New Pyridone-Based Derivatives as Cannabinoid Receptor Type 2 Agonists.

The activation of the human cannabinoid receptor type II (CB2R) is known to mediate analgesic and anti-inflammatory processes without the central adverse effects related to cannabinoid receptor type I (CB1R). In this work we describe the synthesis and evaluation of a novel series of N-aryl-2-pyridone-3-carboxamide derivatives tested as human cannabinoid receptor type II (CB2R) agonists. Different cycloalkanes linked to the N-aryl pyridone by an amide group displayed CB2R agonist activity as determined by intracellular [cAMP] levels. The most promising compound 8d exhibited a non-toxic profile and similar potency (EC50 = 112 nM) to endogenous agonists Anandamide (AEA) and 2-Arachidonoylglycerol (2-AG) providing new information for the development of small molecules activating CB2R. Molecular docking studies showed a binding pose consistent with two structurally different agonists WIN-55212-2 and AM12033 and suggested structural requirements on the pyridone substituents that can satisfy the orthosteric pocket and induce an agonist response. Our results provide additional evidence to support the 2-pyridone ring as a suitable scaffold for the design of CB2R agonists and represent a starting point for further optimization and development of novel compounds for the treatment of pain and inflammation.

Double-blind placebo controlled trial of the anxiolytic effects of a standardized Echinacea extract.

Earlier studies suggested that specific Echinacea preparations might decrease anxiety. To further study the issue, we performed a double blind, placebo controlled trial with a standardized Echinacea angustifolia root extract. Participants were volunteers scoring above 45 points on the state or on the trait subscale of the State Trait Anxiety Inventory (STAI). They were treated with 40 mg Echinacea or with placebo tablets twice daily for 7 days followed by a 3 week-long washout period. Participants were also administered the Beck Depression Inventory (BDI) and the Perceived Stress Scale (PSS). In the Echinacea group, state anxiety scores decreased by approximately 11 points by the end of the treatment period, whereas the decrease was around 3-points in the placebo group (p< 0.01). The effect maintained over the washout period. The difference from placebo was significant from the 7th day of treatment throughout. Changes were less robust with trait anxiety scores, but the preparation performed better than placebo in patients with high baseline anxiety. Neither BDI nor PSS scores were affected by the treatments. Adverse effects were rare and mild, and all were observed in the placebo group. These findings suggest that particular Echinacea preparations have significant beneficial effects on anxiety in humans.

Forsythiaside prevents ß-amyloid-induced hippocampal slice injury by upregulating 2-arachidonoylglycerol via cannabinoid receptor 1-dependent NF-κB pathway.

In the study, the neuroprotectivities of forsythiaside, a main constituent of Forsythia suspensa (Thunb.) Vahl (F. suspensa, Lianqiao in Chinese), were investigated in the hippocampal slices. Forsythiaside suppressed the overexpression of cyclooxygenase-2 (COX-2) and monoacylglycerol lipase (MAGL) proteins induced by ß-amyloid (Aß25-35) to upregulate the levels of 2-arachidonoylglycerol (2-AG), an endogenous endocannabinoids. Then the inhibition of forsythiaside on COX-2 was deeply studied by the molecular docking. Forsythiaside prevented neuroinflammation and apoptosis from Aß25-35 insults, and this action appeared to be mediated via cannabinoid receptor 1 (CB1R)-dependent nuclear factor-κB (NF-κB) signaling pathways. More importantly, forsythiaside functionally improved Aß25-35-induced learning and memory deficits, which was indicated by long term potentiation (LTP). Taken together, forsythiaside may have therapeutic potential for Alzheimer's diseases (AD) by increasing the levels of 2-AG.

A New Generation of Arachidonic Acid Analogues as Potential Neurological Agent Targeting Cytosolic Phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) is an enzyme that releases arachidonic acid (AA) for the synthesis of eicosanoids and lysophospholipids which play critical roles in the initiation and modulation of oxidative stress and neuroinflammation. In the central nervous system, cPLA2 activation is implicated in the pathogenesis of various neurodegenerative diseases that involves neuroinflammation, thus making it an important pharmacological target. In this paper, a new class of arachidonic acid (AA) analogues was synthesized and evaluated for their ability to inhibit cPLA2. Several compounds were found to inhibit cPLA2 more strongly than arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor that is commonly used in the study of cPLA2-related neurodegenerative diseases. Subsequent experiments concluded that one of the inhibitors was found to be cPLA2-selective, non-cytotoxic, cell and brain penetrant and capable of reducing reactive oxygen species (ROS) and nitric oxide (NO) production in stimulated microglial cells. Computational studies were employed to understand how the compound interacts with cPLA2.

