Endogenous cannabinoids: metabolism and their role in reproduction

Over the past two decades a number of endogenous compounds that act as ligands for the cannabinoid receptors has been discovered. In analogy with the "endorphins" these compounds have been called "endocannabinoids". Endocannabinoids have been demonstrated in many mammalian tissues including humans and are widely distributed in the CNS, peripheral nerves, uterus, leukocytes, spleen and testicles. The uterus contains the highest levels of anandamide, the first discovered endocannabinoid, suggesting an important role for this substance in reproduction. Several studies have shown anandamide to be involved in the regulation of implantation and reduced activity of the enzyme that degrades anandamide has been associated with early pregnancy loss in humans. The bulk of the literature concerning endocannabinoids is based upon anandamide related studies; therefore, in this review we focus on the metabolism of anandamide and its role in reproduction.     

Cannabinoid receptors and their ligands

There are at least two types of cannabinoid receptors, CB(1) and CB(2), both coupled to G proteins. CB(1) receptors exist primarily on central and peripheral neurons, one of their functions being to modulate neurotransmitter release. CB(2) receptors are present mainly on immune cells. Their roles are proving more difficult to establish but seem to include the modulation of cytokine release. Endogenous agonists for cannabinoid receptors (endocannabinoids) have also been discovered, the most important being arachidonoyl ethanolamide (anandamide), 2-arachidonoyl glycerol and 2-arachidonyl glyceryl ether. Other endocannabinoids and cannabinoid receptor types may also exist. Although anandamide can act through CB(1) and CB(2) receptors, it is also a vanilloid receptor agonist and some of its metabolites may possess yet other important modes of action. The discovery of the system of cannabinoid receptors and endocannabinoids that constitutes the "endocannabinoid system" has prompted the development of CB(1)- and CB(2)-selective agonists and antagonists/inverse agonists. CB(1)/CB(2) agonists are already used clinically, as anti-emetics or to stimulate appetite. Potential therapeutic uses of cannabinoid receptor agonists include the management of multiple sclerosis/spinal cord injury, pain, inflammatory disorders, glaucoma, bronchial asthma, vasodilation that accompanies advanced cirrhosis, and cancer. Following their release onto cannabinoid receptors, endocannabinoids are removed from the extracellular space by membrane transport and then degraded by intracellular enzymic hydrolysis. Inhibitors of both these processes have been developed. Such inhibitors have therapeutic potential as animal data suggest that released endocannabinoids mediate reductions both in inflammatory pain and in the spasticity and tremor of multiple sclerosis. So too have CB(1) receptor antagonists, for example for the suppression of appetite and the management of cognitive dysfunction or schizophrenia.     

Cannabinoid and GABA modulation of sympathetic nerve activity and blood pressure in the dorsal periaqueductal gray of the rat

Sympathoexcitation and increased blood pressure evoked by central networks integrating defensive behavior are fundamental to the acute stress response. A balance between excitatory glutamatergic and inhibitory GABAergic neurotransmission in the dorsal periaqueductal gray (dPAG) results in a tonic level of activity in the alerting system. Neuromodulators such as endocannabinoids have been shown to influence the sympathoexcitatory and pressor components of acute stress in the dPAG, exemplified by the defense response as a model, but the mechanism of integration remains unknown. The present study examines the role of GABA and its interaction with endocannabinoids in modulating sympathetic nerve activity and blood pressure related to the defense response. Microinjection of the broad-spectrum excitatory amino acid dl-homocysteic acid (DLH) identified sites of the defense pathway in the dPAG from which an increase in renal sympathetic nerve activity and blood pressure could be evoked, and subsequent microinjections were made at the same site through a multibarrelled micropipette. Blockade of GABAA receptors or microinjection of the cannabinoid 1 receptor agonist anandamide elicited a renal sympathoexcitation and pressor response. Prior microinjection of the GABAA receptor antagonist gabazine attenuated the sympathoexcitation and pressor response associated with anandamide microinjection. In contrast, the sympathetic response to DLH was enhanced by GABAA receptor blockade. These data demonstrate that sympathoexcitatory neurons in the dPAG are under tonic inhibition by GABA and that endocannabinoids modulate this GABAergic neurotransmission to help regulate components of the defense response.     

