GTPgammaS Binding Research Articles

Involvement of phosphatidylcholine‐selective phospholipase C in activation of mitogen‐activated protein kinase pathways in imidazoline receptor antisera‐selected protein

Imidazoline receptor antisera-selected protein (IRAS) is considered as a candidate for the I1-imidazoline receptor (I1R), but the signaling pathway mediated by IRAS remains unknown. In our study, the signal transduction pathways of IRAS were investigated in CHO cells stably expressing IRAS (CHO-IRAS), and compared to the native I1R signaling pathways. Rilmenidine or moxonidine (10 nM-100 microM), I1R agonists, failed to stimulate [35S]-GTPgammaS binding in CHO-IRAS cell membrane preparations, suggesting that G protein may not be involved in IRAS signaling pathway. However, incubation of CHO-IRAS with rilmenidine or moxonidine for 5 min could induce an upregulation of phosphatidylcholine-selective phospholipase C (PC-PLC) activity, and an increase in the accumulation of diacylglycerol (DAG), the hydrolysate of PC-PLC, in a concentration-dependent manner. The elevated activation of PC-PLC by rilmenidine or moxonidine (100 nM) could be blocked by efaroxan, a selective I1R antagonist. Cells treated with rilmenidine or moxonidine showed an increased level of extracellular signal-regulated kinase (ERK) phosphorylation in a concentration-dependent manner, which could be reversed by efaroxan or D609, a selective PC-PLC inhibitor. These results suggest that the signaling pathway of IRAS in response to I1R agonists coupled with the activation of PC-PLC and its downstream signal transduction molecule, ERK. These findings are similar to those in the signaling pathways of native I1R, providing some new evidence for the relationship between I1R and IRAS.

The basic secretagogue compound 48/80 activates G proteins indirectly via stimulation of phospholipase D–lysophosphatidic acid receptor axis and 5‐HT1A receptors in rat brain sections

The basic secretagogues, such as compound 48/80 (c48/80) and mastoparans, are widely used histamine-releasing agents and their mechanism of action is commonly attributed to a direct, receptor-bypassing property to activate the G(i/o) class of G proteins. We tested here whether c48/80 could directly stimulate [(35)S]guanosine-5'-[gamma-thio]triphosphate ([(35)S]GTPgammaS) binding to rat brain sections in an attempt to visualize the entire signaling pool of G(i/o) in its native neuroanatomical context. Instead of direct G(i/o) activation, c48/80 (100 microg ml(-1)) from various suppliers stimulated brain phospholipase D (PLD) activity, leading to the generation of endogenous phospholipids capable of activating brain white matter-enriched, G(i/o)-coupled lysophosphatidic acid (LPA) receptors. This response was sensitive to 1-butanol and was potently reversed by the LPA(1)/LPA(3) receptor-selective antagonist Ki16425 (IC(50) 59+/-13 nM, mean+/-s.e.m.), and showed age-dependent decline, closely reflecting known developmental regulation of the PLD-LPA(1) receptor axis in the CNS. In addition, c48/80 was found to modestly activate hippocampal 5-HT(1A) receptors in a pH-dependent and antagonist-sensitive manner. Consistent with the lack of direct G(i/o)-activating properties in brain sections, c48/80 showed no activity in classical membrane [(35)S]GTPgammaS binding assays. Instead, c48/80 from one particular manufacturer elicited non-specific effect in these assays, therefore challenging the previous interpretations regarding the compound's ability to activate G proteins directly. We conclude that c48/80 is not a receptor-bypassing general G protein activator but rather activates PLD, leading to generation of endogenous LPA receptor-activating phospholipids. This property may also contribute to the compound's ability to release histamine from mast cells.

Agonist activity of naloxone benzoylhydrazone at recombinant and native opioid receptors

