Cannabinoid WIN 55,212-2 regulates TRPV1 phosphorylation in sensory neurons

Cannabinoids are known to have multiple sites of action in the nociceptive system, leading to reduced pain sensation. However, the peripheral mechanism(s) by which this phenomenon occurs remains an issue that has yet to be resolved. Because phosphorylation of TRPV1 (transient receptor potential subtype V1) plays a key role in the induction of thermal hyperalgesia in inflammatory pain models, we evaluated whether the cannabinoid agonist WIN 55,212-2 (WIN) regulates the phosphorylation state of TRPV1. Here, we show that treatment of primary rat trigeminal ganglion cultures with WIN led to dephosphorylation of TRPV1, specifically at threonine residues. Utilizing Chinese hamster ovary cell lines, we demonstrate that Thr(144) and Thr(370) were dephosphorylated, leading to desensitization of the TRPV1 receptor. This post-translational modification occurred through activation of the phosphatase calcineurin (protein phosphatase 2B) following WIN treatment. Furthermore, knockdown of TRPA1 (transient receptor potential subtype A1) expression in sensory neurons by specific small interfering RNA abolished the WIN effect on TRPV1 dephosphorylation, suggesting that WIN acts through TRPA1. We also confirm the importance of TRPA1 in WIN-induced dephosphorylation of TRPV1 in Chinese hamster ovary cells through targeted expression of one or both receptor channels. These results imply that the cannabinoid WIN modulates the sensitivity of sensory neurons to TRPV1 activation by altering receptor phosphorylation. In addition, our data could serve as a useful strategy in determining the potential use of certain cannabinoids as peripheral analgesics.     

Investigation of non-CB1, non-CB2 WIN55212-2-sensitive G-protein-coupled receptors in the brains of mammals,birds, and amphibians

Abstract

Purpose: Previous studies have found non-CB₁ non-CB₂ G-protein-coupled receptors in rodents that are activated by the aminoalkylindole cannabinoid agonist WIN55212-2. This work obtained evidence for the presence or absence of similar receptors in the brains of other mammals, birds and amphibians.

Materials and methods: Antagonism of the stimulation of [³⁵S]GTPγS binding by WIN55212-2 and CP55940 was assessed in multiple CNS regions of rat and canine, and in whole brain membranes from shrew, pigeon, frog and newt. A bioinformatics approach searched for orthologs of GRP3, GPR6, and GPR12 (closely related to cannabinoid receptors) in the genomes of these or related species. Orthologs were examined for amino acid motifs known to impart functionality to receptors.

Results: In mammals and pigeon, but not amphibians, a significant fraction of the stimulation of [³⁵S]GTPγS binding by WIN55212-2 was not blocked by the CB₁ antagonist SR141716A. BLAST searches found that GPR3 was restricted to mammals. GPR12 orthologs existed in all species, and they shared identical amino acid motifs. GPR6 orthologs existed all species, but with significant departures in the identity of some critical amino acids in bird, more so in amphibian.

Conclusions: The portion of WIN55212-2-stimulated [³⁵S]GTPγS binding that was antagonized by SR141716A was consistent with stimulation via CB₁ receptors, indicating that antagonist-insensitive activity was via a different G-protein coupled receptor. Pharmacological evidence of this receptor was found in the brains of mammals and pigeon, but not frog or newt. Bioinfomatics results implicate GPR6 as a possible candidate for the additional WIN55212-2-sensitive receptor.     

Protective effects of CB1 receptor agonist WIN 55.212-2 in seizure activity in the model of temporal lobe epilepsy

The influence of a low dose (1 microM, 2 microl) of nonselective agonist of cannabinoid CB1 receptor WIN 55.212-2 on seizure activity in different brain structures was investigated in waking guinea pigs. Changes in spontaneous local field potentials and seizure afterdischarges evoked by the electrical stimulation of the perforant path were recorded simultaneously in the hippocampus, entorhinal cortex, medial septal region, and amygdala after a preliminary intraventricular injection of WIN 55.212-2. It was found that WIN 55.212-2 blocked the stimulation-elicited seizures in 80% of tests. A repeated injection of the agonist within 30 days caused an increase in the amplitude of local field potentials and the power of the theta rhythm in all the structures under study. The infusion of kainic acid provoked the onset of status epilepticus in control animals, whereas guinea pigs injected with the agonist (daily, during 25-30 days) did not develop the status. Possible mechanisms of the protective influence of WIN 55.212-2 are discussed.     

