Type 1 Cannabinoid Receptor Ligands Display Functional Selectivity in a Cell Culture Model of Striatal Medium Spiny Projection Neurons

Modulation of type 1 cannabinoid receptor (CB₁) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB₁ are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC)) on arrestin2-, Gα_i/o-, Gβγ-, Gα_s-, and Gα_q-mediated intracellular signaling in the mouse STHdh^Q7/Q7 cell culture model of striatal medium spiny projection neurons that endogenously express CB₁. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα_i/o and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB₁ levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα_q-dependent pathways. CP55,940 and CBD both signaled through Gα_s. CP55,940, but not CBD, activated downstream Gα_s pathways via CB₁ targets. THC and CP55,940 promoted CB₁ internalization and decreased CB₁ protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB₁ leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.     

Increases in [3H]Muscimol and [3H]Flumazenil Binding in the Dorsolateral Prefrontal Cortex in Schizophrenia Are Linked to α4 and γ2S mRNA Levels Respectively

Background

GABA_A receptors (GABA_AR) are composed of several subunits that determine sensitivity to drugs, synaptic localisation and function. Recent studies suggest that agonists targeting selective GABA_AR subunits may have therapeutic value against the cognitive impairments observed in schizophrenia. In this study, we determined whether GABA_AR binding deficits exist in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia and tested if changes in GABA_AR binding are related to the changes in subunit mRNAs. The GABA orthosteric and the benzodiazepine allosteric binding sites were assessed autoradiographically using [³H]Muscimol and [³H]Flumazenil, respectively, in a large cohort of individuals with schizophrenia (n = 37) and their matched controls (n = 37). We measured, using qPCR, mRNA of β (β1, β2, β3), γ (γ1, γ2, γ2S for short and γ2L for long isoform, γ3) and δ subunits and used our previous measurements of GABA_AR α subunit mRNAs in order to relate mRNAs and binding through correlation and regression analysis.

Results

Significant increases in both [³H]Muscimol (p = 0.016) and [³H]Flumazenil (p = 0.012) binding were found in the DLPFC of schizophrenia patients. Expression levels of mRNA subunits measured did not show any significant difference in schizophrenia compared to controls. Regression analysis revealed that in schizophrenia, the [³H]Muscimol binding variance was most related to α4 mRNA levels and the [³H]Flumazenil binding variance was most related to γ2S subunit mRNA levels. [³H]Muscimol and [³H]Flumazenil binding were not affected by the lifetime anti-psychotics dose (chlorpromazine equivalent).

Conclusions

We report parallel increases in orthosteric and allosteric GABA_AR binding sites in the DLPFC in schizophrenia that may be related to a “shift” in subunit composition towards α4 and γ2S respectively, which may compromise normal GABAergic modulation and function. Our results may have implications for the development of treatment strategies that target specific GABA_AR receptor subunits.     

Identification and characterization of a novel synthetic cannabinoid CP 55,940 binder in rat brain cytosol

We have detected the presence of a specific [³H] CP 55,940 binder in the cytosol of rat cerebral cortex. Competition studies showed that only cold CP 55,940 and to a lesser extent D⁹THC was able to compete with [³H] CP 55,940; little competition was observed with either D⁸;THC or anandamide. Scatchard analysis of the data indicate the presence of two distinct binding components having affinity constants (Kd) of 0.97 ± 0.03 nM, 5.83 ± 0.08 nM, and B_max of 3.31 ± 0.06 pmol/mg protein, 22.2 ± 1.2 pmol/mg protein respectively. The cytosolic CP 55,940 binder was heat stable up to 30øC. Besides the brain cytosol, lesser amounts of binding were also detected in the spleen, and testis. Liver, kidney and muscle cytosol preparations were found to be devoid of this binder. Unlike the previously characterized brain membrane cannabinoid receptor, this binder was found to be salt, sulfhydryl blocking reagents and nucleotide resistant. Interestingly, dithiothreitol (DTT), a protein-disulfide group reducing agent, inhibited the binding of [³H] CP-55,940 to the receptor and approximately 80% binding inhibition was obtained at a 5 mM concentration. Western blot analysis using anti-receptor antibody reveal the presence of a 95-110, 50 and 38 kDa band in the brain, spleen and testis cytosolic preparations. In conclusion, we have identified the presence of a novel CP 55,940 binder in rat cerebral cortex cytosol possessing biochemical properties distinct from those previously observed using rat cerebral cortex membrane cannabinoid receptor.     

