These indications are relevant, but they are open nevertheless to several possible objections. For example, although endogenous cannabimimetic compounds exist, it is conceivable that they may never engage cannabinoid receptors under physiological conditions. Indeed, if these receptors were constitutively active in the absence of agonist, as suggested by studies with heterologous expression models , there may be no need for an intrinsic cannabinoid ‘‘system,’’ intended as a group of interconnected cells that produce and respond to endogenous cannabinoids. These and similar concerns highlight the many gaps left unfilled in our understanding of intrinsic cannabinoid modulation. It is now generally agreed that substances with cannabimimetic properties are released during neuronal activity, and that these substances are inactivated by a set of mechanisms parallel to, but distinct from, those utilized for the elimination of established neurotransmitters. But where in brain and peripheral tissues do these reactions take place? And under what circumstances? Are there discrete cannabinergic pathways comparable to, say, dopaminergic ones? Or do endogenous cannabinoids act as local mediators? What behavioral needs regulate endogenous cannabinoid release?And what physiological adaptations are served by such release? To address these questions critically,microgreen rack for sale we need first to recognize certain biochemical and physiological peculiarities of the cannabinoid signaling system which distinguish it from classical neurotransmitters. The nature of these peculiarities is the key theme that will be developed in the present review.
Early reports of anandamide biosynthesis through energy-independent condensation of nonesterified arachidonate with ethanolamine have been subsequently attributed to a reversal of the anandamide amidohydrolase reaction, which participates in anandamide degradation , or to the artifactual formation of compounds with some chromatographic properties of anandamide . Since anandamide amidohydrolase requires high concentrations of arachidonate and ethanolamine when acting in reverse, much higher than those normally found in cells , this enzyme is unlikely to play a physiological role in anandamide formation . Another model of anandamide is illustrated schematically in Fig. 2. According to this model, anandamide formation proceeds from the cleavage of the phospholipid precursor, N-arachidonyl phosphatidylethanolamine , catalyzed by a phosphodiesterase activity such as phospholipase D. The precursor consumed in this reaction may be rapidly resynthesized by a second enzyme activity, N-acyltransferase, which cleaves arachidonate from the sn-1 glycerol ester position of phospholipids and transfers it to the primary amino group of PE.Under physiological conditions, formation of anandamide and resynthesis of its precursor may be initiated at the same time, when neurons are depolarized and intracellular Ca21 levels are elevated . The anandamide precursor, N-arachidonyl PE, belongs to a family of N-acylated PEs, the ethanolamine moiety of which is linked to different saturated or unsaturated fatty acids. Like N-arachidonyl PE, other N-acyl PEs are also synthesized by N-acyltransferase and, when cleaved by phosphodiesterase, give rise to acylethanolamides. In addition to their lack of activity on CB1 receptors , we know very little about the pharmacological effects of these acylethanolamides and even less about their possible biological functions .
One notable exception may be palmitylethanolamide, which is produced by cleavage of N-palmityl PE. This compound exerts significant analgesic and anti-inflammatory effects in vivo which have been attributed to its ability to interact with a CB2-like receptor sensitive to the compound SR144528 . The molecular identity of this putative receptor sub-type is unknown, although it is likely to be distinct from the cloned CB2 receptor for which palmitylethanolamide shows little or no binding affinity . The fact that both N-acyl PEs and acylethanolamides are also present in plants, where their synthesis is regulated by extracellular stimuli , suggests that this signaling mechanism has been established early in the evolution of multicellular organisms.Textbook lipid biochemistry predicts two most plausible pathways of 2-AG biosynthesis, which are depicted in Fig. 3. Phospholipase C hydrolysis of membrane phospholipids produces 1,2-diacylglycerol, which is converted to 2-AG by diacylglycerol lipase activity. Alternatively, phospholipase A1 generates a lysophospholipid, which is hydrolyzed to 2-AG by lyso-PLC activity . In addition to these phospholipase-mediated pathways, 2-AG accumulation may result from the breakdown of triacylglycerols, catalyzed by neutral lipase activity , or from the dephosphorylation of lysophosphatidic acid . The fact that 2-AG formation in cortical neurons in culture is prevented by various PLC and diacylglycerol lipase inhibitors suggests a predominant involvement of the PLC pathway . However, the phospholipid substrate and PLC isoform implicated in this reaction remain to be discovered. Also, it cannot be excluded that multiple enzyme pathways may participate in generating 2-AG, an event that is not uncommon in lipid metabolism . Regardless of its precise mechanism, 2-AG biosynthesis appears to be triggered by rises in intracellular Ca21 elicited during neuronal activity.
