High-efficacy Agonist Research Articles

Pre- and postsynaptic actions of a partial D2 receptor agonist in reserpinized young rats: Longevity of agonistic effects

Partial D2 receptor agonists (e.g., terguride, preclamol, and aripiprazole) have antagonist-like effects at normosensitive D2 postsynaptic receptors and synthesis modulating autoreceptors. In reserpine-pretreated adult and young rats, however, partial D2 agonists function like high efficacy agonists at D2 postsynaptic receptors and autoreceptors (i.e., terguride increases locomotor activity and decreases dopamine synthesis). The purpose of the present study was to examine the time-course of these pharmacological effects. In all experiments, preweanling rats were given daily injections of reserpine (1 mg/kg, i.p.) or vehicle on postnatal day (PD) 16–PD 20. In the dopamine synthesis experiments, the ability of terguride (0.8 mg/kg) to reduce striatal DOPA accumulation (in NSD-1015 treated rats) was assessed either 5 h or 1, 2, 4, or 8 days (Experiment 1) or 4, 8, 12, 16, 20, or 24 days (Experiment 2) after reserpine pretreatment. In the behavioral experiments, locomotor activity of vehicle or terguride (0.8 mg/kg, i.p.) treated rats was assessed 5 h or 1, 2, 4, or 8 days after the 5-day reserpine regimen. Results from the dopamine synthesis experiments showed that terguride caused agonist-like effects (i.e., decreased DOPA accumulation) at only the 5 h and 1 day time points, although terguride did not induce its normal antagonist-like effects even 20 days after reserpine pretreatment. In the behavioral experiments, terguride stimulated locomotor activity for only the initial 2 days after reserpine pretreatment. The results of the present study show that the agonistic effects of terguride at pre- and postsynaptic receptors are short-lived, but terguride may not exhibit normal antagonistic effects, at least at synthesis modulating autoreceptors, until long after conclusion of reserpine pretreatment.

Development of tolerance and sensitization to different opioid agonists in rats

Despite numerous investigations, the mechanisms underlying the development of opioid tolerance are far from clear. However, several in vitro studies implicated a protective role of agonist-induced micro-opioid receptor endocytosis in the development of opioid tolerance. Moreover, we have recently demonstrated that the high-efficacy agonist etonitazene promotes rapid endocytosis of micro-opioid receptors, whereas the agonist morphine and the low-efficacy agonist buprenorphine fail to promote detectable receptor endocytosis in micro-opioid receptor expressing HEK293 cells. The present study explored the effects of these opioids on the development of tolerance and sensitization in rats in vivo. The opioid effects were quantified using the hot plate, electric tail root stimulation, and the locomotor activity chamber in male Wistar rats. Dose-response curves were generated for each test drug. To induce tolerance, equieffective doses of etonitazene, morphine, and buprenorphine were administered daily for 29 days. We found that chronic treatment with the non-internalizing drugs buprenorphine and morphine resulted in a greater development of tolerance than etonitazene. In addition, the sensitization to the locomotor stimulant effect was high after buprenorphine and morphine, but was lacking after chronic etonitazene application. The results support a role for the endocytotic potency of agonists in the development of tolerance and addiction during long-term opioid treatment.

Continuous opioid agonist treatment dose‐dependently regulates μ‐opioid receptors and dynamin‐2 in mouse spinal cord

Continuous opioid agonist treatment produces tolerance and in some cases mu opioid receptor (muOR) down-regulation. Previous studies indicate that down-regulation of muOR is more likely with high-efficacy opioid agonists (e.g., etorphine), whereas lower efficacy agonists (e.g., morphine) do not regulate muOR density. It has been suggested that muOR down-regulation may depend upon increases in Dynamin-2 (DYN-2) proteins. Therefore, the present study examined the effect of various infusion doses of etorphine on muOR density, DYN-2 protein, and DYN-2 mRNA abundance in mouse spinal cord. Mice were implanted sc with an osmotic pump that infused etorphine (50-250 microg/kg/day). Controls were implanted with inert placebo pellets. At the end of 7 days, mice were sacrificed, spinal cord removed and processed for radioligand binding, quantitative Western blotting, or RT-PCR assay. Results indicate that etorphine induced dose-dependent regulation of muOR density, DYN-2 proteins, and mRNA abundance in mouse spinal cord. Higher infusion doses significantly down-regulated muOR density, increased DYN-2 protein abundance, and decreased DYN-2 mRNA. Analysis of these results indicated a significant correlation between muOR down-regulation and DYN-2 abundance in mouse spinal cord. Taken together, muOR regulation may depend on changes in DYN-2 abundance induced by high-efficacy opioid agonists in mouse spinal cord.

