Ruth A. Ross

Anandamide and vanilloid TRPV1 receptors

A large body of evidence now exists to substantiate that the endocannabinoid, anandamide, activates TRPV1 receptors. It is a low intrinsic efficacy TRPV1 agonist that behaves as a partial agonist in tissues with a low receptor reserve, while in tissues with high receptor reserve and in circumstances associated with certain disease states, it behaves as a full agonist. The efficacy of anandamide as a TRPV1 agonist is influenced by a succession of factors including receptor reserve, phosphorylation, metabolism and uptake, CB1 receptor activation, voltage, temperature, pH and bovine serum albumin. There are indications that the endocannabinoid system may play a role in the modulation of TRPV1 receptor activation. The activation of TRPV1 receptors by anandamide has potential implications in the treatment of inflammatory, respiratory and cardiovascular disorders. The relative importance of anandamide as a physiological and/or pathophysiological TRPV1 receptor agonist in comparison to other potential candidates has yet to be revealed.

Agonist‐inverse agonist characterization at CB1 and CB2 cannabinoid receptors of L759633, L759656 and AM630

We have tested our prediction that AM630 is a CB2 cannabinoid receptor ligand and also investigated whether L759633 and L759656, are CB2 receptor agonists. Binding assays with membranes from CHO cells stably transfected with human CB1 or CB2 receptors using [3H]‐CP55940, confirmed the CB2‐selectivity of L759633 and L759656 (CB2/CB1 affinity ratios=163 and 414 respectively) and showed AM630 to have a Ki at CB2 receptors of 31.2 nM and a CB2/CB1 affinity ratio of 165. In CB2‐transfected cells, L759633 and L759656 were potent inhibitors of forskolin‐stimulated cyclic AMP production, with EC50 values of 8.1 and 3.1 nM respectively and CB1/CB2 EC50 ratios of >1000 and >3000 respectively. AM630 inhibited [35S]‐GTPγS binding to CB2 receptor membranes (EC50=76.6 nM), enhanced forskolin‐stimulated cyclic AMP production in CB2‐transfected cells (5.2 fold by 1 μM), and antagonized the inhibition of forskolin‐stimulated cyclic AMP production in this cell line induced by CP55940. In CB1‐transfected cells, forskolin‐stimulated cyclic AMP production was significantly inhibited by AM630 (22.6% at 1 μM and 45.9% at 10 μM) and by L759633 at 10 μM (48%) but not 1 μM. L759656 (10 μM) was not inhibitory. AM630 also produced a slight decrease in the mean inhibitory effect of CP55940 on cyclic AMP production which was not statistically significant. We conclude that AM630 is a CB2‐selective ligand that behaves as an inverse agonist at CB2 receptors and as a weak partial agonist at CB1 receptors. L759633 and L759656 are both potent CB2‐selective agonists.

Hardwiring the Brain: Endocannabinoids Shape Neuronal Connectivity

The roles of endocannabinoid signaling during central nervous system development are unknown. We report that CB1 cannabinoid receptors (CB1Rs) are enriched in the axonal growth cones of γ-aminobutyric acid–containing (GABAergic) interneurons in the rodent cortex during late gestation. Endocannabinoids trigger CB1R internalization and elimination from filopodia and induce chemorepulsion and collapse of axonal growth cones of these GABAergic interneurons by activating RhoA. Similarly, endocannabinoids diminish the galvanotropism of Xenopus laevis spinal neurons. These findings, together with the impaired target selection of cortical GABAergic interneurons lacking CB1Rs, identify endocannabinoids as axon guidance cues and demonstrate that endocannabinoid signaling regulates synaptogenesis and target selection in vivo.

Cannabidiol displays unexpectedly high potency as an antagonist of CB1 and CB2 receptor agonists in vitro

A nonpsychoactive constituent of the cannabis plant, cannabidiol has been demonstrated to have low affinity for both cannabinoid CB1 and CB2 receptors. We have shown previously that cannabidiol can enhance electrically evoked contractions of the mouse vas deferens, suggestive of inverse agonism. We have also shown that cannabidiol can antagonize cannabinoid receptor agonists in this tissue with a greater potency than we would expect from its poor affinity for cannabinoid receptors. This study aimed to investigate whether these properties of cannabidiol extend to CB1 receptors expressed in mouse brain and to human CB2 receptors that have been transfected into CHO cells.

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Structure-activity relationship for the endogenous cannabinoid, anandamide, and certain of its analogues at vanilloid receptors in transfected cells and vas deferens.

This study was directed at exploring the structure‐activity relationship for anandamide and certain of its analogues at the rat VR1 receptor in transfected cells and at investigating the relative extent to which anandamide interacts with CB1 and vanilloid receptors in the mouse vas deferens. pKi values for displacement of [3H]‐resiniferatoxin from membranes of rVR1 transfected CHO cells were significantly less for anandamide (5.78) than for its structural analogues N‐(4‐hydroxyphenyl)‐arachidonylamide (AM404; 6.18) and N‐(3‐methoxy‐4‐hydroxy)benzyl‐arachidonylamide (arvanil; 6.77). pEC50 values for stimulating 45Ca2+ uptake into rVR1 transfected CHO cells were significantly less for anandamide (5.80) than for AM404 (6.32) or arvanil (9.29). Arvanil was also significantly more potent than capsaicin (pEC50=7.37), a compound with the same substituted benzyl polar head group as arvanil. In the mouse vas deferens, resiniferatoxin was 218 times more potent than capsaicin as an inhibitor of electrically‐evoked contractions. Both drugs were antagonized to a similar extent by capsazepine (pKB=6.93 and 7.18 respectively) but were not antagonized by SR141716A (1 μM). Anandamide was less susceptible than capsaicin to antagonism by capsazepine (pKB=6.02) and less susceptible to antagonism by SR141716A (pKB=8.66) than methanandamide (pKB=9.56). WIN55212 was antagonized by SR141716A (pKB=9.02) but not by capsazepine (10 μM). In conclusion, anandamide and certain of its analogues have affinity and efficacy at the rat VR1 receptor. In the mouse vas deferens, which seems to express vanilloid and CB1 receptors, both receptor types appear to contribute to anandamide‐induced inhibition of evoked contractions.

