Cecilia J. Hillard

Accumulation of N-arachidonoylethanolamine (anandamide) into cerebellar granule cells occurs via facilitated diffusion.

Abstract: N‐Arachidonoylethanolamine (anandamide, AEA) is a putative endogenous ligand of the cannabinoid receptor. Intact cerebellar granule neurons in primary culture rapidly accumulate AEA. [3H]AEA accumulation by cerebellar granule cells is dependent on incubation time (t1/2 of 2.6 ± 0.8 min at 37°C) and temperature. The accumulation of AEA is saturable and has an apparent Km of 41 ± 15 µM and a Vmax of 0.61 ± 0.04 nmol/min/106 cells. [3H]AEA accumulation by cerebellar granule cells is significantly reduced by 200 µM phloretin (57.4 ± 4% of control) in a noncompetitive manner. [3H]AEA accumulation is not inhibited by either ouabain or removal of extracellular sodium. [3H]AEA accumulation is fairly selective for AEA among other naturally occurring N‐acylethanolamines; only N‐oleoylethanolamine significantly inhibited [3H]AEA accumulation at a concentration of 10 µM. The ethanolamides of palmitic acid and linolenic acid were inactive at 10 µM. N‐Arachidonoylbenzylamine and N‐arachidonoylpropylamine, but not arachidonic acid, 15‐hydroxy‐AEA, or 12‐hydroxy‐AEA, compete for AEA accumulation. When cells are preloaded with [3H]AEA, temperature‐dependent efflux occurs with a half‐life of 1.9 ± 1.0 min. Phloretin does not inhibit [3H]AEA efflux from cells. These results suggest that AEA is accumulated by cerebellar granule cells by a protein‐mediated transport process that has the characteristics of facilitated diffusion.

Modulation of the cannabinoid CB2 receptor in microglial cells in response to inflammatory stimuli

The cannabinoid system is known to be important in neuronal regulation, but is also capable of modulating immune function. Although the CNS resident microglial cells have been shown to express the CB2 subtype of cannabinoid receptor during non‐immune‐mediated pathological conditions, little is known about the expression of the cannabinoid system during immune‐mediated CNS pathology. To examine this question, we measured CB2 receptor mRNA expression in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) and, by real‐time PCR, found a 100‐fold increase in CB2 receptor mRNA expression during EAE onset. We next determined whether microglial cells specifically express the CB2 receptor during EAE, and found that activated microglial cells expressed 10‐fold more CB2 receptor than microglia in the resting state. To determine the signals required for the up‐regulation of the CB2 receptor, we cultured microglial cells with combinations of γ‐interferon (IFN‐γ) and granulocyte) macrophage‐colony stimulating factor (GM‐CSF), which both promote microglial cell activation and are expressed in the CNS during EAE, and found that they synergized, resulting in an eight to 10‐fold increase in the CB2 receptor. We found no difference in the amount of the CB2 receptor ligand, 2‐arachidonylglycerol (2‐AG), in the spinal cord during EAE. These data demonstrate that microglial cell activation is accompanied by CB2 receptor up‐regulation, suggesting that this receptor plays an important role in microglial cell function in the CNS during autoimmune‐induced inflammation.

Pharmacological Evaluation of Cannabinoid Receptor Ligands in a Mouse Model of Anxiety: Further Evidence for an Anxiolytic Role for Endogenous Cannabinoid Signaling

Extracts of Cannabis sativa have been used for their calming and sedative effects for centuries. Recent developments in drug discovery have suggested that modulation of neuronal endogenous cannabinoid signaling systems could represent a novel approach to the treatment of anxiety-related disorders while minimizing the adverse effects of direct acting cannabinoid receptor agonists. In this study, we evaluated the effects of direct cannabinoid receptor agonists and antagonists and endocannabinoid-modulating drugs on anxiety-like behavior in mice using the elevated-plus maze. We found that the direct CB1 receptor agonists (1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol (CP 55,940) (0.001-0.3 mg/kg) and 2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo [1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate) (WIN 55212-2) (0.3-10 mg/kg) increased time spent on the open arms (To) at low doses only. At the highest doses tested, both compounds altered overall locomotor activity. In contrast, Δ9-tetrahydrocannabinol (0.25-10 mg/kg) produced a dose-dependent reduction in To. The endocannabinoid uptake/catabolism inhibitor 4-hydroxyphenylarachidonylamide (AM404) (0.3-10 mg/kg) produced an increase in To at low doses and had no effect at the highest dose tested. The fatty acid amide hydrolase inhibitor cyclohexyl carbamic acid 3′-carbamoyl-biphenyl-3-yl ester (URB597) (0.03-0.3 mg/kg) produced a monophasic, dose-dependent increase in To. The CB1 receptor antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide HCl (SR141716) (1-10 mg/kg) and N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) (1-10 mg/kg) produced dose-related decreases in To. These data indicate that activation of CB1 cannabinoid receptors reduces anxiety-like behaviors in mice and further support an anxiolytic role for endogenous cannabinoid signaling. These results suggest that pharmacological modulation of this system could represent a new approach to the treatment of anxiety-related psychiatric disorders.

