Stress stimulates several adaptive hormonal responses. Prominent among these responses are the secretion of catecholamines from the adrenal medulla, corticosteroids from the adrenal cortex, and adrenocorticotropin from the anterior pituitary. A number of complex interactions are involved in the regulation of these hormones. Glucocorticoids regulate catecholamine biosynthesis in the adrenal medulla and catecholamines stimulate adrenocorticotropin release from the anterior pituitary. In addition, other hormones, including corticotropin-releasing factor, vasoactive intestinal peptide, and arginine vasopressin stimulate while the corticosteroids and somatostatin inhibit adrenocorticotropin secretion. Together these agents appear to determine the complex physiologic responses to a variety of stressors.

The neuroprotective actions of cannabidiol and other cannabinoids were examined in rat cortical neuron cultures exposed to toxic levels of the excitatory neurotransmitter glutamate. Glutamate toxicity was reduced by both cannabidiol, a nonpsychoactive constituent of marijuana, and the psychotropic cannabinoid (؊)⌬ 9 -tetrahydrocannabinol (THC). Cannabinoids protected equally well against neurotoxicity mediated by N-methyl-D-aspartate receptors, 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid receptors, or kainate receptors. N-methyl-D-aspartate receptorinduced toxicity has been shown to be calcium dependent; this study demonstrates that 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid͞kainate receptor-type neurotoxicity is also calcium-dependent, partly mediated by voltage sensitive calcium channels. The neuroprotection observed with cannabidiol and THC was unaffected by cannabinoid receptor antagonist, indicating it to be cannabinoid receptor independent. Previous studies have shown that glutamate toxicity may be prevented by antioxidants. Cannabidiol, THC and several synthetic cannabinoids all were demonstrated to be antioxidants by cyclic voltametry. Cannabidiol and THC also were shown to prevent hydroperoxide-induced oxidative damage as well as or better than other antioxidants in a chemical (Fenton reaction) system and neuronal cultures. Cannabidiol was more protective against glutamate neurotoxicity than either ascorbate or ␣-tocopherol, indicating it to be a potent antioxidant. These data also suggest that the naturally occurring, nonpsychotropic cannabinoid, cannabidiol, may be a potentially useful therapeutic agent for the treatment of oxidative neurological disorders such as cerebral ischemia.Cannabinoid components of marijuana are known to exert behavioral and psychotropic effects but also to possess therapeutic properties including analgesia (1), ocular hypotension (2), and antiemesis (3). This report examines another potential therapeutic role for cannabinoids as neuroprotectants and describes their mechanism of action in rat cortical neuronal cultures.During an ischemic episode, large quantities of the excitatory neurotransmitter glutamate are released. This event causes neuronal death by over-stimulating N-methyl-Daspartate receptors (NMDAr) and 2-amino-3-(4-butyl-3-hydroxyisoxazol-5-yl)propionic acid (AMPA) and kainatetype receptors and results in metabolic stress and accumulation of toxic levels of intracellular calcium (4). In vitro and in vivo studies (4,5,6) have demonstrated that such neurotoxicity can be reduced by antioxidants or antagonists to NMDAr and AMPA͞kainate receptors. Antioxidants such as ␣-tocopherol (5, 6) are effective neuroprotectants because of their ability to reduce the toxic reactive oxygen species (ROS) formed during ischemic metabolism. Cannabinoids like (Ϫ)⌬ 9

