Maurice R. Elphick

International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid Receptors and Their Ligands: Beyond CB1 and CB2

There are at least two types of cannabinoid receptors (CB1 and CB2). Ligands activating these G protein-coupled receptors (GPCRs) include the phytocannabinoid Δ9-tetrahydrocannabinol, numerous synthetic compounds, and endogenous compounds known as endocannabinoids. Cannabinoid receptor antagonists have also been developed. Some of these ligands activate or block one type of cannabinoid receptor more potently than the other type. This review summarizes current data indicating the extent to which cannabinoid receptor ligands undergo orthosteric or allosteric interactions with non-CB1, non-CB2 established GPCRs, deorphanized receptors such as GPR55, ligand-gated ion channels, transient receptor potential (TRP) channels, and other ion channels or peroxisome proliferator-activated nuclear receptors. From these data, it is clear that some ligands that interact similarly with CB1 and/or CB2 receptors are likely to display significantly different pharmacological profiles. The review also lists some criteria that any novel “CB3” cannabinoid receptor or channel should fulfil and concludes that these criteria are not currently met by any non-CB1, non-CB2 pharmacological receptor or channel. However, it does identify certain pharmacological targets that should be investigated further as potential CB3 receptors or channels. These include TRP vanilloid 1, which possibly functions as an ionotropic cannabinoid receptor under physiological and/or pathological conditions, and some deorphanized GPCRs. Also discussed are 1) the ability of CB1 receptors to form heteromeric complexes with certain other GPCRs, 2) phylogenetic relationships that exist between CB1/CB2 receptors and other GPCRs, 3) evidence for the existence of several as-yet-uncharacterized non-CB1, non-CB2 cannabinoid receptors; and 4) current cannabinoid receptor nomenclature.

The Endocannabinoid System Controls Key Epileptogenic Circuits in the Hippocampus

Balanced control of neuronal activity is central in maintaining function and viability of neuronal circuits. The endocannabinoid system tightly controls neuronal excitability. Here, we show that endocannabinoids directly target hippocampal glutamatergic neurons to provide protection against acute epileptiform seizures in mice. Functional CB1 cannabinoid receptors are present on glutamatergic terminals of the hippocampal formation, colocalizing with vesicular glutamate transporter 1 (VGluT1). Conditional deletion of the CB1 gene either in cortical glutamatergic neurons or in forebrain GABAergic neurons, as well as virally induced deletion of the CB1 gene in the hippocampus, demonstrate that the presence of CB1 receptors in glutamatergic hippocampal neurons is both necessary and sufficient to provide substantial endogenous protection against kainic acid (KA)-induced seizures. The direct endocannabinoid-mediated control of hippocampal glutamatergic neurotransmission may constitute a promising therapeutic target for the treatment of disorders associated with excessive excitatory neuronal activity.

Localisation of cannabinoid receptors in the rat brain using antibodies to the intracellular C-terminal tail of CB1

The CB1‐type cannabinoid receptor mediates physiologic effects of Δ9‐tetrahydrocannabinol, the psychoactive ingredient of the drug marijuana. In this report, the authors analyse the expression of CB1 in the rat brain by using antibodies to the C‐terminal 13 amino acids of the receptor. Western blot analysis of rat brain membranes revealed a prominent immunoreactive band with a molecular mass (≈53 kDa) consistent with that predicted for CB1 from the rat cDNA sequence. In addition, however, less intense immunoreactive bands corresponding to glycosylated (≈62 kDa) and putative N‐terminally shorter (≈45 kDa) isoforms of CB1 were detected. The distribution of CB1‐immunoreactivity in rat brain was similar to the distribution of binding sites for radiolabelled cannabinoids, with high levels of expression in the olfactory system, the hippocampal formation, the basal ganglia, the cerebellum, and the neocortex. This provides important evidence that CB1 is likely to be largely responsible for mediating effects of cannabinoids in the brain. CB1 immunoreactivity was associated with nerve fibre systems and axon terminals but was not detected in neuronal somata. This is consistent with the presynaptic inhibitory effects of cannabinoids on neurotransmitter release in the brain. Detailed immunocytochemical analysis of anatomically or functionally related regions of the brain revealed the location of CB1 receptors within identified neural circuits. Determination of the cellular and subcellular location of CB1 within known neuronal circuits of the brain provides an anatomic framework for interpretation of the neurophysiologic and behavioural effects of cannabinoids. J. Comp. Neurol. 422:159–171, 2000.

