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RESEARCH COMMUNICATION |
a Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Virginia 23298, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: anandamide • 2-arachidonyl glyceride • CB1 cannabinoid receptors • vasodilation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and interleukin 1ß (1, 2). Although various symptoms of septic shock have been attributed to the LPS-induced release of cytokines from circulating macrophages (1–3), pharmacological antagonism of cytokine effects failed to provide protection from the hypotension of septic shock (4, 5), the mechanism of which remains to be clarified. In addition to their well-known neurobehavioral effects, cannabinoids, including the endogenous ligand arachidonyl ethanolamide (anandamide) (6), can also elicit hypotension mediated via peripherally located CB1 cannabinoid receptors (7–10). Anandamide, originally discovered in brain (6, 11–13), is also present in a macrophage cell line (14); a second endogenous ligand, 2-arachidonyl glyceride (2-AG), has been identified in the gut (15) as well as in brain (16–18). We recently reported that activation of peripheral CB1 receptors in rats by macrophage- and platelet-derived substances contributes to the hypotension in hemorrhagic shock, and that anandamide is generated in activated circulating macrophages but not in platelets (19). The purpose of this study was to test the hypothesis that a similar mechanism may be involved in endotoxic shock and to identify the nature of the platelet-derived factor responsible for eliciting CB1 receptor-mediated hypotension. We report here that rat platelets generate 2-AG, and that LPS stimulates the production of 2-AG in platelets and induces the production of ananamide in macrophages. Furthermore, rat platelets and macrophages obtained from LPS-treated rats or exposed to LPS in vitro trigger CB1 receptor-mediated hypotension in normal recipient rats, which can be also elicited by the administration of synthetic anandamide or 2-AG. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To induce septic shock in anesthetized rats, the animals received an i.v. injection of 15 mg/kg Escherichia coli endotoxin (LPS). This submaximal hypotensive dose was selected because it consistently produced gradually developing, prolonged hypotension associated with tachycardia. Ten minutes before injection of LPS, these rats received an i.v. injection of either vehicle or 3 mg/kg SR141716A, a selective CB1 receptor antagonist (20).
For survival studies, three groups of unanesthetized rats received an i.p. injection of 45 mg/kg LPS. The animals also received two i.p. injections of vehicle (group #1), 3 mg/kg SR141716A (group #2), or 1 mg/kg 
9-tetrahydrocannabinol (THC, group #3) 30 min before and 6 h after injection of LPS. The number of surviving animals was established at 12, 24, 36, and 48 h after LPS treatment.
Preparation of isolated blood cells
Heparinized rat blood was removed via an intraarterial cannula. Mononuclear cells were isolated from whole blood by the method of Böyum (21), as described previously (19). Briefly, 5 ml of heparinized whole blood was carefully layered over 5 ml of Histopaque -1077 solution (Sigma Chemical Co., St. Louis, Mo.) and centrifuged at 400 g for 20 min. The mononuclear fraction formed a white layer at the interface, whereas erythrocytes and granulocytes sedimented and formed a pellet. The mononuclear fraction was aspirated, resuspended in 2 ml of phosphate-buffered saline (PBS), and centrifuged at 800 g for 20 min. The resulting pellet was resuspended in 0.5 ml PBS and used for subsequent experiments. The mononuclear cell fraction prepared from 5 ml of heparinized rat blood contained 1–2 x 107 lymphocytes, ~106 macrophages, and 15–20 platelets per macrophage.
For preparing separate platelet and macrophage cell fractions for in vivo injection into recipients, platelet-rich plasma was removed from 5 ml heparinized blood of a normal control rat by centrifugation at 64 g for 15 min. The mononuclear cell fraction was prepared from the remaining cellular pellet, as described above, after replacing the plasma with PBS. By first removing platelet-rich plasma, the number of contaminating platelets in these preparations was reduced to 3–5 platelets per macrophage. Both preparations were then incubated for 90 min with 200 µg/ml LPS. At the end of the incubation, the preparations were centrifuged (400 g, 15 min), washed twice, and then resuspended in 0.5 ml Krebs' buffer in siliconized glass test tubes for i.v. injection into recipient rats.
