First, motivated by the potential therapeutic applications of cannabis-like molecules, laboratories in academia and the pharmaceutical industry began to develop families of synthetic analogs of delta-9-THC. These agents exerted pharmacological effects that were qualitatively similar to those of delta-9- THC but displayed both greater potency and stereoselectivity. The latter feature cannot be reconciled with nonspecific membrane interactions, providing the first evidence that delta-9-THC exerts its effects by combining with a selective receptor. Second, as a result of these synthetic efforts, it became possible to explore directly the existence of cannabinoid receptors by using standard radioligand binding techniques. In 1988, Howlett and her co-workers described the presence of high-affinity binding sites for cannabinoid agents in brain membranes and showed that these sites are coupled to inhibition of adenylyl cyclase activity. Conclusively supporting these findings, in 1990 Matsuda et al. serendipitously came across a complementary DNA encoding for the first G protein-coupled cannabinoid receptor, now known as CB1. In heterologous expression systems, CB1 receptors were found to be functionally coupled to multiple intracellular signaling pathways, including inhibition of adenylyl cyclase activity,cannabis growing inhibition of voltage-activated calcium channels, and activation of potassium channels . In situ hybridization and immunohistochemical studies have demonstrated that CB1 receptors are abundantly expressed in discrete regions and cell types of the central nervous system but are also present at significant densities in a variety of peripheral organs and tissues .
The selective distribution of CB1 receptors in the CNS provides a clear anatomical correlate for the cognitive, affective, and motor effects of cannabimimetic drugs. The cloning and characterization of CB1 receptors left several important problems unsolved. Since antiquity, it has been known that the actions of Cannabis and delta-9-THC are not restricted to the CNS, but include effects on nonneural tissues such as reduction of inflammation, lowering of intraocular pressure associated with glaucoma, and relief of muscle spasms. Are these peripheral effects all produced by activation of CB1 receptors? An initial answer to this question was provided by the discovery of a second cannabinoid receptor exquisitely expressed in cells of immune origin . This receptor, called CB2, only shares 44% sequence identity with its brain counterpart, implying that the two sub-types diverged long ago in evolution. The intracellular coupling of the CB2 receptor resembles, however, that of the CB1 receptor; for example, in transfected cells, CB2 receptor activation is linked to the inhibition of adenylyl cyclase activity . The experience with opioid receptors and the enkephalins has accustomed scientists to the idea that whenever a receptor is present in the body, endogenous factor that activate this receptor also exist. Not surprisingly, therefore, as soon as cannabinoid receptors were described, a search began to identify their naturally occurring ligand. One way to tackle this problem was based on the premise that, like other neurotransmitters and neuromodulators, an endogenous cannabinoid substance should be released from brain tissue in a calcium dependent manner. Taking this route, Howlett and coworkers incubated rat brain slices in the presence of a calcium ionophore and determined whether the media from these incubations contained a factor that displaced the binding of labeled CP-55940, a cannabinoid agonist, to brain membranes. These studies demonstrated that a cannabinoid-like activity was indeed released from stimulated slices, but the minute amounts of this factor did not allow the elucidation of its chemical structure .
Devane, Mechoulam, and co-workers , at the Hebrew University in Jerusalem, adopted a different strategy. Reasoning that endogenous cannabinoids may be as hydrophobic as delta-9-THC, they subjected porcine brains to organic solvent extraction and fractionated the lipid extract by chromatographic techniques while measuring cannabinoid binding activity. This approach turned out to be highly successful, and the researchers were able to isolate a lipid cannabinoid-like component, which they characterized by mass spectrometry and nuclear magnetic resonance spectroscopy as the ethanolamide of arachidonic acid. They named this novel compound “anandamide” after the sanskrit “ananda,” inner bliss. The chemical synthesis of anandamide confirmed this structural identification and allowed the characterization of its pharmacological properties . In vitro and in vivo tests showed a great similarity of actions between anandamide and cannabinoid drugs. Anandamide reduced the electrogenic contraction of mouse vas deferens and closely mimicked the behavioral responses produced by delta-9-THC in vivo; in the rat, the compound was found to produce analgesia, hypothermia, and hypomotility. However, these effects may not be exclusively due to cannabinoid receptor activation, as anandamide is readily metabolized to arachidonic acid, which can be converted in turn to a variety of biologically active eicosanoid compounds. Subsequent studies demonstrated that anandamide is released from brain neurons in an activity-dependent manner and elucidated the unique biochemical routes of anandamide formation and inactivation in the CNS . Thus anandamide fulfills all key criteria that define an endogenous cannabinoid substance. In their 1992 study, Devane, Mechoulam, and coworkers reported that several lipid fractions from the rat brain contained cannabinoid-binding activity, in addition to anandamide’s. In characterizing these fractions, they discovered that some of them were composed of polyunsaturated fatty acid ethanolamides similar to anandamide , but others were instead constituted of a distinct lipid component, sn-2-arachidonoyl-glycerol . Sugiura et al. arrived independently to the same conclusion.