A NEW PERSPECTIVE ON CANNABINOID SIGNALLING : COMPLEMENTARY LOCALIZATION OF FATTY ACID AMIDE HYDROLASE AND THE CB1 RECEPTOR IN RAT BRAIN

CB1–type cannabinoid receptors in the brain mediate effects of the drug cannabis. Anandamide and sn–2 arachidonylglycerol (2–AG) are putative endogenous ligands for CB1 receptors, but it is not known which cells in the brain produce these molecules. Recently, an enzyme which catalyses hydrolysis of anandamide and 2–AG, known as fatty acid amide hydrolase (FAAH), was identified in mammals. Here we have analysed the distribution of FAAH in rat brain and compared its cellular localization with CB1–type cannabinoid receptors using immunocytochemistry. High concentrations of FAAH activity were detected in the cerebellum, hippocampus and neocortex, regions of the rat brain which are enriched with cannabinoid receptors. Immunocytochemical analysis of these brain regions revealed a complimentary pattern of FAAH and CB1 expression with CB1–immunoreactivity occurring in fibres surrounding FAAH–immunoreactive cell bodies and/or dendrites. In the cerebellum, FAAH was expressed in the cell bodies of Purkinje cells and CB1 was expressed in the axons of granule cells and basket cells, neurons which are presynaptic to Purkinje cells. The close correspondence in the distribution of FAAH and CB1 in rat brain and the complimentary pattern of FAAH and CB1 expression at the cellular level provides important new evidence that FAAH may participate in cannabinoid signalling mechanisms of the brain.

Cannabinoid CB1 receptor expression in rat spinal cord

While evidence implicates the endogenous cannabinoid system as a novel analgesic target at a spinal level, detailed analysis of the distribution of the cannabinoid receptor CB1 in spinal cord has not been reported. Here, immunocytochemical studies were used to characterize the CB1 receptor expression in rat spinal cord. Staining was found in the dorsolateral funiculus, the superficial dorsal horn (a double band of CB1 immunoreactivity (ir) in laminae I and II inner/III transition), and lamina X. Although CB1-ir was present in the same laminae as primary afferent nociceptor markers, there was limited colocalization at an axonal level. Interruption of both primary afferent input by dorsal root rhizotomy and descending input by rostral spinal cord hemisection produced minor changes in CB1-ir. This and colocalization of CB1-ir with interneurons expressing protein kinase C subunit γ-ir suggest that the majority of CB1 expression is on spinal interneurons. These data provide a framework and implicate novel analgesic mechanisms for spinal actions of cannabinoids at the CB1 receptor.

Localisation of cannabinoid receptor 1 in rat dorsal root ganglion using in situ hybridisation and immunohistochemistry

In this study we used in situ hybridisation and double-labelling immunohistochemistry to characterise cannabinoid receptor 1 (CB(1)) expression in rat lumbar dorsal root ganglion (DRG) neurons.Approximately 25% of DRG neurons expressed CB(1) mRNA and displayed immunoreactivity for CB(1). Sixty-nine percent to 82% of CB(1)-expressing cells were also immunoreactive for neurofilament 200, indicative of myelinated A-fibre neurons, which tend to be large- and medium-sized DRG neurons (>600 microm(2)). Approximately 10% of CB1-expressing cells also expressed transient receptor potential vanilloid family ion channel 2 (TRPV2), the noxious heat-transducing channel found in medium to large lightly myelinated Adelta-fibre DRG neurons. Seventeen percent to 26% of CB(1)-expressing cells co-stained using Isolectin B4, 9-10% for calcitonin gene-related peptide and 11-20% for transient receptor potential vanilloid family ion channel 1 (TRPV1), predominantly markers of small non-myelinated C-fibre DRG neurons (<600 microm(2)). These findings suggest that whilst a wide range of DRG neuron phenotypes express CB(1), it is predominantly associated with myelinated fibres.

