anandamide and Shock--Septic

Endocannabinoids are an emerging class of lipid mediators, which include amides and esters of long chain polyunsaturated fatty acids. Anandamide (N-arachidonoylethanolamine, AEA) and 2-arachidonoylglycerol (2-AG) are the main endogenous agonists of cannabinoid receptors. Endotoxic shock is a potentially lethal failure of multiple organs that can be initiated by the inflammatory agent lipopolysaccharide (LPS), present in the outer membrane of gram-negative bacteria. LPS has been recently shown to stimulate the production of AEA in rat macrophages, and of 2-AG in rat platelets. The mechanism responsible for this effect has not been elucidated. On the other hand, mast cells are multifunctional bone marrow-derived cells found in mucosal and connective tissues and in the nervous system, where they play an essential role in inflammation. As yet, little is known about endogenous modulators and mechanisms of mast cell activation. Here, we review recent literature on the role of endocannabinoids in endotoxic shock and inflammation, and report our recent research on the effects of LPS on the production of AEA and 2-AG in human lymphocytes, and on AEA degradation by a specific AEA membrane transporter (AMT) and an AEA-degrading enzyme (fatty acid amide hydrolase, FAAH). We also report the ability of the HMC-1 human mast cells to degrade AEA through a nitric oxide-sensitive AMT and a FAAH. The role of endocannabinoids in HMC-1 degranulation is discussed as well. Taken together, it can be suggested that human lymphocytes and mast cells take part in regulating the peripheral endocannabinoid system, which can affect some activities of these cells.

Topics: Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Endocannabinoids; Fatty Acids, Unsaturated; Glycerides; Humans; Inflammation; Lymphocytes; Mast Cells; Polyunsaturated Alkamides; Shock, Septic

Endotoxin shock is a severe systemic inflammatory response that is caused by the augmented production and release of septic mediators. Among them, inflammatory cytokines such as tumor necrosis factor-alpha, IL-1beta and IL-6 play a pivotal role. In addition, anandamide, an endogenous cannabinoid and high-mobility group box-1 (HMGB1), a non-histone chromosomal protein has recently been recognized as members of septic mediators. We previously reported that cationic antibacterial polypeptide of 11-kDa (CAP11), an antimicrobial cathelicidin peptide (originally isolated from guinea pig neutrophils), potently neutralizes the biological activity of LPS and protects mice from lethal endotoxin shock. In this study, to clarify the protective mechanism of CAP11 against endotoxin shock, we evaluated the effects of CAP11 on the production and release of septic mediators in vitro and in vivo using a murine macrophage cell line RAW264.7 and a D-galactosamine-sensitized murine endotoxin shock model. LPS stimulation induced the production of inflammatory cytokines and anandamide and release of HMGB1 from RAW264.7 cells. Importantly, CAP11 suppressed the LPS-induced production and release of these mediators by RAW264.7 cells. Moreover, LPS administration enhanced the serum levels of HMGB1, anandamide and inflammatory cytokines in the endotoxin shock model. Of note, CAP11 suppressed the LPS-induced increase of these mediators in sera, and LPS binding to CD14-positive cells (peritoneal macrophages), accompanied with the increase of survival rates. Together these observations suggest that the protective action of CAP11 on endotoxin shock may be explained by its suppressive effect on the production and release of septic mediators by CD14-positive cells possibly via the inhibition of LPS binding to the targets.

