No abstract

Two cannabinoid receptors have been identified: CB1, present in the central nervous system (CNS) and to a lesser extent in other tissues, and CB2, present outside the CNS, in peripheral organs. There is evidence for the presence of CB2-like receptors in peripheral nerve terminals. We report now that we have synthesized a CB2-specific agonist, code-named HU-308. This cannabinoid does not bind to CB1 (Ki > 10 M), but does so efficiently to CB2 (Ki ‫؍‬ 22.7 ؎ 3.9 nM); it inhibits forskolin-stimulated cyclic AMP production in CB2-transfected cells, but does so much less in CB1-transfected cells. HU-308 shows no activity in mice in a tetrad of behavioral tests, which together have been shown to be specific for tetrahydrocannabinol (THC)-type activity in the CNS mediated by CB1. However, HU-308 reduces blood pressure, blocks defecation, and elicits anti-inflammatory and peripheral analgesic activity. The hypotension, the inhibition of defecation, the anti-inflammatory and peripheral analgesic effects produced by HU-308 are blocked (or partially blocked) by the CB2 antagonist SR-144528, but not by the CB1 antagonist SR-141716A. These results demonstrate the feasibility of discovering novel nonpsychotropic cannabinoids that may lead to new therapies for hypertension, inflammation, and pain.

The endocannabinoid N-arachidonoyl ethanolamine (anandamide), found both in the CNS and in the periphery, plays a role in numerous physiological systems. One might expect that the chemically related N-arachidonoyl-L-serine (ARA-S) could also be formed alongside anandamide. We have now isolated ARA-S from bovine brain and elucidated its structure by comparison with synthetic ARA-S. Contrary to anandamide, ARA-S binds very weakly to cannabinoid CB 1 and CB2 or vanilloid TRPV1 (transient receptor potential vanilloid 1) receptors. However, it produces endothelium-dependent vasodilation of rat isolated mesenteric arteries and abdominal aorta and stimulates phosphorylation of p44͞42 mitogen-activated protein (MAP) kinase and protein kinase B͞Akt in cultured endothelial cells. ARA-S also suppresses LPS-induced formation of TNF-␣ in a murine macrophage cell line and in wild-type mice, as well as in mice deficient in CB 1 or CB2 receptors. Many of these effects parallel those reported for abnormal cannabidiol (Abn-CBD), a synthetic agonist of a putative novel cannabinoidtype receptor. Hence, ARA-S may represent an endogenous agonist for this receptor.abnormal cannabidiol ͉ anandamide ͉ cannabinoids ͉ endothelium ͉ reactive oxygen intermediates T he identification, structural elucidation, and syntheses of the plant cannabinoids in the early 1960s led to thorough investigations of the chemistry, metabolism, and pharmacology of these compounds, in particular of the psychoactive constituent ⌬ 9 -tetrahydrocannabinol (1, 2). However, until the late 1980s and early 1990s, when specific receptors were identified and shortly thereafter cloned, the mechanism of the numerous cannabinoid actions remained an enigma (3-5). Two main receptors are now known: the CB 1 receptor, found in the CNS, as well as in some peripheral tissues, and the CB 2 receptor, found predominantly in the immune system (6-8). Additional, not yet fully identified receptors are present both in the CNS and in the periphery (6, 9, 10).Because receptors in mammals are not formed to encounter a plant constituent, research was initiated to discover endogenous ligands. In the 1990s two endogenous cannabinoids (endocannabinoids) were identified, N-arachidonoyl ethanolamine (anandamide) (11) and 2-arachidonoyl-glycerol (12, 13). Additional endocannabinoids have been reported, but their biological roles are yet obscure (6, 14). Anandamide and 2-arachidonoyl-glycerol have large spectrum of physiological actions, most of which are associated with the neural and immune systems. However, cardiovascular effects, which are in part CB 1 -mediated (15), are also well established (9,14,16).Anandamide is a product of phosphatidylethanolamine (17). Because phosphatidylserine is found alongside phosphatidylethanolamine in body tissues, one might expect that arachidonoyl-Lserine (ARA-S) is also an endogenous constituent (see Fig. 1A for the structures of anandamide and ARA-S). We report that we have isolated ARA-S from bovine brain and have evaluated some of its biological propertie...

It has been previously shown that heat acclimation leads to an elevated basal level of 72-kDa heat shock protein (HSP72). Augmented expression of HSP72 is considered as a cytoprotective response. This led us to hypothesize that alterations in the heat shock protein (HSP) defense pathway are an integral part of the heat acclimation repertoire. To investigate this, we studied the temporal profile of basal HSP expression upon acclimation and the dynamics of their accumulation subsequent to acute heat stress (HS). In parallel, HSP72 mRNA level before and after HS was measured. For comparison, HSC mRNA [the constitutive member of 70-kDa HSP (HSP70) family] was measured in similar conditions. Heat acclimation was attained by continuous exposure of rats to 34°C for 0, 1, 2, and 30 days. HS was attained by exposure to 41 or 43°C for 2 h. Thermoregulatory capacity of the rats was defined by rectal temperature, heating rate, and the cumulative heat strain invoked during HS. HSP72 and HSP70 gene transcripts were measured in the left ventricle of the heart by means of Western immunoblotting and semiquantitative RT-PCR, respectively. The resultant acclimatory change comprised a higher resting level of the encoded 72-kDa protein (Δ175%, P < 0.0001). After HS, peak HSP72 mRNA level was attained, 40 and 20 min post-HS at 41 and 43°C, respectively, vs. 60 and 40 min in the nonacclimated group. The subsequent HSP synthesis, however, was dependent on the severity of the cumulative heat strain. At the initial phase of heat acclimation, augmented HSP72 transcription unaccompanied by HSP synthesis was observed. It is concluded that upon heat acclimation, the HSP defense pathway is predisposed to a faster response. At the initial phases of heat acclimation, inability to elevate the HSP cytosolic level rules out their direct cytoprotective role.

It has been suggested that reactive oxygen species (ROS) play a role in the pathophysiology of brain damage. A number of therapeutic approaches, based on scavenging these radicals, have been attempted both in experimental models and in the clinical setting. In an experimental rat and mouse model of closed-head injury (CHI), we have studied the total tissue nonenzymatic antioxidant capacity to combat ROS. A major mechanism for neutralizing ROS uses endogenous low-molecular weight antioxidants (LMWA). This review deals with the source and nature of ROS in the brain, along with the endogenous defense mechanisms that fight ROS. Special emphasis is placed on LMWA such as ascorbate, urate, tocopherol, lipoic acid, and histidine-related compounds. A novel electrochemical method, using cyclic voltammetry for the determination of total tissue LMWA, is described. The temporal changes in brain LMWA after CHI, as part of the response of the tissue to high ROS levels, and the correlation between the ability of the brain to elevate LMWA and clinical outcome are addressed. We relate to the beneficial effects observed in heat-acclimated rats and the detrimental effects of injury found in apolipoprotein E-deficient mice. Finally, we summarize the effects of cerebroprotective pharmacological agents including the iron chelator desferal, superoxide dismutase, a stable radical from the nitroxide family, and HU-211, a nonpsychotoropic cannabinoid with antioxidant properties. We conclude that ROS play a key role in the pathophysiology of brain injury, and that their neutralization by endogenous or exogenous antioxidants has a protective effect. It is suggested, therefore, that the brain responds to ROS by increasing LMWA, and that the degree of this response is correlated with clinical recovery. The greater the response, the more favorable the outcome.