Sharon Anavi-Goffer

Actions of cannabinoid receptor ligands on rat cultured sensory neurones: implications for antinociception

Cannabinoids modulate nociceptive processing in models of acute, inflammatory and neuropathic pain. We have investigated the location and function of cannabinoid receptors on cultured neonatal dorsal root ganglion (DRG) neurones and F-11 cells, a dorsal root ganglionxneuroblastoma hybridoma which displays several of the features of authentic DRG neurones. CB(1) receptor immunolabelling was observed on the cell bodies and as fine puncta on processes of both cultured DRG neurones and F-11 cells. Additionally, fluorescence-activated cell sorting (FACS) analysis provided evidence that both CB(1) and CB(2) receptors are expressed on populations of cells within the cultured DRG and F-11 cells. The cannabinoid receptor agonist (+)-WIN55212 (10 and 100 nM) inhibited the mean voltage-activated Ca(2+) current in DRG neurones by 21% and 30%, respectively. The isomer, (-)-WIN55212 (10 and 100 nM) produced significantly less inhibition of 6% and 10% respectively. The CB(1) selective receptor antagonist SR141716A (100 nM) enhanced the peak high voltage-activated Ca(2+) current by 24% and simultaneous application of SR141716A (100 nM) and (+)-WIN55212 (100 nM) resulted in a significant attenuation of the inhibition obtained with (+)-WIN55212 alone. These data give functional evidence for the hypothesis that the analgesic actions of cannabinoids may be mediated by presynaptic inhibition of transmitter release in sensory neurones.

Localisation of cannabinoid CB1 receptor immunoreactivity in the guinea pig and rat myenteric plexus

Activation of cannabinoid CB1 receptors inhibits gastrointestinal motility, propulsion, and transit, whereas selective antagonism of these receptors has the opposite effects, suggesting the presence of endocannabinoid tone. Supporting evidence for presynaptic CB1 receptors on myenteric neurons has been found in vitro. In this study, selective CB1 receptor antibodies and neuronal markers were used to identify and characterise myenteric neurons expressing cannabinoid receptors. Whole mounts of rat and guinea pig myenteric preparations were dually labelled with antibodies against the CB1 receptor and choline acetyltransferase, neurofilament proteins, calbindin, calretinin, synapsin I, microtubule‐associated protein‐2, calcitonin gene‐related peptide, or substance P. The pattern of CB1 receptor labelling and the neurochemical classification of CB1 receptor‐positive cells were markedly influenced by the species and fixation procedure. Virtually all choline acetyltransferase‐immunoreactive myenteric neurons expressed CB1 receptors in ganglia from both species. Subpopulations of neurons identified with calbindin, calretinin, and microtubule‐associated protein‐2 did not express CB1 receptors. A few calcitonin gene‐related peptide‐ and substance P‐positive somata coexpressed CB1 receptor immunoreactivity but showed little colocalisation on individual fibres. There was a close association between CB1 receptor immunoreactivity and fibres labelled for synaptic protein, suggesting a role in the modulation of transmitter release. Functional responses to cannabinoids in the presence of hexamethonium suggest further that CB1 receptors occur on excitatory motoneurons. In conclusion, CB1 receptors are expressed on a variety of cholinergic sensory, interneuronal, and motor neurons in myenteric ganglia. J. Comp. Neurol. 448:410–422, 2002.

Cannabidiol, a non‐psychotropic component of cannabis, attenuates vomiting and nausea‐like behaviour via indirect agonism of 5‐HT1A somatodendritic autoreceptors in the dorsal raphe nucleus

BACKGROUND AND PURPOSE To evaluate the hypothesis that activation of somatodendritic 5‐HT1A autoreceptors in the dorsal raphe nucleus (DRN) produces the anti‐emetic/anti‐nausea effects of cannabidiol (CBD), a primary non‐psychoactive cannabinoid found in cannabis.

A role for L-α-lysophosphatidylinositol and GPR55 in the modulation of migration, orientation and polarization of human breast cancer cells

Background and purpose:  Increased circulating levels of L‐α‐lysophosphatidylinositol (LPI) are associated with cancer and LPI is a potent, ligand for the G‐protein‐coupled receptor GPR55. Here we have assessed the modulation of breast cancer cell migration, orientation and polarization by LPI and GPR55.