Negative allosteric modulators of cannabinoid receptor 2: protein modeling,binding site identification and molecular dynamics simulations in the presence of an orthosteric agonist

Abstract

Selective activation of the cannabinoid receptor subtype 2 (CB2) shows promise for treating pain, inflammation, multiple sclerosis, cancer, ischemic/reperfusion injury and osteoporosis. Target selectivity and off-target side effects are two major limiting factors for orthosteric ligands, and therefore, the search for allosteric modulators (AMs) is a widely used drug discovery approach. To date, only a limited number of negative CB2 AMs have been identified, possessing only micromolar activity at best, and the CB2 receptor’s allosteric site(s) are not well characterized. Herein, we used computational approaches including receptor modeling, site mapping, docking, molecular dynamics (MD) simulations and binding free energy calculations to predict, characterize and validate allosteric sites within the complex of the CB2 receptor with bound orthosteric agonist CP55,940. After docking of known negative CB2 allosteric modulators (NAMs), dihydro-gambogic acid (DHGA) and trans-β-caryophyllene (TBC) (note that TBC also shows agonist activity), at the predicted allosteric sites, the best total complex with CB2, CP55,940 and NAM was embedded into a hydrated lipid bilayer and subjected to a 200 ns MD simulation. The presence of an AM affected the CB2–CP55,940 complex, altering the relative positioning of the toggle switch residues and promoting a strong π–π interaction between Phe117^3.36 and Trp258^6.48. Binding of either TBC or DHGA to a putative allosteric pocket directly adjacent to the orthosteric ligand reduced the binding free energy of CP55,940, which is consistent with the expected effect of a negative AM. The identified allosteric sites present immense scope for the discovery of novel classes of CB2 AMs.     

A Novel Biased Allosteric Compound Inhibitor of Parturition Selectively Impedes the Prostaglandin F2α-mediated Rho/ROCK Signaling Pathway

The prostaglandin F2α (PGF2α) receptor (FP) is a key regulator of parturition and a target for pharmacological management of preterm labor. However, an incomplete understanding of signaling pathways regulating myometrial contraction hinders the development of improved therapeutics. Here we used a peptidomimetic inhibitor of parturition in mice, PDC113.824, whose structure was based on the NH₂-terminal region of the second extracellular loop of FP receptor, to gain mechanistic insight underlying FP receptor-mediated cell responses in the context of parturition. We show that PDC113.824 not only delayed normal parturition in mice but also that it inhibited both PGF2α- and lipopolysaccharide-induced preterm labor. PDC113.824 inhibited PGF2α-mediated, Gα₁₂-dependent activation of the Rho/ROCK signaling pathways, actin remodeling, and contraction of human myometrial cells likely by acting as a non-competitive, allosteric modulator of PGF2α binding. In contrast to its negative allosteric modulating effects on Rho/ROCK signaling, PDC113.824 acted as a positive allosteric modulator on PGF2α-mediated protein kinase C and ERK1/2 signaling. This bias in receptor-dependent signaling was explained by an increase in FP receptor coupling to Gα_q, at the expense of coupling to Gα₁₂. Our findings regarding the allosteric and biased nature of PDC113.824 offer new mechanistic insights into FP receptor signaling relevant to parturition and suggest novel therapeutic opportunities for the development of new tocolytic drugs.     

HU210-Induced Downregulation in Cannabinoid CB1 Receptor Binding Strongly Correlates with Body Weight Loss in the Adult Rat

In vitro autoradiography was used to examine changes in cannabinoid CB1 receptors (targeted with [³H] CP55,940) in rats treated with the potent cannabinoid agonist HU210. Animals were administered with HU210 (25, 50, 100 μg/kg) for 4 or 14 days or received a single 100 μg/kg injection of HU210 and sacrificed 24 h later. The acute dose resulted in a decrease in binding in the caudate putamen and hippocampus. A dose dependent, region-specific reduction (P < 0.0001) in [³H] CP55,940 binding was seen in all brain regions examined after 4 and 14 days treatment. A decrease in body weight was recorded during the first 4 days of treatment but after this animals began to gain weight. Correlations (0.865 < r < 0.659, P < 0.0001) between body weight on day four and CB1 receptor binding were found in all brain regions examined suggesting that downregulation of CB1 receptors may contribute to the induction of tolerance to body weight loss induced by HU210.     

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M₁ muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M₁ mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M₁ mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M₁ mAChRs, we show that once internalized, M₁ mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M₁ mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.     

The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel

The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP₂), we found that PIP₂ increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP₂ or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr³⁷⁰ in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.     

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.     

