Evidence for the involvement of the cannabinoid CB2 receptor and its endogenous ligand 2-arachidonoylglycerol in 12-O-tetradecanoylphorbol-13-acetate-induced acute inflammation in mouse ear

2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors. Two types of cannabinoid receptors have been identified to date. The CB1 receptor is abundantly expressed in the brain, and assumed to be involved in the attenuation of neurotransmission. On the other hand, the physiological roles of the CB2 receptor, mainly expressed in several types of inflammatory cells and immunocompetent cells, have not yet been fully elucidated. In this study, we investigated possible pathophysiological roles of the CB2 receptor and 2-arachidonoylglycerol in acute inflammation in mouse ear induced by the topical application of 12-O-tetradecanoylphorbol-13-acetate. We found that the amount of 2-arachidonoylglycerol was markedly augmented in inflamed mouse ear. In contrast, the amount of anandamide, another endogenous cannabinoid receptor ligand, did not change markedly. Importantly, 12-O-tetradecanoylphorbol-13-acetate-induced ear swelling was blocked by treatment with SR144528, a CB2 receptor antagonist, suggesting that the CB2 receptor is involved in the swelling. On the other hand, the application of AM251, a CB1 receptor antagonist, exerted only a weak suppressive effect. The application of SR144528 also reduced the 12-O-tetradecanoylphorbol-13-acetate-induced production of leukotriene B(4) and the infiltration of neutrophils in the mouse ear. Interestingly, the application of 2-arachidonoylglycerol to the mouse ear evoked swelling, which was abolished by treatment with SR144528. Nitric oxide was suggested to be involved in the ear swelling induced by 2-arachidonoylglycerol. These results suggest that the CB2 receptor and 2-arachidonoylglycerol play crucial stimulative roles during the course of inflammatory reactions.     

Involvement of cannabinoid CB2 receptors in the IgE-mediated triphasic cutaneous reaction in mice

Involvement of cannabinoid CB2 receptors in the IgE-mediated cutaneous reaction was investigated. Epicutaneous challenge with 2,4-dinitrofluorobenzene caused a triphasic swelling in the ear of BALB/c and C57BL/6 mice passively sensitized with anti-dinitrophenol IgE. Peak responses of the ear swelling appeared at 1 h, 24 h, and 8 days after the challenge in both strains of mice. In contrast, cannabinoid CB2 receptor-deficient mice failed to exhibit the obvious triphasic ear swelling observed in wild-type mice. Oral administration of cannabinoid CB2 receptor antagonist/inverse agonists [N-(benzo[1,3]dioxol-5-ylmethyl)-7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxamide] (JTE-907) and {N-[(1S)-endo-1,3,3-trimethylbicyclo[2,2,1]heptan-2yl]5-(4-chloro-3-methyl-phenyl)-1-(4-methylbenzyl)pyrazole-3-carboxamide} (SR144528) at doses of 0.1-10 mg/kg significantly and dose-dependently suppressed all three phases of ear swelling in BALB/c mice. Interestingly, epicutaneous treatment with an ether-linked analogue of endogenous cannabinoids, 2-arachidonoylglycerol, caused an ear swelling that could be detected at 1 h, 24 h, and 8 days after treatment of both BALB/c and C57BL/6 mice. These results suggest that cannabinoid CB2 receptors are involved in induction of the triphasic cutaneous reaction mediated by IgE, and that cannabinoid CB2 receptor antagonist/inverse agonists may serve as anti-allergic agents in the treatment of allergic dermatitis.     

CB(2) cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: role of CB(2) receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.     

Anti-proliferative effect of a putative endocannabinoid, 2-arachidonylglyceryl ether in prostate carcinoma cells

Endocannabinoids (ECs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, 2-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR144528 (a selective CB2R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for 2-AG was high and 2-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR144528 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.     