CB1 Receptor Allosteric Modulators Display Both Agonist and Signaling Pathway Specificity

We have previously identified allosteric modulators of the cannabinoid CB1 receptor (Org 27569, PSNCBAM-1) that display a contradictory pharmacological profile: increasing the specific binding of the CB1 receptor agonist [3H]CP55940 but producing a decrease in CB1 receptor agonist efficacy. Here we investigated the effect one or both compounds in a broad range of signaling endpoints linked to CB1 receptor activation. We assessed the effect of these compounds on CB1 receptor agonist–induced [35S]GTPγS binding, inhibition, and stimulation of forskolin-stimulated cAMP production, phosphorylation of extracellular signal-regulated kinases (ERK), and β-arrestin recruitment. We also investigated the effect of these allosteric modulators on CB1 agonist binding kinetics. Both compounds display ligand dependence, being significantly more potent as modulators of CP55940 signaling as compared with WIN55212 and having little effect on [3H]WIN55212 binding. Org 27569 displays biased antagonism whereby it inhibits: agonist-induced guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding, simulation (Gαs-mediated), and inhibition (Gαi-mediated) of cAMP production and β-arrestin recruitment. In contrast, it acts as an enhancer of agonist-induced ERK phosphorylation. Alone, the compound can act also as an allosteric agonist, increasing cAMP production and ERK phosphorylation. We find that in both saturation and kinetic-binding experiments, the Org 27569 and PSNCBAM-1 appeared to influence only orthosteric ligand maximum occupancy rather than affinity. The data indicate that the allosteric modulators share a common mechanism whereby they increase available high-affinity CB1 agonist binding sites. The receptor conformation stabilized by the allosterics appears to induce signaling and also selectively traffics orthosteric agonist signaling via the ERK phosphorylation pathway.

Modulation of l-α-Lysophosphatidylinositol/GPR55 Mitogen-activated Protein Kinase (MAPK) Signaling by Cannabinoids

Background: The endogenous l-α-lysophosphatidylinositol activates GPR55. Results: Structural analogues of SR141716A act both as agonists alone and as inhibitors of l-α-lysophosphatidylinositol. Certain CB2 receptor agonists also modulate GPR55 activity. Conclusion: Certain cannabinoids can both activate GPR55 and attenuate l-α-lysophosphatidylinositol-mediated phosphorylated ERK1/2 activation. This has mechanistic implications for the antinociceptive effects of certain CB2 agonists. Significance: Cannabinoid ligands have complex interactions with the l-α-lysophosphatidylinositol/GPR55 signaling system. GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.

MODULATION OF L-α-LYSOPHOSPHATIDYLINOSITOL /GPR55 MAP KINASE SIGNALLING BY CANNABINOIDS

Background: The endogenous l-α-lysophosphatidylinositol activates GPR55. Results: Structural analogues of SR141716A act both as agonists alone and as inhibitors of l-α-lysophosphatidylinositol. Certain CB2 receptor agonists also modulate GPR55 activity. Conclusion: Certain cannabinoids can both activate GPR55 and attenuate l-α-lysophosphatidylinositol-mediated phosphorylated ERK1/2 activation. This has mechanistic implications for the antinociceptive effects of certain CB2 agonists. Significance: Cannabinoid ligands have complex interactions with the l-α-lysophosphatidylinositol/GPR55 signaling system. GPR55 is activated by l-α-lysophosphatidylinositol (LPI) but also by certain cannabinoids. In this study, we investigated the GPR55 pharmacology of various cannabinoids, including analogues of the CB1 receptor antagonist Rimonabant®, CB2 receptor agonists, and Cannabis sativa constituents. To test ERK1/2 phosphorylation, a primary downstream signaling pathway that conveys LPI-induced activation of GPR55, a high throughput system, was established using the AlphaScreen® SureFire® assay. Here, we show that CB1 receptor antagonists can act both as agonists alone and as inhibitors of LPI signaling under the same assay conditions. This study clarifies the controversy surrounding the GPR55-mediated actions of SR141716A; some reports indicate the compound to be an agonist and some report antagonism. In contrast, we report that the CB2 ligand GW405833 behaves as a partial agonist of GPR55 alone and enhances LPI signaling. GPR55 has been implicated in pain transmission, and thus our results suggest that this receptor may be responsible for some of the antinociceptive actions of certain CB2 receptor ligands. The phytocannabinoids Δ9-tetrahydrocannabivarin, cannabidivarin, and cannabigerovarin are also potent inhibitors of LPI. These Cannabis sativa constituents may represent novel therapeutics targeting GPR55.

Helix 8 Leu in the CB1 cannabinoid receptor contributes to selective signal transduction mechanisms.