Topics: Animals; Antimicrobial Cationic Peptides; Arachidonic Acids; Cell Line; Cytokines; Down-Regulation; Endocannabinoids; Galactosamine; HMGB1 Protein; Injections, Intraperitoneal; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred BALB C; Polyunsaturated Alkamides; Shock, Septic

Arachidonylethanolamide (AEA) and 2-arachidonylglycerol (2-AG) are endocannabinoids involved in septic shock, and 8-epi prostaglandin F2alpha (F2-isoprostane) is a biomarker of oxidative stress in biological systems. Because the antibiotic polymyxin B absorbs endocannabinoids as well as endotoxins, direct hemoperfusion therapy with polymyxin B-immobilized fibers (PMX-DHP) decreases serum levels of endocannabinoids. To investigate the features of sepsis and determine the proper use of PMX-DHP, we measured the changes in levels of endocannabinoids and F2-isoprostane in patients with septic shock. Twenty-six patients with septic shock, including those with septic shock induced by peritonitis, underwent laparotomy for drainage. Endocannabinoids absorption with PMX-DHP was examined in two groups of patients: patients whose mean arterial blood pressure (mABP) had increased more than 20 mm Hg (responder group; N = 13); and patients iwhose mABP did not increase or had increased no more than 20 mm Hg (non-responder group; N = 13). Levels of AEA did not change after PMX-DHP in either the non-responder or responder groups, whereas levels of 2-AG decreased significantly after PMX-DHP in the responder group, but not in the non-responder group. F2-isoprostane gradually increased after PMX-DHP treatment; on the other hand, levels of F2-isoprostane remained constant in the responder group. Patients with septic shock are under considerable oxidative stress, and 2-AG plays an important role in the cardiovascular status of these patients. The removal of 2-AG by PMX-DHP benefits patients with septic shock by stabilizing cardiovascular status and decreasing long-term oxidative stress.

Topics: Aged; APACHE; Arachidonic Acids; Case-Control Studies; Endocannabinoids; Endotoxins; Female; Follow-Up Studies; Hemoperfusion; Humans; Male; Middle Aged; Polymyxin B; Polyunsaturated Alkamides; Probability; Prospective Studies; Reference Values; Risk Assessment; Sensitivity and Specificity; Severity of Illness Index; Shock, Septic; Survival Rate; Treatment Outcome

An excessive accumulation of anandamide (N-archidonoylethanolamine, AEA) is associated with septic shock. Results of previous studies have suggested that mRNA coding for the AEA degrading enzyme fatty acid amide hydrolase (FAAH), which converts AEA into arachidonic acid and ethanolamine, might be down-regulated in septic shock. We used real-time reverse transcription PCR assays to measure relative FAAH mRNA concentrations in the whole blood of 30 healthy donors and eight sepsis patients to ascertain whether such down-regulation takes place. Our results suggest that concentrations of FAAH mRNA in male and female samples from healthy donors are similar, but that concentrations are significantly lower in sepsis patients. These findings indicate that mRNA expression of FAAH in human whole blood correlates with sepsis, and may be an interesting biomarker for predicting the onset of septic shock.

Topics: Adult; Aged; Amidohydrolases; Arachidonic Acids; Biomarkers; Down-Regulation; Endocannabinoids; Female; Gene Expression Regulation; Humans; Male; Middle Aged; Polyunsaturated Alkamides; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Shock, Septic

Direct hemoperfusion (DHP) with polymixin B-immobilized fiber (PMX) has been reported to be effective for patients with septic shock. The aim of this study was to clarify the mechanism of PMX-DHP effect on septic shock.. The following parameters were measured in septic shock patients who were treated with PMX-DHP: survival rate, sepsis-related organ failure assessment (SOFA) score, acute physiology and chronic health evaluation II (APACHE-II) score, and plasma concentrations of cannabinoids [anandamide (ANA) and 2-arachidonyl glyceride (2-AG)], cytokines [interleukin (IL)-6, IL-8, IL-10], transforming growth factor beta (TGF-beta), and calcitonin gene-related peptide (CGRP)]. The primary end point was mortality from all causes at day 28 after intensive care unit (ICU) admission or discharge.. The survival rate of all patients at 28 days after ICU admission was 37.5% (9/24). The survival group showed significantly lower SOFA and APACHE-II scores than the nonsurvival group after PMX-DHP treatment (P = 0.008 and 0.028, respectively). The improved SOFA score group showed a better survival rate than the nonimproved SOFA score group (71.4% versus 23.5%, P = 0.028). Plasma ANA level significantly decreased after PMX-DHP treatment both in the improved SOFA score group and in the survival group. The level of 2-AG, however, showed no significant change in either group.. ANA, an intrinsic cannabinoid that induces hypotension in septic shock, is inferred to be the main mechanism of the PMX-DHP effect. Removal of ANA by PMX-DHP could be key to successful septic shock treatment.