Activation by 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, of p42/44 mitogen-activated protein kinase in HL-60 cells

2-Arachidonoylglycerol (2-AG), an endogenous cannabinoid receptor ligand, was shown to induce rapid phosphorylation of p42/44 mitogen-activated protein kinase (MAP kinase) in HL-60 cells. We confirmed that the enzyme activity of p42/44 MAP kinase in HL-60 cells was augmented markedly when the cells were stimulated with 2-AG. The addition of SR144528, a cannabinoid CB2 receptor-specific antagonist, to the cells prior to the addition of 2-AG abolished the response induced by 2-AG, indicating that the CB2 receptor is involved in the response. G protein G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells nullified the response induced by 2-AG. CP55940 and anandamide also induced the activation of p42/44 MAP kinase, although the activation by anandamide was less pronounced than that by 2-AG or CP55940. These results suggest that 2-AG may play an important physiological role in this type of cell through the activation of the p42/44 MAP kinase cascade.     

Induction of preferential chemotaxis of unstimulated B-lymphocytes by 2-arachidonoylglycerol in immunized mice

2-Arachidonoylglycerol (2-AG) is an endogenous ligand for cannabinoid receptors. There are two types of cannabinoid receptors, CB1 and CB2. We investigated the chemotactic activity of 2-AG using mouse lymphocytes because cells in the immune system are known to express CB2 . Spleen cell migration toward 2-AG was observed, which was completely inhibited by SR144528, a CB2-specific antagonist. 2-AG has been reported to induce a preferential B cell chemotaxis. We examined whether there is any difference in responsiveness during the activation of B cells. When spleen cells from immunized mice were tested, naive B cells but not germinal center B cells (GL7-positive) were increased in the fraction attracted by 2-AG. Furthermore, when Peyer's patch lymphocytes were tested after oral administration of cholera toxin, the number of IgA* B cells was increased in the fraction attracted by 2-AG. These results suggested that 2-AG preferentially attracts unstimulated naive B cells rather than activated and/or class-switched B cells. This property may influence the structure of B cell compartments in secondary lymphoid tissues.     

The effect of IgE-mediated mast cell degranulation on the expression of experimental contact sensitivity in the mouse

The effects of mast cell activation/degranulation on the elicitation of contact sensitivity (CS) to oxazolone and dinitrofluorobenzene were investigated. Mice were actively sensitized to oxazolone by epicutaneous painting followed by ear challenge. Passive sensitization to DNFB was induced by intradermal injections of dinitrophenol (DNP)-specific cloned T cells in the ears. Mast cells in the challenged ears were activated in various time periods by inducing a passive cutaneous anaphylaxis reaction where passive sensitization with monoclonal IgE anti-DNP antibodies was followed by iv injection of DNP-BSA. This combination of immediate and delayed-type hypersensitivity reactions resulted in a significant increase of ear swelling without any noticeable effect on cellular infiltration when the contact response was evaluated a short time (3-4 hr) after mast cell activation. The very same results were obtained in naive (unsensitized) mice, indicating that this reaction was nonspecific. However, when the CS reaction was evaluated at its peak, i.e., 24 hr post challenge, mast cell activation that had been induced 0.5-11 hr after ear challenge did not have any significant effect on both swelling and cellular infiltration when the latter was evaluated by a radiometric assay. We conclude that in these systems mast cell activation/degranulation makes little or no contribution to the modulation of T-cell activity.     

Regulation of peripheral cannabinoid receptor CB2 phosphorylation by the inverse agonist SR 144528. Implications for receptor biological responses.