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Arachidonylethanolamide (anandamide), a candidate endogenous cannabinoid ligand, has recently been isolated from porcine brain and displayed cannabinoid-like binding activity to synaptosomal membrane preparations and mimicked cannabinoid-induced inhibition of the twitch response in isolated murine vas deferens. In this study, anandamide and several congeners were evaluated as cannabinoid agonists by examining their ability to bind to the cloned cannabinoid receptor, inhibit forskolin-stimulated cAMP accumulation, inhibit N-type calcium channels, and stimulate one or more functional second messenger responses. Synthetic anandamide, and all but one congener, competed for [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the rat cannabinoid receptor. The ability of anandamide to activate receptor-mediated signal transduction was evaluated in Chinese hamster ovary (CHO) cells expressing the human cannabinoid receptor (HCR, termed CHO-HCR cells) and compared to control CHO cells expressing the muscarinic m5 receptor (CHOm5 cells). Anandamide inhibited forskolin-stimulated cAMP accumulation in CHO-HCR cells, but not in CHOm5 cells, and this response was blocked with pertussis toxin. N-type calcium channels were inhibited by anandamide and several active congeners in N18 neuroblastoma cells. Anandamide stimulated arachidonic acid and intracellular calcium release in both CHOm5 and CHO-HCR cells and had no effect on the release of inositol phosphates or phosphatidylethanol, generated after activation of phospholipase C and D, respectively. Anandamide appears to exhibit the essential criteria required to be classified as a cannabinoid/anandamide receptor agonist and shares similar nonreceptor effects on arachidonic acid and intracellular calcium release as other cannabinoid agonists.Both the psychoactive and medicinal properties of marijuana have been known for centuries, but not until the last decade has a clear mechanism of action been ascribed to A9-tetrahydrocannabinol (THC), the active principle of marijuana. It is now known that THC and other more potent synthetic cannabinoid agonists bind to specific cannabinoid receptors and couple functionally to inhibit adenylate cyclase (1, 2) and inhibit N-type calcium channels via a pertussis toxin-sensitive guanine nucleotide binding protein (G protein) (3,4). The existence of the cannabinoid receptor was corroborated with the cloning of a cannabinoid receptor gene from both rat and human (5, 6). To date only a single cannabinoid receptor gene has been identified and its nucleotide sequence indicates that it belongs to the superfamily of G-protein-coupled receptors. Expression studies indicateThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.that the cloned receptor and the native receptor display similar binding and functional coupling to the inhibition of adenylate cyclase (5-7). The ...

When rabbit peritoneal neutrophils were treated with glucocorticoids, their chemotactic response to stimulation by the chemoattractant fMet-Leu-Phe was markedly reduced. Preincubation of cells with glucocorticoids also decreased phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) activity in situ as measured by the release of [l-14C- Glucocorticoids exhibit a variety of biological effects, such as enzyme induction and anti-inflammatory actions (1, 2). The steroids act as a consequence of their binding to cytoplasmic receptors, followed by the translocation of the ligand-receptor complex into the nucleus, which, in turn, affects the transcription of various RNA species (3). The anti-inflammatory action of glucocorticoids is expressed in their ability to inhibit both chemotaxis and release of lysosomal enzymes in phagocytic cells (4-6). Recently, it has been demonstrated that chemotactic peptides enhance the release of arachidonic acid, a product of phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4), in rabbit neutrophils (7). Steroids possibly exert their anti-inflammatory action by preventing the release of arachidonic acid from phospholipids and its conversion to prostaglandins (8). These observations prompted a search for a steroid-induced inhibitor of phospholipase A2 in neutrophils. Here, we report the existence of a protein in rabbit neutrophils that inhibits phospholipase A2 and whose synthesis is induced by glucocorticoids.METHODS AND MATERIALS Assay of Phospholipase A2 Activities in Situ and in Vitro. Rabbit peritoneal neutrophils were obtained as described (7) and treated with various glucocorticoids in RPMI 1640 medium (GIBCO) for 16 hr at 37°C under a humid atmosphere of 95% 02/5% CO2. Phospholipase A2 activity was measured by the release of ["4C]arachidonic acid from the cellular lipids, mainly phospholipids, with a slight modification of the method described (7). Bovine serum albumin (1%) was included in Gey's balanced salt solution buffered with 10 mM Hepes, pH 7.4 (modified Gey's buffer). The cells (8-11 X 106 cells per ml) were preincubated in a total volume of 5 ml with 1.25 ,uCi of [1-'4C]arachidonic acid (55.5 mCi/mmol) at 370C for 1 hr (1 Ci = 3.7 X 1010 becquerels). The cells were washed twice with 5 ml of modified Gey's buffer and resuspended in 5 ml of the same buffer. For arachidonic acid release, the cells were stimulated with 10 nM fMet-Leu-Phe for 10 min at 370C (7). 