The GPR55 ligand l-α-lysophosphatidylinositol promotes RhoA-dependent Ca2+ signaling and NFAT activation

The endogenous phospholipid l‐α‐lyso‐phosphatidylinositol (LPI) was recently identified as a novel ligand for the orphan G protein‐coupled receptor 55 (GPR55). In this study we define the downstream signaling pathways activated by LPI in a human embryonic kidney (HEK) 293 cell line engineered to stably express recombinant human GPR55. We find that treatment with LPI induces marked GPR55 internalization and stimulates a sustained, oscillatory Ca2+ release pathway, which is dependent on Gα13 and requires RhoA activation. We then establish that this signaling cascade leads to the efficient activation of NFAT (nu‐clear factor of activated T cells) family transcription factors and their nuclear translocation. Analysis of cannabinoid ligand activity at GPR55 revealed no clear effect of the endocannabinoids anandamide and 2‐arachidonoylglycerol;however, the classical CB1 antagonist AM251 evoked GPR55‐mediated Ca2+ signaling. Thus, LPI is a potent and efficacious ligand at GPR55, which is likely to be a key plasma membrane mediator of LPI‐mediated signaling events and changes in gene expression.—Henstridge, C. M., Balenga, N. A. B., Ford, L. A., Ross, R. A., Waldhoer, M., Irving, A. J. The GPR55 ligand l‐α‐lysophosphatidylinositol promotes RhoA‐dependent Ca2+ signaling and NFAT activation. FASEB J. 23, 183‐193 (2009)

The putative cannabinoid receptor GPR55 affects osteoclast function in vitro and bone mass in vivo

GPR55 is a G protein-coupled receptor recently shown to be activated by certain cannabinoids and by lysophosphatidylinositol (LPI). However, the physiological role of GPR55 remains unknown. Given the recent finding that the cannabinoid receptors CB1 and CB2 affect bone metabolism, we examined the role of GPR55 in bone biology. GPR55 was expressed in human and mouse osteoclasts and osteoblasts; expression was higher in human osteoclasts than in macrophage progenitors. Although the GPR55 agonists O-1602 and LPI inhibited mouse osteoclast formation in vitro, these ligands stimulated mouse and human osteoclast polarization and resorption in vitro and caused activation of Rho and ERK1/2. These stimulatory effects on osteoclast function were attenuated in osteoclasts generated from GPR55−/− macrophages and by the GPR55 antagonist cannabidiol (CBD). Furthermore, treatment of mice with this non-psychoactive constituent of cannabis significantly reduced bone resorption in vivo. Consistent with the ability of GPR55 to suppress osteoclast formation but stimulate osteoclast function, histomorphometric and microcomputed tomographic analysis of the long bones from male GPR55−/− mice revealed increased numbers of morphologically inactive osteoclasts but a significant increase in the volume and thickness of trabecular bone and the presence of unresorbed cartilage. These data reveal a role of GPR55 in bone physiology by regulating osteoclast number and function. In addition, this study also brings to light an effect of both the endogenous ligand, LPI, on osteoclasts and of the cannabis constituent, CBD, on osteoclasts and bone turnover in vivo.

Evidence for the presence of CB2-like cannabinoid receptors on peripheral nerve terminals.

We have investigated whether there are cannabinoid CB2 receptors that can mediate cannabinoid-induced inhibition of electrically evoked contractions in the mouse vas deferens or guinea-pig myenteric plexus-longitudinal muscle preparation. Our results showed that mouse vas deferens and guinea-pig whole gut contain cannabinoid CB1 and CB2-like mRNA whereas the myenteric plexus preparation seemed to contain only cannabinoid CB1 mRNA. JWH-015 (1-propyl-2-methyl-3-( -naphthoyl)indole) and JWH-051 (1-deoxy-11-hydroxy-delta8-tetrahydrocannabinol-dimethylheptyl+ ++), which have higher affinities for CB2 than CB1 cannabinoid binding sites, inhibited electrically evoked contractions of both tissues in a concentration related manner. This inhibition was attenuated by 31.62 nM of the cannabinoid CB1 receptor selective antagonist SR141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-me thyl-1H-pyrazole-3-carboxamide hydrochloride] only in the myenteric plexus preparation. Vasa deferentia from delta9-tetrahydrocannabinol-pretreated mice (20 mg/kg i.p. once daily for two days) showed reduced sensitivity to JWH-015 and JWH-051. The results suggest that these compounds exert their inhibitory effects through cannabinoid CB1 receptors in the myenteric plexus preparation, but mainly through CB2-like cannabinoid receptors in the vas deferens.

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.