Molecular-interaction and signaling profiles of AM3677, a novel covalent agonist selective for the cannabinoid 1 receptor.

The cannabinoid 1 receptor (CB1R) is one of the most abundant G protein-coupled receptors (GPCRs) in the central nervous system. CB1R involvement in multiple physiological processes, especially neurotransmitter release and synaptic function, has made this GPCR a prime drug discovery target, and pharmacological CB1R activation has been demonstrated to be a tenable therapeutic modality. Accordingly, the design and profiling of novel, drug-like CB1R modulators to inform the receptor's ligand-interaction landscape and molecular pharmacology constitute a prime contemporary research focus. For this purpose, we report utilization of AM3677, a designer endocannabinoid (anandamide) analogue derivatized with a reactive electrophilic isothiocyanate functionality, as a covalent, CB1R-selective chemical probe. The data demonstrate that reaction of AM3677 with a cysteine residue in transmembrane helix 6 of human CB1R (hCB1R), C6.47(355), is a key feature of AM3677's ligand-binding motif. Pharmacologically, AM3677 acts as a high-affinity, low-efficacy CB1R agonist that inhibits forskolin-stimulated cellular cAMP formation and stimulates CB1R coupling to G protein. AM3677 also induces CB1R endocytosis and irreversible receptor internalization. Computational docking suggests the importance of discrete hydrogen bonding and aromatic interactions as determinants of AM3677's topology within the ligand-binding pocket of active-state hCB1R. These results constitute the initial identification and characterization of a potent, high-affinity, hCB1R-selective covalent agonist with utility as a pharmacologically active, orthosteric-site probe for providing insight into structure-function correlates of ligand-induced CB1R activation and the molecular features of that activation by the native ligand, anandamide.

Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons.

The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB1 receptor activity. Here we investigated the possibility of a negative regulation by CB1 receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2'-chloroethylamide (ACEA) in a CB1-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL(-/-) mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB1 activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.

Metabolic conversion of C20 polymethylene-interrupted polyunsaturated fatty acids to essential fatty acids.

Polymethylene-interrupted (PMI)-polyunsaturated fatty acids (PUFA) are fatty acids present largely in gymnosperm. Sciadonic acid (SciA, 20:3 Δ-5,11,14) and juniperonic acid (JA, 20:4 Δ-5,11,14,17) are typical C20 PMI-PUFA with an isolated double bond at Δ5. Previously, we found that SciA and JA are converted to linoleic acid (LNA) and α-linolenic acid (ΑLA), respectively. The conversion process includes chain-shortening step by peroxisomal ß-oxidation for elimination a double bond at Δ5, and subsequent chain-elongation step in microsomes. In this study, we examined the substrate specificity of this metabolism in rodent and human cells. Supplementation of SciA, eicosadienoic acid (EDA, 20:2 Δ-11,14) or JA to CHO-K1 cells (wild type) induced an accumulation of LNA, LNA or ALA, respectively, in cellular lipids. These changes were not observed in the peroxisomes-deficient CHO cells, indicating involvement of peroxisomes in the metabolism. Two types of human cells (MKN74 and HepG2) also converted the C20 PMI-PUFA and EDA to the respective essential fatty acids. In contrast, no chain-shortened metabolite of pinolenic acid (18:3 Δ-5,9,12) was detected in any cell lines tested. From these results, C20 PMI-PUFA and EDA, but not C18 PMI-PUFA, are suggested as being effectively converted to essential fatty acids by the fatty acid remodeling system in rodent and human cells.

Chemical approaches to therapeutically target the metabolism and signaling of the endocannabinoid 2-AG and eicosanoids.