Kidney Dysfunction in Adult Offspring Exposed In Utero to Type 1 Diabetes Is Associated with Alterations in Genome-Wide DNA Methylation

Background

Fetal exposure to hyperglycemia impacts negatively kidney development and function.

Objective

Our objective was to determine whether fetal exposure to moderate hyperglycemia is associated with epigenetic alterations in DNA methylation in peripheral blood cells and whether those alterations are related to impaired kidney function in adult offspring.

Design

Twenty nine adult, non-diabetic offspring of mothers with type 1 diabetes (T1D) (case group) were matched with 28 offspring of T1D fathers (control group) for the study of their leukocyte genome-wide DNA methylation profile (27,578 CpG sites, Human Methylation 27 BeadChip, Illumina Infinium). In a subset of 19 cases and 18 controls, we assessed renal vascular development by measuring Glomerular Filtration Rate (GFR) and Effective Renal Plasma Flow (ERPF) at baseline and during vasodilatation produced by amino acid infusion.

Results

Globally, DNA was under-methylated in cases vs. controls. Among the 87 CpG sites differently methylated, 74 sites were less methylated and 13 sites more methylated in cases vs. controls. None of these CpG sites were located on a gene known to be directly involved in kidney development and/or function. However, the gene encoding DNA methyltransferase 1 (DNMT1)—a key enzyme involved in gene expression during early development–was under-methylated in cases. The average methylation of the 74 under-methylated sites differently correlated with GFR in cases and controls.

Conclusion

Alterations in methylation profile imprinted by the hyperglycemic milieu of T1D mothers during fetal development may impact kidney function in adult offspring. The involved pathways seem to be a nonspecific imprinting process rather than specific to kidney development or function.     

Acute effects of the cannabinoid receptor agonist WIN55212-2 on dopamine release in rat: an in vivo electrochemical study

The aim of this study was to investigate the effects of the cannabinoid receptor agonist, WIN55212-2, and the cannabinoid receptor antagonist, SR141716A, on dopamine (DA) release evoked by KC1 (120 mM) microinjected into the striatum. The cannabinoid agonist WIN55212-2 (1 and 5 mg/kg, i.p.) dose-dependently attenuated DA release in the striatum, whereas the cannabinoid receptor antagonist SR141716A (3 mg/kg, i.p.) produced the opposite effect. SR141716A (3 mg/kg, i.p.) blocked the effects on DA release by WIN55212-2 (5 mg/kg, i.p.). Vehicle alone did not change DA release. These results suggest that cannabinoids modulate DA release in the striatum.     

Negative Regulation of Leptin-induced Reactive Oxygen Species (ROS) Formation by Cannabinoid CB1 Receptor Activation in Hypothalamic Neurons