For biochemical analyses, platelet-rich plasma and the platelet-depleted mononuclear cell preparation were separately plated in 10 cm plastic culture dishes. Nonadherent cells were removed after 1 h by aspiration, and the remaining adherent cells were maintained in RPMI 1640 medium containing 10% fetal calf serum under standard cell culture conditions (19).
Biochemical analysis of anandamide and 2-arachidonyl glyceride
For the identification of anandamide, platelets and macrophages were isolated from a total of 100 ml of rat blood. The cells (2–3x1010 pure platelets or 2–3x107 macrophages contaminated with ~108 platelets, respectively) were incubated for 90 min with 200 µg/ml LPS or vehicle. The medium was then replaced with serum-free RPMI 1640 medium containing 200 µM phenylmethylsulfonylfluoride (PMSF) to prevent the breakdown of anandamide (22). Thirty minutes later, the cells + medium were extracted three times with 2 volumes of ethylacetate; the organic phase was dried under nitrogen and resuspended in ethanol. The extract was fractionated by reverse phase high-performance liquid chromatography (HPLC), using a Waters Guard-pak precolumn module, a RCM-100 radial compression module fitted with an 8 x 100 mm Radial Pak A cartridge containing 4 µm C-18 packing, a PM-80 solvent delivery system (Bioanalytical Systems, W. Lafayette, Ind.), and an acidified methanol:water gradient, as described elsewhere (23). The fractions corresponding to the elution time of authentic anandamide (95 to 103 min) were pooled, dried under nitrogen, and derivatized with bis[trimethylsilyl]trifluoroacetamide (BSTFA) for 1 h at 70°C. Samples were dried again and resuspended in n-hexane. The trimethylsilylether derivatives were subjected to gas chromatography/mass spectrometry as described previously (19). Briefly, the samples were injected in 'spitless' mode into a gas chromatograph (GC, Hewlett-Packard 5890) equipped with an HP-1 capillary column (25 mx0.2 mmx0.33 µm) and interfaced with a mass spectrometer (Hewlett-Packard 5988A). Four minutes after injection, the oven temperature was ramped from 90°C to 300°C at the rate of 10°C/min. Helium was used as carrier gas. The injector and detector temperatures were 260°C, and the source temperature was 200°C. Anandamide eluted from the GC column at ~23.2 min. Based on the electron impact spectrum of anandamide, selected ion monitoring analyses were performed on m/z 85, m/z 116, and m/z 404.
For analysis of 2-AG, macrophages and platelets were obtained from 30 ml rat blood. Incubation of the cells with LPS and their subsequent processing were as described above, including the use of PMSF to inhibit the degradation of 2-AG (24). Using the same reverse phase HPLC system as for anandamide, authentic 2-AG eluted between 105 and 115 min. These fractions were pooled, dried under nitrogen, and derivatized with BSTFA, as described above. GC/MS analysis of 2-AG was done under the same conditions, except a different GC column (SPB-5, 30mx0.25 mmx0.25 µm) was used. 2-AG eluted from this GC column at ~26.9–27.0 min. Based on the electron impact spectrum of authentic 2-AG, selected ion monitoring analyses were performed on m/z 103, m/z 129, m/z 147, m/z 432, m/z 451, and m/z 507.