The endocannabinoid system, most popularly known as the target of the psychoactive component of marijuana, Δ(9)-tetrahydrocannabinol (THC), is a signaling network that modulates a diverse range of physiological processes including nociception, behavior, cognitive function, appetite, metabolism, motor control, memory formation, and inflammation. While THC and its derivatives have garnered notoriety in the eyes of the public, the endocannabinoid system consists of two endogenous signaling lipids, 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (anandamide), which activate cannabinoid receptors CB1 and CB2 in the nervous system and peripheral tissues. This review will focus on the recent efforts to chemically manipulate 2-AG signaling through the development of inhibitors of the 2-AG-synthesizing enzyme diacylglycerol lipase (DAGL) or the 2-AG-degrading enzyme monoacylglycerol lipase (MAGL), and assessing the therapeutic potential of DAGL and MAGL inhibitors in pain, inflammation, degenerative diseases, tissue injury, and cancer.

Palmitoylethanolamide: from endogenous cannabimimetic substance to innovative medicine for the treatment of cannabis dependence.

Palmitoylethanolamide (PEA) is a fatty acid amide showing some pharmacodynamic similarities with Δ9-tetrahydrocannabinol, the principal psychoactive compound present in the cannabis plant. Like Δ9-tetrahydrocannabinol, PEA can produce a direct or indirect activation of cannabinoid receptors. Furthermore, it acts as an agonist at TRPV1 receptor. The hypothesis is that PEA has anti-craving effects in cannabis dependent patients, is efficacious in the treatment of withdrawal symptoms, produces a reduction of cannabis consumption and is effective in the prevention of cannabis induced neurotoxicity and neuro-psychiatric disorders.

Hypothalamic 2-arachidonoylglycerol regulates multistage process of high-fat diet preferences.

BACKGROUND: In this study, we examined alterations in the hypothalamic reward system related to high-fat diet (HFD) preferences. We previously reported that hypothalamic 2-arachidonoylglycerol (2-AG) and glial fibrillary acid protein (GFAP) were increased after conditioning to the rewarding properties of a HFD. Here, we hypothesized that increased 2-AG influences the hypothalamic reward system. METHODS: The conditioned place preference test (CPP test) was used to evaluate HFD preferences. Hypothalamic 2-AG was quantified by gas chromatography-mass spectrometry. The expression of GFAP was examined by immunostaining and western blotting. RESULTS: Consumption of a HFD over either 3 or 7 days increased HFD preferences and transiently increased hypothalamic 2-AG levels. HFD consumption over 14 days similarly increased HFD preferences but elicited a long-lasting increase in hypothalamic 2-AG and GFAP levels. The cannabinoid 1 receptor antagonist O-2050 reduced preferences for HFDs after 3, 7, or 14 days of HFD consumption and reduced expression of GFAP after 14 days of HFD consumption. The astrocyte metabolic inhibitor Fluorocitrate blocked HFD preferences after 14 days of HFD consumption. CONCLUSIONS: High levels of 2-AG appear to induce HFD preferences, and activate hypothalamic astrocytes via the cannabinoid system. We propose that there may be two distinct stages in the development of HFD preferences. The induction stage involves a transient increase in 2-AG, whereas the maintenance stage involves a long lasting increase in 2-AG levels and activation of astrocytes. Accordingly, hypothalamic 2-AG may influence the development of HFD preferences.

Glycerophospholipids and glycerophospholipid-derived lipid mediators: a complex meshwork in Alzheimer's disease pathology.

An increasing body of evidence suggested that intracellular lipid metabolism is dramatically perturbed in various cardiovascular and neurodegenerative diseases with genetic and lifestyle components (e.g., dietary factors). Therefore, a lipidomic approach was also developed to suggest possible mechanisms underlying Alzheimer's disease (AD). Neural membranes contain several classes of glycerophospholipids (GPs), that not only constitute their backbone but also provide the membrane with a suitable environment, fluidity, and ion permeability. In this review article, we focused our attention on GP and GP-derived lipid mediators suggested to be involved in AD pathology. Degradation of GPs by phospholipase A(2) can release two important brain polyunsaturated fatty acids (PUFAs), e.g., arachidonic acid and docosahexaenoic acid, linked together by a delicate equilibrium. Non-enzymatic and enzymatic oxidation of these PUFAs produces several lipid mediators, all closely associated with neuronal pathways involved in AD neurobiology, suggesting that an interplay among lipids occurs in brain tissue. In this complex GP meshwork, the search for a specific modulating enzyme able to shift the metabolic pathway towards a neuroprotective role as well as a better knowledge about how lipid dietary modulation may act to slow the neurodegenerative processes, represent an essential step to delay the onset of AD and its progression. Also, in this way it may be possible to suggest new preventive or therapeutic options that can beneficially modify the course of this devastating disease.