We recently demonstrated that the selective cannabinoid receptor antagonist SR 144528 acts as an inverse agonist that blocks constitutive mitogen-activated protein kinase activity coupled to the spontaneous autoactivated peripheral cannabinoid receptor (CB2) in the Chinese hamster ovary cell line stably transfected with human CB2. In the present report, we studied the effect of SR 144528 on CB2 phosphorylation. The CB2 phosphorylation status was monitored by immunodetection using an antibody specific to the COOH-terminal CB2 which can discriminate between phosphorylated and non-phosphorylated CB2 isoforms at serine 352. We first showed that CB2 is constitutively active, phosphorylated, and internalized at the basal level. By blocking autoactivated receptors, inverse agonist SR 144528 treatment completely inhibited this phosphorylation state, leading to an up-regulated CB2 receptor level at the cell surface, and enhanced cannabinoid agonist sensitivity for mitogen-activated protein kinase activation of Chinese hamster ovary-CB2 cells. After acute agonist treatment, serine 352 was extensively phosphorylated and maintained in this phosphorylated state for more than 8 h after agonist treatment. The cellular responses to CP-55,940 were concomitantly abolished. Surprisingly, CP-55,940-induced CB2 phosphorylation was reversed by SR 144528, paradoxically leading to a non-phosphorylated CB2 which could then be fully activated by CP-55,940. The process of CP-55,940-induced receptor phosphorylation followed by SR 144528-induced receptor dephosphorylation kept recurring many times on the same cells, indicating that the agonist switches the system off but the inverse agonist switches the system back on. Finally, we showed that autophosphorylation and CP-55, 940-induced serine 352 CB2 phosphorylation involve an acidotropic GRK kinase, which does not use Gibetagamma. In contrast, SR 144528-induced CB2 dephosphorylation was found to involve an okadaic acid and calyculin A-sensitive type 2A phosphatase.     

SUBCUTANEOUS OR ORAL ADMINISTRATION OF rhIL-11 MODULATES THE CONTACT HYPERSENSITIVITY RESPONSE

Recombinant human interleukin 11 (rhIL-11) is a multifunctional cytokine with immunomodulatory activity on both T cells and macrophages. The effects of rhIL-11 in a murine model of contact hypersensitivity (CHS) response have been studied. The CHS response is a T cell-mediated response directed against chemically modified self-proteins following epidermal exposure to haptens. CHS is generated in two phases. The sensitization phase involves dermal dendritic cell recognition of haptenized proteins and antigen presentation. The effector phase involves T cell recognition and activation. In mice sensitized with oxazolone, CHS was induced by secondary challenge to the right ear and measured by ear swelling 24 h later. rhIL-11 significantly suppressed CHS as measured by ear swelling and tissue myeloperoxidase activity when injected subcutaneously for 5 days from the day of sensitization or when administered only on the day before and the day of challenge, but was not effective when administered prior to or on the day of sensitization. These results indicate that subcutaneously administered rhIL-11 may modulate the effector phase of CHS. Administration of rhIL-11 as an oral gavage prior to sensitization also reduced CHS. However oral administration of rhIL-11 after sensitization had no effect. These results suggest that orally and subcutaneously administered rhIL-11 may act through different mechanisms to affect CHS.     

Depolarization-induced rapid generation of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, in rat brain synaptosomes

2-arachidonoylglycerol (2-AG) is an endogenous ligand for the cannabinoid receptors with a variety of potent biological activities. In this study, we first examined the effects of potassium-induced depolarization on the level of 2-AG in rat brain synaptosomes. We found that a significant amount of 2-AG was generated in the synaptosomes following depolarization. Notably, depolarization did not affect the levels of other molecular species of monoacylglycerols. Furthermore, the level of anandamide was very low and did not change markedly following depolarization. It thus appeared that the depolarization-induced accelerated generation is a unique feature of 2-AG. We obtained evidence that phospholipase C is involved in the generation of 2-AG in depolarized synaptosomes: U73122, a phospholipase C inhibitor, markedly reduced the depolarization-induced generation of 2-AG, and the level of diacylglycerol was rapidly elevated following depolarization. A significant amount of 2-AG was released from synaptosomes upon depolarization. Interestingly, treatment of the synaptosomes with SR141716A, a CB1 receptor antagonist, augmented the release of glutamate from depolarized synaptosomes. These results strongly suggest that the endogenous ligand for the cannabinoid receptors, i.e. 2-AG, generated through increased phospholipid metabolism upon depolarization, plays an important role in attenuating glutamate release from the synaptic terminals by acting on the CB1 receptor.     