Drugs
Anandamide (arachidonyl ethanolamide) was synthesized by Dr. Raj Razdan (Organix Inc., Woburn, Mass.); SR141716A (N-[piperidine-1-yl]-5-[4-chlorophenyl]-1-[2,4-dichlorophenyl]-4-methyl-1H-pyrazole-3-carboxamide HCl) was provided by Dr. John Lowe (Pfizer Central Research). 2-AG was obtained from Deva Biotech Inc. (Hatboro, Pa). THC was obtained from the National Institute on Drug Abuse. E. coli lipopolysaccharide (0127:B8) was from Sigma Chemical Co. The vehicle for anandamide, THC, and SR141716A was emulphor:ethanol:saline 1:1:18. Emulphor is a polyoxyphenylated vegetable oil. 2-AG was originally received in n-hexane, which was evaporated; the drug was redissolved in ethanol and diluted in emulphor:ethanol:saline 1:1:8 immediately before its i.v. administration.
Statistical analyses
Time-dependent changes in mean blood pressure and heart rate in the different treatment groups were compared by analysis of variance, followed by Bonferroni's post hoc test. Survival data were analyzed by using the chi square test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fig. 1).
This dose of SR141716A causes similar, near-maximal inhibition of the hypotensive (7, 8) as well as the neurobehavioral effects of both THC and anandamide (20), which are thought to be mediated by CB1 receptors (8, 20). In contrast, similar doses of SR141716A did not antagonize the ability of the cannabinoid agonist CP-55,940 to suppress mitogen-induced splenocyte proliferation and natural killer activity (25), thought to be mediated by CB2 receptors (26). In agreement with our earlier findings (7, 27), 3 mg/kg SR141716A alone caused no consistent change in basal blood pressure (-4±3 mmHg) and no change in heart rate. In other animals, the maximal hypotensive response to the same dose of LPS was unaffected by pretreatment with intracisternally administered SR141716A, 300 µg/kg (BP: -29±5 mmHg, n=4, compared to -22±2 mmHg in the absence of SR141716A, n=26, P>0.1). This suggests that the hypotensive but not the tachycardic response to LPS is due to activation of peripherally located CB1 receptors, possibly by an endogenous cannabinoid.
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Since circulating macrophages are targets for LPS (1, 2) and can generate anandamide (19), it is plausible that they are the source of the endogenous cannabinoid mediating the hypotensive response to LPS. To test this, 5 ml of arterial blood was withdrawn from LPS-treated rats at the peak of their hypotensive re~sponse for preparation of the mononuclear cell fraction. These cells caused prolonged hypotension and tachycardia when injected into untreated control rats, but only tachycardia when injected into recipient rats pretreated with SR141716A ( Fig. 2).
Mononuclear cells from untreated donors caused only minimal changes in blood pressure (-7±1 mmHg) or heart rate (+24±9 beats/min). The mononuclear cell preparation contained macrophages, lymphocytes, and contaminating platelets. Lymphocytes from LPS-treated rats, separated by aspiration after allowing the macrophages and platelets to adhere to a plastic culture dish, did not cause any change in blood pressure or heart rate in recipient rats. Therefore, macrophages and/or platelets appear to be responsible for the CB1 receptor-mediated hypotension in response to LPS treatment.
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To examine the respective roles of macrophages and platelets in LPS-induced hypotension, platelet-rich plasma was removed from 5 ml of control rat blood before isolating the mononuclear cells, and both fractions were then incubated in vitro with LPS. LPS is known to be able to directly activate both macrophages (1, 2) and platelets (28), and such direct activation also provides the advantage of avoiding possible indirect effects of in vivo treatment with LPS. Injection of either the LPS-treated platelets or mononuclear cells into normal recipients caused hypotension and tachycardia ( Fig. 3).
In both cases hypotension was prevented by SR141716A pretreatment, whereas the tachycardia was unaffected (platelets) or partially reduced (macrophages).