Dietary combination effects of conjugated linoleic acid and flaxseed or fish oil on the concentration of linoleic and arachidonic acid in poultry meat.

This study was conducted to determine the effects of the combination of dietary conjugated linoleic acid (CLA) and n-3 fatty acids on the linoleic acid (C18:2n-6) and arachidonic acid (C20:4n-6) concentrations of broiler chicken breast and thigh muscles. One hundred and twenty broilers were raised to 6 wk of age. All chicks were fed a basal corn-soybean meal diet containing 5 different fat sources at an inclusion level of 2% total fat: 1) CLA, 2) flaxseed oil, 3) menhaden fish oil, 4) CLA and flaxseed oil, and 5) CLA and menhaden fish oil. Eight broilers from each treatment were processed at 4 and 6 wk of age. Breast and thigh muscle samples were collected and analyzed for total fat content and fatty acid composition. The results showed that broilers from the CLA and fish oil treatment had lower arachidonic acid concentrations in both breast and thigh muscles than those fed the flaxseed oil diet or the CLA and flaxseed oil diet (P < 0.05). The arachidonic acid concentration and n-6:n-3 ratio of breast and thigh samples from the menhaden fish oil diet were similar to those of the CLA and fish oil diet (P > 0.05), but the inclusion of linoleic acid into chicken thigh muscles of broilers fed the CLA and menhaden fish oil diet improved significantly when compared with that of the diet containing menhaden fish oil only. Thus, the combination of CLA and menhaden fish oil is recommended to reduce the concentrations of linoleic and arachidonic acids in broiler chicken breast and thigh muscles.

Fast ultrasound-assisted extraction of polar (phenols) and nonpolar (lipids) fractions in Heterotheca inuloides Cass.

INTRODUCTION: Heterotheca inuloides Cass., also known as "arnica", is used in traditional medicine in Mexico. OBJECTIVE: Development of fast methods for the extraction of lipidic and phenolic fractions from arnica plants and their subsequent characterization. METHODOLOGY: Ultrasound was applied to accelerate extraction of the target compounds from this plant and reduce the use of organic solvents as compared with conventional methods. Gas chromatography-ion trap mass spectrometry and liquid chromatography with diode-array detection were used for the characterization of the lipidic and phenolic fractions, respectively. RESULTS: Under optimal extraction conditions, 9 and 55 min were necessary to complete extraction of the lipidic and phenolic fractions, respectively. The fatty acids present at the highest concentrations in H. inuloides were eicosatetraenoic n3 (24.6 µg/g), cis-9-hexadecenoic n7 (23.1 µg/g), exacosanoic (22.7 µg/g) and cis-9-octadecenoic acid (21.3 µg/g), while the rest were in the range 7.6-1.3 µg/g. The most concentrated phenols were guaiacol (41.5 µg/g), catechin (38.7 µg/g), ellagic acid (35.9 µg/g), carbolic acid (24.2 µg/g) and p-coumaric acid (19.5 µg/g), while the rest were in the range 5.1-0.4 µg/g. CONCLUSION: Ultrasound reduces the time necessary to complete the extraction 160 and 26 times, the extraction volume 2.5 and 4 times, and increases the extraction efficiency 5 and 3 times for lipidic and phenolic fractions, respectively, in comparison with conventional extraction methods. In addition, the characterization of the lipidic and phenolic fractions constitutes a first approach to the H. inuloides metabolome.

Cannabinoids and the gut: new developments and emerging concepts.

