH flavone at a dose of 100 microM inhibition of lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced events via apoptosis induction is identified. Furthermore, lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate, but not lipopolysaccharide or 12-o-tetradecanoylphorbol 13-acetate, induces tumoral invasion and migration in vitro and in vivo, and those are blocked by kaempferol and wogonin addition. These data suggest that combination of lipopolysaccharide and 12-o-tetradecanoylphorbol 13-acetate promotes tumoral progression via activating metalloproteinase 9 enzyme activity and inducible nitric oxide synthase gene expression, which is located downstream of mitogen-activated protein kinases activation, in rat glioma cells C6. Kaempferol and wogonin exhibit effective inhibitory effects on lipopolysaccharide/12-o-tetradecanoylphorbol 13-acetate-induced events, and thus possess the potential for further development.

Animals↗

PubMed2000

The effects of delayed treatment with sialyl Lewis X against lipopolysaccharide-induced acute lung injury in rabbits.

The therapeutic effects of a selectin inhibitor against lipopolysaccharide-induced acute lung injury were studied in rabbits by using sialyl Lewis X-oligosaccharide. Lipopolysaccharide-induced acute lung injury, as characterized by an impairment of pulmonary gas exchange, clinically resembles that of the acute respiratory distress syndrome. Delayed treatments with sialyl Lewis X-oligosaccharide (55 mg kg(-1) i.v. bolus injection 0.5, 1 or 2 h after lipopolysaccharide administration+36 mg kg(-1) h(-1) i.v. infusion for 5.5, 5 or 4 h, respectively) prevented the lipopolysaccharide-induced impairments in pulmonary gas exchange, as well as the accumulation of polymorphonuclear leukocytes in the lung tissue. In contrast, this agent had no significant effects on lipopolysaccharide-induced systemic hypotension, the decrease in the number of circulating white blood cells and platelets or the decline in blood pH. This is the first demonstration that sialyl Lewis X-oligosaccharide is effective against the impairments in pulmonary gas exchange even if administered 0.5, 1 or 2 h following the lipopolysaccharide injection.

Animals↗

PubMed2000

Blockade of lipopolysaccharide-induced fever by a mu-opioid receptor-selective antagonist in rats.

The endogenous opioid system has been found to be involved in fever caused by pyrogens. In the present study, we have investigated the role of the mu-opioid receptor in the brain in fever induced by lipopolysaccharide. Rats were microinjected with 1 microg of the mu-opioid receptor-selective antagonist, cyclic D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP), into the preoptic anterior hypothalamus. Thirty minutes later, lipopolysaccharide (50 microg/kg) was injected intraperitoneally (i.p.). CTAP reduced by 1 degrees C the fever induced by lipopolysaccharide. However, it did not affect lipopolysaccharide fever when it was given 3 h after lipopolysaccharide injection. These data indicate that mu-opioid receptors within the preoptic anterior hypothalamus mediate the initiation of lipopolysaccharide fever and suggest that the opioid system is involved in the pathogenesis of fever in rats.

Amino Acid Sequence↗

PubMed2002

Baclofen influences lipopolysaccharide-mediated interleukin-6 release from murine pituicytes.

Pituicytes, the glial cells of the neurohypophysis, secrete interleukin-6 upon stimulation with various inflammatory mediators, i.e. lipopolysaccharide. Previous studies have identified several receptors on pituicytes. This study investigates the effect of GABA(B) receptor activation on interleukin-6 release from pituicytes. Cultured murine pituicytes were stimulated for 24 h with lipopolysaccharide (0.5 ng/ml) to give a significant interleukin-6 release compared to control. The interleukin-6 release was significantly potentiated by the GABA(B) receptor agonist (R)-4-amino-3-(4-chlorophenyl) butanoic acid (R-baclofen; 10, 100 or 500 microM). However, R-baclofen itself (10, 100 or 500 microM) did not stimulate the interleukin-6 secretion. Furthermore, the potent GABA(B) receptor antagonists 3-[[(3,4-Dichlorophenyl)methyl]amino]propyl]diethoxymethyl) phosphinic acid (CGP52432; 30 or 300 microM) and (RS)-3-Amino-2-(4-chlorophenyl)-2-hydroxypropyl-sulphonic acid (2-OH-saclofen; 10 or 100 microM) did not remove the effect of R-baclofen (100 microM). Gamma-amino butyric acid (GABA; 30 or 300 microM) did not alter the lipopolysaccharide-mediated interleukin-6 response. After 30 min, intracellular cyclic AMP (cAMP) was higher in cells stimulated with lipopolysaccharide compared to control, and R-baclofen significantly inhibited this increase in cAMP. Nevertheless, neither lipopolysaccharide nor R-baclofen had any effect on intracellular cAMP after 24 h of stimulation. The results suggest that the effect of R-baclofen on lipopolysaccharide-stimulated interleukin-6 secretion is independent of GABA(B) receptors.