This was shown in hippocampal slices by applying electrical stimulations to the Schaffer collaterals, a glutamatergic fiber tract in the Ammon’s horn region that projects from CA3 to CA1 neurons. High-frequency stimulation of these fibers produced a fourfold increase in 2-AG accumulation compared to controls, which was prevented by the Na1 channel blocker tetrodotoxin or by removing Ca21 from the superfusing solution. Noteworthy, the local concentrations reached by 2-AG after stimulation were calculated to be approximately 1–2 µM . Since 2-AG binds to CB1 receptors with a Kd of 0.7–2 µM , the levels of 2-AG found after stimulation are expected to cause a substantial activation of the dense CB1 receptor population expressed in hippocampus .An essential feature of these models is that both anandamide and 2-AG may be produced and released upon demand, through a mechanism that may not require vesicle neurosecretion. In the case of anandamide, this hypothesis is supported by a variety of experimental evidence. First, the concentration of anandamide in neurons is exceedingly low under basal conditions , about 100 to 10,000 times lower than those of amino acid and amine neurotransmitters which are stored in synaptic vesicles . Second, stimulus-dependent release of anandamide from neurons is associated with anandamide formation and with de novo N-arachidonyl PE biosyn-thesis . Third, the release of anandamide may be dissociated experimentally from that of classical neurotransmitters; for example, although striatal neurons in culture rapidly take up radioactively labeled anandamide, they do not release it in a Ca21 -dependent manner, as it can be readily demonstrated with labeled amino acids or bio-genic amines . A parsimonious interpretation of these findings is that anandamide may be produced when need arises and immediately dispatched outside cells, without an intermediate step of vesicle storage.How is newly formed anandamide released, and how does it reach its cellular targets? Water-soluble neurotransmitters that are released by secretion can diffuse unhindered through the aqueous compartment of the synaptic cleft to their postsynaptic receptors. But this is probably not the case with anandamide, the hydrophobic nature of which may favor its association with lipid membranes and introduce considerable constraints to its extracellular movements. Nevertheless,cannabis grow facility layout we know that anandamide does exit neurons because it can be found in incubation media of brain slices or perfusates of brain microdialysis experiments . We also know that certain cells, such as striatal astrocytes, respond to anandamide but do not have the enzymatic machinery to produce it . This implies that anandamide may travel from one cell to another, overcoming its tendency to partition in the lipid bilayer. We do not know yet how this may occur, but enough clues are available to offer the working hypothesis illustrated in Fig. 4.This indicates that anandamide may be produced within the cell membrane and may be able to move into the extracellular space immediately after cleavage of Narachidonyl PE has taken place. As with other lipid compounds, the actual release step may be mediated either by membrane transporters or by lipid-binding proteins . The latter may also facilitate the movement of anandamide in the aqueous environment surrounding cells and help it attain its cellular targets.A close link between activity-dependent formation and extracellular release is a feature that distinguishes anandamide from classical neurotransmitters and underscores certain functional properties that may be characteristic of this endogenous cannabinoid. From a kinetic standpoint, anandamide release is unlikely to be as rapid and discrete as that of established neurotransmitters, implying that anandamide may act as a slow messenger molecule. Moreover, since anandamide release does not appear to be mediated by vesicle secretion, it is unlikely to be exclusively localized to synaptic nerve endings. As a result, anandamide may serve both synaptic and extrasynaptic signaling functions, a possibility that finds support in the presence of CB1 cannabinoid receptors in neuronal cell bodies and dendrites .
Slow and diffuse local effects are not unique to anandamide. To a certain extent they are also seen with neuroactive peptides, but they are especially characteristic of the growing family of phospholipid-derived bioactive mediators. It is perhaps not surprising that compounds like the eicosanoids and LPA would share with anandamide a number of functional properties such as release upon demand and spatially circumscribed actions. More intriguing is the fact that LPA and CB1 receptors exhibit a significant degree of sequence homology , which suggests the existence of a connection between these two apparently unrelated messenger systems. The functional significance of this link, if any, remains at present unknown. The dissimilarities with classical neurotransmitters on the one hand, and the analogies with lipid mediators on the other, emphasize the idea that anandamide may act primarily as a local modulatory substance. In agreement with this, as we have seen, may be the time course of anandamide release and the extrasynaptic range of its biological effects. But also the possibility that many cell types that express cannabinoid receptors may also be capable to synthesize anandamide , which is consistent with a localized feedback action of this lipid messenger. A critical test of this hypothesis is still lacking, however. This will have to come from the anatomical localization of the enzymes involved in anandamide formation, as well as from detailed studies on the kinetics and distribution of anandamide release and inactivation in vivo.Neurons and astrocytes in primary culture avidly internalize exogenous anandamide through a process that meets all key criteria of a carrier-mediated transport. These criteria have been defined in detail by experiments with other membrane transporters , and include time and temperature dependence as well as high substrate affinity and selectivity . Strong support to the existence of an anandamide transporter has also come from the development of a compound, N–arachidonylethanolamide , which blocks anandamide transport competitively . Using this inhibitor, it has been possible to demonstrate that high-affinity transport participates in the inactivation of exogenously administered anandamide both in vitro and in vivo . It is unclear whether AM404 also blocks the biological disposition of endogenously released anandamide; if this were the case, AM404 may be helpful to understand the physiological functions of anandamide, and may serve as a scaffold to develop therapeutic agents acting as indirect agonists at central and peripheral cannabinoid receptors. A mechanistic feature of anandamide transport is its lack of Na1 dependence, which suggests that anandamide is accumulated in cells via a carrier-mediated diffusion process driven by the concentration gradient of anandamide across the lipid bilayer. This feature differentiates anandamide from all known neurotransmitters but associates it with prostaglandin E2, the membrane transporter of which is also Na1 independent . Thus, even from the standpoint of its inactivation route, anandamide appears to behave more as a local mediator than as a bona fifide neurotransmitter. In addition, the fact that anandamide may be transported by facilitated diffusion suggests that the kinetics of its biodisposition may be slower than that of amino acid or amine transmitters. To be able to address this question, however, we must await the cloning and functional expression of the anandamide transporter protein.If cellular energy does not propel the transfer of anandamide across cell membranes, what does? Intracellular degradation may be one possibility. Indeed, a membrane-bound enzyme that catalyzes the breakdown of anandamide to arachidonic acid and ethanolamine has been identified and cloned . The intracellular localization of this enzyme, termed anandamide amidohydrolase or fatty acid amide hydrolase, is supported by studies with subcellular membrane fractions , by its deduced amino acid sequence , and by the fact that anandamide hydrolysis takes place after this lipid has been accumulated in cells .