Endocannabinoids and related fatty acid derivatives in pain modulation

The brain produces at least five compounds that possess sub-micromolar affinity for cannabinoid receptors: anandamide, 2-arachidonoylglycerol, noladin ether, virodhamine, and N-arachidonoyldopamine (NADA). One function of these and/or related compounds is to suppress pain sensitivity. Much evidence supports a role of endocannabinoids in pain modulation in general, and some evidence points to the role of particular endocannabinoids. Related endogenous fatty acid derivatives such as oleamide, palmitoylethanolamide, 2-lineoylglycerol, 2-palmitoylglycerol, and a family of arachidonoyl amino acids may interact with endocannabinoids in the modulation of pain sensitivity.     

Endocannabinoids and the gut

In the digestive tract, there is evidence for the presence of high amounts of endocannabinoids (anandamide and 2-arachidonylglycerol) and of mechanisms for endocannabinoid metabolism and possibly endocannabinoid uptake. Pharmacological studies have shown that anandamide inhibits excitatory transmission and peristalsis in the isolated guinea-pig ileum and reduces intestinal motility in the mouse in vivo; all these effects are mediated by CB(1) receptors, which are located on enteric nerves. Conversely, the selective CB(1) receptor antagonist SR141716A increased intestinal motility and this effect is likely due to the displacement of endocannabinoids rather than to its inverse agonist properties. Interestingly, inhibitory effects of anandamide via non-CB(1) receptors and stimulatory effects via vanilloid receptors have also been proposed.     

Depolarizing and calcium-mobilizing stimuli fail to enhance synthesis and release of endocannabinoids from rat brain cerebral cortex slices

The concentrations of the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonylethanolamine (anandamide) were examined in rat brain cerebral cortex slices and surrounding medium. Basal concentrations of endocannabinoids were similar to those identified previously in rat brain, with anandamide content being much lower (19 pmol/g) than that of 2-AG (7300 pmol/g). In contrast, basal concentrations in the surrounding medium were proportionally much lower for 2-arachidonoylglycerol (16 pmol/mL) compared to anandamide (0.6 pmol/mL). Incubation of slices with glutamate receptor agonists, depolarizing concentrations of KCl, or ionomycin failed to alter tissue concentrations of endocannabinoids, while endocannabinoids in the medium were unaltered by elevated KCl. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester, an inhibitor of fatty acid amide hydrolase, significantly enhanced tissue concentrations of anandamide (and related N-acylethanolamines), without altering 2-AG, while evoking proportional elevations of anandamide in the medium. Removal of extracellular calcium ions failed to alter tissue concentrations of anandamide, but significantly reduced 2-AG in the tissue by 90% and levels in the medium to below the detection limit. Supplementation of the medium with 50 μM N-oleoylethanolamine only raised tissue concentrations of N-oleoylethanolamine in the presence of cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester and failed to alter either tissue or medium anandamide or 2-AG concentrations. These results highlight the ongoing turnover of endocannabinoids, and the importance of calcium ions in maintaining 2-AG concentrations in this tissue.     

An optimized GC-MS method detects nanomolar amounts of anandamide in mouse brain

The endocannabinoids anandamide and 2-arachidonoylglycerol, as well as several anandamide-related N-acylethanolamines, belong to a family of lipid transmitter that regulate fundamental physiological processes, including neurotransmission and neuroinflammation. Their precise quantification in biological matrices can be achieved by gas chromatography-mass spectrometry (GC-MS), but this method typically requires multiple time-consuming purification steps such as solid-phase extraction followed by HPLC. Here we report a novel solid-phase extraction procedure allowing for single-step, and thus higher throughput, purification of endocannabinoids and N-acylethanolamines before GC-MS quantification. We determined the minimal amount of mouse brain tissue required to reliably detect endocannabinoids and N-acylethanolamines when using this approach and provide direct evidence for quantification accuracy by using radioactive and deuterated standards spiked into mouse brain samples. Using this method, we found that mouse brain contains much higher levels of anandamide (>1 nmol/g tissue) than previously reported, whereas levels of 2-arachidonoylglycerol and other N-acylethanolamines are well within the range of previous reports. In addition, we show that mouse brain amounts of endocannabinoids and N-acylethanolamines differ depending on animal gender as well as on whether the tissue was fixed or not. Our study shows that endocannabinoid and N-acylethanolamine levels quantified in mouse brain by GC-MS depend closely on tissue amount and preparation as well as on animal gender and that, depending on such parameters, anandamide levels could be underestimated.     

