Dale G. Deutsch

Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist

Enzymatic activities have been identified which catalyze both the hydrolysis and synthesis of arachidonylethanolamide (anandamide). Anandamide was taken up by neuroblastoma and glioma cells in culture, but it did not accumulate since it was rapidly degraded by an amidase activity that resided mainly in the membrane fractions. This amidase activity was expressed in brain and the majority of cells and tissues tested. Phenylmethylsulfonyl fluoride (PMSF) was found to be a potent inhibitor of this amidase. A catalytic activity for the biosynthesis of anandamide from ethanolamine and arachidonic acid was readily apparent in incubations of rat brain homogenates. The stability of anandamide in serum and its rapid breakdown in cells and tissues are consistent with the observation that it is active when administered systemically, and its duration of action will be regulated by its rate of degradation in cells.

Production and physiological actions of anandamide in the vasculature of the rat kidney.

The endogenous cannabinoid receptor agonist anandamide is present in central and peripheral tissues. As the kidney contains both the amidase that degrades anandamide and transcripts for anandamide receptors, we characterized the molecular components of the anandamide signaling system and the vascular effects of exogenous anandamide in the kidney. We show that anandamide is present in kidney homogenates, cultured renal endothelial cells (EC), and mesangial cells; these cells also contain anandamide amidase. Reverse-transcriptase PCR shows that EC contain transcripts for cannabinoid type 1 (CB1) receptors, while mesangial cells have mRNA for both CB1 and CB2 receptors. EC exhibit specific, high-affinity binding of anandamide (Kd = 27.4 nM). Anandamide (1 microM) vasodilates juxtamedullary afferent arterioles perfused in vitro; the vasodilation can be blocked by nitric oxide (NO) synthase inhibition with L-NAME (0.1 mM) or CB1 receptor antagonism with SR 141716A (1 microM), but not by indomethacin (10 microM). Anandamide (10 nM) stimulates CB1-receptor-mediated NO release from perfused renal arterial segments; a similar effect was seen in EC. Finally, anandamide (1 microM) produces a NO-mediated inhibition of KCl-stimulated [3H]norepinephrine release from sympathetic nerves on isolated renal arterial segments. Hence, an anandamide signaling system is present in the kidney, where it exerts significant vasorelaxant and neuromodulatory effects.

Evidence against the presence of an anandamide transporter

On the basis of temperature dependency, saturability, selective inhibition, and substrate specificity, it has been proposed that an anandamide transporter exists. However, all of these studies have examined anandamide accumulation at long time points when downstream effects such as metabolism and intracellular sequestration are operative. In the current study, we have investigated the initial rates (<1 min) of anandamide accumulation in neuroblastoma and astrocytoma cells in culture and have determined that uptake is not saturable with increasing concentrations of anandamide. However, anandamide hydrolysis, after uptake in neuroblastoma cells, was saturable at steady-state time points (5 min), suggesting that fatty acid amide hydrolase (FAAH) may be responsible for observed saturation of uptake at long time points. In general, arvanil, olvanil, and N-(4-hydroxyphenyl)arachidonylamide (AM404) have been characterized as transport inhibitors in studies using long incubations. However, we found these “transport inhibitors” did not inhibit anandamide uptake in neuroblastoma and astrocytoma cells at short time points (40 sec or less). Furthermore, we confirmed that these inhibitors in vitro were actually inhibitors of FAAH. Therefore, the likely mechanism by which the transport inhibitors raise anandamide levels to exert pharmacological effects is by inhibiting FAAH, and they should be reevaluated in this context. Immunofluorescence has indicated that FAAH staining resides mainly on intracellular membranes of neuroblastoma cells, and this finding is consistent with our observed kinetics of anandamide hydrolysis. In summary, these data suggest that anandamide uptake is a process of simple diffusion. This process is driven by metabolism and other downstream events, rather than by a specific membrane-associated anandamide carrier.

Fatty acid amide hydrolase is located preferentially in large neurons in the rat central nervous system as revealed by immunohistochemistry

The distribution in the rat brain of fatty acid amide hydrolase (FAAH) an enzyme that catalyzes the hydrolysis of the endogenous cannabinoid anandamide was studied by immunohistochemistry. An immunopurified, polyclonal antibody to the C terminal region of FAAH was used in these studies. The large principal neurons, such as pyramidal cells in the cerebral cortex, the pyramidal cells the hippocampus, Purkinje cells in the cerebellar cortex and the mitral cells in the olfactory bulb, showed the strongest FAAH immunoreactivity. These FAAH-containing principal neurons except the mitral cells in the olfactory bulb are in close proximity with cannabinoid CB1 receptors as revealed by our previous immunohistochemical study. Moderately or lightly stained FAAH-containing neurons were also found in the amygdala, the basal ganglia, the deep cerebellar nuclei, the ventral posterior nuclei of the thalamus, the optic layer and the intermediate white layer of the superior colliculus and the red nucleus in the midbrain, and motor neurons of the spinal cord. These data demonstrate that FAAH is heterogeneously distributed and this distribution exhibits considerable, although not complete, overlap with the distribution of cannabinoid CB1 receptors in rat brain.