That polyunsaturated fatty acid ethanolamides should mimic anandamide, to which they are structurally very similar, does not come as a great surprise. Moreover, the pharmacological properties of these fatty acid ethanolamides, essentially indistinguishable from those of anandamide, and their scarcity in brain relegate them, at least for the moment, to a position secondary to anandamide’s. We cannot say the same for 2-AG. This lipid, considered until now a mere intermediate in glycerophospholipid turnover , is present in the brain at concentrations that are 170-fold greater than those of anandamide and possesses two pharmacological properties that make it crucially different from the latter: it binds to both CB1 and CB2 cannabinoid receptors with similar affinities, and it activates CB1 receptors as a full agonist, whereas anandamide acts as a partial agonist.We have learned much over the past 10 years on the behavioral effects of these molecules, on how these lipid mediators are produced physiologically, and on the functional roles that they may serve. A major step was the discovery that depolarization-induced suppression of inhibition , a type of short-term synaptic plasticity originally discovered in the cerebellum and the hippocampus ,cannabis grow equipment is mediated by endocannabinoids . This discovery allowed the results of over a decade of research on retrograde synaptic signaling in these networks to be considered as functional characteristics of endocannabinoid signaling. The substrate of retrograde signaling and DSI is the predominantly presynaptic distribution of CB1 receptors on axon terminals in the hippocampus , as well as throughout the brain, where activation of CB1 by endocannabinoids can efficiently veto neurotransmitter release in many distinct types of synapses . The conditions of synthesis, release, distance of diffusion, duration of effect, and site of action were all extensively characterized for the mediator of DSI that turned out to be an endocannabinoid . The fact that neurons are able to control the efficacy of their own synaptic input in an activity-dependent manner is functionally very important, since this mechanism may sub-serve several functions in information processing by neuronal networks from temporal coding and oscillations to group selection and the fine tuning of signal-to-noise ratio.
The crucial involvement of endocannabinoids in these functions just began to emerge from recent studies, which are reviewed in section V. Due to the exceptionally rapid expansion of this field in recent years , we decided to focus the present review on questions related to the composition of the endocannabinoid system and its physiological roles in controlling brain activity at the regional and cellular levels as synaptic signal molecules. We did not aim to provide detailed accounts of studies dealing with other, similarly important, aspects of cannabinoid research, which have been dealt with in excellent recent reviews, e.g., about the relation of the endocannabinoid system to pain modulation , the immune system , neuroprotection , and addiction . The final message of the present review is that to understand the possible physiological functions of the endogenous cannabinoids, their roles in normal and pathological brain activity, pharmacological agents targeting the cascade of anandamide and 2-AG formation, release, uptake, and degradation will have to be developed. Such drugs, which undoubtedly will become invaluable research tools to study the potential functions listed above, may also provide novel therapeutic approaches to diseases whose clinical, biochemical, and pharmacological features suggest a link with the endogenous cannabinoid system.A basic principle that has emerged from the last two decades of research on cellular signaling is that simple phospholipids such as phosphatidylcholine or phosphatidylinositol should be regarded not only as structural components of the cell membrane, but also as precursors for transmembrane signaling molecules. Intracellular second messengers like 1,2-diacylglycerol and ceramide are familiar examples of this concept. Along with their intracellular roles, however, lipid compounds may also serve important functions in the exchange of information between cells. Indeed, biochemical mechanisms analogous to those involved in the generation of DAG or ceramide give rise to biologically active lipids that leave their cell of origin to activate G protein-coupled receptors located on the surface of neighboring cells. Traditionally overshadowed by amino acid, amine, and peptide transmitters, biologically active lipids are now emerging as essential mediators of cell-to-cell communication within the CNS, where G protein-coupled receptors for multiple families of such compounds, including lysophosphatidic acid and eicosanoids, have been identified . In this section, we discuss the biochemical properties of endogenous lipids that activate brain cannabinoid receptors. These compounds share two common structural motifs: a polyunsaturated fatty acid moiety and a polar head group consisting of ethanolamine or glycerol . Because of these features, endocannabinoid substances seemingly resemble the eicosanoids, ubiquitous bio-active lipids generated through the enzymatic oxygenation of arachidonic acid. However, the endocannabinoids are clearly distinguished from the eicosanoids by their different bio-synthetic routes, which do not involve oxidative metabolism. The two best characterized endocannabinoids, anandamide and 2-AG , may be produced instead through cleavage of phospholipid precursors present in the membranes of neurons, glia, and other cells. In the following sections, we will first focus on the biochemical pathways that lead to the formation of endocannabinoids in neurons and then turn to the mechanism by which these compounds are deactivated.Anandamide formation via energy-independent condensation of arachidonic acid and ethanolamine was described in brain tissue homogenates soon after the discovery of anandamide and was attributed to an enzymatic activity that was termed “anandamide synthase” . Subsequent work has demonstrated, however, that this reaction is in fact catalyzed by fatty acid amide hydrolase , the primary enzyme of anandamide hydrolysis, acting in reverse . Since FAAH requires high concentrations of arachidonate and ethanolamine to synthesize anandamide, higher than those normally found in cells, this enzyme is unlikely to play a role in the physiological formation of anandamide . Another model for anandamide biosynthesis is illustrated schematically in Figure 2. According to this model, anandamide may be produced via hydrolysis of the phospholipid precursor N-arachidonoyl phosphatidylethanolamine , catalyzed by a phospholipase D -type activity . The precursor consumed in this reaction may be resynthesized by a separate enzyme activity, N-acyltransferase , which may transfer an arachidonate group from the sn-1 glycerol ester position of phospholipids to the primary amino group of PE . The validity of this model was initially questioned, because previous studies had failed to detect N-arachidonoyl PE in mammalian tissues . More recent chromatographic and mass spectrometric analyses have unambiguously shown, however, that N-arachidonyl PE is present in brain and other tissues, where it may serve as a physiological precursor for anandamide . Although biochemically distinct, anandamide formation and N-arachidonoyl PE synthesis are thought to proceed in parallel. Both reactions may be initiated by intracellular Ca2 rises and/or by activation of neurotransmitter receptors . For example, administration of dopamine D2-receptor agonists to rats in vivo causes a profound stimulation of anandamide release in the striatum , which is likely mediated by de novo anandamide synthesis . Unfortunately, the two key enzyme activities responsible for these reactions, PLD and NAT, have only been partially characterized, and their molecular properties are still unknown .