Mechanisms for recycling and biosynthesis of endogenous cannabinoids anandamide and 2-arachidonylglycerol

The mechanisms of endogenous cannabinoid biosynthesis are not completely understood. We hypothesized that anandamide could be recycled by the cell to form new endocannabinoid molecules and released into the extracellular space. We determined that new endocannabinoids derived from exogenous anandamide or arachidonic acid were synthesized and released from RBL-2H3 cells in response to ionomycin. Treatment of RBL-2H3 cells with nystatin and progesterone, agents that disrupt organization of lipid raft/caveolae, resulted in the attenuation of anandamide and 2-arachidonyl glycerol synthesis and/or release in response to stimulation with ionomycin suggesting a role for these membrane microdomains in endocannabinoid biosynthesis. Furthermore, anandamide synthesis may be independent of N-acyl phosphatidylethanolamine phospholipase D as expression of the enzyme was not detected in RBL-2H3 cells. We also established that extracellular calcium is necessary for endocannabinoid biosynthesis because release of intracellular calcium stores alone does not promote endocannabinoid biosynthesis. Next, we examined the role of calcium as a 'switch' to activate the synthesis of anandamide and simultaneously reduce uptake. Indeed, [(3)H] anandamide uptake was reduced in the presence of calcium. Our findings suggest a mechanism indicative of calcium-modulated activation of anandamide synthesis and simultaneous termination of uptake.     

Enhanced levels of endogenous cannabinoids in the globus pallidus are associated with a reduction in movement in an animal model of Parkinson's disease.

In recent years, cannabinoid receptors and their endogenous ligands (endocannabinoids) have been identified within the brain. The high density of CB1 cannabinoid receptors within the basal ganglia suggests a potential role for endocannabinoids in the control of voluntary movement and in basal ganglia-related movement disorders such as Parkinson's disease. However, whether endocannabinoids play a role in regulating motor behavior in health and disease is unknown. Here we report the presence in two regions of the basal ganglia, the globus pallidus and substantia nigra, of the endocannabinoids 2-arachidonoylglycerol (2AG) and anandamide. The levels of the latter compound are approximately threefold higher than those previously reported in any other brain region. In the reserpine-treated rat, an animal model of Parkinson's disease, suppression of locomotion is accompanied by a sevenfold increase in the levels of the 2AG in the globus pallidus, but not in the other five brain regions analyzed. Stimulation of locomotion in the reserpine-treated rat by either of the two selective agonists of D2 and D1 dopamine receptors, quinpirole and R-(+/-)-3-allyl-6-chloro-7, 8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (Cl-APB), respectively, results in the reduction of both anandamide and 2AG levels in the globus pallidus. Finally, full restoration of locomotion in the reserpine-treated rat is obtained by coadministration of quinpirole and the selective antagonist of the cannabinoid CB1 receptor subtype, SR141716A. These findings indicate a link between endocannabinoid signaling in the globus pallidus and symptoms of Parkinson's disease in the reserpine-treated rat, and suggest that modulation of the endocannabinoid signaling system might prove useful in treating this or other basal ganglia-related movement disorders.     

Identification of endocannabinoids and related compounds in human fat cells

Objective: Recently, an activation of the endocannabinoid system during obesity has been reported. More particularly, it has been demonstrated that hypothalamic levels of both endocannabinoids, 2‐arachidonoylglycerol and anandamide (N‐arachidonoylethanolamine), are up‐regulated in genetically obese rodents. Circulating levels of both endocannabinoids were also shown to be higher in obese compared with lean women. Yet, the direct production of endocannabinoids by human adipocytes has never been demonstrated. Our aim was to evaluate the ability of human adipocytes to produce endocannabinoids. Research Methods and Procedures: The production of endocannabinoids by human adipocytes was investigated in a model of human white subcutaneous adipocytes in primary culture. The effects of leptin, adiponectin, and peroxisome proliferator‐activated receptor (PPAR)‐γ activation on endocannabinoid production by adipocytes were explored. Endocannabinoid levels were determined by high‐performance liquid chromatography (HPLC)‐atmospheric pressure chemical ionization (APCI)‐mass spectrometry (MS) analysis, leptin and adiponectin secretion measured by enzyme‐linked immunosorbent assay (ELISA), and PPAR‐γ protein expression examined by Western blotting. Results: We show that 2‐arachidonoylglycerol, anandamide, and both anandamide analogs, N‐palmitoylethanolamine and N‐oleylethanolamine, are produced by human white subcutaneous adipocytes in concentrations ranging from 0.042 ± 0.004 to 0.531 ± 0.048 pM/mg lipid extract. N‐palmitoylethanolamine is the most abundant cannabimimetic compound produced by human adipocytes, and its levels are significantly down‐regulated by leptin but not affected by adiponectin and PPAR‐γ agonist ciglitazone. N‐palmitoylethanolamine itself does not affect either leptin or adiponectin secretion or PPAR‐γ protein expression in adipocytes. Discussion: This study has led to the identification of human adipocytes as a new source of endocannabinoids and related compounds. The biological significance of these adipocyte cannabimimetic compounds and their potential implication in obesity should deserve further investigations.     