Animals↗

PubMed1999

Modulation by endothelin-1 of lipopolysaccharide-induced cyclooxygenase 2 expression in mouse peritoneal macrophages.

We investigated the modulation by endothelin-1 of lipopolysaccharide-induced cyclooxygenase 2 expression and prostaglandin E2 production by mouse peritoneal macrophages. Our previous report showed that endothelin-1 at concentrations above 10(-11) M induced cyclooxygenase 2 expression through mainly endothelin ET(B) receptors and that an endothelin ET(B) receptor-mediated process was not involved in cyclooxygenase 2 activation in macrophages stimulated by lipopolysaccharide for 4 h. In the present study, when macrophages were stimulated by lipopolysaccharide for 12 h in the presence of endothelin-1 (10(-15) to 10(-8) M), cyclooxygenase 2 expression and prostaglandin E2 production were enhanced by 1.2- to 1.6-fold. The endothelin ET(B) receptor selective antagonist, BQ788 (N-cis-2,6-dimethylpiperidino-carbonyl-L-gamma-methyl-leucyl-D-L-m ethoxycarbonyl-tryptophanyl-norleucine), significantly inhibited this synergistic effect of endothelin-1. In addition, the cyclooxygenase 2-selective inhibitor, NS398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide), also suppressed this effect. Western blot analysis showed that the endothelin ET(B) receptor was up-regulated by lipopolysaccharide in a time- and concentration-dependent manner, and that this up-regulation was inhibited by NS398. From these results, we conclude that endothelin-1 promotes lipopolysaccharide-induced cyclooxygenase 2 activation in the delayed phase through endothelin ET(B) receptors up-regulated by lipopolysaccharide.

Animals↗

PubMed1999

Lipopolysaccharides and cytokines downregulate the angiotensin II type 2 receptor in rat cardiac fibroblasts.

The present study examines the effect of lipopolysaccharides and proinflammatory cytokines on the expression of the second isoform of the angiotensin II receptor (AT(2)), which may have a role in lowering collagen deposition in cardiac tissue. Cardiac fibroblasts express high levels of both angiotensin II type 1 (AT(1)) and type 2 receptors. Incubation with lipopolysaccharides for 24 h dose- and time-dependently decreased angiotensin II AT(2) receptor expression with no apparent difference in the affinity. Actinomycin D, cycloheximide, N(omega)-nitro-L-arginine methyl ester and the protein tyrosine kinase inhibitor herbimycin A, but not the protein kinase C inhibitors bisindolylmaleimide and calphostin C, abolished the inhibitory action of lipopolysaccharides. The cytokines interleukin-1beta and tissue necrosis factor-alpha mimicked the effect of lipopolysaccharides. All three compounds induced inducible nitric oxide synthase (iNOS). The nitric oxide donor sodium nitroprusside and the cGMP analog 8-bromoguanosine cyclic monophosphate downregulated angiotensin II AT (2) receptor expression. The findings are consistent with the pathway in which lipopolysaccharides or cytokines induce iNOS. The data suggest that lipopolysaccharide- or cytokine-dependent induction of iNOS and resultant production of nitric oxide leads to the production of cGMP, which in turn downregulates expression of the angiotensin II AT (2) receptor in cardiac fibroblasts.