1. In the present study, we examined the pharmacological activity of the putative kappa3-opioid receptor agonist naloxone benzoylhydrazone (NalBzoH) at recombinant human opioid receptors individually expressed in Chinese hamster ovary (CHO) cells and native opioid receptors present in rat striatum. 2. At the mu-opioid receptor (MOR), NalBzoH stimulated guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding (pEC50=8.59) and inhibited cyclic AMP accumulation (pEC50=8.74) with maximal effects (Emax) corresponding to 55 and 65% of those obtained with the MOR agonist DAMGO, respectively. The MOR antagonist CTAP blocked the stimulatory effects of NalBzoH and DAMGO with similar potencies. 3. At the kappa-opioid receptor (KOR), NalBzoH stimulated [35S]GTPgammaS binding (pEC50=9.70) and inhibited cyclic AMP formation (pEC50=9.45) as effectively as the selective KOR agonist (-)-U-50,488. The NalBzoH effect was blocked by the KOR antagonist nor-binaltorphimine (nor-BNI) (pKi=10.30). 4. In CHO cells expressing the delta-opioid receptor (DOR), NalBzoH increased [35S]GTPgammaS binding (pEC50=8.49) and inhibited cyclic AMP formation (pEC50=8.61) almost as effectively as the DOR agonist DPDPE. Naltrindole (NTI), a selective DOR antagonist, completely blocked the response to NalBzoH (pKi of 10.40). 5. In CHO cells expressing the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), NalBzoH failed to exert agonist effects and antagonized the agonist-induced receptor activation. 6. When compared to other opioid receptor ligands, NalBzoH showed an efficacy that was lower than that of morphine at MOR, but higher at KOR and DOR. 7. In rat striatum, NalBzoH enhanced [35S]GTPgammaS binding and inhibited adenylyl cyclase activity. These effects were antagonized by either CTAP, nor-BNI or NTI, each antagonist blocking a fraction of the NalBzoH response. 8. These data demonstrate that NalBzoH displays agonist activity at MOR, DOR and KOR expressed either in a heterologous cell system or in a native environment.

5-HT-stimulated [35S]guanosine-5′-O-(3-thio)triphosphate binding as an assay for functional activation of G proteins coupled with 5-HT1B receptors in rat striatal membranes

Receptor-mediated guanine nucleotide-binding regulatory protein (G protein) activation or functional coupling between receptors and G proteins has been investigated by means of agonist-induced [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTPgammaS) binding, especially for the receptor subtypes negatively coupled to adenylyl cyclase through Gi type G proteins. In the present study, 5-HT-stimulated [35S]GTPgammaS binding to rat stritatal membranes was pharmacologically characterized in detail with the help of an extensive series of 5-HT receptor ligands. The optimum experimental conditions for the concentrations of GDP, MgCl2 and NaCl in the assay buffer were initially determined, and the standard assay was performed with 20 microM GDP, 5 mM MgCl2 and 100 mM NaCl. The specific [35S]GTPgammaS binding was stimulated by several compounds that had been shown to be agonists at 5-HT(1B/1D) receptors. The negative logarithmic values of the concentration eliciting half-maximal effect (pEC50) for these agonists were significantly correlated with their pKi's reported in the previous study of 5-HT1B receptor binding in rat frontal cortical membranes. The increase in specific [35S]GTPgammaS binding in response to 1 microM 5-HT was potently inhibited by several 5-HT(1B/1D) receptor antagonists as well as beta-adrenoceptor antagonists such as S(-)-cyanopindolol. On the other hand, 3-[4-(4-chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol HCl (BRL15572), a selective antagonist against human 5-HT1D receptors, was inactive as an antagonist at least up to 1 microM. Additionally, the concentration-response curve for 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine (L694247) was shifted rightward in parallel by the addition of S(-)-cyanopindolol at concentrations of 10 and 100 nM, indicative of the competitive inhibitory manner. The specific [35S]GTPgammaS binding was reduced by 1'-methyl-5-([2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl)-2,3,6,7-tetrahydrospirospiro(furo[2,3-f]indole-3,4'-piperidine) (SB224289) and methiothepin in a concentration-dependent manner. The inhibitory curve by either compound was shifted to the right by 10 and 100 nM S(-)-cyanopindolol, suggesting that these two drugs behaved as inverse agonists at 5-HT1B receptors in the present functional assay system. 5-HT-stimulated [35S]GTPgammaS binding to rat striatal membranes serves as a simple but useful method of investigating the functional interaction between the native 5-HT1B receptors and their coupled G proteins in this brain region.