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LPS induces the production of anandamide in circulating macrophages
We examined the ability of LPS to induce the production of anandamide in isolated macrophages and platelets. Each treatment group consisted of cells isolated from a total of 100 ml rat blood. The cells were plated, treated in vitro with LPS or vehicle, and extracted with ethylacetate, as described in Materials and Methods. The extract was then fractionated by reverse phase HPLC, and the fractions corresponding to the elution profile of authentic anandamide were pooled and subjected to GC/MS analysis to test for the presence of anandamide. As illustrated in Fig. 4A, C
, anandamide could be clearly identified by its GC elution time and m/z ratios in macrophages treated in vitro with LPS. To estimate the amount of anandamide in cells, a calibration curve was generated from the areas under the GC elution curves obtained with different amounts of authentic anandamide. Using this curve, the amount of anandamide in macrophages was estimated to be 130 ng, or about 20 amol/cell. There was no detectable anandamide (<1 ng) in the same number of macrophages treated with vehicle ( Fig. 4B), nor was there detectable anandamide in either LPS-treated ( Fig. 4D) or control platelets (not shown).
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Cardiovascular effects of 2-arachidonyl glyceride
2-AG has slightly lower affinity for CB1 receptors than anandamide (15, 16), but its cardiovascular actions are unknown. We tested the effects of 2-AG on the blood pressure and heart rate of eight urethane-an~esthetized rats. Bolus injection of 2-AG (1 and 3 mg/kg i.v.) caused dose-dependent hypotension (peak change: -12±2 and -34±5 mmHg, respectively) and tachycardia (+22±4 and +39±9 beats/min, respectively). The hypotensive response to both doses of 2-AG was significantly reduced when tested 10 min after treatment of the rats with 3 mg/kg SR141716A (-6±2 mmHg, P<0.03, and -19±3 mmHg, P<0.001, respectively), whereas the tachycardia remained unchanged (+20±3 and +37±9 beats/min, respectively). As this response pattern is similar to that caused by LPS-activated macrophages and platelets, we tested whether 2-AG is present in these cells.
LPS stimulates the production of 2-arachidonyl glyceride in platelets
For identifying 2-AG in platelets and macrophages, cells from a total of 30 ml rat blood were used. Cells were prepared, treated with LPS, then incubated in the presence of PMSF, extracted with ethylacetate, and fractionated by reverse phase HPLC in the same way as for anandamide assays. Using the same solvent gradient, 2-AG is eluted immediately after anandam~ide in this reverse phase HPLC system (between 105 and 115 min). The pooled HPLC fractions were then subjected to GC/MS analysis. As seen in Fig. 5,
2-AG could be clearly identified both in control and LPS-treated platelets, the amount in the latter being about threefold higher than in controls. 2-AG was also identified in control and LPS-treated macrophage preparations (not shown). However, since contaminating platelets cannot be removed from the mononuclear cell fraction and their numbers are variable, the cellular source of 2-AG cannot be clearly identified in these latter preparations.
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Macrophage-induced, CB1 receptor-mediated hypotension is independent of sympathetic tone or nitric oxide
These findings support the hypothesis that LPS-induced hypotension is mediated, at least in part, by macrophage- and platelet-derived cannabinoids acting at peripheral CB1 receptors, but do not indicate the location of these receptors. Exogenous anandamide can reduce sympathetic vasoconstrictor tone by acting at presynaptic CB1 cannabinoid receptors located on peripheral sympathetic nerve terminals (27, 29, 30), but can also cause vasodilation by a direct action on vascular smooth muscle (31–33). To determine which of these two mechanisms is involved in the effect of macrophage- and platelet-derived cannabinoids, we compared the hypotensive action of LPS-treated mononuclear cells in control rats vs. in three rats pretreated with phentolamine (2 mg/kg i.v.) to remove sympathetic vasoconstrictor tone, then continuously infused with vasopressin (2 µg·kg-1·min-1 i.v.) to restore basal blood pressure to control levels. The hypotensive effect of LPS-treated cells in the latter group (-33±4 mmHg) was not different from that in controls (-26±5 mmHg), which suggests that the vasodilator effect of macrophage-derived cannabinoids is independent of sympathetic tone. We also tested whether endothelial NO is involved in this vasodilator effect. In six rats, the i.v. injection of the irreversible nitric oxide synthase inhibitor, L-NAME (20 mg/kg), caused a sustained increase in basal blood pressure (+24±3 mmHg), but the hypotensive and tachycardic responses to the subsequent administration of LPS-treated mononuclear cells (-18±4 mmHg, +81±8 beats/min) were not significantly different from the same responses in untreated controls (see 
Fig. 3). This indicates that, as in hemorrhagic shock (19), the hypotension elicited by activated macrophages/platelets is independent of vascular NO.