The mechanism of action of the Pseudomonas aeruginosa-encoded type III cytotoxin, ExoU.

Pseudomonas aeruginosa delivers the toxin ExoU to eukaryotic cells via a type III secretion system. Intoxication with ExoU is associated with lung injury, bacterial dissemination and sepsis in animal model and human infections. To search for ExoU targets in a genetically tractable system, we used controlled expression of the toxin in Saccharomyces cerevisiae. ExoU was cytotoxic for yeast and caused a vacuolar fragmentation phenotype. Inhibitors of human calcium‐independent (iPLA2) and cytosolic phospholipase A2 (cPLA2) lipase activity reduce the cytotoxicity of ExoU. The catalytic domains of patatin, iPLA2 and cPLA2 align or are similar to ExoU sequences. Site‐specific mutagenesis of predicted catalytic residues (ExoUS142A or ExoUD344A) eliminated toxicity. ExoU expression in yeast resulted in an accumulation of free palmitic acid, changes in the phospholipid profiles and reduction of radiolabeled neutral lipids. ExoUS142A and ExoUD344A expressed in yeast failed to release palmitic acid. Recombinant ExoU demonstrated lipase activity in vitro, but only in the presence of a yeast extract. From these data we conclude that ExoU is a lipase that requires activation or modification by eukaryotic factors.

Cannabinoid CB1 receptor of cat cerebral arterial muscle functions to inhibit L-type Ca2+ channel current

The CB1 subtype of the cannabinoid receptor is present on neurons in the brain and mediates the perceptual effects of Delta9-tetrahydrocannabinol and other cannabinoids. We found that cat cerebral arterial smooth muscle cells (VSMC) contain the protein for the CB1 receptor and express a cDNA that has >98% amino acid homology to the CB1 cDNA expressed in rat and human neurons. Activation of the CB1 cannabinoid receptor has been shown to decrease the opening of N-type voltage-gated Ca2+ channels in neurons through a pertussis toxin-sensitive GTP-binding protein. In the present study we tested the hypothesis that activation of the cannabinoid CB1 receptor in cerebral VSMC inhibits voltage-gated Ca2+ channels and results in cerebral vasodilation. The predominant Ca2+ current identified in cat cerebral VSMC is a voltage-gated, dihydropyridine-sensitive, L-type Ca2+ current. The cannabimimetic drug WIN-55,212-2 (10-100 nM) induced concentration-dependent inhibition of peak L-type Ca2+ current, which reached a maximum of 82 +/- 4% at 100 nM (n = 14). This effect was mimicked by the putative endogenous CB1-receptor agonist anandamide, which produced a concentration-related reduction of peak L-type Ca2+ current with a maximum inhibition (at 300 nM) of 39 +/- 4% (n = 12). The inhibitory effects of both ligands on peak L-type Ca2+ currents were abolished by pertussis toxin pretreatment and application of the CB1-receptor antagonist SR-141716A (100 nM, n = 5). Both WIN-55,212-2 and anandamide produced concentration-dependent relaxation of preconstricted cerebral arterial segments that was abolished by SR-141716A. These results indicate that the CB1 receptor is expressed in cat cerebral VSMC and that the cerebral vasculature is one of the targets for endogenous cannabinoids. These findings suggest that the CB1 receptor and its endogenous ligand may play a fundamental role in the regulation of cerebral arterial tone and reactivity by modulating the influx of Ca2+ through L-type Ca2+ channels.

Direct suppression of CNS autoimmune inflammation via the cannabinoid receptor CB1 on neurons and CB2 on autoreactive T cells.

The cannabinoid system is immunomodulatory and has been targeted as a treatment for the central nervous system (CNS) autoimmune disease multiple sclerosis. Using an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we investigated the role of the CB1 and CB2 cannabinoid receptors in regulating CNS autoimmunity. We found that CB1 receptor expression by neurons, but not T cells, was required for cannabinoid-mediated EAE suppression. In contrast, CB2 receptor expression by encephalitogenic T cells was critical for controlling inflammation associated with EAE. CB2-deficient T cells in the CNS during EAE exhibited reduced levels of apoptosis, a higher rate of proliferation and increased production of inflammatory cytokines, resulting in severe clinical disease. Together, our results demonstrate that the cannabinoid system within the CNS plays a critical role in regulating autoimmune inflammation, with the CNS directly suppressing T-cell effector function via the CB2 receptor.