Extracellular Calcium Modulates Actions of Orthosteric and Allosteric Ligands on Metabotropic Glutamate Receptor 1α

Metabotropic glutamate receptor 1α (mGluR1α), a member of the family C G protein-coupled receptors, is emerging as a potential drug target for various disorders, including chronic neuronal degenerative diseases. In addition to being activated by glutamate, mGluR1α is also modulated by extracellular Ca²⁺. However, the underlying mechanism is unknown. Moreover, it has long been challenging to develop receptor-specific agonists due to homologies within the mGluR family, and the Ca²⁺-binding site(s) on mGluR1α may provide an opportunity for receptor-selective targeting by therapeutics. In the present study, we show that our previously predicted Ca²⁺-binding site in the hinge region of mGluR1α is adjacent to the site where orthosteric agonists and antagonists bind on the extracellular domain of the receptor. Moreover, we found that extracellular Ca²⁺ enhanced mGluR1α-mediated intracellular Ca²⁺ responses evoked by the orthosteric agonist l-quisqualate. Conversely, extracellular Ca²⁺ diminished the inhibitory effect of the mGluR1α orthosteric antagonist (S)-α-methyl-4-carboxyphenylglycine. In addition, selective positive (Ro 67-4853) and negative (7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester) allosteric modulators of mGluR1α potentiated and inhibited responses to extracellular Ca²⁺, respectively, in a manner similar to their effects on the response of mGluR1α to glutamate. Mutations at residues predicted to be involved in Ca²⁺ binding, including E325I, had significant effects on the modulation of responses to the orthosteric agonist l-quisqualate and the allosteric modulator Ro 67-4853 by extracellular Ca²⁺. These studies reveal that binding of extracellular Ca²⁺ to the predicted Ca²⁺-binding site in the extracellular domain of mGluR1α modulates not only glutamate-evoked signaling but also the actions of both orthosteric ligands and allosteric modulators on mGluR1α.     

Effects of dietary glucose and fructose upon cannabinoid CB1 receptor functionality in the rat brain: A pilot study

Aims

A high consumption of fructose leads not only to peripheral changes in insulin sensitivity and vascular function, but also to central changes in several brain regions. Given the role of the endogenous cannabinoid system in the control of energy intake, we undertook a pilot study to determine whether a high fructose diet produced changes in brain CB₁ receptor functionality.

Main methods

Male rats given access ad libitum to normal chow were given either water, glucose or fructose solutions to drink. CB₁ receptor functionality was measured autoradiographically as the increase in [³⁵S]GTPγS binding produced by the agonist CP55,940.

Key findings

Seven regions were investigated: the prefrontal cortex, caudate–putamen, hippocampal CA1–CA3, dentate gyrus, amygdala, and dorsomedial and ventromedial hypothalami. Two-way robust Wilcoxon analyses for each brain region indicated that the dietary treatment did not produce significant main effects upon agonist-stimulated [³⁵S]GTPγS binding in any of the regions, in contrast to a significant main effect upon both leptin and adiponectin levels in the blood. However, a MANCOVA of the data controlling for leptin and adiponectin as co-variables identified a significant effect of glucose and fructose treatment for five weeks upon the [³⁵S]GTPγS response in the ventromedial hypothalamus, a region of importance for regulation of appetite.

Significance

It is concluded from this pilot study that palatable solutions do not produce overt changes in brain CB₁ receptor functionality, although subtle changes in discrete brain regions may occur.     

Two Distinct Allosteric Binding Sites at α4β2 Nicotinic Acetylcholine Receptors Revealed by NS206 and NS9283 Give Unique Insights to Binding Activity-associated Linkage at Cys-loop Receptors

Positive allosteric modulators (PAMs) of α4β2 nicotinic acetylcholine receptors have the potential to improve cognitive function and alleviate pain. However, only a few selective PAMs of α4β2 receptors have been described limiting both pharmacological understanding and drug-discovery efforts. Here, we describe a novel selective PAM of α4β2 receptors, NS206, and compare with a previously reported PAM, NS9283. Using two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, NS206 was observed to positively modulate acetylcholine (ACh)-evoked currents at both known α4β2 stoichiometries (2α:3β and 3α:2β). In the presence of NS206, peak current amplitudes surpassed those of maximal efficacious ACh stimulations (E_max(ACh)) with no or limited effects at potencies and current waveforms (as inspected visually). This pharmacological action contrasted with that of NS9283, which only modulated the 3α:2β receptor and acted by left shifting the ACh concentration-response relationship. Interestingly, the two modulators can act simultaneously in an additive manner at 3α:2β receptors, which results in current levels exceeding E_max(ACh) and a left-shifted ACh concentration-response relationship. Through use of chimeric and point-mutated receptors, the binding site of NS206 was linked to the α4-subunit transmembrane domain, whereas binding of NS9283 was shown to be associated with the αα-interface in 3α:2β receptors. Collectively, these data demonstrate the existence of two distinct modulatory sites in α4β2 receptors with unique pharmacological attributes that can act additively. Several allosteric sites have been identified within the family of Cys-loop receptors and with the present data, a detailed picture of allosteric modulatory mechanisms of these important receptors is emerging.     