Animals↗

PubMed1998

Translocation of platelets into Disse spaces and their entry into hepatocytes in response to lipopolysaccharides, interleukin-1 and tumour necrosis factor: the role of Kupffer cells.

BACKGROUND/AIMS: Injection into mice of a small dose of either a lipopolysaccharide or interleukin-1 induces a slowly developing accumulation of 5-hydroxytryptamine, predominantly in the liver. We have established that this 5-hydroxytryptamine accumulation is the result of the translocation of platelets to hepatic sinusoidal spaces and, further, into Disse spaces, and that the platelets make direct contact with hepatocytes. In the present study, we report our recent findings on this phenomenon. METHODS: Platelets contain a large amount of 5-hydroxytryptamine, but the 5-hydroxytryptamine content of the liver is normally very small. Therefore, the translocation of platelets to the liver was assessed by measuring 5-hydroxytryptamine as in previous studies, and it was also analysed by electron microscopy. RESULTS: Anti-platelet agents, such as heparin and inhibitors of prostaglandin synthesis, were ineffective in preventing the lipopolysaccharide-induced accumulation of 5-hydroxytryptamine in the liver. Of the various cytokines tested, only interleukin-1 and tumour necrosis factor induced such an accumulation of 5-hydroxytryptamine. Intravenous injection of liposomes encapsulating dichloromethylene bisphosphonate resulted in an almost complete depletion of macrophages from the liver. The lipopolysaccharide- and cytokine-induced hepatic accumulations of 5-hydroxytryptamine were abolished almost completely in such macrophage-depleted mice. Electron microscopy revealed no accumulation of platelets in the liver after injection of lipopolysaccharide into the macrophage-depleted mice. Surprisingly, in normal mice injected with lipopolysaccharide, several platelets were found inside some hepatocytes, even though there was no visible damage to these hepatocytes. In fact, there were many polysomes around the degranulated platelets within the hepatocytes, suggesting an enhanced protein synthesis. CONCLUSION: These results suggest that, in response to lipopolysaccharide, interleukin-1 or tumour necrosis factor, platelets translocate into the liver in a way that is different from aggregation, and that some, at least, enter hepatocytes. During these processes, hepatic macrophages play an essential role.

Animals↗

PubMed1999

Splenectomy attenuates superoxide anion release into the hepatic sinusoids after lipopolysaccharide challenge.

BACKGROUND/AIMS: The aim of this study was to determine whether the spleen contributes to superoxide anion release into the hepatic sinusoids and subsequent damage to endothelial cells of the hepatic sinusoids after lipopolysaccharide challenge. METHODS: Rats were given 2 mg/kg body weight lipopolysaccharide. Three hours after the treatment, superoxide anion release into the hepatic sinusoids was examined in a liver perfusion model using the cytochrome C method. Damage to endothelial cells of the hepatic sinusoids was assessed from the purine nucleoside phosphorylase/glutamic-pyruvic transaminase ratio in the liver perfusate. To further characterize the mechanisms behind these changes, these studies were done in rats given superoxide dismutase or an anti-TNFalpha antibody. To study whether the spleen plays a role in the mechanisms, experiments with splenectomized rats were performed. RESULTS: Lipopolysaccharide challenge resulted in superoxide anion release into the hepatic sinusoids and damage to endothelial cells of the hepatic sinusoids. These changes were significantly attenuated by the treatments with superoxide dismutase or an antibody against TNFalpha, as well as by splenectomy. The hepatic macrophage and Kupffer cell populations after lipopolysaccharide challenge were significantly smaller in the rats given splenectomy than in those given a sham operation. There were no significant differences in the neutrophil populations between the two groups. Levels of TNFalpha were significantly lower in the former than the latter, whereas there were no significant differences in levels of Interleukin-8 between the two groups. CONCLUSIONS: Splenectomy reduced the superoxide anion release into the hepatic sinusoids caused by the lipopolysaccharide challenge and subsequent damage to endothelial cells of the hepatic sinusoids. This supports the view that splenectomy has a protective effect in lipopolysaccharide-induced liver injury.

Animals↗