The adipocyte-derived, anorectic hormone leptin was recently shown to owe part of its regulatory effects on appetite-regulating hypothalamic neuropeptides to the elevation of reactive oxygen species (ROS) levels in arcuate nucleus (ARC) neurons. Leptin is also known to exert a negative regulation on hypothalamic endocannabinoid levels and hence on cannabinoid CB₁ receptor activity. Here we investigated the possibility of a negative regulation by CB₁ receptors of leptin-mediated ROS formation in the ARC. Through pharmacological and molecular biology experiments we report data showing that leptin-induced ROS accumulation is 1) blunted by arachidonyl-2′-chloroethylamide (ACEA) in a CB₁-dependent manner in both the mouse hypothalamic cell line mHypoE-N41 and ARC neuron primary cultures, 2) likewise blocked by a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist, troglitazone, in a manner inhibited by T0070907, a PPAR-γ antagonist that also inhibited the ACEA effect on leptin, 3) blunted under conditions of increased endocannabinoid tone due to either pharmacological or genetic inhibition of endocannabinoid degradation in mHypoE-N41 and primary ARC neuronal cultures from MAGL^−/− mice, respectively, and 4) associated with reduction of both PPAR-γ and catalase activity, which are reversed by both ACEA and troglitazone. We conclude that CB₁ activation reverses leptin-induced ROS formation and hence possibly some of the ROS-mediated effects of the hormone by preventing PPAR-γ inhibition by leptin, with subsequent increase of catalase activity. This mechanism might underlie in part CB1 orexigenic actions under physiopathological conditions accompanied by elevated hypothalamic endocannabinoid levels.     

A CB2 Receptor Agonist Reduces the Production of Inflammatory Mediators and Improves Locomotor Activity in Experimental Autoimmune Encephalomyelitis

Background:Cannabinoids (CBs) have been found to regulate the immune system, affect innate and adaptive immune responses, and reduce inflammatory reactions. This study assessed the therapeutic effects of GW-405833 synthetic CB2 agonist on inflammatory factors as well as locomotor activity in experimental autoimmune encephalomyelitis (EAE).Methods:In this experimental study, 48 adult male C57BL/6 mice were randomly and equally assigned to eight groups. By injecting 250 mg of MOG35-55 peptide, EAE was induced. Every other day for 17 days after EAE onset, EAE-afflicted mice in groups 1–3 received an intraperitoneal injection of GW-405833 at a dose of 3, 10, and 30 mg/kg, respectively. Clinical status and locomotor activity, measured using the beam walking assay, were assessed every other day during the first 17 days after EAE onset. Mice were euthanized in day 17th of treatment and the serum levels of the IL-1β, IL-12, CRP, and TNF-α proinflammatory cytokines as well as IL-4 and TGF-β anti-inflammatory cytokines were measured by ELISA method.Results:Clinical manifestations of EAE in groups 2 and 3 were significantly milder than group 4 and locomotor activity in groups 1–3 was significantly better than group 4 in days 5–17 (p< 0.05). GW-405833 also significantly decreased the levels of IL-12, TNF-α, and CRP and significantly increased the levels of IL-4 and TGF-β but had no significant effects on the level of IL-1β. GW-405833 was not associated with significant side effects.Conclusion:The CB2 receptor agonist GW-405833, improves clinical conditions and reduces inflammation in mice with EAE.Key Words: Clinical evaluation, Experimental autoimmune encephalomyelitis, GW-405833, Locomotor activity, Multiple sclerosis, Proinflammatory cytokines     

Agonist efficacy and receptor efficiency in heterozygous CB1 knockout mice: relationship of reduced CB1 receptor density to G-protein activation

Heterozygous CB1 receptor knockout mice were used to examine the effect of reduced CB1 receptor density on G-protein activation in membranes prepared from four brain regions: cerebellum, hippocampus, striatum/globus pallidus (striatum/GP) and cingulate cortex. Results showed that CB1 receptor levels were approximately 50% lower in heterozygous mice in all regions examined. However, maximal stimulation of [(35)S]guanosine-5'-(gamma-O-thio) triphosphate ([(35)S]GTPgammaS) binding by the high efficacy agonist WIN 55,212-2 was reduced by only 20-25% in most brain regions, with the exception of striatum/GP where the decrease in stimulation was as predicted (approximately 50%). Furthermore, although the efficacies of the cannabinoid partial agonists, methanandamide and (9)-tetrahydrocannabinol, were similarly lower in heterozygous mice, their relative efficacies compared with WIN 55,212-2 were generally unchanged. Saturation analysis of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding showed that decreased stimulation by WIN 55,212-2 in striatum/GP of heterozygous mice was caused by a decrease in the apparent affinity of net-stimulated [(35)S]GTPgammaS binding. The apparent maximal number of binding sites (B(max)) values of net WIN 55,212-2-stimulated [(35)S]GTPgammaS binding were unchanged in cerebellum and striatum/GP of heterozygous mice, but decreased in cingulate cortex, with a similar trend in hippocampus. Moreover, in every region except cingulate cortex, the maximal number of net-stimulated [(35)S]GTPgammaS binding sites per receptor was significantly increased in heterozygous mice. These results indicate region-dependent increases in the apparent efficiency of CB1 receptor-mediated G-protein activation in heterozygous CB1 knockout mice.     