Endocannabinoids in the immune system and cancer

The present review focuses on the role of the endogenous cannabinoid system in the modulation of immune response and control of cancer cell proliferation. The involvement of cannabinoid receptors, endogenous ligands and enzymes for their biosynthesis and degradation, as well as of cannabinoid receptor-independent events is discussed. The picture arising from the recent literature appears very complex, indicating that the effects elicited by the stimulation of the endocannabinoid system are strictly dependent on the specific compounds and cell types considered. Both the endocannabinoid anandamide and its congener palmitoylethanolamide, exert a negative action in the onset of a variety of parameters of the immune response. However, 2-arachidonoylglycerol appears to be the true endogenous ligand for peripheral cannabinoid receptors, although its action as an immunomodulatory molecule requires further characterization. Modulation of the endocannabinoid system interferes with cancer cell proliferation either by inhibiting mitogenic autocrine/paracrine loops or by directly inducing apoptosis; however, the proapoptotic effect of anandamide is not shared by other endocannabinoids and suggests the involvement of non-cannabinoid receptors, namely the VR1 class of vanilloid receptors. In conclusion, further investigations are needed to elucidate the function of endocannabinoids as immunosuppressant and antiproliferative/cytotoxic agents. The experimental evidence reviewed in this article argues in favor of the therapeutic potential of these compounds in immune disorders and cancer.     

Astrocytes in culture produce anandamide and other acylethanolamides

Anandamide (arachidonylethanolamide) is an endocannabinoid that belongs to the acylethanolamide lipid family. It is produced by neurons in a calcium-dependent manner and acts through cannabinoid CB1 receptors. Other members of the acylethanolamide lipid family are also produced by neurons and act through G-protein-coupled receptors: homo-gamma-linolenylethanolamide (HEA) and docosatetraenylethanolamide (DEA) act through CB1 receptors, palmitylethanolamide (PEA) acts through CB2-like receptors, and oleylethanolamide (OEA) acts through receptors that have not yet been cloned. Although it is clear that anandamide and other acylethanolamides play a major role in neuronal signaling, whether astrocytes also produce these lipids is unknown. We developed a chemical ionization gas chromatography/mass spectrometry method that allows femtomole detection and quantification of anandamide and other acylethanolamides. Using this method, we unambiguously detected and quantified anandamide, HEA, DEA, PEA, and OEA in mouse astrocytes in culture. Stimulation of mouse astrocytes with ionomycin, a calcium ionophore, enhanced the production of anandamide, HEA, and DEA, whereas PEA and OEA levels were unchanged. Endothelin-1, a peptide known to act on astrocytes, enhanced the production of anandamide, without affecting the levels of other acylethanolamides. These results show that astrocytes produce anandamide, HEA, and DEA in a calcium-dependent manner and that anandamide biosynthesis can be selectively stimulated under physiologically relevant conditions. The relative levels of acylethanolamides in astrocytes from rat and human were different from the relative levels of acylethanolamides in mouse astrocytes, indicating that the production of these lipids differs between species. Because astrocytes are known to express CB1 receptors and inactivate endocannabinoids, our finding strongly suggests the existence of a functional endocannabinoid signaling system in these cells.     

