BfCBR: a cannabinoid receptor ortholog in the cephalochordate Branchiostoma floridae (Amphioxus)

A gene encoding an ortholog of vertebrate CB(1)/CB(2) cannabinoid receptors was recently identified in the urochordate Ciona intestinalis (CiCBR; [Elphick, M.R., Satou, Y., Satoh, N., 2003. The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis. Gene 302, 95-101.]). Here a cannabinoid receptor ortholog (BfCBR) has been identified in the cephalochordate Branchiostoma floridae. BfCBR is encoded by a single exon and is 410 amino acid residue protein that shares 28% sequence identity with CiCBR and 23% sequence identity with human CB(1) and human CB(2). The discovery of BfCBR and CiCBR and the absence of cannabinoid receptor orthologs in non-chordate invertebrates indicate that CB(1)/CB(2)-like cannabinoid receptors originated in an invertebrate chordate ancestor of urochordates, cephalochordates and vertebrates. Furthermore, analysis of the relationship of BfCBR and CiCBR with vertebrate CB(1) and CB(2) receptors indicates that the gene/genome duplication that gave rise to CB(1) and CB(2) receptors occurred in the vertebrate lineage. Identification of BfCBR, in addition to CiCBR, paves the way for comparative analysis of the expression and functions of these proteins in Branchiostoma and Ciona, respectively, providing an insight into the ancestral functions of cannabinoid receptors in invertebrate chordates prior to the emergence of CB(1) and CB(2) receptors in vertebrates.     

CB(2) cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: role of CB(2) receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB(2) cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB(2) receptors in the brain need to be clarified. The aim of our work was to study the mu-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB(2) receptor antagonist SR144528 in brainstem of mice deficient in either CB(1) or CB(2) receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB(2) cannabinoid antagonist SR144528, suggesting a CB(2) receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [(35)S]GTPgammaS binding assay to analyze the capability of mu-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB(1) wild-type and CB(1) knockout mice after a single injection of SR144528 at 0.1mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB(1) wild-type and CB(1) knockout mice. In vitro addition of 1microM SR144528 caused a decrease in the maximal stimulation of DAMGO in [(35)S]GTPgammaS binding assays in CB(2) wild-type brainstem membranes whereas no significant changes were observed in CB(2) receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB(2) cannabinoid receptors.     

The pseudokinase tribbles homologue-3 plays a crucial role in cannabinoid anticancer action

Δ⁹-Tetrahydrocannabinol (THC), the major active ingredient of marijuana, and other cannabinoids inhibit tumor growth in animal models of cancer. This effect relies, at least in part, on the up-regulation of several endoplasmic reticulum stress-related proteins including the pseudokinase tribbles homologue-3 (TRIB3), which leads in turn to the inhibition of the AKT/mTORC1 axis and the subsequent stimulation of autophagy-mediated apoptosis in tumor cells. Here, we took advantage of the use of cells derived from Trib3-deficient mice to investigate the precise mechanisms by which TRIB3 regulates the anti-cancer action of THC. Our data show that RasV¹²/E1A-transformed embryonic fibroblasts derived from Trib3-deficient mice are resistant to THC-induced cell death. We also show that genetic inactivation of this protein abolishes the ability of THC to inhibit the phosphorylation of AKT and several of its downstream targets, including those involved in the regulation of the AKT/mammalian target of rapamycin complex 1 (mTORC1) axis. Our data support the idea that THC-induced TRIB3 up-regulation inhibits AKT phosphorylation by regulating the accessibility of AKT to its upstream activatory kinase (the mammalian target of rapamycin complex 2; mTORC2). Finally, we found that tumors generated by inoculation of Trib3-deficient cells in nude mice are resistant to THC anticancer action. Altogether, the observations presented here strongly support that TRIB3 plays a crucial role on THC anti-neoplastic activity. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.     

In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB1 receptor antagonist for the management of obesity

Cannabinoid CB₁ receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB₁ receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB₁ receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB₁ receptors in both binding (K_i = 0.7 nM) and functional assays (K_i = 0.2 nM). The compound has low affinity (K_i = 7600 nM) for human CB₂ receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10 mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB₁ receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB₁ receptor competitive antagonist that may further our understanding of the endocannabinoid system.     