Opioid agonist and antagonist treatment differentially regulates immunoreactive μ-opioid receptors and dynamin-2 in vivo

Opioid agonists and antagonists can regulate the density of μ-opioid receptors in whole animal and in cell culture. High intrinsic efficacy agonists (e.g., etorphine), but not lower intrinsic efficacy agonists (e.g., morphine), produce μ-opioid receptor down-regulation and can alter the abundance of μ-opioid receptor mRNA. Conversely, opioid antagonists substantially increase the density of μ-opioid receptors without changing its mRNA. μ-Opioid receptor up-regulation has been associated with decreases in the trafficking protein dynamin-2, whereas μ-opioid receptor down-regulation produces an increase in dynamin-2 abundance. To probe the differences between opioid agonist and antagonist-induced μ-opioid receptor regulation, the current study determined changes in μ-opioid receptor density using a combined radioligand binding ([ 3H] DAMGO) and quantitative Western blotting approach in mouse spinal cord. Furthermore, the differences between intermittent and continuous dosing protocols were evaluated. Continuous (7–8 days) s.c. infusions of naloxone (5 mg/kg/day) or naltrexone (15 mg s.c. implant pellet) increased μ-opioid receptor density in radioligand binding assays (≈+80%) in mouse spinal cord and down-regulated dynamin-2 abundance (≈−30%), but had no effect on the abundance of immunoreactive μ-opioid receptor. Continuous (7 days) s.c. infusion of etorphine (200 μg/kg/day) decreased immunoreactive μ-opioid receptor (≈−35%) and [ 3H] DAMGO binding (≈−30%), and concurrently increased dynamin-2 abundance (≈+40%). Continuous (7 days) morphine infusion (40 mg/kg/day plus 25 mg s.c. implant pellet) had no effect on any outcome measure. Delivery of the same daily dose of etorphine or naloxone using intermittent (every 24 h for 7 days) s.c. administration had no effect on immunoreactive μ-opioid receptor, [ 3H] DAMGO binding or dynamin-2 abundance. These data indicate that μ-opioid receptor density, determined in radioligand binding assays, and immunoreactive dynamin-2 abundance are regulated by continuous, but not intermittent, opioid ligand treatment. Furthermore, the differential regulation of μ-opioid receptor abundance by agonists and antagonists in immunoblotting assays contrasts with changes in [ 3H] DAMGO binding. Taken together, these results suggest that etorphine-induced down-regulation may depend upon μ-opioid receptor degradation and changes in dynamin-2-mediated receptor trafficking. Conversely, antagonist-induced up-regulation does not require an increase in μ-opioid receptor synthesis and may entail conversion of receptors to an appropriate conformation to bind ligand, as well as changes in receptor trafficking.

Effects of dopamine D1 ligands on eye blinking in monkeys: efficacy, antagonism, and D1/D2 interactions.

Dopamine D1 ligands have been classified and ordered according to efficacy in both in vitro and in vivo studies. In the present experiments, dopamine D1 ligands reported to differ in in vitro efficacy were evaluated for efficacy-related effects on eye blinking in squirrel monkeys. Additional comparisons were made with the effects of D2 receptor agonists and indirect dopamine agonists. The results show that D1 agonists increased eye blinking in an efficacy-related manner, whereas the D1 receptor blocker SCH 39166 [(-)-trans-6,7,7alpha,8,9,13beta-hexahydro-3-chloro-2-hydroxy-N-methyl-5H-benzo[d]naphtho[2,1-b]azepine] only decreased rates of eye blinking. D1 high-efficacy agonists induced rates of eye blinking that were 2- to 3-fold greater than observed with dopamine D2 agonists and indirect agonists. In drug combination experiments, increases in eye blinking induced by the D1 high-efficacy agonist R-(+)-6-Br-APB [R-(+)-6-bromo-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide] were antagonized by both the D1 antagonist SCH 39166 and the lower efficacy agonist SKF 83959 [6-chloro-7,8-dihydroxy-3-methyl-1-(3-methylphenyl)-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide], consistent with dopamine D1 receptor mediation of these behavioral effects. The dopamine D2 agonist (+)-PHNO [(+)-N-propyl-hydroxynaphthoxazine], which selectively activates dopamine D2 receptors, also attenuated D1 agonist-induced increase in eye blinking, suggesting that D2 receptor actions may inhibit D1-mediated increases in eye blinking. Overall, eye blink rate appears to be a robust behavioral measure that can be used to measure changes in dopaminergic D1 signaling and as a functional assay of agonist efficacy at dopamine D1 receptors.