Effects of SR141716A and THC on survival from endotoxic shock
Since SR141716A was able to prevent LPS-induced hypotension, we tested its ability to modify the rate of survival from LPS-induced shock. As illustrated in Fig. 6,
treatment with SR141716A (see Materials and Methods) significantly increased the survival rate at all four time points tested. Treatment with the cannabinoid agonist THC also increased the rate of survival during the first 24 h after LPS administration.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous studies have demonstrated that anandamide can elicit CB1 receptor-mediated hypotension in rats (7–10). Anandamide was undetectable in control macrophages ( Fig. 4B), whereas substantial amounts could be identified in LPS-treated cells ( Fig. 4A), which makes it a plausible mediator of the CB1 receptor-mediated hypotension induced by activated macrophages. 2-AG has long been postulated as a transient byproduct of the generation of arachidonic acid from phosphatidylinositol in platelets, although its presence has not been documented (34). Here we clearly identify, for the first time, the presence of 2-AG in platelets and show that it is substantially increased in response to LPS. Since platelets do not contain detectable levels of anandamide and the hypotensive action of 2-AG is attenuated by SR141716A, it is likely that 2-AG is responsible for the ability of LPS-treated platelets to induce CB1 receptor-mediated hypotension. Although platelet contamination of 2-AG precluded its unequivocal identification in circulating rat macrophages (see Results), the presence of 2-AG in the J774 macrophage cell line (V. Di Marzo, personal communication) makes it likely that macrophages may produce both of these endogenous cannabinoids.
Both 2-AG and LPS-treatment also elicit tachycardia resistant to SR141716A, which suggests that 2-AG could be a mediator of this effect. The resistance of the tachycardia to blockade by SR141716A may suggest the involvement of CB2 receptors, which could be tested by using a novel, selective CB2 receptor antagonist (35). Alternatively, both anandamide and 2-AG are rapidly degraded to yield arachidonic acid (18, 22), and arachidonic acid elicits tachycardia in rats by a mechanism not involving cannabinoid receptors (K. Varga and G. Kunos, unpublished observations). Additional studies are in progress to determine the mechanism of the tachycardia in response to LPS treatment.
Although 2-AG could be detected in control platelets, these cells do not trigger hypotension in recipient rats, and there is no evidence for tonic activation of hypotensive CB1 receptors under control conditions (7, 8, 10, 19). Attachment of macrophages and platelets to the vascular wall is known to increase in both hemorrhagic (36) and septic shock (37) through the increased expression of selectins (38, 39) and other cell adhesion molecules. Cannabinoids released from these adherent cells may act as `juxtacrine' mediators at CB1 receptors in the vasculature. It is plausible that 2-AG released from nonadherent platelets is diluted in plasma, whereas activation of platelets by LPS not only increases the cellular levels of 2-AG ( Fig. 5), but the adherence of platelets to the vascular wall (38) greatly enhances the local concentration of platelet-derived 2-AG at vascular receptors. A high-affinity uptake system for 2-AG in platelets, similar to that documented for anandamide in neurons (40, 41) and macrophages (42), could further limit the free concentration of 2-AG in plasma. However, no evidence for facilitated diffusion of 2-AG was found in a recent study in rat basophilic leukemia and mouse neuroblastoma cells (43).
