Two types of endogenous cannabinoid-receptor agonists have been identified thus far. They are the ethanolamides of polyunsaturated fatty acids-arachidonoyl ethanolamide (anandamide) is the best known compound in the amide series-and 2-arachidonoyl glycerol, the only known endocannabinoid in the ester series. We report now an example of a third, ether-type endocannabinoid, 2-arachidonyl glyceryl ether (noladin ether), isolated from porcine brain. The structure of noladin ether was determined by mass spectrometry and nuclear magnetic resonance spectroscopy and was confirmed by comparison with a synthetic sample. It binds to the CB1 cannabinoid receptor (Ki ؍ 21.2 ؎ 0.5 nM) and causes sedation, hypothermia, intestinal immobility, and mild antinociception in mice. It binds weakly to the CB2 receptor (Ki > 3 M).W e have reported the isolation and identification of two types of endogenous cannabinoids that bind and activate the known cannabinoid receptors CB 1 and CB 2 . Arachidonoyl ethanolamide (anandamide; ref. 1) and later two more polyunsaturated fatty acid ethanol amides (2) were found in porcine brain. An ester, 2-arachidonoyl glycerol (2-AG) was isolated by us from canine gut (3) and by Sugiura et al. from brain (4). Anandamide and 2-AG have been the objects of numerous investigations in various areas of biology and have been found to affect processes in the nervous, cardiovascular, immune, and reproductive systems (5-8). They interact with many neurotransmitters and affect hormone levels (9-10). This ubiquity of effects led us to look for additional endocannabinoids.We report now that we have isolated from porcine brain a third endocannabinoid, 2-arachidonyl glyceryl ether, which we have named noladin ether ( Fig. 1). Materials and MethodsIsolation of Noladin Ether. Porcine brain (100 g, approximately a single brain) was added to a mixture of chloroform (200 ml) and methanol (200 ml) and mixed in a blender for 2 min. Water (100 ml) was added, and the mixing process was continued for another minute. The mixture was filtered. Two layers were formed. The water-methanol layer was separated and evaporated under reduced pressure. The residue obtained was extracted with methanol. The above isolation was repeated numerous times (from a total of 2.4 kg porcine brain). The extract obtained was chromatographed on a gravity column (i.d. 2.5 cm, height 28 cm, 82 g ICN Silica TSC, 60 A) with hexane͞acetone initially in a ratio of 10:1 (vol͞vol) (400 ml), then 9:1 (100 ml), and finally 4:1 (100 ml). The fractions eluted were monitored for binding to the CB 1 receptor from rat brain synaptosomes (prepared as described below; ref. 11) on the basis of displacement of the potent labeled agonist [ 3 H]HU-243 purchased from Tocris (Bristol, U.K.). The material eluted in fractions 45-54 (10-ml each) was found to bind to the receptor. These fractions were combined and purified further by HPLC (see below). A polar-active compound developed on a TLC plate (silica gel 60 F 254 , Merck) in a hexane͞ acetone (4:1) solvent system gave ...
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 background knowledge leading to the isolation and identification of anandamide and 2-arachidonoyl glycerol, the principal endocannabinoids is described. The structure-activity relationships of these lipid derivatives are summarized. Selected biochemical and pharmacological topics in this field are discussed, the main ones being levels of endocannabinoids in unstimulated tissue and cells, biosynthesis, release and inactivation of endocannabinoids, the effects of 'entourage' compounds on the activities of anandamide and 2-arachidonoyl glycerol, their signaling mechanisms and effects in animals.
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