The effects of beta-arrestin1 deletion on acute cannabinoid activity,brain cannabinoid receptors and tolerance to cannabinoids in mice

Abstract

Context: Previous studies have indicated a role for beta-arrestin2 in the regulation of brain cannabinoid effects and cannabinoid CB₁ receptors, but whether beta-arrestin1 has a role has not been investigated. Objective: To determine the role of beta-arrestin1 in cannabinoid activity. Materials and methods: Beta-arrestin1 ?/? mice and their wild-type (+/+) counterparts were assayed for antinociceptive and temperature-decreasing effects of two ligands, Δ⁹-tetrahydrocannabinol (THC) and CP55940, after both single and repeated administration. In vitro assays examined the effects of deletion on CB₁ receptor density, agonist-binding and G-protein activation. Results: Deletion of beta-arrestin1 diminished the effects of CP55940 in both antinociception (latency to tail withdrawal) and temperature-depression assays in mice. However, deleting beta-arrestin1 had no effect on the actions of THC in either assay. Antagonist radioligand ([³H]SR141716A) saturation binding indicated no difference between beta-arrestin1 +/+ and ?/? mice in the density or affinity for cannabinoid CB₁ receptors in brain membranes. CP55940 agonist binding in brain membranes from beta-arrestin1 +/+ mice exhibited high- and intermediate-affinity sites, but beta-arrestin1 ?/? membranes exhibited an additional site with low affinity. CP55940 produced greater stimulation of [³⁵S]GTPγS binding to membranes from whole brain of beta-arrestin1 ?/? than +/+ mice. The rates of the development of tolerance to chronic THC or CP55940 administration did not appear to be affected by genotype. Discussion: Beta-arrestin1 appeared to mediate the actions of CP55940, but did not affect the activity of THC. Conclusion: Beta-arrestin1 regulates cannabinoid CB₁ receptor sensitivity in an agonist-selective manner, but may not be the primary mediator of tolerance to cannabinoid agonists.     

G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors

We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT_2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT_2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT_2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB₂ receptors, β-arrestin 2, and ERK1/2 signaling because treatment with CB₂ shRNA lentiviral particles, β-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT_2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB₂ receptors and increased interaction between β-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB₂ receptors, which up-regulates 5-HT_2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB₂ receptors; and the β-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure.     

Evidence for a cannabinoid receptor in sea urchin sperm and its role in blockade of the acrosome reaction

Delta-9-tetrahydrocannabinol ((?)δ⁹ THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [ H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [ H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [ H]CP-55,940 to sperm, defined as total binding displaced by (?)δ⁹ THC, was saturable: K_D 5.16 ± 1.02 nM; Hill coefficient 0.98 ± 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 ± 122 cannabinoid receptors per cell. Binding of [ H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (?)δ⁹ THC, and (+)δ⁹ THC. The rank order of potency to inhibit binding of [ H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (?)δ⁹THC > (+)δ⁹THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [ H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation. © 1993 Wiley-Liss, Inc.     

Co-Expression of GRK2 Reveals a Novel Conformational State of the μ-Opioid Receptor

Agonists at the µ-opioid receptor are known to produce potent analgesic responses in the clinical setting, therefore, an increased understanding of the molecular interactions of ligands at this receptor could lead to improved analgesics. As historically morphine has been shown to be a poor recruiter of β-arrestin in recombinant cell systems and this can be overcome by the co-expression of GRK2, we investigated the effects of GRK2 co-expression, in a recombinant µ-opioid receptor cell line, on ligand affinity and intrinsic activity in both β-arrestin recruitment and [³⁵S]GTPγS binding assays. We also investigated the effect of receptor depletion in the β-arrestin assay. GRK2 co-expression increased both agonist Emax and potency in the β-arrestin assay. The increase in agonist potency could not be reversed using receptor depletion, supporting that the effects were due to a novel receptor conformation not system amplification. We also observed a small but significant effect on agonist K_L values. Potency values in the [³⁵S]GTPγS assay were unchanged; however, inverse agonist activity became evident with GRK2 co-expression. We conclude that this is direct evidence that the µ-opioid receptor is an allosteric protein and the co-expression of signalling molecules elicits changes in its conformation and thus ligand affinity. This has implications when describing how ligands interact with the receptor and how efficacy is determined.     