Reciprocal inhibition of G-protein signaling is induced by CB(1) cannabinoid and GABA(B) receptor interactions in rat hippocampal membranes

Cannabinoid CB(1) and the metabotropic GABA(B) receptors have been shown to display similar pharmacological effects and co-localization in certain brain regions. Previous studies have reported a functional link between the two systems. As a first step to investigate the underlying molecular mechanism, here we show cross-inhibition of G-protein signaling between GABA(B) and CB(1) receptors in rat hippocampal membranes. The CB(1) agonist R-Win55,212-2 displayed high potency and efficacy in stimulating guanosine-5'-O-(3-[(35)S]thio)triphosphate, [(35)S]GTPgammaS binding. Its effect was completely blocked by the specific CB(1) antagonist AM251 suggesting that the signaling was via CB(1) receptors. The GABA(B) agonists baclofen and SKF97541 also elevated [(35)S]GTPgammaS binding by about 60%, with potency values in the micromolar range. Phaclofen behaved as a low potency antagonist with an ED(50) approximately 1mM. However, phaclofen at low doses (1 and 10nM) slightly but significantly attenuated maximal stimulation of [(35)S]GTPgammaS binding by the CB(1) agonist R-Win55,212-2. The observation that higher concentrations of phaclofen had no such effect rule out the possibility of its direct action on CB(1) receptors. The pharmacologically inactive stereoisomer S-Win55,212-3 had no effect either alone or in combination with phaclofen establishing that the interaction is stereospecific in hippocampus. The specific CB(1) antagonist AM251 at a low dose (1 nM) also inhibited the efficacy of G-protein signaling of the GABA(B) receptor agonist SKF97541. Cross-talk of the two receptor systems was not detected in either spinal cord or cerebral cortex membranes. It is speculated that the interaction might occur via an allosteric interaction between a subset of GABA(B) and CB(1) receptors in rat hippocampal membranes. Although the exact molecular mechanism of the reciprocal inhibition between CB(1) and GABA(B) receptors will have to be explored by future studies it is intriguing that the cross-talk might be involved in balance tuning the endocannabinoid and GABAergic signaling in hippocampus.     

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.     

Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach

Agonist activation of a G protein-coupled receptor (GPCR) results in the redistribution of the receptor protein away from the cell surface into internal cellular compartments through a process of endocytosis known as internalization. Visualization of receptor internalization has become experimentally practicable by using fluorescent reagents such as green fluorescent protein (GFP). In this study, we examined whether the ligand-mediated internalization of a GPCR can be exploited for pharmacological evaluations. We acquired fluorescent images of cells expressing GFP-labeled GPCRs and evaluated the ligand-mediated internalization quantitatively by image processing. Using beta2-adrenoceptor and vasopressin V1a receptor as model GPCRs that couple to Gs and Gq, respectively, we first examined whether these GFP-tagged GPCRs exhibited appropriate pharmacology. The rank order of receptor internalization potency for a variety of agonists and antagonists specific to each receptor corresponded well with that previously observed in ligand binding studies. In addition to chemical ligand-induced internalization, this cell-based fluorescence imaging system successfully monitored the internalization of the proton-sensing GPCR TDAG8, and that of the free fatty acid-sensitive GPCR GPR120. The results show that monitoring receptor internalization can be a useful approach for pharmacological characterization of GPCRs and in fishing for ligands of orphan GPCRs.     

Cobalt and the iron acquisition pathway: competition towards interaction with receptor 1