Downregulation of Endocannabinoid Signaling in the Hippocampus Following Chronic Unpredictable Stress

Deficits in cognitive functioning and flexibility are seen following both chronic stress and modulation of endogenous cannabinoid (eCB) signaling. Here, we investigated whether alterations in eCB signaling might contribute to the cognitive impairments induced by chronic stress. Chronic stress impaired reversal learning and induced perseveratory behavior in the Morris water maze without significant effect on task acquisition. These cognitive impairments were reversed by exogenous cannabinoid administration, suggesting deficient eCB signaling underlies these phenomena. In line with this hypothesis, chronic stress downregulated CB1 receptor expression and significantly reduced the content of the endocannabinoid 2-arachidonylglycerol within the hippocampus. CB1 receptor density and 2-arachidonylglycerol content were unaffected in the limbic forebrain. These data suggest that stress-induced downregulation of hippocampal eCB signaling contributes to problems in behavioral flexibility and could play a role in the development of perseveratory and ruminatory behaviors in stress-related neuropsychiatric disorders.

Inhibition of an equilibrative nucleoside transporter by cannabidiol: A mechanism of cannabinoid immunosuppression

The plant-derived cannabinoids Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) both have immunosuppressive effects; although some effects of THC are mediated by the CB2 receptor, CB2 binds CBD weakly. In examining the effects of THC and CBD on microglial proliferation, we found that these compounds potently inhibit [3H]thymidine incorporation into a murine microglial cell line with no effect on cell cycle. Treatment with THC and CBD decreased [3H]thymidine uptake into microglia, with IC50 values that match inhibition of [3H]thymidine incorporation into DNA. CBD and, less potently, THC decreased uptake of [3H]adenosine to a similar extent as [3H]thymidine in both murine microglia and RAW264.7 macrophages. Binding studies confirm that CBD binds to the equilibrative nucleoside transporter 1 with a Ki < 250 nM. Because adenosine agonists have antiinflammatory effects, and because uptake of adenosine is a primary mechanism of terminating adenosine signaling, we tested the hypothesis that CBD is immunosuppressive because it enhances endogenous adenosine signaling. In vivo treatment with a low dose of CBD decreases TNFα production in lipopolysaccharide-treated mice; this effect is reversed with an A2A adenosine receptor antagonist and abolished in A2A receptor knockout mice. These studies demonstrate that CBD has the ability to enhance adenosine signaling through inhibition of uptake and provide a non-cannabinoid receptor mechanism by which CBD can decrease inflammation.

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.

Inhibition of restraint stress-induced neural and behavioural activation by endogenous cannabinoid signalling.

The role of endocannabinoid (eCB) signalling in restraint stress‐induced neuronal activation was studied. Male mice exposed to 30 min of restraint exhibit increased Fos protein within prefrontal cortex (PFC), lateral septum (LS), nucleus accumbens (Acb) and medial amygdala. SR141716 (2 mg/kg) itself had no effect on Fos but pretreatment with SR141716 significantly potentiated restraint‐induced Fos expression in cingulate, LS and Acb. SR141716 also significantly increased the time spent in active escape behaviours during the restraint. In restraint‐habituated mice (mice exposed to four previous restraint episodes), the fifth restraint exposure resulted in decreased expression of active escape behaviours compared to the first exposure and only induced Fos protein in the central and medial amygdala. Administration of SR141716 prior to the fifth restraint episode resulted in greater potentiation of restraint‐induced Fos induction than the first; significant increases occurred within all regions of PFC examined, LS and Acb. Brain regional eCB content was measured immediately after restraint. N‐arachidonylethanolamine content within the amygdala was significantly decreased after both restraint episodes. 2‐Arachidonylglycerol content was significantly increased in both the limbic forebrain and amygdala after the fifth restraint but not the first. Restraint had no effect on cerebellar eCB content. These data suggest that eCB activation of CB1 receptors opposes the behavioural and neuronal responses to aversive stimuli. Because repeated homotypic stress increased both limbic 2‐AG and resulted in a greater effect of SR141716 on limbic Fos expression, we hypothesize that increased CB1 receptor activity contributes to the expression of habituation to homotypic stress.