Subcutaneous or oral administration of rhIL-11 modulated the contact hypersensitivity response

Recombinant human interleukin 11 (rhIL-11) is a multifunctional cytokine with immunomodulatory activity on both T cells and macrophages. The effects of rhIL-11 in a murine model of contact hypersensitivity (CHS) response have been studied. The CHS response is a T cell-mediated response directed against chemically modified self-proteins following epidermal exposure to haptens. CHS is generated in two phases. The sensitization phase involves dermal dendritic cell recognition of haptenized proteins and antigen presentation. The effector phase involves T cell recognition and activation. In mice sensitized with oxazolone, CHS was induced by secondary challenge to the right ear and measured by ear swelling 24 h later. rhIL-11 significantly suppressed CHS as measured by ear swelling and tissue myeloperoxidase activity when injected subcutaneously for 5 days from the day of sensitization or when administered only on the day before and the day of challenge, but was not effective when administered prior to or on the day of sensitization. These results indicate that subcutaneously administered rhIL-11 may modulate the effector phase of CHS. Administration of rhIL-11 as an oral gavage prior to sensitization also reduced CHS. However oral administration of rhIL-11 after sensitization had no effect. These results suggest that orally and subcutaneously administered rhIL-11 may act through different mechanisms to affect CHS.     

Autocrine and paracrine regulation of lymphocyte CB2 receptor expression by TGF-beta.

The marijuana-derived cannabinoid Delta(9)-tetrahydrocannabinol (THC) has been shown to be immunosuppressive. We report that THC induces the immunosuppressive cytokine TGF-beta by human peripheral blood lymphocytes (PBL). The ability of THC to stimulate TGF-beta production was blocked by the CB2 receptor specific antagonist SR144528 but not by the CB1 specific antagonist AM251. Furthermore, our data suggest that TGF-beta actively regulates lymphocyte CB2 receptor expression in an autocrine and paracrine manner. Whereas the addition of recombinant TGF-beta to PBL cultures downregulated CB2 receptor expression, anti-TGF-beta antibody treatment increased CB2 receptor expression. We conclude that one mechanism by which THC contributes to immune suppression is by stimulating an enhanced production of lymphocyte TGF-beta.     

Inhibitory effect of anandamide on resiniferatoxin-induced sensory neuropeptide release in vivo and neuropathic hyperalgesia in the rat

Anandamide (AEA) is an endogenous cannabinoid ligand acting predominantly on the cannabinoid 1 (CB(1)) receptor, but it is also an agonist on the capsaicin VR(1)/TRPV(1) receptor. In the present study we examined the effects of AEA and the naturally occurring cannabinoid 2 (CB(2)) receptor agonist palmitylethanolamide (PEA) on basal and resiniferatoxin (RTX)-induced release of calcitonin gene-related peptide (CGRP) and somatostatin in vivo. Since these sensory neuropeptides play important role in the development of neuropathic hyperalgesia, the effect of AEA and PEA was also examined on mechanonociceptive threshold changes after partial ligation of the sciatic nerve. Neither AEA nor PEA affected basal plasma peptide concentrations, but both of them inhibited RTX-induced release. The inhibitory effect of AEA was prevented by the CB(1) receptor antagonist SR141716A. AEA abolished and PEA significantly decreased neuropathic mechanical hyperalgesia 7 days after unilateral sciatic nerve ligation, which was antagonized by SR141716A and the CB(2) receptor antagonist SR144528, respectively. Both SR141716A and SR144528 increased hyperalgesia, indicating that endogenous cannabinoids acting on CB(1) and peripheral CB(2)-like receptors play substantial role in neuropathic conditions to diminish hyperalgesia. AEA and PEA exert inhibitory effect on mechanonociceptive hyperalgesia and sensory neuropeptide release in vivo suggesting their potential therapeutical use to treat chronic neuropathic pain.     

Cannabinoid receptor interactions with the antagonists SR 141716A and SR 144528.