During iron acquisition by the cell, complete homodimeric transferrin receptor 1 in an unknown state (R1) binds iron-loaded human serum apotransferrin in an unknown state (T) and allows its internalization in the cytoplasm. T also forms complexes with metals other than iron. Are these metals incorporated by the iron acquisition pathway and how can other proteins interact with R1? We report here a four-step mechanism for cobalt(III) transfer from CoNtaCO(3)(2-) to T and analyze the interaction of cobalt-loaded transferrin with R1. The first step in cobalt uptake by T is a fast transfer of Co(3+) and CO(3)(2-) from CoNtaCO(3)(2-) to the metal-binding site in the C-lobe of T: direct rate constant, k(1)=(1.1+/-0.1) x 10(6) M(-1) s(-1); reverse rate constant, k(-1)=(1.9+/-0.6) x 10(6) M(-1) s(-1); and equilibrium constant, K=1.7+/-0.7. This step is followed by a proton-assisted conformational change of the C-lobe: direct rate constant, k(2)=(3+/-0.3) x 10(6) M(-1) s(-1); reverse rate constant, k(-2)=(1.6+/-0.3) x 10(-2) s(-1); and equilibrium constant, K(2a)=5.3+/-1.5 nM. The two final steps are slow changes in the conformation of the protein (0.5 h and 72 h), which allow it to achieve its final thermodynamic state and also to acquire second cobalt. The cobalt-saturated transferrin in an unknown state (TCo(2)) interacts with R1 in two different steps. The first is an ultra-fast interaction of the C-lobe of TCo(2) with the helical domain of R1: direct rate constant, k(3)=(4.4+/-0.6)x10(10) M(-1) s(-1); reverse rate constant, k(-3)=(3.6+/-0.6) x 10(4) s(-1); and dissociation constant, K(1d)=0.82+/-0.25 muM. The second is a very slow interaction of the N-lobe of TCo(2) with the protease-like domain of R1. This increases the stability of the protein-protein adduct by 30-fold with an average overall dissociation constant K(d)=25+/-10 nM. The main trigger in the R1-mediated iron acquisition is the ultra-fast interaction of the metal-loaded C-lobe of T with R1. This step is much faster than endocytosis, which in turn is much faster than the interaction of the N-lobe of T with the protease-like domain. This can explain why other metal-loaded transferrins or a protein such as HFE-with a lower affinity for R1 than iron-saturated transferrin but with, however, similar or higher affinities for the helical domain than the C-lobe-competes with iron-saturated transferrin in an unknown state towards interaction with R1.     

Expression,surface immobilization,and characterization of functional recombinant cannabinoid receptor CB2

Human peripheral cannabinoid receptor CB₂, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB₂ may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB₂ and immobilization at solid interfaces. Expression of CB₂ as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB₂ was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB₂ was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB₂ from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB₂ and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB₂.     

CB₁ receptor activation inhibits neuronal and astrocytic intermediary metabolism in the rat hippocampus

Cannabinoid CB₁ receptor (CB₁R) activation decreases synaptic GABAergic and glutamatergic transmission and it also controls peripheral metabolism. Here we aimed at testing with ¹³C NMR isotopomer analysis whether CB₁Rs could have a local metabolic role in brain areas having high CB₁R density, such as the hippocampus. We labelled hippocampal slices with the tracers [2-¹³C]acetate, which is oxidized in glial cells, and [U-¹³C]glucose, which is metabolized both in glia and neurons, to evaluate metabolic compartmentation between glia and neurons. The synthetic CB₁R agonist WIN55212-2 (1 μM) significantly decreased the metabolism of both [2-¹³C]acetate (−11.6 ± 2.0%) and [U-¹³C]glucose (−11.2 ± 3.4%) in the tricarboxylic acid cycle that contributes to the glutamate pool. WIN55212-2 also significantly decreased the metabolism of [U-¹³C]glucose (−11.7 ± 4.0%) but not that of [2-¹³C]acetate contributing to the pool of GABA. These effects of WIN55212-2 were prevented by the CB₁R antagonist AM251 (500 nM). These results thus suggest that CB₁Rs might be present also in hippocampal astrocytes besides their well-known neuronal localization. Indeed, confocal microscopy analysis revealed the presence of specific CB₁R immunoreactivity in astrocytes and pericytes throughout the hippocampus.In conclusion, CB₁Rs are able to control hippocampal intermediary metabolism in both neuronal and glial compartments, which suggests new alternative mechanisms by which CB₁Rs control cell physiology and afford neuroprotection.     