Nitric oxide synthesis and action in an invertebrate brain

Nitric oxide (NO) is synthesized in mammalian neurons by Ca2+/calmodulin activated NO synthase and functions as a signalling molecule by activating soluble guanylyl cyclases in target cells. We demonstrate here that both NO synthase and NO-activated guanylyl cyclase are present in the brain of the locust Schistocerca gregaria. Our observations indicate, for the first time, that the NO-cyclic GMP signalling pathway exists in invertebrate nervous systems.

MRAP and MRAP2 are bidirectional regulators of the melanocortin receptor family

The melanocortin receptor (MCR) family consists of 5 G protein-coupled receptors (MC1R–MC5R) with diverse physiologic roles. MC2R is a critical component of the hypothalamic–pituitary–adrenal axis, whereas MC3R and MC4R have an essential role in energy homeostasis. Mutations in MC4R are the single most common cause of monogenic obesity. Investigating the way in which these receptors signal and traffic to the cell membrane is vital in understanding disease processes related to MCR dysfunction. MRAP is an MC2R accessory protein, responsible for adrenal MC2R trafficking and function. Here we identify MRAP2 as a unique homologue of MRAP, expressed in brain and the adrenal gland. We report that MRAP and MRAP2 can interact with all 5 MCRs. This interaction results in MC2R surface expression and signaling. In contrast, MRAP and MRAP2 can reduce MC1R, MC3R, MC4R, and MC5R responsiveness to [Nle4,D-Phe7]alpha-melanocyte-stimulating hormone (NDP-MSH). Collectively, our data identify MRAP and MRAP2 as unique bidirectional regulators of the MCR family.

Minocycline treatment inhibits microglial activation and alters spinal levels of endocannabinoids in a rat model of neuropathic pain

Activation of spinal microglia contributes to aberrant pain responses associated with neuropathic pain states. Endocannabinoids (ECs) are present in the spinal cord, and inhibit nociceptive processing; levels of ECs may be altered by microglia which modulate the turnover of endocannabinoids in vitro. Here, we investigate the effect of minocycline, an inhibitor of activated microglia, on levels of the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and the related compound N-palmitoylethanolamine (PEA), in neuropathic spinal cord. Selective spinal nerve ligation (SNL) in rats resulted in mechanical allodynia and the presence of activated microglia in the ipsilateral spinal cord. Chronic daily treatment with minocycline (30 mg/kg, ip for 14 days) significantly reduced the development of mechanical allodynia at days 5, 10 and 14 post-SNL surgery, compared to vehicle-treated SNL rats (P < 0.001). Minocycline treatment also significantly attenuated OX-42 immunoreactivity, a marker of activated microglia, in the ipsilateral (P < 0.001) and contralateral (P < 0.01) spinal cord of SNL rats, compared to vehicle controls. Minocycline treatment significantly (P < 0.01) decreased levels of 2-AG and significantly (P < 0.01) increased levels of PEA in the ipsilateral spinal cord of SNL rats, compared to the contralateral spinal cord. Thus, activation of microglia affects spinal levels of endocannabinoids and related compounds in neuropathic pain states.

The SALMFAmides: A New Family of Neuropeptides Isolated from an Echinoderm

We have isolated two novel related neuropeptides from the radial nerve cords of the starfishes Asterias rubens and Asterias forbesi. One is an octapeptide with the amino acid sequence Gly-Phe-Asn-Ser-Ala-Leu-Met-Phe-NH2 and the other is a dodecapeptide with the amino acid sequence Ser-Gly-Pro-Tyr-Ser-Phe-Asn-Ser-Gly-Leu-Thr-Phe-NH2. The peptides were purified using high performance liquid chromatography (HPLC) and a radioimmunoassay for the molluscan FMRFamide-related neuropeptide, pQDPFLRFamide. Both peptides share minimal sequence identity with members of the family of FMRFamide-like peptides so we have designated them as founder members of a new family, the SALMFamides. We refer to the octapeptide as SALMFamide 1 (S1) and the dodecapeptide as SALMFamide 2 (S2). S1 and S2 are the first neuropeptides identified in species belonging to the phylum Echinodermata.