Population-Based Input Function Modeling for [18F]FMPEP-d 2, an Inverse Agonist Radioligand for Cannabinoid CB1 Receptors: Validation in Clinical Studies

Background

Population-based input function (PBIF) may be a valid alternative to full blood sampling for quantitative PET imaging. PBIF is typically validated by comparing its quantification results with those obtained via arterial sampling. However, for PBIF to be employed in actual clinical research studies, its ability to faithfully capture the whole spectrum of results must be assessed. The present study validated a PBIF for [¹⁸F]FMPEP-d ₂, a cannabinoid CB₁ receptor radioligand, in healthy volunteers, and also attempted to utilize PBIF to replicate three previously published clinical studies in which the input function was acquired with arterial sampling.

Methods

The PBIF was first created and validated with data from 42 healthy volunteers. This PBIF was used to assess the retest variability of [¹⁸F]FMPEP-d ₂, and then to quantify CB₁ receptors in alcoholic patients (n = 18) and chronic daily cannabis smokers (n = 29). Both groups were scanned at baseline and after 2–4 weeks of monitored drug abstinence.

Results

PBIF yielded accurate results in the 42 healthy subjects (average Logan-distribution volume (V _T) was 13.3±3.8 mL/cm³ for full sampling and 13.2±3.8 mL/cm³ for PBIF; R² = 0.8765, p<0.0001) and test-retest results were comparable to those obtained with full sampling (variability: 16%; intraclass correlation coefficient: 0.89). PBIF accurately replicated the alcoholism study, showing a widespread ∼20% reduction of CB₁ receptors in alcoholic subjects, without significant change after abstinence. However, a small PBIF-V _T bias of −9% was unexpectedly observed in cannabis smokers. This bias led to substantial errors, including a V _T decrease in regions that had shown no downregulation in the full input function. Simulated data showed that the original findings could only have been replicated with a PBIF bias between −6% and +4%.

Conclusions

Despite being initially well validated in healthy subjects, PBIF may misrepresent clinical protocol results and be a source of variability between different studies and institutions.     

Developmental and Visual Input-Dependent Regulation of the CB1 Cannabinoid Receptor in the Mouse Visual Cortex

The mammalian visual system exhibits significant experience-induced plasticity in the early postnatal period. While physiological studies have revealed the contribution of the CB1 cannabinoid receptor (CB1) to developmental plasticity in the primary visual cortex (V1), it remains unknown whether the expression and localization of CB1 is regulated during development or by visual experience. To explore a possible role of the endocannabinoid system in visual cortical plasticity, we examined the expression of CB1 in the visual cortex of mice. We found intense CB1 immunoreactivity in layers II/III and VI. CB1 mainly localized at vesicular GABA transporter-positive inhibitory nerve terminals. The amount of CB1 protein increased throughout development, and the specific laminar pattern of CB1 appeared at P20 and remained until adulthood. Dark rearing from birth to P30 decreased the amount of CB1 protein in V1 and altered the synaptic localization of CB1 in the deep layer. Dark rearing until P50, however, did not influence the expression of CB1. Brief monocular deprivation for 2 days upregulated the localization of CB1 at inhibitory nerve terminals in the deep layer. Taken together, the expression and the localization of CB1 are developmentally regulated, and both parameters are influenced by visual experience.     

Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity

Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest—in this case CB2—but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.     

Cannabinoid receptor CB1-like and glutamic acid decarboxylase-like immunoreactivities in the brain of Xenopus laevis

Investigation of the cannabinoid system in a vertebrate group phylogenetically distant from mammals might improve understanding of its physiological role. Thus, in the present study, the distribution of the cannabinoid CB1 receptor has been investigated in the brain of Xenopus laevis (anuran amphibians) by immunohistochemistry, using both light and confocal laser-scanning microscopy. Immunostained neuronal perikarya and terminals were found in the olfactory bulb, dorsal and medial pallium, striatum, and amygdala. Varicosities and nerve terminals containing CB1-like immunoreactivity were also seen in the thalamus and hypothalamus. A number of stained cells were observed in the pars distalis of the pituitary gland. Positive nerve fibers were distributed throughout mesencephalic tegmentum, and in the cerebellum immunolabeling was observed in some Purkinje and possibly Golgi cells. The confocal microscopic analysis of CB1-like and glutamic acid decarboxylase-like immunoreactivities in both the medial pallium of the telencephalon and the olfactory bulbs showed a wide codistribution of the two markers. The present results indicate that distribution of CB1 is conserved in the course of phylogeny. Furthermore, the close relationship between CB1-like and glutamic acid decarboxylase-like immunolabelings point toward the existence of a functional link between cannabinergic and GABAergic innervations also in amphibian brain.     

Cannabinoid (CB2) receptor deficiency reduces the susceptibility of macrophages to oxidized LDL/oxysterol-induced apoptosis

Macrophage apoptosis is an important process in the pathophysiology of atherosclerosis. Oxidized low-density lipoproteins (OxLDL) are a major component of lesions and potently induce macrophage apoptosis. Cannabinoid receptor 2 (CB2), the predominant macrophage cannabinoid receptor, modulates several macrophage processes associated with ongoing atherosclerosis; however, the role of CB2 in macrophage apoptosis is unknown. To determine if CB2 influences a macrophage apoptotic pathway relevant to atherosclerosis, we examined the effect of CB2 deficiency on OxLDL-induced macrophage apoptosis. In situ terminal transferase-mediated dUTP nick end labeling (TUNEL) analysis of resident peritoneal macrophages detected significantly fewer apoptotic CB2(-/-) macrophages than CB2(+/+) macrophages after incubation with OxLDL (27.9 +/- 4.7% vs. 61.9 +/- 8.5%, P < 0.001) or 7-ketocholesterol (7KC) (18.9 +/- 10.5% vs. 54.1 +/- 6.9%, P < 0.001), an oxysterol component of OxLDL. Caspase-3 activity; proteolytic conversion of procaspase-3; and cleavage of a caspase-3 substrate, PARP, were also diminished in 7KC-treated CB2(-/-) macrophages. Furthermore, the deactivation of the prosurvival kinase, Akt, in response to 7KC was impaired in CB2(-/-) macrophages. These results suggest that CB2 expression increases the susceptibility of macrophages to OxLDL-induced apoptosis, in part, by modulating the effect of oxysterols on the Akt survival pathway and that CB2 may influence atherosclerosis by modulating lesional macrophage apoptosis.     

Localization and Function of the Cannabinoid CB1 Receptor in the Anterolateral Bed Nucleus of the Stria Terminalis

Background

The bed nucleus of the stria terminalis (BNST) is involved in behaviors related to natural reward, drug addiction and stress. In spite of the emerging role of the endogenous cannabinoid (eCB) system in these behaviors, little is known about the anatomy and function of this system in the anterolateral BNST (alBNST). The aim of this study was to provide a detailed morphological characterization of the localization of the cannabinoid 1 (CB1) receptor a necessary step toward a better understanding of the physiological roles of the eCB system in this region of the brain.