Cannabis has been used to treat gastrointestinal (GI) conditions that range from enteric infections and inflammatory conditions to disorders of motility, emesis and abdominal pain. The mechanistic basis of these treatments emerged after the discovery of Delta(9)-tetrahydrocannabinol as the major constituent of Cannabis. Further progress was made when the receptors for Delta(9)-tetrahydrocannabinol were identified as part of an endocannabinoid system, that consists of specific cannabinoid receptors, endogenous ligands and their biosynthetic and degradative enzymes. Anatomical, physiological and pharmacological studies have shown that the endocannabinoid system is widely distributed throughout the gut, with regional variation and organ-specific actions. It is involved in the regulation of food intake, nausea and emesis, gastric secretion and gastroprotection, GI motility, ion transport, visceral sensation, intestinal inflammation and cell proliferation in the gut. Cellular targets have been defined that include the enteric nervous system, epithelial and immune cells. Molecular targets of the endocannabinoid system include, in addition to the cannabinoid receptors, transient receptor potential vanilloid 1 receptors, peroxisome proliferator-activated receptor alpha receptors and the orphan G-protein coupled receptors, GPR55 and GPR119. Pharmacological agents that act on these targets have been shown in preclinical models to have therapeutic potential. Here, we discuss cannabinoid receptors and their localization in the gut, the proteins involved in endocannabinoid synthesis and degradation and the presence of endocannabinoids in the gut in health and disease. We focus on the pharmacological actions of cannabinoids in relation to GI disorders, highlighting recent data on genetic mutations in the endocannabinoid system in GI disease.

A complex between 6-iodolactone and the peroxisome proliferator-activated receptor type gamma may mediate the antineoplastic effect of iodine in mammary cancer.

Recently we and other groups have shown that molecular iodine (I(2)) exhibits potent antiproliferative and apoptotic effects in mammary cancer models. In the human breast cancer cell line MCF-7, I(2) treatment generates iodine-containing lipids similar to 6-iodo-5-hydroxy-eicosatrienoic acid and the 6-iodolactone (6-IL) derivative of arachidonic acid (AA), and it significantly decreases cellular proliferation and induces caspase-dependent apoptosis. Several studies have shown that AA is a natural ligand of the peroxisome proliferator-activated receptors (PPARs), which are nuclear transcription factors thought to participate in regulating cancer cell proliferation. Our results show that in MCF-7 cells: (1) 6-IL binds specifically and with high affinity to PPAR proteins (EMSA assays), (2) 6-IL activates both transfected (by transactivation assays) and endogenous (by lipid accumulation) peroxisome proliferator response elements, and (3) 6-IL supplementation increases PPAR gamma and decreases PPAR alpha expression. These results implicate PPARs in a molecular mechanism by which I(2), through formation of 6-IL, inhibits the growth of human breast cancer cells.

Effects of non-methylene-interrupted polyunsaturated fatty acid, sciadonic (all-cis-5,11,14-eicosatrienoic acid) on lipid metabolism in rats.

We investigated effects of the non-methylene-interrupted polyunsaturated fatty acid, sciadonic acid (all-cis-5,11,14-eicosatrienoic acid), on the lipid metabolism in rats, to identify the mechanism for the plasma and hepatic triacylglycerol-lowering effects of Japanese torreya (Torreya nucifera) seed oil. Sciadonic acid was isolated from torreya seed oil by the combination of urea-adduct with lipase-esterification. Sprague-Dawley (SD) male rats were fed with experimental diets containing 5% and 10% sciadonic acid based on corn oil for 2 weeks. The serum and liver triacylglycerol levels were lower in the rats fed with sciadonic acid. Considerable amounts of sciadonic acid were detected in the triacylglycerol and phospholipid in both the serum and liver of the rats fed with sciadonic acid. These observations demonstrate that sciadonic acid could modify the lipid metabolism in rats.

Novel, potent THC/anandamide (hybrid) analogs.

The structure-activity relationship (SAR) of the end pentyl chain in anandamide (AEA) has been established to be very similar to that of Delta(9)-tetrahydrocannabinol (Delta(9)-THC). In order to broaden our understanding of the structural similarities between AEA and THC, hybrid structures 1-3 were designed. In these hybrids the aromatic ring of THC-DMH was linked to the AEA moiety through an ether linkage with the oxygen of the phenol of THC. Hybrid 1 (O-2220) was found to have very high binding affinity to CB1 receptors (K(i)=8.5 nM), and it is interesting to note that the orientation of the side chain with respect to the oxygen in the phenol is the same as in THCs. To further explore the SAR in this series the terminal carbon of the side chain was modified by adding different substituents. Several such analogs were synthesized and tested for their CB1 and CB2 binding affinities and in vivo activity (tetrad tests). The details of the synthesis and the biological activity of these compounds are described.