Cell signaling by endocannabinoids and their congeners: questions of selectivity and other challenges

The major endocannabinoids, anandamide (N-arachidonoylethanolamide, 20:4n-6 N-acylethanolamine) and 2-arachidonoylglycerol (2-AG) are structurally and functionally similar, but they are produced by different metabolic pathways and their levels must therefore be regulated by different mechanisms. Both endocannabinoids are accompanied by cannabinoid receptor-inactive, saturated and mono- or di-unsaturated congeners which can influence their metabolism and function. Here we review published data on the presence and production of anandamide and 2-AG and their congeners in mammalian cells and discuss this information in terms of their proposed signaling functions.     

Modulation of sweet responses of taste receptor cells

Taste receptor cells play a major role in detection of chemical compounds in the oral cavity. Information derived from taste receptor cells, such as sweet, bitter, salty, sour and umami is important for evaluating the quality of food components. Among five basic taste qualities, sweet taste is very attractive for animals and influences food intake. Recent studies have demonstrated that sweet taste sensitivity in taste receptor cells would be affected by leptin and endocannabinoids. Leptin is an anorexigenic mediator that reduces food intake by acting on leptin receptor Ob-Rb in the hypothalamus. Endocannabinoids such as anandamide [N-arachidonoylethanolamine (AEA)] and 2-arachidonoyl glycerol (2-AG) are known as orexigenic mediators that act via cannabinoid receptor 1 (CB₁) in the hypothalamus and limbic forebrain to induce appetite and stimulate food intake. At the peripheral gustatory organs, leptin selectively suppresses and endocannabinoids selectively enhance sweet taste sensitivity via Ob-Rb and CB₁ expressed in sweet sensitive taste cells. Thus leptin and endocannabinoids not only regulate food intake via central nervous systems but also modulate palatability of foods by altering peripheral sweet taste responses. Such reciprocal modulation of leptin and endocannabinoids on peripheral sweet sensitivity may play an important role in regulating energy homeostasis.     

Cannabinoids and brain injury: therapeutic implications

Mounting in vitro and in vivo data suggest that the endocannabinoids anandamide and 2-arachidonoyl glycerol, as well as some plant and synthetic cannabinoids, have neuroprotective effects following brain injury. Cannabinoid receptor agonists inhibit glutamatergic synaptic transmission and reduce the production of tumour necrosis factor-alpha and reactive oxygen intermediates, which are factors in causing neuronal damage. The formation of the endocannabinoids anandamide and 2-arachidonoyl glycerol is strongly enhanced after brain injury, and there is evidence that these compounds reduce the secondary damage incurred. Some plant and synthetic cannabinoids, which do not bind to the cannabinoid receptors, have also been shown to be neuroprotective, possibly through their direct effect on the excitatory glutamate system and/or as antioxidants.     

Endocannabinoids effect on oxidative status of human platelets

Reactive oxygen species (ROS) are known to regulate platelet activation. Since endocannabinoids behave as platelet agonists, we investigated the effect of two endocannabinoids, 2-arachidonoylglycerol (2AG) and anandamide (AEA) on the oxidative status of human platelets. We have demonstrated that 2AG and AEA stimulate ROS production, superoxide anion formation and lipid peroxidation. The effect is dose and time dependent and mainly occurs through the involvement of cannabinoid receptor 1 (CB1) since all tested parameters are greatly reduced by SR141716, the CB1 specific inhibitor. The specific inhibitor of cannabinoid receptor 2 (CB2) SR144528 produces a very small inhibition. The involvement of syk/PI3K/AKT/mTor pathway in oxidative stress induced by endocannabinoids is shown. Nicotinamide adenine dinucleotide phosphate oxidase seems to be poorly involved in the endocannabinoids effect. Concerning the aerobic metabolism, it has been demonstrated that endocannabinoids reduce the oxygen consumption and adenosine triphosphate synthesis, both in the presence of pyruvate + malate or succinate. In addition, endocannabinoids inhibit the activity of respiratory complexes II, III and IV and increase the activity of respiratory complex I. The endocannabinoids effect on aerobic metabolism seems to be also a CB1 mediated mechanism. Thus, in human platelets oxidative stress induced by endocannabinoids, mainly generated in the respiratory chain through the activation of complex I and the inhibition of complex II, III and IV, may lead to thrombotic events, contributing to cardiovascular diseases.     