16 hr. After three washings with 0.84% NaCl solution buffered with 10 mM sodium phosphate buffer, pH 7.4, the cells (8 X 106 cells) were lysed in 3 ml of distilled water. After centrifugation, at 27,000 X g for 50 min, the precipitates were solubilized with 0.5 ml of 2% Nonidet P40. Samples (0.5 ml) of 2533The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Many types of cells methylate phospholipids using two methyltransferase enzymes that are asymmetrically distributed in membranes. As the phospholipids are successively methylated, they are translocated from the inside to the outside of the membrane. When catecholamine neurotransmitters, lectins, immunoglobulins or chemotaxic peptides bind to the cell surface, they stimulate the methyltransferase enzymes and reduce membrane viscosity. The methylation of phospholipids is coupled to Ca2+ influx and the release of arachidonic acid, lysophosphatidylcholine, and prostaglandins. These closely associated biochemical changes facilitate the transmission of many signals through membranes, resulting in the generation of adenosine 3',5'-monophophate in many cell types, release of histamine in mast cells and basophils, mitogenesis in lymphocytes, and chemotaxis in neutrophils.

Anandamide (arachidonylethanolamide) is a novel lipid neurotransmitter first isolated from porcine brain which has been shown to be a functional agonist for the cannabinoid CB1 and CB2 receptors. Anandamide has never been isolated from human brain or peripheral tissues and its role in human physiology has not been examined. Anandamide was measured by LC/MS/MS and was found in human and rat hippocampus (and human parahippocampal cortex), striatum, and cerebellum, brain areas known to express high levels of CB1 cannabinoid receptors. Significant levels of anandamide were also found in the thalamus which expresses low levels of CB1 receptors. Anandamide was also found in human and rat spleen which expresses high levels of the CB2 cannabinoid receptor. Small amounts of anandamide were also detected in human heart and rat skin. Only trace quantities were detected in pooled human serum, plasma, and CSF. The distribution of anandamide in human brain and spleen supports its potential role as an endogenous agonist in central and peripheral tissues. The low levels found in serum, plasma, and CSF suggest that it is metabolized in tissues where it is synthesized, and that its action is probably not hormonal in nature.Key words: Anandamide; Cannabis; Cannabinoid receptor; Marijuana porcine brain and found to be a lipid of novel structure [7]. Anandamide displayed specific binding to the CBI receptor and inhibited a prototypical twitch response in mouse vas deferens. Anandamide has also been shown to induce similar behavioral [8,9], pharmacological [10,11], and signal transduction effects [12] as classical cannabinoid agonists, but high concentrations were required to induce these effects. Levels of anandamide were first estimated to occur at 0.4 pmol/g (133 pg/g) in whole porcine brain [7], and recently quantitated in porcine and bovine brain at 173 pmol/g (60 ng/g) and 101 pmol/g (35 ng/g) respectively [13]. A recent study reports levels of anandamide in rat testis to be considerably lower (6 pmol/ g, 2.1 ng/g) [14]. However, anandamide has never been isolated from human tissue or fluids. Furthermore, levels of anandamide have not been measured in regions of rat brain or in tissues such as spleen where CB2 receptors have been shown to be expressed at high levels. Studies of anandamide distribution should help elucidate the physiologic role of anandamide as a cannabimimetic eicosanoid and possibly broader functions. In this study we report the isolation and quantitation of anandamide by liquid chromatography/mass spectrometry in various tissues and fluids from postmortem human and rat.