The GABAA receptor is an FMRP target with therapeutic potential in fragile X syndrome

Previous research indicates that the GABA_Aergic system is involved in the pathophysiology of the fragile X syndrome, a frequent form of inherited intellectual disability and associated with autism spectrum disorder. However, the molecular mechanism underlying GABA_Aergic deficits has remained largely unknown. Here, we demonstrate reduced mRNA expression of GABA_A receptor subunits in the cortex and cerebellum of young Fmr1 knockout mice. In addition, we show that the previously reported underexpression of specific subunits of the GABA_A receptor can be corrected in YAC transgenic rescue mice, containing the full-length human FMR1 gene in an Fmr1 knockout background. Moreover, we demonstrate that FMRP directly binds several GABA_A receptor mRNAs. Finally, positive allosteric modulation of GABA_A receptors with the neurosteroid ganaxolone can modulate specific behaviors in Fmr1 knockout mice, emphasizing the therapeutic potential of the receptor.     

Functional relevance of the cannabinoid receptor 2 — heme oxygenase pathway: A novel target for the attenuation of portal hypertension

Aims

In liver cirrhosis, inflammation triggers portal hypertension. Kupffer cells (KC) produce vasoconstrictors upon activation by bacterial constituents. Here, we hypothesize that the anti-inflammatory action of the cannabinoid receptor 2 (CB₂) agonists JWH-133 and GP 1a attenuate portal hypertension.

Main methods

In vivo measurements of portal pressures and non-recirculating liver perfusions were performed in rats 4 weeks after bile duct ligation (BDL). Zymosan (150 μg/ml, isolated liver perfusion) or LPS (4 mg/kg b.w., in vivo) was infused to activate the KC in the absence or presence of JWH-133 (10 mg/kg b.w.), GP 1a (2.5 mg/kg b.w.) or ZnPP IX (1 μM). Isolated KC were treated with Zymosan (0.5 mg/ml) in addition to JWH-133 (5 μM). The thromboxane (TX) B₂ levels in the perfusate and KC media were determined by ELISA. Heme oxygenase-1 (HO-1) and CB₂ were analyzed by Western blot or confocal microscopy.

Key findings

JWH-133 or GP 1a pre-treatment attenuated portal pressures following KC activation in all experimental settings. In parallel, HO-1 expression increased with JWH-133 pre-treatment. However, the inhibition of HO-1 enhanced portal hypertension, indicating the functional role of this novel pathway. In isolated KC, the expression of CB₂ and HO-1 increased with Zymosan, LPS and JWH-133 treatment while TXB₂ production following KC activation was attenuated by JWH-133 pre-treatment.

Significance

JWH-133 or GP 1a treatment attenuates portal hypertension. HO-1 induction by JWH-133 plays a functional role. Therefore, the administration of JWH-133 or GP 1a represents a promising new treatment option for portal hypertension triggered by microbiological products.     

Oxidative stress and cannabinoid receptor expression in type‐2 diabetic rat pancreas following treatment with Δ9‐THC

The objectives of study were (a) to determine alteration of feeding, glucose level and oxidative stress and (b) to investigate expression and localization of cannabinoid receptors in type‐2 diabetic rat pancreas treated with Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC). Rats were randomly divided into four groups: control, Δ⁹‐THC, diabetes and diabetes + Δ⁹‐THC groups. Diabetic rats were treated with a single dose of nicotinamide (85 mg/kg) 15 min before injection of streptozotocin (65 mg/kg). Δ⁹‐THC was administered intraperitoneally at 3 mg/kg/day for 7 days. Body weights and blood glucose level of rats in all groups were measured on days 0, 7, 14 and 21. On day 15 after the Δ⁹‐THC injections, pancreatic tissues were removed. Blood glucose levels and body weights of diabetic rats treated with Δ⁹‐THC did not show statistically significant changes when compared with the diabetic animals on days 7, 14 and 21. Treatment with Δ⁹‐THC significantly increased pancreas glutathione levels, enzyme activities of superoxide dismutase and catalase in diabetes compared with non‐treatment diabetes group. The cannabinoid 1 receptor was found in islets, whereas the cannabinoid 2 receptor was found in pancreatic ducts. Their localization in cells was both nuclear and cytoplasmic. We can suggest that Δ⁹‐THC may be an important agent for the treatment of oxidative damages induced by diabetes. However, it must be supported with anti‐hyperglycaemic agents. Furthermore, the present study for the first time emphasizes that Δ⁹‐THC may improve pancreatic cells via cannabinoid receptors in diabetes. The aim of present study was to elucidate the effects of Δ⁹‐THC, a natural cannabinoid receptor agonist, on the expression and localization of cannabinoid receptors, and oxidative stress statue in type‐2 diabetic rat pancreas. Results demonstrate that the cannabinoid receptors are presented in both Langerhans islets and duct regions. The curative effects of Δ⁹‐THC can be occurred via activation of cannabinoid receptors in diabetic rat pancreas. Moreover, it may provide a protective effect against oxidative damage induced by diabetes. Thus, it is suggested that Δ⁹‐THC can be a candidate for therapeutic alternatives of diabetes symptoms. Copyright © 2014 John Wiley & Sons, Ltd.     