Endocannabinoids and endocannabinoid-related mediators: Targets,metabolism and role in neurological disorders

The endocannabinoid system (ECS) is composed of two G protein-coupled receptors (GPCRs), the cannabinoid CB1 and CB2 receptors, and the two main endogenous lipid ligands of such receptors (also known as the “endocannabinoids”), anandamide and 2-arachidonoyl-glycerol. The ECS is a pleiotropic signalling system involved in all aspects of mammalian physiology and pathology, and for this reason it represents a potential target for the design and development of new therapeutic drugs. However, the endocannabinoids as well as some of their congeners also interact with a much wider range of receptors, including members of the Transient Receptor Potential (TRP) channels, Peroxisome Proliferator-Activated Receptors (PPARs), and other GPCRs. Indeed, following the discovery of the endocannabinoids, endocannabinoid-related lipid mediators, which often share the same metabolic pathways of the endocannabinoids, have also been identified or rediscovered. In this review article, we discuss the role of endocannabinoids and related lipids during physiological functions, as well as their involvement in some of the most common neurological disorders.     

Anandamide, but not 2-arachidonoylglycerol, accumulates during in vivo neurodegeneration

Endogenous cannabinoid receptor ligands (endocannabinoids) may rescue neurons from glutamate excitotoxicity. As these substances also accumulate in cultured immature neurons following neuronal damage, elevated endocannabinoid concentrations may be interpreted as a putative neuroprotective response. However, it is not known how glutamatergic insults affect in vivo endocannabinoid homeostasis, i.e. N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as other constituents of their lipid families, N-acylethanolamines (NAEs) and 2-monoacylglycerols (2-MAGs), respectively. Here we employed three in vivo neonatal rat models characterized by widespread neurodegeneration as a consequence of altered glutamatergic neurotransmission and assessed changes in endocannabinoid homeostasis. A 46-fold increase of cortical NAE concentrations (anandamide, 13-fold) was noted 24 h after intracerebral NMDA injection, while less severe insults triggered by mild concussive head trauma or NMDA receptor blockade produced a less pronounced NAE accumulation. By contrast, levels of 2-AG and other 2-MAGs were virtually unaffected by the insults employed, rendering it likely that key enzymes in biosynthetic pathways of the two different endocannabinoid structures are not equally associated to intracellular events that cause neuronal damage in vivo. Analysis of cannabinoid CB(1) receptor mRNA expression and binding capacity revealed that cortical subfields exhibited an up-regulation of these parameters following mild concussive head trauma and exposure to NMDA receptor blockade. This may suggest that mild to moderate brain injury may trigger elevated endocannabinoid activity via concomitant increase of anandamide levels, but not 2-AG, and CB(1) receptor density.     

Endocannabinoids in the central nervous system--an overview

Many aspects of the physiology and pharmacology of anandamide (arachidonoyl ethanol amide), the first endogenous cannabinoid ligand ("endocannabinoid") isolated from pig brain, have been studied since its discovery in 1992. Ethanol amides from other fatty acids have also been identified as endocannabinoids with similar in vivo and in vitro pharmacological properties. 2-Arachidonoyl glycerol and noladin ether (2-arachidonyl glyceryl ether), isolated in 1995 and 2001, respectively, so far, display pharmacological properties in the central nervous system, similar to those of anandamide. The endocannabinoids are widely distributed in brain, they are synthesized and released upon neuronal stimulation, undergo reuptake and are hydrolyzed intracellularly by fatty acid amide hydrolase (FAAH). For therapeutic purposes, inhibitors of FAAH may provide more specific cannabinoid activities than direct agonists, and several such molecules have already been developed.Pharmacological effects of the endocannabinoids are very similar, yet not identical, to those of the plant-derived and synthetic cannabinoid receptor ligands. In addition to pharmacokinetic explanations, direct or indirect interactions with other receptors have been considered to explain some of these differences, including activities at serotonin and GABA receptors. Binding affinities for other receptors such as the vanilloid receptor, have to be taken into account in order to fully understand endocannabinoid physiology. Moreover, possible interactions with receptors for the lysophosphatidic acids deserve attention in future studies.Endocannabinoids have been implicated in a variety of physiological functions. The areas of central activities include pain reduction, motor regulation, learning/memory, and reward. Finally, the role of the endocannabinoid system in appetite stimulation in the adult organism, and perhaps more importantly, its critical involvement in milk ingestion and survival of the newborn, may not only further our understanding of the physiology of food intake and growth, but may also find therapeutic applications in wasting disease and infant's "failure to thrive".