GTPgammaS Binding Research Articles

Decrease of D2 receptor binding but increase in D2‐stimulated G‐protein activation, dopamine transporter binding and behavioural sensitization in brains of mice treated with a chronic escalating dose ‘binge’ cocaine administration paradigm

Understanding the neurobiology of the transition from initial drug use to excessive drug use has been a challenge in drug addiction. We examined the effect of chronic 'binge' escalating dose cocaine administration, which mimics human compulsive drug use, on behavioural responses and the dopaminergic system of mice and compared it with a chronic steady dose (3 x 15 mg/kg/day) 'binge' cocaine administration paradigm. Male C57BL/6J mice were injected with saline or cocaine in an escalating dose paradigm for 14 days. Locomotor and stereotypy activity were measured and quantitative autoradiographic mapping of D(1) and D(2) receptors, dopamine transporters and D(2)-stimulated [(35)S]GTPgammaS binding was performed in the brains of mice treated with this escalating and steady dose paradigm. An initial sensitization to the locomotor effects of cocaine followed by a dose-dependent increase in the duration of the locomotor effect of cocaine was observed in the escalating but not the steady dose paradigm. Sensitization to the stereotypy effect of cocaine and an increase in cocaine-induced stereotypy score was observed from 3 x 20 to 3 x 25 mg/kg/day cocaine. There was a significant decrease in D(2) receptor density, but an increase in D(2)-stimulated G-protein activity and dopamine transporter density in the striatum of cocaine-treated mice, which was not observed in our steady dose paradigm. Our results document that chronic 'binge' escalating dose cocaine treatment triggers profound behavioural and neurochemical changes in the dopaminergic system, which might underlie the transition from drug use to compulsive drug use associated with addiction, which is a process of escalation.

Neuropeptide Y Receptors in Primary Human Brain Tumors

Peptide receptors are often overexpressed in tumors, and they may be targeted in vivo. We evaluated neuropeptide Y (NPY) receptor expression in 131 primary human brain tumors, including gliomas, embryonal tumors, meningiomas, and pituitary adenomas, by in vitro receptor autoradiography using the 125I-labeled NPY receptor ligand peptide YY in competition with NPY receptor subtype-selective analogs. Receptor functionality was investigated in selected cases using [35S]GTPgammaS-binding autoradiography. World Health Organization Grade IV glioblastomas showed a remarkably high expression of the NPY receptor subtype Y2 with respect to both incidence (83%) and density (mean, 4,886 dpm/mg tissue); astrocytomas World Health Organization Grades I to III and oligodendrogliomas also exhibited high Y2 incidences but low Y2 densities. In glioblastomas, Y2 agonists specifically stimulated [35S]GTPgammaS binding, suggesting that tumoral Y2 receptors were functional. Furthermore, nonneoplastic nerve fibers containing NPY peptide were identified in glioblastomas by immunohistochemistry. Medulloblastomas, primitive neuroectodermal tumors of the CNS, and meningiomas expressed Y1 and Y2 receptor subtypes in moderate incidence and density. In conclusion, Y2 receptors in glioblastomas that are activated by NPY originating from intratumoral nerve fibers might mediate functional effects on the tumor cells. Moreover, identification of the high expression of NPY receptors in high-grade gliomas and embryonal brain tumors provides the basis for in vivo targeting.

G Protein Coupling and Signaling Pathway Activation by M1Muscarinic Acetylcholine Receptor Orthosteric and Allosteric Agonists