The G protein-coupled cannabinoid receptor subtypes CB1 and CB2 have been cloned from several species. The CB1 receptor is highly conserved across species, whereas the CB2 receptor shows considerable cross-species variations. The two human receptors share only 44% overall identity, ranging from 35% to 82% in the transmembrane regions. Despite this structural disparity, the most potent cannabinoid agonists currently available are largely undiscriminating and are therefore unsatisfactory tools for investigating the architecture of ligand binding sites. However, the availability of two highly specific antagonists, SR 141716A for the CB1 receptor and SR 144528 for the CB2 receptor, has allowed us to adopt a systematic approach to defining their respective binding sites through the use of chimeric CB1 receptor/CB2 receptor constructs, coupled with site-directed mutagenesis. We identified the region encompassed by the fourth and fifth transmembrane helices as being critical for antagonist specificity. Both the wild type human receptors overexpressed in heterologous systems are autoactivated; SR 141716A and SR 144528 exhibit classical inverse agonist properties with their respective target receptors. In addition, through its interaction with the CB1 receptor SR 141716A blocks the Gi protein-mediated activation of mitogen-activated protein kinase stimulated by insulin or insulin-like growth factor I. An in-depth analysis of this discovery has led to a modified three-state model for the CB1 receptor, one of which implicates the SR 141716A-mediated sequestration of Gi proteins, with the result that the growth factor-stimulated intracellular pathways are effectively impeded.     

The CB1 endocannabinoid system modulates adipocyte insulin sensitivity

Mounting evidence suggests that the endocannabinoid system regulates energy metabolism through direct effects on peripheral tissues as well as central effects that regulate appetite. Here we examined the effect of cannabinoid receptor 1 (CB1) signaling on insulin action in fat cells. We examined effects of the natural CB1 agonist, 2-Arachidonoylglycerol (2-AG), and the synthetic CB1 antagonist, SR141716, on insulin action in cultured adipocytes. We used translocation of glucose transporter GLUT4 to plasma membrane (PM) as a measure of insulin action. 2-AG activation of the CB1 receptor promoted insulin sensitivity whereas antagonism by SR141716 reduced insulin sensitivity. Neither drug affected GLUT4 translocation in the absence of insulin or with high doses of insulin. Consistent with these results we found that insulin-stimulated phosphorylation of the protein kinase Akt was increased by 2-AG, attenuated by SR141716, and unaffected in the absence of insulin or by addition of high-dose insulin. These data provide a functional and molecular link between the CB1 receptor and insulin sensitivity, because insulin-stimulated phosphorylation of Akt is required for GLUT4 translocation to the PM. The sensitizing effects of 2-AG were abrogated by SR141716 and Pertussis toxin, indicating that the effects are mediated by CB1 receptor. Importantly, neither 2-AG nor SR141716 alone or in combination with maximal dose of insulin had effects on GLUT4 translocation and Akt phosphorylation. These data are consistent with a model in which the endocannabinoid system sets the sensitivity of the insulin response in adipocytes rather than directly regulating the redistribution of GLUT4 or Akt phosphorylation.     