The fatty acid amide hydrolase (FAAH)

The fatty acid amide hydrolase (FAAH), is the enzyme responsible for the hydrolysis of anandamide, an endocannabinoid. The FAAH knockout, the assays for FAAH, the activity of its substrates, its reversibility and its cloning from rat, mouse, human, and pig are covered in this review. The conserved regions of FAAH are described in terms of sequence and function, including the domains that contains the serine catalytic nucleophile, the hydrophobic domain important for self-association, the proline rich domain region which may be important for subcellular localization and the fatty acid chain binding domain. The FAAH mouse promoter region was characterized in terms of its transcription start site and its activity in different cell types. The distribution of FAAH in the major organs in the body is described as well as regional distribution in the brain and its correlation with cannabinoid receptors. Since FAAH is recognized as a drug target, a large number of inhibitors have been synthesized and tested since 1994 and these are reviewed in terms of reversibility, potency, and specificity for FAAH.

Methyl arachidonyl fluorophosphonate: a potent irreversible inhibitor of anandamide amidase

Anandamide amidase (EC 3.5.1.4) is responsible for the hydrolysis of arachidonoyl ethanolamide (anandamide). Relatively selective and potent enzyme reversible inhibitors effective in the low micromolar range, such as arachidonyl trifluoromethyl ketone (Arach-CF3), have been described (Koutek et al., J Biol Chem 269: 22937-22940, 1994). In the current study, methyl arachidonyl fluorophosphonate (MAFP), an arachidonyl binding site directed phosphonylation reagent, was tested as an inhibitor of anandamide amidase and as a ligand for the CB1 cannabinoid receptor. MAFP was 800 times more potent than Arach-CF3 and phenylmethylsulfonyl fluoride (PMSF) as an amidase inhibitor in rat brain homogenates. In intact neuroblastoma cells, MAFP was also approximately 1000-fold more potent than Arach-CF3. MAFP demonstrated selectivity towards anandamide amidase for which it was approximately 3000 and 30,000-fold more potent than it was towards chymotrypsin and trypsin, respectively. MAFP displaced [3H]CP-55940 binding to the CB1 cannabinoid receptor with an IC50 of 20 nM vs 40 nM for anandamide. It bound irreversibly and prevented subsequent binding of the cannabinoid radioligand [3H]CP-55940 at that locus. These studies suggest that MAFP is a potent and specific inhibitor of anandamide amidase and, in addition, can interact with the cannabinoid receptors at the cannabinoid binding site. This is the first report of a potent and relatively selective irreversible inhibitor of arachidonoyl ethanolamide amidase.

Identification of intracellular carriers for the endocannabinoid anandamide

The endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) is an uncharged neuromodulatory lipid that, similar to many neurotransmitters, is inactivated through its cellular uptake and subsequent catabolism. AEA is hydrolyzed by fatty acid amide hydrolase (FAAH), an enzyme localized on the endoplasmic reticulum. In contrast to most neuromodulators, the hydrophilic cytosol poses a diffusional barrier for the efficient delivery of AEA to its site of catabolism. Therefore, AEA likely traverses the cytosol with the assistance of an intracellular carrier that increases its solubility and rate of diffusion. To study this process, AEA uptake and hydrolysis were examined in COS-7 cells expressing FAAH restricted to the endoplasmic reticulum, mitochondria, or the Golgi apparatus. AEA hydrolysis was detectable at the earliest measurable time point (3 seconds), suggesting that COS-7 cells, normally devoid of an endocannabinoid system, possess an efficient cytosolic trafficking mechanism for AEA. Three fatty acid binding proteins (FABPs) known to be expressed in brain were examined as possible intracellular AEA carriers. AEA uptake and hydrolysis were significantly potentiated in N18TG2 neuroblastoma cells after overexpression of FABP5 or FABP7, but not FABP3. Similar results were observed in COS-7 cells stably expressing FAAH. Consistent with the roles of FABP as AEA carriers, administration of the competitive FABP ligand oleic acid or the selective non-lipid FABP inhibitor BMS309403 attenuated AEA uptake and hydrolysis by ≈50% in N18TG2 and COS-7 cells. Taken together, FABPs represent the first proteins known to transport AEA from the plasma membrane to FAAH for inactivation and may therefore be novel pharmacological targets.