The presence of anandamide and 2-AG in circulating blood cells from animals in different forms of shock provides a plausible explanation for the CB1 receptor-mediated hypotension under these conditions. However, one cannot exclude the alternative (or additional) possibility that these activated cells trigger hypotension indirectly by releasing endogenous cannabinoids from another source, such as the vascular endothelium. Such a mechanism has recently been proposed to account for the ability of SR141716A to inhibit the NO-independent component of carbachol-induced vasodilation in an isolated, buffer-perfused mesenteric vascular bed preparation (33), although this mechanism has been questioned (44, 45). In either case, a likely target of the released cannabinoid is the vascular smooth muscle cell, which has been shown to contain functional CB1 receptors (32). The message for CB1 cannabinoid receptors is also present in postganglionic sympathetic neurons (29), and activation of presynaptic CB1 receptors on such neurons has been shown to inhibit stimulation-induced norepinephrine release both in vitro (29) and in vivo (30). However, the present finding that the hypotension induced by activated macrophages and platelets is unchanged in the absence of sympathetic tone indicates that presynaptic CB1 receptors are not the targets of macrophage- and platelet-derived cannabinoids.
It is possible that macrophage-and platelet-derived cannabinoids also subserve noncardiovascular functions in shock. The immunosuppressive action of cannabinoids is thought to be mediated by CB2 receptors located primarily on macrophages (26, 46). 2-AG is known to be able to activate such receptors (47) and may contribute to the depressed immune response present in both hemorrhagic (48) and septic shock (49).
Pretreatment of animals with the CB1 antagonist SR141716A not only prevented LPS-induced hypotension, but also improved survival. This observation opposes recent findings about hemorrhagic shock where pretreatment with SR141716A shortened sur~vival (19). In this latter model, the same degree of hypotension was induced by controlled bleeding in the absence or presence of SR141716A, and it is possible that removal of cannabinoid-mediated vasodilation further compromised tissue perfusion. In contrast, prevention of LPS-induced hypotension by SR141716A would help to maintain adequate tissue perfusion. Pretreatment with the cannabinoid agonist THC improved survival both in hemorrhagic (19) and endotoxic shock ( Fig. 6). By stimulating vascular CB1 receptors, THC may help to counteract excessive sympathetic vasoconstriction triggered by the hypotension. THC will also stimulate CB2 receptors on macrophages, which could suppress the LPS-induced release of cytokines known to contribute to the multiorgan failure of septic shock.
In summary, the present findings document a novel paracrine mechanism of vasodilation in endotoxic shock, where macrophage-derived anandamide and platelet-derived 2-AG are likely responsible for the activation of vascular CB1 cannabinoid receptors. Unlike the tonically active vascular NO system, the vasodilator activity of endogenous cannabinoids is evident only under certain pathological conditions, and it appears to be independent of NO. The activation of this mechanism in hemorrhagic (19) and septic shock represents a novel physiological function of the endogenous cannabinoid system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Abbreviations: HPLC, high-performance liquid chromatography; LPS, lipopolysaccharide; 2-AG, 2-arachidonyl glyceride; THC, 9-tetrahydrocannabinol; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonylfluoride; GC, gas chromatography; BSTFA, bis[trimethylsilyl]trifluoroacetamide; anandamide, arachidonyl ethanolamide; SR141716A, N-[piperidine-1-yl]-5-[4-chloro-phenyl]-1-[2,4-di-chloro-phenyl]-4-methyl-1H-pyrazole-3-carboxamide HCl.
Received for publication January 30, 1998. Accepted for publication February 27, 1998.