The intracellular C-terminal helix 8 (H8) of the CB1 cannabinoid receptor deviates from the highly conserved NPXXY(X)5,6F G-protein-coupled receptor motif, possessing a Leu instead of a Phe. We compared the signal transduction capabilities of CB1 with those of an L7.60F mutation and an L7.60I mutation that mimics the CB2 sequence. The two mutant receptors differed from wild type (WT) in their ability to regulate G-proteins in the [35S]guanosine 5′-3-O-(thio)triphosphate binding assay. The L7.60F receptor exhibited attenuated stimulation by agonists WIN-55,212-2 and CP-55,940 but not HU-210, whereas the L7.60I receptor exhibited impaired stimulation by all agonists tested as well as by the inverse agonist rimonabant. The mutants internalized more rapidly than WT receptors but could equally sequester G-proteins from the somatostatin receptor. Both the time course and maximal N-type Ca2+ current inhibition by WIN-55,212-2 were reduced in the mutants. Reconstitution experiments with pertussis toxin-insensitive G-proteins revealed loss of coupling to Gαi3 but not Gα0A in the L7.60I mutant, whereas the reduction in the time course for the L7.60F mutant was governed by Gαi3. Furthermore, Gαi3 but not Gα0A enhanced basal facilitation ratio, suggesting that Gαi3 is responsible for CB1 tonic activity. Co-immunoprecipitation studies revealed that both mutant receptors were associated with Gαi1 or Gαi2 but not with Gαi3. Molecular dynamics simulations of WT CB1 receptor and each mutant in a 1-palmitoyl-2-oleoylphosphatidylcholine bilayer suggested that the packing of H8 is different in each. The hydrogen bonding patterns along the helix backbones of each H8 also are different, as are the geometries of the elbow region of H8 (R7.56(400)-K7.58(402)). This study demonstrates that the evolutionary modification to NPXXY(X)5,6L contributes to maximal activity of the CB1 receptor and provides a molecular basis for the differential coupling observed with chemically different agonists.

Cellular distribution of vanilloid VR1 receptor immunoreactivity in the guinea-pig myenteric plexus.

Recent investigations suggest that vanilloid receptor-1 (VR1) immunoreactivity occurs in the intestine. We have determined and quantified this immunoreactivity in the myenteric plexus with respect to cholinergic and neurofilament protein-positive neurones. Guinea-pig and rat preparations were dual-labelled with specific antibodies raised in rabbit or goat against vanilloid receptor-1 and against other neurochemical markers. In the rat ileum, both vanilloid receptor antibodies were co-distributed, whereas in the guinea-pig ileum and colon, tertiary fibres were also detected with the goat antibody. In the guinea-pig, all vanilloid receptor-1-immunoreactive cell bodies were choline acetyltransferase-immunopositive (100%) and showed some immunoreactivity to neurofilament proteins (NFP-200 kDa (79%) or triplet (10.8%)) or calretinin. Immunoreactive fibres in the secondary plexus co-localised with calcitonin gene-related peptide (CGRP) and with substance P, calretinin and synapsin I in the tertiary plexus. Subpopulations of cholinergic neurones including sensory, interneuronal and secretory neurones express vanilloid receptor-1. Co-localisation with substance P and calretinin in fibres suggests that vanilloid receptor-1 may be expressed by excitatory motor neurones. The association of vanilloid receptors with calcitonin gene-related peptide and synaptic protein in fibres implies a role for vanilloid receptors in neurotransmitter/neuropeptide release. Although it is likely that at least some of the vanilloid receptor-bearing fibres originate in immunopositive myenteric soma, the origin of all these fibres cannot be identified in the present study.

Vanilloid receptor type 1-immunoreactivity is expressed by intrinsic afferent neurones in the guinea-pig myenteric plexus

The enteric sensory nervous system consists of extrinsic and intrinsic components. The cellular distribution of vanilloid receptor type 1 (VR1) and its relation to the intrinsic sensory neurones were studied in myenteric plexus-longitudinal muscle preparations of rat ileum and guinea-pig ileum and colon. VR1-immunoreactivity was localized on fine fibres and expressed by ganglionic cells. In the guinea-pig myenteric plexus, a proportion of VR1-immunoreactive cells co-localized with calbindin, a marker for intrinsic afferent neurones. Reverse transcription-polymerase chain reaction with rat VR1-specific primers detected VR1 mRNA in rat but not in guinea-pig preparations. We conclude that VR1 is expressed on fibres and by myenteric neurones. In the guinea-pig, VR1 is expressed by intrinsic afferent neurones but its mRNA may differ from the rat sequence in the region of the primers.