The Cellular Uptake of Anandamide Is Coupled to Its Breakdown by Fatty-acid Amide Hydrolase

Anandamide is an endogenous compound that acts as an agonist at cannabinoid receptors. It is inactivated via intracellular degradation after its uptake into cells by a carrier-mediated process that depends upon a concentration gradient. The fate of anandamide in those cells containing an amidase called fatty-acid amide hydrolase (FAAH) is hydrolysis to arachidonic acid and ethanolamine. The active site nucleophilic serine of FAAH is inactivated by a variety of inhibitors including methylarachidonylfluorophosphonate (MAFP) and palmitylsulfonyl fluoride. In the current report, the net uptake of anandamide in cultured neuroblastoma (N18) and glioma (C6) cells, which contain FAAH, was decreased by nearly 50% after 6 min of incubation in the presence of MAFP. Uptake in laryngeal carcinoma (Hep2) cells, which lack FAAH, is not inhibited by MAFP. Free anandamide was found in all MAFP-treated cells and in control Hep2 cells, whereas phospholipid was the main product in N18 and C6 control cells when analyzed by TLC. The intracellular concentration of anandamide in N18, C6, and Hep2 cells was up to 18-fold greater than the extracellular concentration of 100 nm, which strongly suggests that it is sequestered within the cell by binding to membranes or proteins. The accumulation of anandamide and/or its breakdown products was found to vary among the different cell types, and this correlated approximately with the amount of FAAH activity, suggesting that the breakdown of anandamide is in part a driving force for uptake. This was shown most clearly in Hep2 cells transfected with FAAH. The uptake in these cells was 2-fold greater than in vector-transfected or untransfected Hep2 cells. Therefore, it appears that FAAH inhibitors reduce anandamide uptake by cells by shifting the anandamide concentration gradient in a direction that favors equilibrium. Because inhibition of FAAH increases the levels of extracellular anandamide, it may be a useful target for the design of therapeutic agents.

Morphine and Anandamide Stimulate Intracellular Calcium Transients in Human Arterial Endothelial Cells: Coupling to Nitric Oxide Release

Both morphine and anandamide significantly stimulated cultured endothelial intracellular calcium level increases in a concentration-dependent manner in cells pre-loaded with fura 2/AM. Morphine is more potent than anandamide (approximately 275 vs. 135 nM [Ca]i), and the [Ca]i for both ligands was blocked by prior exposure of the cells to their respective receptor antagonist, i.e., naloxone and SR 171416A. Various opioid peptides did not exhibit this ability, indicating a morphine-mu3-mediated process. In comparing the sequence of events concerning morphines and anandamides action in stimulating both [Ca]i and nitric oxide production in endothelial cells, we found that the first event precedes the second by 40+/-8 sec. The opiate and cannabinoid stimulation of [Ca]i was attenuated in cells leeched of calcium, strongly suggesting that intracellular calcium levels regulate cNOS activity.

Immunocytochemical localization of cannabinoid CB1 receptor and fatty acid amide hydrolase in rat retina.

Cannabinoids have major effects on central nervous system function. Recent studies indicate that cannabinoid effects on the visual system have a retinal component. Immunocytochemical methods were used to localize cannabinoid CB1 receptor immunoreactivity (CB1R‐IR) and an endocannabinoid (anandamide and 2‐arachidonylglycerol) degradative enzyme, fatty acid amide hydrolase (FAAH)‐IR, in the rat retina. Double labeling with neuron‐specific markers permitted identification of cells that were labeled with CB1R‐IR and FAAH‐IR. CB1R‐IR was observed in all cells that were protein kinase C‐immunoreactive (rod bipolar cells and a subtype of GABA‐amacrine cell) as well as horizontal cells (identified by calbindin‐IR). There was also punctate CB1R‐IR in the distal one‐third of the inner plexiform layer (IPL) that could not be assigned to a cell type. FAAH‐IR was most prominent in large ganglion cells, whose dendrites projected to a narrow band in the proximal IPL. Weaker FAAH‐IR was observed in the soma of horizontal cells (identified by calbindin‐IR); the soma of large, but not small, dopamine amacrine cells (identified by tyrosine hydroxylase‐IR); and dendrites of orthotopic‐ and displaced‐starburst amacrine cells (identified by choline acetyltransferase‐IR) but in less than 50% of the starburst amacrine cell somata. The extensive distribution of CB1R‐IR on horizontal cells and rod bipolar cells indicates a role of endocannabinoids in scotopic vision, whereas the more widespread distribution of FAAH‐IR indicates a complex control of endocannabinoid release and degradation in the retina. J. Comp. Neurol. 415:80–90, 1999.