The M(1) muscarinic acetylcholine (mACh) receptor is among a growing number of G protein-coupled receptors that are able to activate multiple signaling cascades. AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine) is an allosteric agonist that can selectively activate the M(1) mACh receptor in the absence of an orthosteric ligand. Allosteric agonists have the potential to stabilize unique receptor conformations, which may in turn cause differential activation of signal transduction pathways. In the present study, we have investigated the signaling pathways activated by AC-42, its analog 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone), and a range of orthosteric muscarinic agonists [oxotremorine-M (oxo-M), arecoline, and pilocarpine] in Chinese hamster ovary cells recombinantly expressing the human M(1) mACh receptor. Each agonist was able to activate Galpha(q/11)-dependent signaling, as demonstrated by an increase in guanosine 5'-O-(3-thiotriphosphate) ([(35)S]GTPgammaS) binding to Galpha(q/11) proteins and total [(3)H]inositol phosphate accumulation assays in intact cells. All three orthosteric agonists caused significant enhancements in [(35)S]GTPgammaS binding to Galpha(i1/2) subunits over basal; however, neither allosteric ligand produced a significant response. In contrast, both orthosteric and allosteric agonists are able to couple to the Galpha(s)/cAMP pathway, enhancing forskolin-stimulated cAMP accumulation. These data provide support for the concept that allosteric and orthosteric mACh receptor agonists both stabilize receptor conformations associated with Galpha(q/11)- and Galpha(s)-dependent signaling; however, AC-42 and 77-LH-28-1, unlike oxo-M, arecoline, and pilocarpine, do not seem to promote M(1) mACh receptor-Galpha(i1/2) coupling, suggesting that allosteric agonists have the potential to activate distinct subsets of downstream effectors.

Effects of Δ9‐tetrahydrocannabivarin on [35S]GTPγS binding in mouse brain cerebellum and piriform cortex membranes

We have recently shown that the phytocannabinoid Delta9-tetrahydrocannabivarin (Delta9-THCV) and the CB1 receptor antagonist AM251 increase inhibitory neurotransmission in mouse cerebellum and also exhibit anticonvulsant activity in a rat piriform cortical (PC) model of epilepsy. Possible mechanisms underlying cannabinoid actions in the CNS include CB1 receptor antagonism (by displacing endocannabinergic tone) or inverse agonism at constitutively active CB1 receptors. Here, we investigate the mode of cannabinoid action in [35S]GTPgammaS binding assays. Effects of Delta9-THCV and AM251 were tested either alone or against WIN55,212-2-induced increases in [35S]GTPgammaS binding in mouse cerebellar and PC membranes. Effects on non-CB receptor expressing CHO-D2 cell membranes were also investigated. Delta9-THCV and AM251 both acted as potent antagonists of WIN55,212-2-induced increases in [35S]GTPgammaS binding in cerebellar and PC membranes (Delta9-THCV: pA2=7.62 and 7.44 respectively; AM251: pA2=9.93 and 9.88 respectively). At micromolar concentrations, Delta9-THCV or AM251 alone caused significant decreases in [35S]GTPgammaS binding; Delta9-THCV caused larger decreases than AM251. When applied alone in CHO-D2 membranes, Delta9-THCV and AM251 also caused concentration-related decreases in G protein activity. Delta9-THCV and AM251 act as CB1 receptors antagonists in the cerebellum and PC, with AM251 being more potent than Delta9-THCV in both brain regions. Individually, Delta9-THCV or AM251 exhibited similar potency at CB1 receptors in the cerebellum and the PC. At micromolar concentrations, Delta9-THCV and AM251 caused a non-CB receptor-mediated depression of basal [35S]GTPgammaS binding.

Subnanomolar dopamine D3 receptor antagonism coupled to moderate D2 affinity results in favourable antipsychotic-like activity in rodent models: I. neurochemical characterisation of RG-15

RG-15 (trans-N-[4-[2-[4-(3-cyano-5-trifluoromethyl-phenyl)-piperazine-1-yl]-ethyl]-cyclohexyl]-3-pyridinesulfonic amide dihydrochloride) displayed subnanomolar affinity to human and rat dopamine D3 receptors (pKi 10.49 and 9.42, respectively) and nanomolar affinity to human and rat D2 receptors (pKi 8.23 and 7.62, respectively). No apparent interactions were found with the other 44 receptors and four channel sites tested in this study. RG-15 inhibited dopamine-stimulated [35S]GTPgammaS binding in membranes from rat striatum, in murine A9 cells expressing human D2L receptors and in CHO cells expressing human D3 receptors (IC50 values were 21.2, 36.7 and 7.2 nM, respectively). In these tests RG-15 showed the highest affinity toward D3 receptors when compared to amisulpride, haloperidol and SB-277011. RG-15, similar to haloperidol and amisulpride, dose-dependently inhibited in vivo [3H]raclopride binding in mouse striatum, enhanced dopamine turnover and synthesis rate in mouse and rat striatum and olfactory tubercle. SB-277011 did not change [3H]raclopride binding in mouse striatum nor biosynthesis or turnover rates in either region in mice or rats. RG-15 and haloperidol, but not SB-277011, antagonised dopamine synthesis inhibition induced by the D3/D2 full agonist 7-OH-DPAT in GBL-treated mice. RG-15, but not SB-277011, elevated plasma prolactin levels. In vitro receptor binding and functional experiments demonstrated that RG-15 had an antagonist profile on both D3 and D2 receptors. with high selectivity for dopamine D3 receptors over D2 receptors. However, in vivo, its neurochemical actions were similar to those of D2 receptor antagonists. Neurochemical comparison of RG-15 with antagonists having a different affinity and selectivity toward D3 and D2 receptors indicate that D3 receptors have little, if any, role in the control of presynaptic dopamine biosynthesis/release in dopaminergic terminal regions.