Tryptamine-based human β3-adrenergic receptor agonists. Part 2: SAR of the methylene derivatives

A series of tryptamine derivatives with modified sulfonamide were designed, synthesized, and evaluated for their ability to stimulate cAMP accumulation in CHO cells expressing the cloned human β₃-adrenergic receptor (AR). For this series of compounds, our objective was to symmetrize the α-position of the tryptamine moiety maintaining its activity and reducing the cost of production. Compound 11h, having m-aminobenzene, exhibited excellent agonistic activity for β₃-AR with excellent subtype selectivity.     

N-terminal extended conjugates of the agonists and antagonists of both bradykinin receptor subtypes: structure-activity relationship, cell imaging using ligands conjugated with fluorophores and prospect for functionally active cargoes

Peptide agonists and antagonists of both bradykinin (BK) B(1) and B(2) receptors (B(1)R, B(2)R) are known to tolerate to a certain level N-terminal sequence extensions. Using this strategy, we produced and characterized the full set of fluorescent ligands by extending both agonists and antagonist peptides at both receptor subtypes with 5(6)-carboxyfluorescein (CF) and the ε-aminocaproyl (ε-ACA) optional spacer. Alternatively, kinin receptor ligands were extended with another carboxylic acid cargo (chlorambucil, biotinyl, pentafluorocinnamoyl, AlexaFluor-350 (AF350), ferrocenoyl, cetirizine) or with fluorescein isothiocyanate. N-terminal extension always reduced receptor affinity, more importantly for bulkier substituents and more so for the agonist version compared to the antagonist. This loss was generally alleviated by the presence of the spacer and modulated by the species of origin for the receptor. We report and review the pharmacological properties of these N-terminally extended peptides and the use of fluorophore-conjugated ligands in imaging of cell receptors and of angiotensin converting enzyme (ACE) in intact cells. Antagonists (B(1)R: B-10376: CF-ε-ACA-Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; B(2)R: B-10380: CF-ε-ACA-D-Arg-[Hyp(3), Igl(5), D-Igl(7), Oic(8)]-BK and fluorescein-5-thiocarbamoyl (FTC)-B-9430) label the plasma membrane of cells expressing the cognate receptors. The B(2)R agonists CF-ε-ACA-BK, AF350-ε-ACA-BK and FTC-B-9972 are found in endosomes and model the endosomal degradation of BK in a complementary manner. The uneven surface fluorescence associated to the B(1)R agonist B-10378 (CF-ε-ACA-Lys-des-Arg(9)-BK) is compatible with a particular form of agonist-induced receptor translocation. CF-ε-ACA-BK binds to the carboxydipeptidase ACE with an affinity identical to that of BK. Metal- or drug-containing cargoes further show the prospect of ligands that confer special signaling to kinin receptors.     

Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y₁ receptor (P2Y₁R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A₃ and A₁ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y₁R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

     

Design of novel α7-subtype-preferring nicotinic acetylcholine receptor agonists: Application of docking and MM-PBSA computational approaches, synthetic and pharmacological studies

In the search for nicotinic acetylcholine receptor (nAChRs) agonists with a selective affinity for the homomeric α7 channels, we carried out the virtual screening of a test set of potential nicotinic ligands, and adopted a simplified MM-PBSA approach to estimate their relative binding free energy values. By means of this procedure, previously validated by a training set of compounds, we reached a realistic compromise between computational accuracy and calculation rate, and singled out a small group of novel structurally related derivatives characterized by a promising theoretical affinity for the α7 subtype. Among them, five new compounds were synthesized and assayed in binding experiments at neuronal α7 as well as α4β2 nAChRs.     