Methodology/Principal Findings

We have combined anatomical approaches at the confocal and electron microscopy level to ex-vivo electrophysiological techniques. Here, we report that CB1 is localized on presynaptic membranes of about 55% of immunopositive synaptic terminals for the vesicular glutamate transporter 1 (vGluT1), which contain abundant spherical, clear synaptic vesicles and make asymmetrical synapses with alBNST neurons. About 64% of vGluT1 immunonegative synaptic terminals show CB1 immunolabeling. Furthermore, 30% and 35% of presynaptic boutons localize CB1 in alBNST of conditional mutant mice lacking CB1 mainly from GABAergic neurons (GABA-CB1-KO mice) and mainly from cortical glutamatergic neurons (Glu-CB1-KO mice), respectively. Extracellular field recordings and whole cell patch clamp in the alBNST rat brain slice preparation revealed that activation of CB1 strongly inhibits excitatory and inhibitory synaptic transmission.

Conclusions/Significance

This study supports the anterolateral BNST as a potential neuronal substrate of the effects of cannabinoids on stress-related behaviors.     

Imaging the Brain Marijuana Receptor: Development of a Radioligand that Binds to Cannabinoid CB1 Receptors In Vivo

Abstract: The major active ingredient of marijuana, (−)-Δ⁹-tetrahydrocannabinol, exerts its psychoactive effects via binding to cannabinoid CB1 receptors, which are widely distributed in the brain. Radionuclide imaging of CB1 receptors in living human subjects would help explore the presently unknown physiological roles of this receptor system, as well as the neurochemical consequences of marijuana dependence. Currently available cannabinoid receptor radioligands are exceedingly lipophilic and unsuitable for in vivo use. We report the development of a novel radioligand, [¹²³I]AM281{ N -(morpholin-4-yl)-5-(4-[¹²³I]iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide}, that is structurally related to the CB1-selective antagonist SR141716A [ N -(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H -pyrazole-3-carboxamide]. Baboon single photon emission computed tomography studies, mouse brain dissection studies, and ex vivo autoradiography in rat brain demonstrated rapid passage of [¹²³I]AM281 into the brain after intravenous injection, appropriate regional brain specificity of binding, and reduction of binding after treatment with SR141716A. AM281 has an affinity in the low nanomolar range for cerebellar binding sites labeled with [³H]SR141716A in vitro, and binding of [¹²³I]AM281 is inhibited by several structurally distinct cannabinoid receptor ligands. We conclude that [¹²³I]AM281 has appropriate properties for in vivo studies of cannabinoid CB1 receptors and is suitable for imaging these receptors in the living human brain.     

The Endocannabinoid/Endovanilloid N-Arachidonoyl Dopamine (NADA) and Synthetic Cannabinoid WIN55,212-2 Abate the Inflammatory Activation of Human Endothelial Cells

Although cannabinoids, such as Δ⁹-tetrahydrocannabinol, have been studied extensively for their psychoactive effects, it has become apparent that certain cannabinoids possess immunomodulatory activity. Endothelial cells (ECs) are centrally involved in the pathogenesis of organ injury in acute inflammatory disorders, such as sepsis, because they express cytokines and chemokines, which facilitate the trafficking of leukocytes to organs, and they modulate vascular barrier function. In this study, we find that primary human ECs from multiple organs express the cannabinoid receptors CB₁R, GPR18, and GPR55, as well as the ion channel transient receptor potential cation channel vanilloid type 1. In contrast to leukocytes, CB₂R is only minimally expressed in some EC populations. Furthermore, we show that ECs express all of the known endocannabinoid (eCB) metabolic enzymes. Examining a panel of cannabinoids, we demonstrate that the synthetic cannabinoid WIN55,212-2 and the eCB N-arachidonoyl dopamine (NADA), but neither anandamide nor 2-arachidonoylglycerol, reduce EC inflammatory responses induced by bacterial lipopeptide, LPS, and TNFα. We find that endothelial CB₁R/CB₂R are necessary for the effects of NADA, but not those of WIN55,212-2. Furthermore, transient receptor potential cation channel vanilloid type 1 appears to counter the anti-inflammatory properties of WIN55,212-2 and NADA, but conversely, in the absence of these cannabinoids, its inhibition exacerbates the inflammatory response in ECs activated with LPS. These data indicate that the eCB system can modulate inflammatory activation of the endothelium and may have important implications for a variety of acute inflammatory disorders that are characterized by EC activation.     