Guanidine and 2-Aminoimidazoline Aromatic Derivatives as α2-Adrenoceptor Antagonists. 2. Exploring Alkyl Linkers for New Antidepressants

The preparation of a number of (bis)guanidine and (bis)2-aminoimidazoline derivatives as potential alpha 2-adrenoceptor antagonists for the treatment of depression is presented. Human brain tissue was used to measure their affinity toward the alpha 2-adrenoceptors in vitro. Compounds 6b, 8b, 9b, 10b, 15b, 17b, 18b, 20b, and 21b displayed a good affinity (pKi > 7) and were evaluated in in vitro functional [(35)S]GTPgammaS binding assays in human prefrontal cortex to determine their agonistic or antagonistic activity. Among these compounds, 17b and 20b showed the expected behavior for an antagonist and were subject to in vivo microdialysis experiments in rats. Significantly, these experiments confirmed the antagonistic properties of 17b and 20b, and therefore both compounds can be considered as potential antidepressants.

Bioisosteric Heterocyclic Versions of 7-{[2-(4-Phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol: Identification of Highly Potent and Selective Agonists for Dopamine D3 Receptor with Potent in Vivo Activity

In the current report, we extend the SAR study on our hybrid structure 7-{[2-(4-phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol further to include heterocyclic bioisosteric analogues. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors with tritiated spiperone to evaluate inhibition constants (Ki). Functional activity of selected compounds in stimulating GTPgammaS binding was assessed with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. The highest binding affinity and selectivity for D3 receptors were exhibited by (-)-34 (Ki=0.92 nM and D2/D3=253). In the functional GTPgammaS binding assay, (-)-34 exhibited full agonist activity with picomolar affinity for D3 receptor with high selectivity (EC50=0.08 nM and D2/D3=248). In the in vivo rotational study, (-)-34 exhibited potent rotational activity in 6-OH-DA unilaterally lesioned rats with long duration of action, which indicates its potential application in neuroprotective treatment of Parkinson's disease.

Development of a universal high-throughput calcium assay for G-protein-coupled receptors with promiscuous G-protein Gα15/16

To develop a universal high-throughput screening assay based on Galpha15/16- mediated calcium mobilization for the identification of novel modulators of Gprotein- coupled receptors (GPCR). In the present study, CHO-K1 or HEK293 cells were co-transfected with plasmids encoding promiscuous G-protein Galpha15/16 and various receptors originally coupled to Galphas, Galphai, or Galphaq pathways. Intracellular calcium change was monitored with fluorescent dye Fluo-4. We found out for all the receptors tested, Galpha15/16 could shift the receptorso coupling to the calcium mobilization pathway, and the EC50 values of the ligands generated with this method were comparable with reported values that were obtained using traditional methods. This assay was validated and optimized with the zeta-opioid receptor, which originally coupled to Galphai and was recently found to play important roles in neurodegenerative and autoimmune diseases. A largescale screening of 48 000 compounds was performed based on this system. Several new modulators were identified and confirmed with the traditional GTPgammaS binding assay. This cell-based calcium assay was proved to be robust and easy to automate, and could be used as a universal method in searching for GPCR modulators.