The ‘allosteric modulator’ SCH‐202676 disrupts G protein‐coupled receptor function via sulphydryl‐sensitive mechanisms

1. Previous studies suggest that the thiadiazole compound SCH-202676 (N-(2,3-diphenyl-1,2,4-thiadiazol-5-(2H)-ylidene)methanamine) acts as an allosteric modulator of a variety of structurally distinct G protein-coupled receptors (GPCRs). It was postulated that SCH-202676 would directly bind a structural motif in the receptor molecule common to divergent members of the GPCR family. The molecular mechanisms of such a promiscuous action, however, remain obscure. 2. To clarify the mechanism of SCH-202676 action, we used the functional approach of [35S]GTPgammaS autoradiography with rat brain cryostat sections together with classical membrane [35S]GTPgammaS binding assays to evaluate how the thiadiazole affects G protein activity mediated by various receptors linked to the Gi-family of G proteins. 3. We found that in the absence of dithiotreitol (DTT), SCH-202676 (10(-7)-10(-5) M) elicits nonspecific effects in the [35S]GTPgammaS-based G protein activation assays, thereby severely compromising interpretations on the compounds ability to allosterically inhibit receptor-mediated G protein activity. Such a nonspecific behaviour was fully reversed upon addition of DTT (1 mM), revealing thiol-based mechanism of action. 4. In routine incubations containing DTT, SCH-202676 had no effect on receptor-driven G protein activity, as assessed for adenosine A1, alpha2-adrenergic, cannabinoid CB1, lysophosphatidic acid LPA1, muscarinic M2/M4, purinergic P2Y12 or sphingosine 1-phosphate receptors, suggesting that the thiadiazole does not act as an allosteric modulator of GPCR function. 5. 1H NMR analysis indicated that SCH-202676 underwent structural changes after incubation with the reducing agent DTT or with brain tissue. 6. We conclude that SCH-202676 modulates GPCRs via thiol modification rather than via true allosteric mechanisms.

Comparison of the Relative Efficacy and Potency of μ-Opioid Agonists to Activate Gαi/oProteins Containing a Pertussis Toxin-Insensitive Mutation

Pertussis toxin (PTX)-insensitive mutants of Galpha(i/o) proteins expressed in C6mu cells were used to examine the hypothesis that there are agonist-specific conformational states of the mu-opioid receptor with coupling preferences to different Galpha(i/o) subtypes, as measured by the degree of stimulation of [(35)S]guanosine 5'-O-(3-thio)triphosphate (GTPgammaS) binding. Binding of [(35)S]GTPgammaS to endogenous Galpha(i/o) proteins stimulated by the full mu-opioid agonist [d-Ala(2),MePhe(4),Gly(5)-ol]enkephalin (DAMGO) was completely blocked by overnight treatment with 100 ng/ml PTX. Treatment for 4 h with lower concentrations led to a PTX-dependent reduction in the maximal effect of DAMGO but no alteration in the potency of DAMGO or morphine nor in the relative maximal effect (relative efficacy) of the partial agonists morphine and buprenorphine compared with the full agonist DAMGO. Using PTX-insensitive Galpha mutants in which the PTX-sensitive cysteine was replaced with isoleucine, the potency for a series of mu-opioid agonists was highest in cells expressing Galpha(i3) and Galpha(o) and lowest with Galpha(i1) and Galpha(i2), with no significant change in the order of potency, namely, etorphine >> endomorphin-1 = DAMGO = endomorphin-2 = fentanyl = morphine >> meperidine. The order of agonist relative efficacy, etorphine = DAMGO = endomorphin-1 = endomorphin-2 = fentanyl > or = morphine > or = meperidine > buprenorphine > or = nalbuphine, was also the same across all of the PTX-insensitive Galpha(i/o) subtypes. Highest relative efficacy to stimulate [(35)S]GTPgammaS binding was seen with Galpha(i3). Consequently, reported observations of agonist-directed trafficking at mu-opioid receptors most likely involve non-PTX-sensitive Galpha protein mechanisms.

Synthesis and Activity of 1,3,5-Triphenylimidazolidine-2,4-diones and 1,3,5-Triphenyl-2-thioxoimidazolidin-4-ones: Characterization of New CB1 Cannabinoid Receptor Inverse Agonists/Antagonists

Obesity and metabolic syndrome, along with drug dependence (nicotine, alcohol, opiates), are two of the major therapeutic applications for CB(1) cannabinoid receptor antagonists and inverse agonists. In the present study, we report the synthesis and structure-affinity relationships of 1,5-diphenylimidazolidine-2,4-dione and 1,3,5-triphenylimidazolidine-2,4-dione derivatives. These new 1,3,5-triphenylimidazolidine-2,4-dione derivatives and their thio isosteres were obtained by an original pathway and exhibited interesting affinity and selectivity for the human CB(1) cannabinoid receptor. A [(35)S]-GTPgammaS binding assay revealed the inverse agonist properties of the compounds at the CB(1) cannabinoid receptor. Furthermore, molecular modeling studies were conducted in order to delineate the binding mode of this series of derivatives into the CB(1) cannabinoid receptor. 1,3-Bis(4-bromophenyl)-5-phenylimidazolidine-2,4-dione (25) and 1,3-bis(4-chlorophenyl)-5-phenylimidazolidine-2,4-dione (23) are the imidazolidine-2,4-dione derivatives possessing the highest affinity for the human CB(1) cannabinoid receptor reported to date.