Self-assembling cannabinomimetics: supramolecular structures of N-alkyl amides.

Certain fatty acid N-alkyl amides from the medicinal plant Echinacea activate cannabinoid type-2 (CB2) receptors. In this study we show that the CB2-binding Echinacea constituents dodeca-2E,4E-dienoic acid isobutylamide (1) and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (2) form micelles in aqueous medium. In contrast, micelle formation is not observed for undeca-2E-ene-8,10-diynoic acid isobutylamide (3), which does not bind to CB2, or structurally related endogenous cannabinoids, such as arachidonoyl ethanolamine (anandamide). The critical micelle concentration (CMC) range of 1 and 2 was determined by fluorescence spectroscopy as 200-300 and 7400-10000 nM, respectively. The size of premicelle aggregates, micelles, and supermicelles was studied by dynamic light scattering. Microscopy images show that compound 1, but not 2, forms globular and rod-like supermicelles with radii of approximately 75 nm. The self-assembling N-alkyl amides partition between themselves and the CB2 receptor, and aggregation of N-alkyl amides thus determines their in vitro pharmacological effects. Molecular mechanics by Monte Carlo simulations of the aggregation process support the experimental data, suggesting that both 1 and 2 can readily aggregate into premicelles, but only 1 spontaneously assembles into larger aggregates. These findings have important implications for biological studies with this class of compounds.

The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins.

Central nervous system responses to cannabis are primarily mediated by CB(1) receptors, which couple preferentially to G(i/o) G proteins. Here, we used calcium photometry to monitor the effect of CB(1) activation on intracellular calcium concentration. Perfusion with 5 microM CB(1) aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB(1) and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB(1) antagonist, SR141716A, and was absent in nontransfected human embryonic kidney 293 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including Delta(9)-tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G(i/o), because it was present in pertussis toxin-treated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by G(q) G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative G(q) or treated with the phospholipase C inhibitors U73122 and ET-18-OCH(3) and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1'-diheptyl-4,4'-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB(1) receptors in a conformation that enables G(q) signaling, thus shifting the G protein specificity of the receptor.

Crystal structure of arachidonic acid bound to a mutant of prostaglandin endoperoxide H synthase-1 that forms predominantly 11-hydroperoxyeicosatetraenoic acid.

Kinetic studies and analysis of the products formed by native and mutant forms of ovine prostaglandin endoperoxide H synthase-1 (oPGHS-1) have suggested that arachidonic acid (AA) can exist in the cyclooxygenase active site of the enzyme in three different, catalytically competent conformations that lead to prostaglandin G2 (PGG2), 11R-hydroperoxyeicosatetraenoic acid (HPETE), and 15R,S-HPETE, respectively. We have identified an oPGHS-1 mutant (V349A/W387F) that forms predominantly 11R-HPETE. Thus, the preferred catalytically competent arrangement of AA in the cyclooxygenase site of this double mutant must be one that leads to 11-HPETE. The crystal structure of Co3+-protoporphyrin IX V349A/W387F oPGHS-1 in a complex with AA was determined to 3.1 A. Significant differences are observed in the positions of atoms C-3, C-4, C-5, C-6, C-10, C-11, and C-12 of bound AA between native and V349A/W387F oPGHS-1; in comparison, the positions of the side chains of cyclooxygenase active site residues are unchanged. The structure of the double mutant presented here provides structural insight as to how Val349 and Trp387 help position C-9 and C-11 of AA so that the incipient 11-peroxyl radical intermediate is able to add to C-9 to form the 9,11 endoperoxide group of PGG2. In the V349A/W387F oPGHS-1.AA complex the locations of C-9 and C-11 of AA with respect to one another make it difficult to form the endoperoxide group from the 11-hydroperoxyl radical. Therefore, the reaction apparently aborts yielding 11R-HPETE instead of PGG2. In addition, the observed differences in the positions of carbon atoms of AA bound to this mutant provides indirect support for the concept that the conformer of AA shown previously to be bound within the cyclooxygenase active site of native oPGHS-1 is the one that leads to PGG2.