Pharmacological Assessment of M1Muscarinic Acetylcholine Receptor-Gq/11Protein Coupling in Membranes Prepared from Postmortem Human Brain Tissue

Using a selective Galpha(q/11) protein antibody capture guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding approach, it has been possible to perform a quantitative pharmacological examination of the functional activity of the M(1) muscarinic acetylcholine receptor (mAChR) in membranes prepared from human postmortem cerebral cortex. Oxotremorine-M caused a > or = 2-fold increase in [35S]GTPgammaS-Galpha(q/11) binding with a pEC(50) of 6.06 +/- 0.16 in Brodmann's areas 23 and 25 that was almost completely inhibited by preincubation of membranes with the M(1) mAChR subtype-selective antagonist muscarinic toxin-7. In addition, the orthosteric and allosteric agonists, xanomeline [3(3-hexyloxy-1,2,5-thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine] and AC-42 (4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine hydrogen chloride), increased [35S]-GTPgammaS-Galpha(q/11) binding, but with reduced intrinsic activities, inducing maximal responses that were 42 +/- 1 and 44 +/- 2% of the oxotremorine-M-induced response, respectively. These data indicate that the M(1) receptor is the predominant mAChR subtype coupling to the Galpha(q/11) G protein in these brain regions and that it is possible to quantify the potency and intrinsic activity of full and partial M(1) mAChR receptor agonists in postmortem human brain using a selective Galpha(q/11) protein antibody capture [35S]GTPgammaS binding assay.

Purification of a Yellow Fluorescent Protein‐Tagged Mu Opioid Receptor and Functional Incorporation into rHDL Particles

Despite extensive characterization of the opioid receptor much is unknown about the biochemical properties of the isolated receptor. Here we present the purification of the mu‐opioid receptor and demonstrate functional reconstitution into phospholipid bilayers. We constructed a human mu‐opioid receptor fused to yellow fluorescent protein at its amino‐terminus (YMOR). Baculovirus‐mediated expression of YMOR in insect cells yields receptors with high affinity [3H]diprenorphine (DPN) binding (Kd=0.5 nM). Dodecylmaltoside‐solubilized (with cholesterol hemisuccinate) YMOR was purified using a series of chromatography steps. Purified YMOR in detergent micelles showed impaired binding to [3H]DPN (Kd=4.14 nM), consistent with the behavior of many GPCRs in detergent. Incorporation of purified YMOR into a phospholipid bilayer in the form of reconstituted high density lipoprotein (rHDL) particles was able to restore high affinity [3H]DPN binding (0.26 nM). YMOR in rHDL bound naltrexone, naloxone and beta‐funaltrexamine with high affinity (Ki = 1.64, 11.9 and 2.8 nM, respectively). DAMGO‐stimulated [35S]GTPgammaS binding to Gi or Go is observed when YMOR is reconstituted into HDL. These data illustrate that the mu‐opioid receptor can be purified to homogeneity and its functional properties restored when incorporated into rHDL particles. This work was supported by NIGMS (GM068603).

Synthesis and In Vitro Opioid Receptor Functional Antagonism of Analogues of the Selective Kappa Opioid Receptor Antagonist (3R)-7-Hydroxy-N-((1S)-1-{[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic)

In previous structure-activity relationship (SAR) studies, we identified (3 R)-7-hydroxy- N-((1 S)-1-{[(3 R,4 R)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidinyl]methyl}-2-methylpropyl)-1,2,3,4-tetrahydro-3-isoquinolinecarboxamide (JDTic, 1) as the first potent and selective kappa opioid receptor antagonist from the trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine class of opioid antagonist. In the present study, we report the synthesis and in vitro opioid receptor functional antagonism of a number of analogues of 1 using a [ (35) S]GTPgammaS binding assay. The results from the studies better define the pharmacophore for this class of kappa opioid receptor antagonist and has identified new potent and selective kappa antagonist. (3 R)-7-Hydroxy- N-[(1 S,2 S)-1-{[(3 R,4 R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]methyl}-2-methylbutyl]-1,2,3,4-tetrahydroisoquinoline-3-carboxamide ( 3) with a K e value of 0.03 nM at the kappa receptor and 100- and 793-fold selectivity relative to the mu and delta receptors was the most potent and selective kappa opioid receptor antagonist identified.