Distribution of nociceptin and Ro64-6198 activated [35S]-GTPγS binding in the rat brain

The peptide, nociceptin, was discovered as the endogenous ligand for the opioid-like receptor, ORL1. Since its discovery, this peptide has been shown to modulate the perception of pain, modulate feeding and produce behavioral effects in rodent models of mood disorders. Recently, the non-peptide agonist {(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one} (Ro64-6198) of the ORL1 receptor has been reported in the literature. In the present study, we compared the distribution and potency of Ro64-6198 with nociceptin for their ability to stimulate [(35)S]-GTPgammaS binding to sections of rat brain. In initial studies, Ro64-6198 inhibited (125)I-nociceptin binding to the hORL1 receptors with a K(i) of 1.75 nM compared with 0.20 nM for nociceptin. To assess agonist potency in a whole cell assay, a cell line expressing the hORL1 receptor and G(alpha15) was created and used for calcium mobilization studies. In this assay system, Ro64-6198 increased intracellular calcium with an EC(50) of 52nM compared with 24 nM for nociceptin. Having verified the agonist properties of Ro64-6198, we then assessed the potency and distribution of ORL1 receptor activation in rat brain sections. In dose-response studies, Ro64-6198 increased [(35)S]-GTPgammaS binding to a variety of brain regions with EC(50) values ranging from 84.9 to 2,143 nM depending on the brain regions evaluated. These potencies were similar to that seen for nociceptin, but substantially lower than values established using [(125)I] nociceptin binding to the cloned human ORL1 receptor. In general, the brain distribution of agonist stimulated [(35)S]-GTPgammaS binding was similar when either Ro64-6198 or nociceptin were used. Using these techniques, we have demonstrated, for the first time that Ro64-6198 activates [(35)S]-GTPgammaS binding to rat brain sections and this compound stimulates a similar population of receptors as nociceptin.

In vitro characterization of SLV308 (7-[4-methyl-1-piperazinyl]-2(3H)-benzoxazolone, monohydrochloride): A novel partial dopamine D2 and D3 receptor agonist and serotonin 5-HT1A receptor agonist

Present Parkinson's disease treatment strategies are far from ideal for a variety of reasons; it has therefore been suggested that partial dopamine receptor agonism might be a potential therapeutic approach with potentially fewer side effects. In the present study, we describe the in vitro characterization of the nonergot ligand SLV308 (7-[4-methyl-1-piperazinyl]-2(3H)-benzoxazolonemonohydrochloride). SLV308 binds to dopamine D(2), D(3), and D(4) receptors and 5-HT(1) (A) receptors and is a partial agonist at dopamine D(2) and D(3) receptors and a full agonist at serotonin 5-HT(1) (A) receptors. At cloned human dopamine D(2,L) receptors, SLV308 acted as a potent but partial D(2) receptor agonist (pEC(50) = 8.0 and pA(2) = 8.4) with an efficacy of 50% on forskolin stimulated cAMP accumulation. At human recombinant dopamine D(3) receptors, SLV308 acted as a partial agonist in the induction of [(35)S]GTPgammaS binding (intrinsic activity of 67%; pEC(50) = 9.2) and antagonized the dopamine induction of [(35)S]GTPgammaS binding (pA(2) = 9.0). SLV308 acted as a full 5-HT(1) (A) receptor agonist on forskolin induced cAMP accumulation at cloned human 5-HT(1) (A) receptors but with low potency (pEC(50) = 6.3). In rat striatal slices SLV308 concentration-dependently attenuated forskolin stimulated accumulation of cAMP, as expected for a dopamine D(2) and D(3) receptor agonist. SLV308 antagonized the inhibitory effect of quinpirole on K(+)-stimulated [(3)H]-dopamine release from rat striatal slices (pA(2) = 8.5). In the same paradigm, SLV308 had antagonist properties in the presence of quinpirole (pA(2) = 8.5), but the partial D(2) agonist terguride had much stronger antagonistic properties. In conclusion, SLV308 combines high potency partial agonism at dopamine D(2) and D(3) receptors with full efficacy low potency serotonin 5-HT(1) (A) receptor agonism and is worthy of profiling in in vivo models of Parkinson's disease.