Detection of Cannabinoid CB1, Adenosine A1, Muscarinic Acetylcholine, and GABAB Receptor-Dependent G Protein Activity in Transducin-Deactivated Membranes and Autoradiography Sections of Rat Retina

1. Several G-protein-coupled receptors (GPCRs) have been localized to various layers of the vertebrate retina, using autoradiographic and immunohistochemical techniques, but the functional data concerning G protein activation are limited. Here, we establish optimized assay conditions to detect receptor-dependent G protein activity in membranes and tissue sections of the rat retina. 2. Agonist-stimulated [35S]GTPgammaS-binding responses were characterized for the Gi/o-linked adenosine A1, cannabinoid CB1, m2/m4 muscarinic acetylcholine, and GABA(B) receptors. Initial assumption was that G protein activity under "basal conditions" is high due to enrichment and activity of rhodopsin and transducin in this tissue. 3. We found that pretreatment of retina membranes with hydroxylamine (10 mM), a rhodopsin-inactivating drug, substantially (up to 60%) reduced basal G protein activity, thereby improving signal-to-noise ratio to detect agonist-stimulated G protein activation for all studied receptors. [35S]GTPgammaS autoradiography revealed that hydroxylamine specifically reduced basal binding in the transducin-enriched photoreceptor layer. In contrast, hydroxylamine did not affect GPCR signaling in brain membranes, indicating specific action on retinal transducin. 4. For all studied receptors, [35S]GTPgammaS autoradiography allowed localization of G protein activity to different retinal layers, with the bulk of signal detected in the ganglion cell layer. Strongest responses were observed for adenosine and muscarinic receptor agonists. Additional G protein activity was detected in the inner plexiform layer. 5. Responses to all tested agonists were reversed in the presence of appropriate receptor-selective antagonists, indicating receptor-mediated G protein activation.     

Cannabinoid CB2 receptors are upregulated via bivalent histone modifications and control primary afferent input to the spinal cord in neuropathic pain

Type-2 cannabinoid receptors (CB2, encoded by the Cnr2 gene) are mainly expressed in immune cells, and CB2 agonists normally have no analgesic effect. However, nerve injury upregulates CB2 in the dorsal root ganglion (DRG), following which CB2 stimulation reduces neuropathic pain. It is unclear how nerve injury increases CB2 expression or how CB2 activity is transformed in neuropathic pain. In this study, immunoblotting showed that spinal nerve ligation (SNL) induced a delayed and sustained increase in CB2 expression in the DRG and dorsal spinal cord synaptosomes. RNAscope in situ hybridization also showed that SNL substantially increased CB2 mRNA levels, mostly in medium and large DRG neurons. Furthermore, we found that the specific CB2 agonist JWH-133 significantly inhibits the amplitude of dorsal root–evoked glutamatergic excitatory postsynaptic currents in spinal dorsal horn neurons in SNL rats, but not in sham control rats; intrathecal injection of JWH-133 reversed pain hypersensitivity in SNL rats, but had no effect in sham control rats. In addition, chromatin immunoprecipitation–qPCR analysis showed that SNL increased enrichment of two activating histone marks (H3K4me3 and H3K9ac) and diminished occupancy of two repressive histone marks (H3K9me2 and H3K27me3) at the Cnr2 promoter in the DRG. In contrast, SNL had no effect on DNA methylation levels around the Cnr2 promoter. Our findings suggest that peripheral nerve injury promotes CB2 expression in primary sensory neurons via epigenetic bivalent histone modifications and that CB2 activation reduces neuropathic pain by attenuating nociceptive transmission from primary afferent nerves to the spinal cord.