Activities of mixed NOP and μ‐opioid receptor ligands

Compounds that activate both NOP and mu-opioid receptors might be useful as analgesics and drug abuse medications. Studies were carried out to better understand the biological activity of such compounds. Binding affinities were determined on membranes from cells transfected with NOP and opioid receptors. Functional activity was determined by [(35)S]GTPgammaS binding on cell membranes and using the mouse vas deferens preparation in vitro and the tail flick antinociception assay in vivo. Compounds ranged in affinity from SR14150, 20-fold selective for NOP receptors, to buprenorphine, 50-fold selective for mu-opioid receptors. In the [(35)S]GTPgammaS assay, SR compounds ranged from full agonist to antagonist at NOP receptors and most were partial agonists at mu-opioid receptors. Buprenorphine was a low efficacy partial agonist at mu-opioid receptors, but did not stimulate [(35)S]GTPgammaS binding through NOP. In the mouse vas deferens, each compound, except for SR16430, inhibited electrically induced contractions. In each case, except for N/OFQ itself, the inhibition was due to mu-opioid receptor activation, as determined by equivalent results in NOP receptor knockout tissues. SR14150 showed antinociceptive activity in the tail flick test, which was reversed by the opioid antagonist naloxone. Compounds that bind to both mu-opioid and NOP receptors have antinociceptive activity but the relative contribution of each receptor is unclear. These experiments help characterize compounds that bind to both receptors, to better understand the mechanism behind their biological activities, and identify new pharmacological tools to characterize NOP and opioid receptors.

Analyzing and modeling the inhibitory effect of phosphatidic acid on the GTP‐γ‐S binding activity of Goα

G proteins are the molecular switches of G-protein-coupled signal transmembrane transduction, which plays a pivotal role in diverse cellular processes. The guanine nucleotide binding states of Galpha-subunits are considered key factors for their functions. We report here that phosphatidic acid (PA) inhibits the [(35)S]-GTPgammaS binding activity of Goalpha. To elucidate this inhibitory effect, biochemical analyses are carried out and a structure-based model is proposed. The experimental results show that PA particularly inhibits the activity of the Goalpha in a dose-dependent manner, whereas other lipids tested do not. Further analysis on the effects of PA analogs demonstrate that a phosphate head group together with at least one fatty acid chain is necessary for the inhibition. Using a lipid-protein binding assay, it is shown that Goalpha specifically and directly interacts with PA. In addition to these experimental studies, a 3D structure of Goalpha is constructed, based on sequence homology greater than 70% to E. coli Gialpha(1). Molecular docking is performed with PA and PA analogs, and the results are compared and analyzed. Collectively, the results of this investigation provide direct experimental evidence for an inhibitory effect of PA on GTP binding activity of Goalpha, and also suggest a structural model for the inhibitory mechanism. The lipid-protein model suggests that PA may occupy the channel for exchanging guanine nucleotides, thus leading to the inhibition. These findings reveal a potential new drug target for the diseases caused by genetic G-protein abnormalities.

Mapping the Structural Requirements in the CB1Cannabinoid Receptor Transmembrane Helix II for Signal Transduction

Amino acid residues in the transmembrane domains of the CB(1) receptor are important for ligand recognition and signal transduction. We used site-directed mutagenesis to identify the role of two novel and adjacent residues in the transmembrane helix II domain, Ile2.62 and Asp2.63. We investigated the role of the conserved, negatively charged aspartate at position 2.63 in cannabinoid receptor (CB(1)) function by substituting it with asparagine (D2.63N) and glutamate (D2.63E). In addition, the effect of the mutant I2.62T alone and in combination with D2.63N (double mutant) on the affinity and potency of structurally diverse ligands was investigated. Recombinant human CB(1) receptors, stably expressed in human embryonic kidney 293 cells, were assayed for ligand affinity and agonist-stimulated guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding. The charge-conserved mutant D2.63E behaved similar to wild type. The charge-neutralization mutation D2.63N attenuated the potency of (-)-3-[2-hydroxyl-4-(1,1-dimethylheptyl)phenyl]-4-[3-hydroxylpropyl] cyclohexan-1-ol (CP,55940), (R)-(-)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl](1-naphthalenyl)methanone (WIN55212-2), (-)-11beta-hydroxy-3-(1',1'-dimethylheptyl) hexahydrocannabinol (AM4056), and (-)-11-hydroxyldimethylheptyl-Delta(8)-tetrahydrocannabinol (HU210) for the stimulation of GTPgammaS binding, without affecting their binding affinities. Likewise, the I2.62T mutant selectively altered agonist potency without altering agonist affinity. It was surprising to note that the double mutant (I2.62T-D2.63N) displayed a drastic and synergistic increase (by approximately 50-fold) in the EC(50) for agonist-mediated activation. The profound loss of function in the I2.62T-D2.63N double mutant suggests that, although these residues are not obligatory for agonist recognition, they play a synergistic and crucial role in modulating signal transduction.