Topics: Adult; Aged; APACHE; Arachidonic Acids; Calcitonin Gene-Related Peptide; Endocannabinoids; Female; Hemoperfusion; Humans; Male; Middle Aged; Multiple Organ Failure; Polymyxin B; Polyunsaturated Alkamides; Prognosis; Prospective Studies; Shock, Septic; Survival Rate; Transforming Growth Factor beta

Toxic shock syndrome is a rare but potentially fatal toxin-mediated febrile illness. We report a case of toxic shock syndrome complicated by life-threatening organ dysfunction with high toxin-1 and staphylococcus enterotoxin type A levels that were successfully reduced by early introduction of plasma exchanges. The report shows the time course of the concentrations of anandamide and 2-arachidonyl glyceride and confirms that early introduction of plasma exchange can result in a rapid reduction of circulating toxins and mediators in the treatment of life-threatening multiple organ dysfunction.

Topics: Adolescent; Arachidonic Acids; Bacterial Toxins; Cannabinoid Receptor Modulators; Diglycerides; Endocannabinoids; Enterotoxins; Humans; Male; Plasma Exchange; Polyunsaturated Alkamides; Shock, Septic; Staphylococcal Infections; Staphylococcus aureus; Superantigens

The aim of the present experiments was to study the effects of exogenously added anandamide on transient norepinephrine (NE)-induced contractions in mesenteric beds isolated from adult male Sprague-Dawley rats 6 h after the i.p. administration of 5 mg kg(-1) lipopolysaccharide (LPS). LPS treatment induced a 3-fold increase in total nitric-oxide synthase (NOS) activity without modifying either the systolic blood pressure or the vascular reactivity to NE of the isolated mesenteric bed. The endocannabinoid anandamide (0.01-10 microM) caused concentration-dependent reductions of the contractile responses to NE in the isolated mesenteric bed. This effect was significantly potentiated after LPS treatment compared with the controls. Anandamide-induced reductions of the contractile responses to NE in mesenteric beds isolated from LPS-treated rats were unmodified by endothelium removal but significantly diminished by either the anandamide amidase inhibitor phenylmethylsulfonyl fluoride (200 microM) or the vanilloid receptor antagonist capsazepine (1 microM). The vanilloid receptor agonist capsaicin (0.01-100 nM) also caused concentration-dependent relaxations that were potentiated in mesenteric beds from LPS-treated rats. Nevertheless, they were unmodified by 1 microM capsazepine. It is concluded that the potentiation of anandamide relaxations after LPS treatment, which are evident at early stages of endotoxic shock, could involve the participation of an anandamide metabolite and might be mediated, at least in part, through a vanilloid receptor.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Blood Pressure; Cannabinoid Receptor Modulators; Capsaicin; Endocannabinoids; Endotoxemia; Enzyme Inhibitors; Lipopolysaccharides; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Norepinephrine; Phenylmethylsulfonyl Fluoride; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Receptors, Drug; Shock, Septic; Splanchnic Circulation; Vasoconstrictor Agents

Anandamide (AEA), an endogenous cannabinoid, is generated by macrophages during shock conditions, and is thought to be a causative mediator of septic shock. Thus, we hypothesized that AEA plays a crucial role in endothelial cell (EC) injury. Here, we demonstrate that AEA induces apoptosis in a time-and dose-dependent manner in human umbilical vein endothelial cells (HUVECs). AEA triggered phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38 mitogen activated protein kinase. AEA also showed a marked increase of interleukin Ibeta- converting enzyme (ICE)CED-3 family protease (caspase-3) activity. AEA-induced EC death was inhibited by a selective vanilloid receptor 1 (VR1) antagonist, capsazepine, and was enhanced by a VR1 agonist, capsaicin, indicating that AEA induces apoptosis in ECs via VR1. In conclusion, we propose that AEA may play a crucial role in EC injury under conditions of shock, and that the use of inhibitors of the AEA regulation system may have a therapeutic effect under these conditions.