Activation by 2-arachidonoylglycerol of platelet p38MAPK/cPLA2 pathway

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is described as a platelet agonist able to induce aggregation and to increase intracellular calcium. In the present report we have confirmed these data and demonstrated that the inhibitor of p38MAPK SB203580 and the inhibitor of cPLA(2) metabolism ETYA affect both these parameters. Thus, we aimed to define the role of p38MAPK/cytosolic phospholipase A(2) (cPLA(2)) pathway in 2-AG-induced human platelet activation. p38MAPK activation was assayed by phosphorylation. cPLA(2) activation was assayed by phosphorylation and as arachidonic acid release and thromboxane B(2) formation. It was shown that 2-AG in a dose- and time-dependent manner activates p38MAPK peaking at 10 μM after 1 min of incubation. The 2-AG effect on p38MAPK was not impaired by apyrase, indomethacin or RGDS peptide but it was significantly reduced by SR141716, specific inhibitor of type-1 cannabinoid receptor and unaffected by the specific inhibitor of type-2 cannabinoid receptor SR144528. Moreover, the incubation of platelets with 2-AG led to the phosphorylation of cPLA(2) and its activation. Platelet pretreatment with SB203580, inhibitor of p38MAPK, abolished both cPLA(2) phosphorylation and activation. In addition SR141716 strongly impaired cPLA(2) phosphorylation, arachidonic acid release and thromboxane B(2) formation, whereas SR144528 did not change these parameters. Finally platelet stimulation with 2-AG led to an increase in free oxygen radical species. In conclusion, data provide insight into the mechanisms involved in platelet activation by 2-AG, indicating that p38MAPK/cPLA(2) pathway could play a relevant role in this complicated process.     

Alkylamides from Echinacea are a new class of cannabinomimetics. Cannabinoid type 2 receptor-dependent and -independent immunomodulatory effects

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor alpha mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 approximately 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and pi-pi interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor alpha, IL-1beta, and IL-12p70 expression (5-500 nM) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding.     

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

We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca(2+) concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca(2+) concentrations in HL-60 cells. The response was affected by neither cyclooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca(2+) transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmitoylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system.     

Modulation of cutaneous inflammation by angiotensin-converting enzyme

Cutaneous neurogenic inflammation is a complex biological response of the host immune system to noxious stimuli. Present evidence suggests that zinc metalloproteases may play an important role in the regulation of neurogenic inflammation by controlling the local availability of neuropeptides, such as substance P (SP), that are capable of initiating or amplifying cutaneous inflammation after release from sensory nerves. To address the hypothesis that the dipeptidyl carboxypeptidase angiotensin-converting enzyme (ACE) is capable of modulating skin inflammation, we have analyzed murine allergic contact dermatitis (ACD) and irritant contact dermatitis (ICD) using wild-type C57BL/6J (ACE(+/+)) or genetically engineered mice with a heterozygous deletion of somatic ACE (ACE(+/-)). In 2,4-dinitro-1-fluorobenzene-sensitized ACE(+/-) mice, ACD was significantly augmented in comparison to ACE(+/+) controls as determined by the degree of ear swelling after exposure to hapten. Likewise, systemic treatment of ACE(+/+) mice with the ACE inhibitor captopril before sensitization or elicitation of ACD significantly augmented the ACD response. In contrast, local damage and neuropeptide depletion of sensory nerves following capsaicin, injection of a bradykinin B(2), or a SP receptor antagonist before sensitization significantly inhibited the augmented effector phase of ACD in mice with functionally absent ACE. However, in contrast to ACD, the response to the irritant croton oil was not significantly altered in ACE(+/-) compared with ACE(+/+) mice. Thus, ACE by degrading bradykinin and SP significantly controls cutaneous inflammatory responses to allergens but not to irritants, which may explain the frequently observed exacerbation of inflammatory skin disease in patients under medication with ACE inhibitors.     

Increase of the heart arrhythmogenic resistance and decrease of the myocardial necrosis zone during activation of cannabinoid receptors

We have found that intravenous administration of cannabinoid receptor (CB) agonist HU-210 (0.05 mg/kg), increases cardiac resistance against arrhythmogenic effect of epinephrine, aconitine, coronary artery occlusion and reperfusion in rats. Pretreatment with CB2-receptor antagonist, SR144528 (1 mg/kg), completely abolished the antiarrhythmic effect of HU-210. However this effect of HU-210 was not attenuated by pretreatment with CB1-receptor antagonist, SR141716A (3 mg/kg). We also found that HU-210 (0.05 mg/kg) decreased the relationship between infarction size and area of ischemia. It is concluded that CB2 receptor stimulation promotes an increase in the cardiac resistance against arrhythmogenic influences and probably increases myocardial tolerance of both ischemic and reperfusion damages in rats.