Serotonin‐1A receptor function in the dorsal raphe nucleus following chronic administration of the selective serotonin reuptake inhibitor sertraline

Serotonin-1A (5-HT(1A) receptors in the dorsal raphe nucleus (DRN) function as somatodendritic autoreceptors, and therefore play a critical role in controlling serotonergic cell firing and serotonergic neurotransmission. We hypothesized that a decrease in the capacity of 5-HT(1A) receptors to activate G proteins was a general mechanism by which 5-HT(1A) receptors in the DRN are desensitized following chronic administration of selective serotonin reuptake inhibitors (SSRIs). Using in vivo microdialysis, we found that the ability of the 5-HT(1A) receptor agonist 8-hydroxydipropylaminotetralin hydrobromide (8-OH-DPAT) (0.025 mg/kg, s.c.) to decrease extracellular 5-HT levels in striatum was attenuated following chronic treatment of rats with the SSRIs sertraline or fluoxetine. This apparent desensitization of somatodendritic 5-HT(1A) autoreceptor function was not accompanied by a decrease in 5-HT(1A) receptor sites in the coupled, high-affinity agonist state as measured by the binding of [3H]8-OH-DPAT. In marked contrast to what was observed following chronic administration of fluoxetine, 5-HT(1A) receptor-stimulated [(35)S]GTPgammaS binding in the DRN was not altered following chronic sertraline treatment. Thus, desensitization of 5-HT(1A) somatodendritic autoreceptor function following chronic sertraline administration appears not to be due to a decrease in the capacity 5-HT(1A) receptors to activate G proteins in the DRN. Our findings suggest that the SSRIs may not be a homogeneous class of antidepressant drug with regard to the mechanism by which the function of somatodendritic 5-HT(1A) autoreceptors is regulated.

Effects of chronic treatment with citalopram on cannabinoid and opioid receptor-mediated G-protein coupling in discrete rat brain regions

There is growing interest in investigating the mechanisms of action of selective serotonin reuptake inhibitors (SSRIs), beyond their association with the serotonergic system, due to their wide therapeutic potential for disorders including depression, pain and addiction. The aim of this study was to investigate whether chronic treatment with the SSRI, citalopram, alters the functional coupling of G(i/o)-associated cannabinoid type 1 (CB(1)) and mu-opioid receptors in selected areas of rat brain implicated in psychiatric disorders and pain. Using an autoradiographic approach, the effects of the cannabinoid receptor agonist, HU210 (in the presence or absence of the CB(1) receptor antagonist AM251), or the mu-opioid receptor agonist, [D: -Ala(2),N-Me-Phe4,Gly(5)-ol]-enkephalin (DAMGO; in the presence or absence of the mu-opioid receptor antagonist D: -Phe-Cys-Tyr-D: -Trp-Orn-Thr-Pen-Thr-NH(2)), on [(35)S]GTPgammaS binding in discrete brain regions of citalopram-treated (10 mg kg(-1) day(-1) for 14 days by subcutaneous minipump) and control rats were investigated. The HU210-induced increase in [(35)S]GTPgammaS binding observed in the hypothalamic paraventricular nucleus of control rats was abolished after chronic treatment with citalopram. Reduced response to HU210 in rats receiving chronic treatment with citalopram was also observed in the hippocampus and medial geniculate nucleus. Citalopram had no significant effect on DAMGO-induced [(35)S]GTPgammaS binding in the brain regions investigated, with the exception of the medial geniculate nucleus where a modest impairment was observed. These data provide evidence for reduced cannabinoid receptor-mediated G-protein coupling in the hypothalamus, hippocampus and medial geniculate nucleus of rats chronically treated with citalopram, effects which may, in part, underlie the mechanism of action of SSRIs.