Topics: Apoptosis; Arachidonic Acids; Caspase 3; Caspases; Cells, Cultured; Endocannabinoids; Endothelial Cells; Endothelium, Vascular; Humans; JNK Mitogen-Activated Protein Kinases; Kinetics; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Polyunsaturated Alkamides; Receptors, Drug; Shock, Septic; Umbilical Veins

This article describes the overall procedure for the simultaneous determination of endocannabinoids (arachidonylethanolamide and 2-arachidonyglycerol) and isoprostane by gas chromatography-mass spectrometry in the selected-ion monitoring SIM mode (GC-MS-SIM) for medical samples. It also describes the general points of this method which a scientist who wants to assay a new, unidentified prostanoids and related compounds in medical samples would need to be clarified. The similar structures of prostaglandins, thromboxane, their metabolites, isoprostane, and arachidonyl compounds, allow them to be assayed after the simultaneous preparation of a single sample. The dimethyl isopropylsilyl ether forms of derivatized compounds are suitable for multiple GC-MS-SIM assay because of their molecular stability, and because they produce positive, strong, and large fragments on MS.

Topics: Arachidonic Acids; Endocannabinoids; Gas Chromatography-Mass Spectrometry; Glycerides; Humans; Isoprostanes; Polyunsaturated Alkamides; Sensitivity and Specificity; Shock, Septic

Anandamide (ANA) and 2-arachidonoylglycerol (2-AG), two endogenous cannabinoids, can be generated by activated macrophages and platelets, respectively, in the context of endotoxic shock, and are proposed to play a crucial role in the induction of the shock-related hypotension. Taking advantage of our recently discovered function of polymyxin B (PMB) binding to ANA and 2-AG, we developed a new method for measuring ANA and 2-AG by applying PMB-immobilized beads to selectively adsorb them in biological fluids, instead of organic solvent extraction. The eluate from beads can be directly fractionated by reverse-phase high-performance liquid chromatography (HPLC), and the fractionations corresponding to authentic ANA and 2-AG are collected and derivatized with fluorogenic reagent and subsequently quantified by HPLC with fluorometric detection. The calibration graphs of ANA and 2-AG were linear over a range of 1 to 500 pmol/ml. The limits of detection for ANA and 2-AG were 20 and 50 fmol, respectively. Intraassay precision was 2.24-4.25 and 3.47-5.44%, and interassay was 4.05-6.14 and 4.92-7.28% for ANA and 2-AG, respectively. Using this method, we first determined a 4-fold and 3-fold higher level of ANA and 2-AG, respectively, in the sera of patients with endotoxic shock than in normal serum. This finding should help in elucidating the role of the endogenous cannabinoids in the hypotension of human endotoxic shock. This method is rapid, sensitive, and reliable for simultaneously quantifying ANA and 2-AG in biological fluids, and has potential for clinical usage.

Topics: Adsorption; Animals; Arachidonic Acids; Calibration; Cell Line; Chromatography, High Pressure Liquid; Endocannabinoids; Glycerides; Humans; Mice; Polymyxin B; Polyunsaturated Alkamides; Reproducibility of Results; Shock, Septic; Spectrometry, Fluorescence

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)-immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.

Topics: Adsorption; Animals; Arachidonic Acids; Blood Proteins; Cannabinoids; Cell Death; Chromatography, Affinity; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Endocannabinoids; Humans; Hypotension; Lipopolysaccharides; Microspheres; PC12 Cells; Polymyxin B; Polyunsaturated Alkamides; Protein Binding; Rats; Shock, Septic; Surface Plasmon Resonance