Alanine Scan of α-Conotoxin RegIIA Reveals a Selective α3β4 Nicotinic Acetylcholine Receptor Antagonist

Activation of the α3β4 nicotinic acetylcholine receptor (nAChR) subtype has recently been implicated in the pathophysiology of various conditions, including development and progression of lung cancer and in nicotine addiction. As selective α3β4 nAChR antagonists, α-conotoxins are valuable tools to evaluate the functional roles of this receptor subtype. We previously reported the discovery of a new α4/7-conotoxin, RegIIA. RegIIA was isolated from Conus regius and inhibits acetylcholine (ACh)-evoked currents mediated by α3β4, α3β2, and α7 nAChR subtypes. The current study used alanine scanning mutagenesis to understand the selectivity profile of RegIIA at the α3β4 nAChR subtype. [N11A] and [N12A] RegIIA analogs exhibited 3-fold more selectivity for the α3β4 than the α3β2 nAChR subtype. We also report synthesis of [N11A,N12A]RegIIA, a selective α3β4 nAChR antagonist (IC₅₀ of 370 nm) that could potentially be used in the treatment of lung cancer and nicotine addiction. Molecular dynamics simulations of RegIIA and [N11A,N12A]RegIIA bound to α3β4 and α3β2 suggest that destabilization of toxin contacts with residues at the principal and complementary faces of α3β2 (α3-Tyr⁹², Ser¹⁴⁹, Tyr¹⁸⁹, Cys¹⁹², and Tyr¹⁹⁶; β2-Trp⁵⁷, Arg⁸¹, and Phe¹¹⁹) may form the molecular basis for the selectivity shift.     

Neonicotinoid Binding,Toxicity and Expression of Nicotinic Acetylcholine Receptor Subunits in the Aphid Acyrthosiphon pisum

Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [³H]-IMI (Kd of 0.16±0.04 nM and 41.7±5.9 nM) and for the nicotinic antagonist [¹²⁵I]-α-bungarotoxin (Kd of 0.008±0.002 nM and 1.135±0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [¹²⁵I]-α-bungarotoxin binding sites while CLT affinity was similar for both [¹²⁵I]-α-bungarotoxin and [³H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC₅₀ = 0.038 µg/ml) and TMX (LC₅₀ = 0.034 µg/ml) were more toxic than CLT (LC₅₀ = 0.118 µg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.     

Up-regulation of Brain N-Methyl- -aspartate Receptors Following Multiple Intracerebro- ventricular Injections of [ -Pen, -Pen] Enkephalin and [ -Ala, Glu]deltorphin II in Mice

Bhargava, H. N., S. Kumar and J. T. Bian. Up-regulation of brain N-methyl- -aspartate receptors following multiple intracerebroventricular injections of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II in mice. Peptides 18(10) 1609–1613, 1997.—The effects of chronic administration of [ -Pen², -Pen⁵]enkephalin and [ -Ala², Glu⁴]deltorphin II, the selective agonists of the δ₁- and δ₂-opioid receptors, on the binding of [³H]MK-801, a noncompetitive antagonist of the N-methyl- -aspartate receptor, were determined in several brain regions of the mouse. Male Swiss-Webster mice were injected intracerebroventricularly (i.c.v.) with [ -Pen², -Pen⁵]enkephalin or [ -Ala², Glu⁴]deltorphin II (20 μg/mouse) twice a day for 4 days. Vehicle injected mice served as controls. Previously we have shown that the above treatment results in the development of tolerance to their analgesic activity. The binding of [³H]MK-801 was determined in brain regions (cortex, midbrain, pons and medulla, hippocampus, striatum, hypothalamus and amygdala). At 5 nM concentration, the binding of [³H]MK-801 was increased in cerebral cortex, hippocampus, and pons and medulla of [ -Pen², -Pen⁵]enkephalin treated mice. In [ -Ala², Glu⁴]deltorphin II treated mice, the binding of [³H]MK-801 was increased in cerebral cortex and hippocampus. The changes in the binding were due to increases in the B_max value of [³H]MK-801. It is concluded that tolerance to δ₁- and δ₂-opioid receptor agonists is associated with up-regulation of brain N-methyl- -aspartate receptors, however, some brain areas affected differ with the two treatments. The results are consistent with the recent observation from this laboratory that N-methyl- -aspartate receptors antagonists block tolerance to the analgesic action of δ₁- and δ₂-opioid receptor agonists.