Further Structure–Activity Relationships Study of Hybrid 7-{[2-(4-Phenylpiperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol Analogues: Identification of a High-Affinity D3-Preferring Agonist with Potent in Vivo Activity with Long Duration of Action

This paper describes an extended structure-activity relationships study of aminotetralin-piperazine-based hybrid molecules developed earlier for dopamine D2/D3 receptors. Various analogues as positional isomers have been developed where location of the phenolic hydroxyl group has been varied on the aromatic ring. Between two catechol derivatives, compound 6e with a two methylene linker length exhibited higher affinity and selectivity for D3 over D2 receptors over compound 6f with four methylene linkers (D2/D3 = 50.6 vs 7.51 for 6e and 6f, respectively). In general, the (-)-isomer was more potent than the (+)-isomeric counterpart. Binding results indicated highest selectivity for D3 receptors in compound (-)- 10 ( K i = 0.35 nM; D2/D3 = 71). In the 5-hydroxy series, highest selectivity for D3 receptors was exhibited by compound (-)- 25 ( K i = 0.82 nM; D2/D3 = 31.5). Most potent compounds exhibited binding and functional affinities at the sub-nanomolar level for the D3 receptor. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors by using tritiated spiperone as radioligand for competition studies to evaluate inhibition constants ( K i). A functional assay of selected compounds for stimulating GTPgammaS binding was carried out with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. The functional assay results indicated partial to full agonist characteristics of test compounds. Compound (-)- 25 was selected further for in vivo study to evaluate its potency in producing contralateral rotations in rats with unilateral lesion in the nigrostriatal region induced by neurotoxin 6-OHDA, a Parkinsonian animal model. Compound (-)- 25 at 5 micromol/kg exhibited rotational activity that lasted beyond 12 h, whereas at a 1 micromol/kg dose the rotations lasted beyond 8 h.

The orphan receptor GPR55 is a novel cannabinoid receptor

The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes. Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways. We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1. These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).

Influence of positive allosteric modulators on GABAB receptor coupling in rat brain: a scintillation proximity assay characterisation of G protein subtypes

Little is known concerning coupling of cerebral GABA(B) receptors to G protein subtypes, and the influence of positive allosteric modulators (PAMs) has not been evaluated. These questions were addressed by an antibody-capture/scintillation proximity assay strategy. GABA concentration-dependently enhanced the magnitude of [(35)S]GTPgammaS binding to Galphao and, less markedly, Galphai(1/3) in cortex, whereas Gq and Gs/olf were unaffected. (R)-baclofen and SKF97581 likewise activated Galphao and Galphai(1/3), expressing their actions more potently than GABA. Similar findings were acquired in hippocampus and cerebellum, and the GABA(B) antagonist, CGP55845A, abolished agonist-induced activation of Galphao and Galphai(1/3) in all structures. The PAMs, GS39783, CGP7930 and CGP13501, inactive alone, enhanced efficacy and potency of agonist-induced [(35)S]GTPgammaS binding to Galphao in all regions, actions abolished by CGP55845A. In contrast, they did not modify efficacies at Galphai(1/3). Similarly, in human embryonic kidney cells expressing GABA(B(1a+2)) or GABA(B(1b+2)) receptors, allosteric modulators did not detectably enhance efficacy of GABA at Galphai(1/3), though they increased its potency. To summarise, GABA(B) receptors coupled both to Galphao and to Galphai, but not Gq and Gs/olf, in rat brain. PAMs more markedly enhanced efficacy of coupling to Go versus Gi(1/3). It will be of interest to confirm these observations employing complementary techniques and to evaluate their potential therapeutic significance.

C-terminal truncated cannabinoid receptor 1 coexpressed with G protein trimer in Sf9 cells exists in a precoupled state and shows constitutive activity

We have investigated the existence of a precoupled form of the distal C-terminal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production levels obtained in our expression system were double the values reported in the literature. We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization, as was predicted in the literature. Using fluorescence resonance energy transfer, we found that CB1-417 and Galpha(i1)beta(1)gamma(2) proteins were colocalized in the cells. GTPgammaS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Galpha(i1) protein in the absence of agonists. A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor. We found that the CB1-417/Galpha(i1)beta(1)gamma(2) complex could be solubilized from Sf9 cell membranes and coimmunoprecipitated. In this study, we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells, and that the protein complex is stable in detergent solution. Thus, our system can be used to produce sufficient quantities of the protein complex to start structural studies.