A selective metabotropic glutamate receptor 7 agonist: activation of receptor signaling via an allosteric site modulates stress parameters in vivo.

Metabotropic glutamate receptor (mGluR) subtypes (mGluR1 to mGluR8) act as important pre- and postsynaptic regulators of neurotransmission in the CNS. These receptors consist of two domains, an extracellular region containing the orthosteric agonist site and a transmembrane heptahelical domain involved in G protein activation and recognition of several recently synthesized pharmacological modulators. The presynaptic receptor mGluR7 shows the highest evolutionary conservation within the family, but no selective pharmacological tool was known. Here we characterize an mGluR7-selective agonist, N,N'-dibenzhydrylethane-1,2-diamine dihydrochloride (AMN082), which directly activates receptor signaling via an allosteric site in the transmembrane domain. At transfected mammalian cells expressing mGluR7, AMN082 potently inhibits cAMP accumulation and stimulates GTPgammaS binding (EC50-values, 64-290 nM) with agonist efficacies comparable with those of L-2-amino-4-phosphonobutyrate (L-AP4) and superior to those of L-glutamate. AMN082 (< or = 10 microM) failed to show appreciable activating or inhibitory effects at other mGluR subtypes and selected ionotropic GluRs. Chimeric receptor studies position the binding site of AMN082 in the transmembrane region of mGluR7, and we demonstrate that this allosteric agonist has little, if any, effect on the potency of orthosteric ligands. Here we provide evidence for full agonist activity mediated by the heptahelical domain of family 3 G protein-coupled receptors (which have mGluR-like structure) that may lead to drug development opportunities. Further, AMN082 is orally active, penetrates the blood-brain barrier, and elevates the plasma stress hormones corticosterone and corticotropin in an mGluR7-dependent fashion. Therefore, AMN082 is a valuable tool for unraveling the role of mGluR7 in stress-related CNS disorders.

Differential coupling of the P2Y 1 receptor to Gα 14 and Gα q/11 proteins during the development of the rat salivary gland

In rat submandibular gland (SMG), the P2Y1 receptor (P2Y1R) mediates increases in the intracellular calcium concentration, [Ca2+]i that diminish as the animal ages from 1 to 4-6 weeks. However, P2Y1R mRNA levels do not change with age, suggesting that the age-dependent decrease in the [Ca2+]i response to P2Y1R agonists may be due to alterations in the activity of a component of the P2Y1R signalling pathway. To assess whether the decrease in P2Y1R-mediated intracellular calcium signalling in SMG cells as rats age is due to a decrease in P2Y1R coupling to G proteins or to a decrease in the expression of a cognate G protein. SMG cells were isolated from Sprague-Dawley rats. P2Y1R function was assessed by measuring 2-MeSADP-induced increases in [Ca2+]i and ERK1/2 activation. P2Y(1)R-mediated activation of G proteins was determined by the [35S]GTPgammaS binding assay. Gq protein expression was determined by RT-PCR, Northern, and Western analysis. In SMG cells from 1-week-old rats, two bands (52 and 42kDa) were detected using anti-Galpha14 antibody, whereas in SMG cells from 4- to 6-week-old rats only the 42 kDa band was detected. Furthermore, 2-MeSADP-induced GTPgamma35S binding to Galpha14 and Galphaq/11 decreases in SMG cells from 4- to 6-week-old rats as compared to 1-week-old rats. These findings suggest that the age-dependent decrease in P2Y1R-mediated intracellular calcium signalling in rat SMG cells is due to a loss of 52 kDa Galpha14 and indicate the differential coupling of the P2Y1R to Galpha14 and Galphaq/11 as the gland develops.