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 S. Kishimoto, M. Muramatsu, M. Gokoh, S. Oka, K. Waku, and T. Sugiura Endogenous Cannabinoid Receptor Ligand Induces the Migration of Human Natural Killer Cells J. Biochem., February 1, 2005; 137(2): 217 - 223. [Abstract] [Full Text] [PDF]
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S. M. Gardiner, T. Bennett, G. Kunos, S. Batkai, P. Pacher, J. A. Wagner, and Z. Jarai Cannabinoids and Endotoxemia Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H451 - H451. [Abstract] [Full Text] [PDF]
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 S. Oka, S. Ikeda, S. Kishimoto, M. Gokoh, S. Yanagimoto, K. Waku, and T. Sugiura 2-Arachidonoylglycerol, an endogenous cannabinoid receptor ligand, induces the migration of EoL-1 human eosinophilic leukemia cells and human peripheral blood eosinophils J. Leukoc. Biol., November 1, 2004; 76(5): 1002 - 1009. [Abstract] [Full Text] [PDF]
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 E. Deusch, H. G. Kress, B. Kraft, and S. A. Kozek-Langenecker The Procoagulatory Effects of Delta-9-Tetrahydrocannabinol in Human Platelets Anesth. Analg., October 1, 2004; 99(4): 1127 - 1130. [Abstract] [Full Text] [PDF]
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S. Batkai, P. Pacher, Z. Jarai, J. A. Wagner, and G. Kunos Cannabinoid antagonist SR-141716 inhibits endotoxic hypotension by a cardiac mechanism not involving CB1 or CB2 receptors Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H595 - H600. [Abstract] [Full Text] [PDF]
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 P. Pacher, S. Batkai, and G. Kunos Haemodynamic profile and responsiveness to anandamide of TRPV1 receptor knock-out mice J. Physiol., July 15, 2004; 558(2): 647 - 657. [Abstract] [Full Text] [PDF]
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 Y. Koura, A. Ichihara, Y. Tada, Y. Kaneshiro, H. Okada, C. J. Temm, M. Hayashi, and T. Saruta Anandamide Decreases Glomerular Filtration Rate through Predominant Vasodilation of Efferent Arterioles in Rat Kidneys J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1488 - 1494. [Abstract] [Full Text] [PDF]
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S. Kishimoto, Y. Kobayashi, S. Oka, M. Gokoh, K. Waku, and T. Sugiura 2-Arachidonoylglycerol, an Endogenous Cannabinoid Receptor Ligand, Induces Accelerated Production of Chemokines in HL-60 Cells J. Biochem., April 1, 2004; 135(4): 517 - 524. [Abstract] [Full Text] [PDF]
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J. Liu, S. Batkai, P. Pacher, J. Harvey-White, J. A. Wagner, B. F. Cravatt, B. Gao, and G. Kunos Lipopolysaccharide Induces Anandamide Synthesis in Macrophages via CD14/MAPK/Phosphoinositide 3-Kinase/NF-{kappa}B Independently of Platelet-activating Factor J. Biol. Chem., November 7, 2003; 278(45): 45034 - 45039. [Abstract] [Full Text] [PDF]
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S. Kishimoto, M. Gokoh, S. Oka, M. Muramatsu, T. Kajiwara, K. Waku, and T. Sugiura 2-Arachidonoylglycerol Induces the Migration of HL-60 Cells Differentiated into Macrophage-like Cells and Human Peripheral Blood Monocytes through the Cannabinoid CB2 Receptor-dependent Mechanism J. Biol. Chem., June 27, 2003; 278(27): 24469 - 24475. [Abstract] [Full Text] [PDF]
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L. Offertaler, F.-M. Mo, S. Batkai, J. Liu, M. Begg, R. K. Razdan, B. R. Martin, R. D. Bukoski, and G. Kunos Selective Ligands and Cellular Effectors of a G Protein-Coupled Endothelial Cannabinoid Receptor Mol. Pharmacol., March 1, 2003; 63(3): 699 - 705. [Abstract] [Full Text] [PDF]