Neuroprotective roles of the P2Y2 receptor

Purinergic signaling plays a unique role in the brain by integrating neuronal and glial cellular circuits. The metabotropic P1 adenosine receptors and P2Y nucleotide receptors and ionotropic P2X receptors control numerous physiological functions of neuronal and glial cells and have been implicated in a wide variety of neuropathologies. Emerging research suggests that purinergic receptor interactions between cells of the central nervous system (CNS) have relevance in the prevention and attenuation of neurodegenerative diseases resulting from chronic inflammation. CNS responses to chronic inflammation are largely dependent on interactions between different cell types (i.e., neurons and glia) and activation of signaling molecules including P2X and P2Y receptors. Whereas numerous P2 receptors contribute to functions of the CNS, the P2Y(2) receptor is believed to play an important role in neuroprotection under inflammatory conditions. While acute inflammation is necessary for tissue repair due to injury, chronic inflammation contributes to neurodegeneration in Alzheimer's disease and occurs when glial cells undergo prolonged activation resulting in extended release of proinflammatory cytokines and nucleotides. This review describes cell-specific and tissue-integrated functions of P2 receptors in the CNS with an emphasis on P2Y(2) receptor signaling pathways in neurons, glia, and endothelium and their role in neuroprotection.     

Co-administration of dopamine D1 and D2 agonists additively decreases daily food intake, body weight and hypothalamic neuropeptide Y level in rats

This study investigated whether co-administration of dopamine D1 and D2 agonists might additively inhibit the feeding effect and whether this effect was mediated by the action on hypothalamic neuropeptide Y (NPY). The D1 agonist SKF 38393 (SKF) and D2 agonists apomorphine (APO) or quinpirole (QNP) were administered, alone or in combination, to examine this possibility. In single administration, decreases of daily food intake were observed only in rats treated twice a day with a higher dose of SKF, APO or QNP. However, combined administration of D1 and D2 agonists, with each agent at a dose that alone did not induce anorexia in one daily treatment, exerted a significant effect. These results reveal that co-activation of D1 and D2 receptors can additively reduce daily food intake and body weight. The same treatment also decreased the level of hypothalamic NPY 24 h post-treatment. These results suggest an additive effect during combined activation of D1 and D2 receptor subtypes to decrease food intake and body weight that are mediated by the action of hypothalamic NPY. Similar to the effects seen in healthy rats, combined D1/D2 administration was also effective in the reduction of food intake in diabetic rats, revealing the efficiency of D1/D2 agonist in the improvement of hyperphasia in diabetic animals.     

Effects of morphine-3-glucuronide on the antinociceptive activity of peptide and nonpeptide opioid receptor agonists in mice

The effects of morphine-3-glucuronide (M3G), a metabolite of morphine, were determined on the antinociceptive actions, as measured by the tail flick test, of morphine, a μ-opioid receptor agonist, of U-50,488H, a κ-opioid receptor agonist, of [ -Pen², -Pen⁵]enkephalin (DPDPE), a δ₁-opioid receptor agonist, and of [ -Ala²,Glu⁴]deltorphin II (deltorphin II), a δ₂-opioid receptor agonist in mice. Morphine administered ICV (2.5 μg/mouse) or SC (10 mg/kg), U-50,488H (25 mg/kg, IP), DPDPE (15 μg/mouse; ICV), and deltorphin II (15 μg/mouse, ICV) produced antinociception in mice. Intraperitoneal or ICV injections of M3G did not produce any effect on the tail flick latency nor did it affect the antinociception-induced by morphine, U-50,488H, DPDPE, or deltorphin II. Previously M3G has been shown to antagonize the antinociceptive effects of morphine in the rat. It is concluded that in the mouse, M3G neither produces hyperalgesia nor modifies the actions of μ-, κ-, δ₁-, or δ₂-opioid receptor agonists.