Synthesis and Preliminary In vitro Investigation of Bivalent Ligands Containing Homo- and Heterodimeric Pharmacophores at μ, δ, and κ Opioid Receptors

A series of homo- and heterodimeric ligands containing kappa agonist and mu agonist/antagonist pharmacophores joined by a linker chain of varying lengths was synthesized and evaluated in vitro by their binding affinity at mu, delta, and kappa opioid receptors. The functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. The data suggest that the stereochemistry of the pharmacophores, the N-substituents of the pharmacophore, ester linkages, and the spacer length were crucial factors for optimum interactions of such ligands at opioid receptor binding sites. These novel ligands as well as their pharmacological properties will serve as the basis for our continuing investigation of such bivalent ligands as probes of the opioid receptor oligomerization phenomena and for in vivo studies as analgesics.

Evidence that the plant cannabinoid Δ9‐tetrahydrocannabivarin is a cannabinoid CB1 and CB2 receptor antagonist

Delta9-tetrahydrocannabivarin (THCV) displaced [(3)H]CP55940 from specific binding sites on mouse brain and CHO-hCB(2) cell membranes (K(i)=75.4 and 62.8 nM, respectively).THCV (1 microM) also antagonized CP55940-induced stimulation of [(35)S]GTPgammaS binding to these membranes (apparent K(B)=93.1 and 10.1 nM, respectively). In the mouse vas deferens, the ability of Delta9-tetrahydrocannabinol (THC) to inhibit electrically evoked contractions was antagonized by THCV, its apparent K(B)-value (96.7 nM) approximating the apparent K(B)-values for its antagonism of CP55940- and R-(+)-WIN55212-induced stimulation of [(35)S]GTPgammaS binding to mouse brain membranes. THCV also antagonized R-(+)-WIN55212, anandamide, methanandamide and CP55940 in the vas deferens, but with lower apparent K(B)-values (1.5, 1.2, 4.6 and 10.3 nM, respectively).THCV (100 nM) did not oppose clonidine, capsaicin or (-)-7-hydroxy-cannabidiol-dimethylheptyl-induced inhibition of electrically evoked contractions of the vas deferens. Contractile responses of the vas deferens to phenylephrine hydrochloride or beta,gamma-methylene-ATP were not reduced by 1microM THCV or R-(+)-WIN55212, suggesting that THCV interacts with R-(+)-WIN55212 at prejunctional sites. At 32 microM, THCV did reduce contractile responses to phenylephrine hydrochloride and beta,gamma-methylene-ATP, and above 3 microM it inhibited electrically evoked contractions of the vas deferens in an SR141716A-independent manner. In conclusion, THCV behaves as a competitive CB(1) and CB(2) receptor antagonist. In the vas deferens, it antagonized several cannabinoids more potently than THC and was also more potent against CP55940 and R-(+)-WIN55212 in this tissue than in brain membranes. The bases of these agonist- and tissue-dependent effects remain to be established.

Cell migration activated by platelet‐derived growth factor receptor is blocked by an inverse agonist of the sphingosine 1‐phosphate receptor‐1

We have previously identified a novel complex between the platelet-derived growth factor (PDGF)beta receptor and the sphingosine 1-phosphate receptor-1 (S1P1). The complex permits the utilization of active G-protein subunits (made available by constitutively active S1P1 receptor) by the PDGFbeta receptor kinase to transmit signals to p42/p44 MAPK in response to PDGF. Therefore, an inverse agonist of the S1P1 receptor is predicted to reduce signal transduction from PDGFbeta receptor tyrosine kinase by blocking the constitutive activity of the G-protein coupled receptor. SB649146 is a novel inverse agonist of the S1P1 receptor. First, SB649146 displaced the S1P1 receptor agonist dihydrosphingosine 1-phosphate from membranes expressing the recombinant S1P1 receptor. Second, SB649146 reduced basal recombinant S1P1 receptor-induced GTPgammaS binding and S1P-induced GTPgammaS binding in membranes. Third, SB649146 blocked the S1P-induced activation of p42/p44 MAPK in airway smooth muscle cells, a response that is mediated by the S1P1 receptor. We now report that inverse agonism of the S1P1 receptor with SB649146 reduced the endocytosis of the PDGFbeta receptor-S1P1 receptor complex and the stimulation of p42/p44 MAPK and cell migration in response to PDGF. These findings are the first to report that a GPCR inverse-agonist reduces growth factor-induced receptor tyrosine kinase signaling, fundamentally broadening their mechanism of action. The data obtained with SB649146 also suggest that the constitutively active endogenous S1P1 receptor enhances PDGF-induced cell migration.