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R. Sancho, M. A. Calzado, V. Di Marzo, G. Appendino, and E. Munoz Anandamide Inhibits Nuclear Factor-kappa B Activation through a Cannabinoid Receptor-Independent Pathway Mol. Pharmacol., February 1, 2003; 63(2): 429 - 438. [Abstract] [Full Text] [PDF]
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M. L. Orliac, R. Peroni, S. M Celuch, and E. Adler-Graschinsky Potentiation of Anandamide Effects in Mesenteric Beds Isolated from Endotoxemic Rats J. Pharmacol. Exp. Ther., January 1, 2003; 304(1): 179 - 184. [Abstract] [Full Text] [PDF]
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J. A. Wagner, K. Hu, J. Bauersachs, J. Karcher, M. Wiesler, S. K. Goparaju, G. Kunos, and G. Ertl Endogenous cannabinoids mediate hypotension after experimental myocardial infarction J. Am. Coll. Cardiol., December 1, 2001; 38(7): 2048 - 2054. [Abstract] [Full Text] [PDF]
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 E. V. BERDYSHEV, P. C. SCHMID, R. J. KREBSBACH, and H. H. O. SCHMID Activation of PAF receptors results in enhanced synthesis of 2-arachidonoylglycerol (2-AG) in immune cells FASEB J, October 1, 2001; 15(12): 2171 - 2178. [Abstract] [Full Text] [PDF]
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H. Iwamura, H. Suzuki, Y. Ueda, T. Kaya, and T. Inaba In Vitro and in Vivo Pharmacological Characterization of JTE-907, a Novel Selective Ligand for Cannabinoid CB2 Receptor J. Pharmacol. Exp. Ther., April 13, 2001; 296(2): 420 - 425. [Abstract] [Full Text]
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N. Garcia Jr., Z. Jarai, F. Mirshahi, G. Kunos, and A. J. Sanyal Systemic and portal hemodynamic effects of anandamide Am J Physiol Gastrointest Liver Physiol, January 1, 2001; 280(1): G14 - G20. [Abstract] [Full Text] [PDF]
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C. J. Hillard Endocannabinoids and Vascular Function J. Pharmacol. Exp. Ther., July 1, 2000; 294(1): 27 - 32. [Abstract] [Full Text]
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 Z. Jarai, J. A. Wagner, S. K. Goparaju, L. Wang, R. K. Razdan, T. Sugiura, A. M. Zimmer, T. I. Bonner, A. Zimmer, and G. Kunos Cardiovascular Effects of 2-Arachidonoyl Glycerol in Anesthetized Mice Hypertension, February 1, 2000; 35(2): 679 - 684. [Abstract] [Full Text] [PDF]
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T. Sugiura, S. Kondo, S. Kishimoto, T. Miyashita, S. Nakane, T. Kodaka, Y. Suhara, H. Takayama, and K. Waku Evidence That 2-Arachidonoylglycerol but Not N-Palmitoylethanolamine or Anandamide Is the Physiological Ligand for the Cannabinoid CB2 Receptor. COMPARISON OF THE AGONISTIC ACTIVITIES OF VARIOUS CANNABINOID RECEPTOR LIGANDS IN HL-60 CELLS J. Biol. Chem., January 7, 2000; 275(1): 605 - 612. [Abstract] [Full Text] [PDF]
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L. Hanus, A. Breuer, S. Tchilibon, S. Shiloah, D. Goldenberg, M. Horowitz, R. G. Pertwee, R. A. Ross, R. Mechoulam, and E. Fride HU-308: A specific agonist for CB2, a peripheral cannabinoid receptor PNAS, December 7, 1999; 96(25): 14228 - 14233. [Abstract] [Full Text] [PDF]
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J. A. Wagner, K. Varga, Z. Jarai, and G. Kunos Mesenteric Vasodilation Mediated by Endothelial Anandamide Receptors Hypertension, January 1, 1999; 33(1): 429 - 434. [Abstract] [Full Text] [PDF]
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, n=26) or SR141716A-pretreated rats (
, n=9). SR141716A (3 mg/kg i.v.) was given 10 min prior to LPS, which was injected at 0 min. Vertical bars represent SEM. *Significant difference (P<0.05) from corresponding control value.
, SR141716A+LPS), or 13 (
, THC+LPS). *Significant difference from corresponding value in rats treated with LPS only.