Design, Synthesis, and Action of Oxotremorine-Related Hybrid-Type Allosteric Modulators of Muscarinic Acetylcholine Receptors

A novel series of muscarinic receptor ligands of the hexamethonio-type was prepared which contained, on one side, the phthalimidopropane or 1,8-naphthalimido-2,2-dimethylpropane moiety typical for subtype selective allosteric antagonists and, on the other, the acetylenic fragment typical for the nonselective orthosteric muscarinic agonists oxotremorine, oxotremorine-M, and related muscarinic agonists. Binding experiments in M(2) receptors using [(3)H]N-methylscopolamine as an orthosteric probe proved an allosteric action of both groups of hybrids, 7a-10a and 8b-10b. The difference in activity between a-group and b-group hybrids corresponded with the activity difference between the allosteric parent compounds. In M(1)-M(3) muscarinic isolated organ preparations, most of the hybrids behaved as subtype selective antagonists. [(35)S]GTPgammaS binding assays using human M(2) receptors overexpressed in CHO cells revealed that a weak intrinsic efficacy was preserved in 8b-10b. Thus, attaching muscarinic allosteric antagonist moieties to orthosteric muscarinic agonists may lead to hybrid compounds in which functions of both components are mixed.

Comparison of the Antinociceptive Response to Morphine and Morphine-Like Compounds in Male and Female Sprague-Dawley Rats

Male rats are more sensitive to the antinociceptive effects of morphine than female rats. This difference is seen across several rat strains using a variety of nociceptive stimuli. However, the literature in regard to sex differences in antinociceptive responses to mu-opioids other than morphine is less consistent. The present study was designed to examine whether there is a structure-activity rationale that determines which mu-opioids will show a differential antinociceptive response between male and female rats. A series of morphinans closely related in structure to morphine, namely, codeine, heroin, hydrocodone, hydromorphone, oxymorphone, and oxycodone, were examined for their antinociceptive activity in male and female Sprague-Dawley rats and compared with the structurally unrelated mu-opioid agonists methadone and fentanyl. Antinociception was measured by the warm-water tail-withdrawal assay. The results show that morphine is more potent in males compared with females > hydromorphone = hydrocodone = oxymorphone, but there was no observable sex difference in the antinociceptive potency of codeine, heroin, oxycodone, methadone, or fentanyl. The potency to stimulate guanosine 5'-O-(3-[35 S]thio)triphosphate ([35S]GTPgammaS) binding and binding affinity of the various morphinans was compared in rat glioma C6 cells expressing the rat mu-opioid receptor; relative efficacy was also compared by stimulation of [35S]GTPgammaS binding in slices of rat brain thalamus. The presence of a sex difference in antinociceptive responsiveness was not related to drug potency, efficacy, or affinity. Consequently, it is likely that differential metabolism of the opioid, possibly by glucuronidation, determines the presence or absence of a sex difference.

Differential in Vivo Potencies of Naltrexone and 6β-Naltrexol in the Monkey

6beta-Naltrexol is the major metabolite of the opioid receptor antagonist, naltrexone, in humans. However, there are no functional studies of 6beta-naltrexol in primates. The aim of this study was to compare the in vitro and in vivo potencies of naltrexone and 6beta-naltrexol in rhesus monkeys. Affinity and potency were determined using radioligand displacement and stimulation of 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding in monkey brain membranes. In vivo apparent pA(2) analysis was applied to compare the mu-opioid receptor (MOR) antagonist potency of both compounds in nondependent monkeys. In addition, the potencies of both compounds were determined in precipitating withdrawal manifested by increased respiratory parameters in acute morphine-dependent monkeys. In vitro assays revealed that naltrexone displayed 2-fold higher affinity and potency than 6beta-naltrexol for the MOR binding site and for MOR agonist-stimulated [(35)S]GTPgammaS binding, respectively. 6beta-Naltrexol (0.32-3.2 mg/kg) dose-dependently produced parallel rightward shifts of the dose-response curve of alfentanil-induced antinociception. Nevertheless, the apparent pA(2) value of 6beta-naltrexol (6.5) was 100-fold less potent than that of naltrexone (8.5) determined previously. 6beta-Naltrexol was also less potent than naltrexone in antagonizing other MOR-mediated effects including respiratory depression and itch/scratching. Naltrexone (0.0032-0.032 mg/kg) and 6beta-naltrexol (0.32-3.2 mg/kg) retained the same potency difference in precipitating withdrawal to a similar degree. Furthermore, 6beta-naltrexol failed to block naltrexone-precipitated withdrawal in morphine-dependent monkeys. These results indicate that naltrexone and 6beta-naltrexol display similar pharmacological actions with a large in vivo potency difference in monkeys such that 6beta-naltrexol may play a minimal role in the therapeutic or antagonist effects of naltrexone in primates.

Expression of Functional G Protein-Coupled Receptors in Photoreceptors of TransgenicXenopus laevis

G protein-coupled receptors (GPCRs) constitute the largest superfamily of transmembrane signaling proteins; however, the only known GPCR crystal structure is that of rhodopsin. This disparity reflects the difficulty in generating purified GPCR samples of sufficient quantity and quality. Rhodopsin, the light receptor of retinal rod neurons, is produced in large amounts of homogeneous quality in the vertebrate retina. We used transgenic Xenopus laevis to convert these retina rod cells into bioreactors to successfully produce 20 model GPCRs. The receptors accumulated in rod outer segments and were homogeneously glycosylated. Ligand and [(35)S]GTPgammaS binding assays of the 5HT(1A) and EDG(1) GPCRs confirmed that they were properly folded and functional. 5HT(1A)R was highly purified by taking advantage of the rhodopsin C-terminal immunoaffinity tag common to all GPCR constructs. We have also developed an automated system that can generate hundreds of transgenic tadpoles per day. This expression approach could be extended to other animal model systems and become a general method for the production of large numbers of GPCRs and other membrane proteins for pharmacological and structural studies.

κ‐ and δ‐opioid receptor functional activities are increased in the caudate putamen of cannabinoid CB1 receptor knockout mice

The purpose of this study was to examine the functional interaction between endogenous opioid and cannabinoid receptor systems in the caudate putamen and nucleus accumbens. We therefore examined by autoradiography the functional activity and density of micro-, kappa- and delta-opioid receptors in both brain regions of cannabinoid CB1 receptor knockout mice. Functional activity was estimated by measuring agonist-stimulated [35S]GTPgammaS binding. Results showed that deletion of the CB1 cannabinoid receptor markedly increased kappa-opioid (50%) and delta-opioid (42%) receptor activities whereas no differences were found in micro-opioid receptor in the caudate putamen. In contrast, binding autoradiography showed a similar density of micro-, kappa- and delta-opioid receptors between mutant and wild-type mice. No differences were found in densities or activities of micro-, kappa- and delta-opioid receptors between mutant and wild-type mice in the nucleus accumbens. Taken together, our results revealed that deletion of CB1 cannabinoid receptors produced a pronounced increase in the activity of kappa- and delta-opioid receptors in the caudate putamen. This endogenous interaction between opioid and cannabinoid receptors may be relevant to further understand a variety of neuroadaptative processes involving the participation of opioid receptors, such as motor behaviour, emotional responses and drug dependence.

Subchronic haloperidol increases CB1 receptor binding and G protein coupling in discrete regions of the basal ganglia

The present study was designed to test whether chronic neuroleptic treatment, which is known to alter both expression and density of dopamine D(2) receptors in striatal regions, has effects upon function and binding level of the cannabinoid CB(1) receptor in the basal ganglia by using receptor autoradiography. As predicted, subchronic haloperidol treatment resulted in increased binding of (3)H-raclopride and quinpirole-induced guanosine 5'-O-(gamma-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) in the striatum when compared to that measured in control animals. This increased D(2) receptor binding and function after 3 days washout was normalized after a 2-week washout period. Effect of haloperidol treatment was studied for CB(1) receptor binding and CP55,940-stimulated [(35)S]GTPgammaS in the striatum, globus pallidus, and substantia nigra. (3)[H]CP55,940 binding levels were found in rank order from highest to lowest in substantia nigra > globus pallidus > striatum. Furthermore, subchronic haloperidol treatment resulted in elevated binding levels of (3)[H]CP55,940 in the striatum and the substantia nigra and CB(1) receptor-stimulated [(35)S]GTPgammaS bindings in the substantia nigra after 3 days washout. These increased binding levels were normalized at 1-4 weeks after termination of haloperidol treatment. Haloperidol treatment had no significant effect on CB(1) receptor or [(35)S]GTPgammaS binding levels in globus pallidus. The results help to elucidate the underlying biochemical mechanism of CB(1) receptor supersensitivity after haloperidol treatment.