Effects of Opioids in Morphine-Treated Pigeons Trained to Discriminate among Morphine, the Low-Efficacy Agonist Nalbuphine, and Saline

In opioid-dependent subjects, the low-efficacy mu agonist nalbuphine generally precipitates withdrawal or withdrawal-like stimulus effects. To provide a more complete characterization of the discriminative stimulus effects of nalbuphine in opioid-treated subjects, seven White Carneux pigeons were treated daily with 10 mg/kg morphine i.m. and trained 6 h later to discriminate among 10 mg/kg morphine, 1.0 mg/kg nalbuphine, and saline by responding on one of three different keys. When tested, morphine produced morphine-key responding and nalbuphine produced nalbuphine-key responding. Replacing the daily morphine injection with saline produced nalbuphine-key responding, and this effect was reversed by the administration of morphine. In substitution tests with other compounds, the antagonists naltrexone (i.m.) and CTAP (D-Phe-Cys-Tyr-D-Tryp-Lys-Thr-Pen-Thr-NH2) (i.c.v.) produced nalbuphine-key responding. High-efficacy agonists fentanyl and etorphine produced morphine-key responding. The intermediate-efficacy agonists buprenorphine, dezocine, and butorphanol produced a pattern of morphine-, saline-, and/or nalbuphine-key responding that differed across individual pigeons. The lower efficacy agonists nalorphine and levallorphan produced predominantly nalbuphine-key responding. The kappa agonists spiradoline and U50,488 [trans-3,4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl)benzeneacetamide methanesulfonate], the nonopioid d-amphetamine, and saline produced predominantly saline-key responding. Naltrexone and nalbuphine dose dependently reversed the morphine-key responding produced by the training dose of morphine. Together, these data suggest that the discriminative-stimulus effects of the low-efficacy micro agonist nalbuphine in morphine-treated pigeons are similar to those of other low-efficacy agonists, naltrexone, and the termination of daily morphine treatment.

Characterisation of G s activation by dopamine D1 receptors using an antibody capture assay: antagonist properties of clozapine

Herein, we examined the direct coupling of human dopamine D1 receptors to G s proteins using an antibody capture assay together with a detection technique employing scintillation proximity assay beads. Using a specific antibody, dopamine (DA) and the selective dopamine D1 receptor agonists, 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF81297) and 3-allyl-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF82958), behaved as high-efficacy agonists (∼100%) in stimulating guanosine-5′- O-(3-[ 35S]thio)-triphosphate ([ 35S]GTPγS) binding to G s in L-cells, whereas 2,3,4,5,-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (SKF38393) displayed partial agonist properties (70%). The action of dopamine was specifically mediated by human dopamine D1 receptors inasmuch as the selective human dopamine D1 receptor antagonist, ( R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7-ol (SCH23390), blocked dopamine-induced [ 35S]GTPγS binding to G s with a p K B (9.29) close to its p K i (9.33). The antipsychotic agents, clozapine and haloperidol, displayed no intrinsic activity when tested alone and inhibited dopamine-stimulated G s activation with p K B's of 6.7 and 7.3, respectively, values close to their p K i values at these sites. In conclusion, the use of an anti-G s protein immunoprecipitation assay coupled to scintillation proximity assays allows direct evaluation of the functional activity of dopamine D1 receptors ligands at the G protein level. Employing this novel technique, the typical and atypical antipsychotics, clozapine and haloperidol, respectively, both exhibited antagonist properties at dopamine D1 receptors.

μ-Opioid Receptor Desensitization in Mature Rat Neurons: Lack of Interaction between DAMGO and Morphine

Mu-opioid receptors (MORs) exhibit rapid desensitization and internalization during exposure to various opioid agonists. In some studies, however, morphine has been observed to produce little MOR desensitization or internalization. We examined desensitization in mature rat locus ceruleus (LC) neurons and confirmed that morphine is a very poor desensitizing agent, whereas [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO), a high-efficacy agonist, and methadone, an agonist we observed to be of equivalent efficacy to morphine, produced profound rapid desensitization. Similarly, by measuring plasma membrane receptor levels in HEK293 cells stably expressing T7-epitope-tagged rat MOR1 at near physiological levels (HEK293-MOR1 cells), DAMGO and methadone but not morphine caused rapid MOR internalization. It has been reported that a low concentration of DAMGO, coapplied with morphine, caused morphine to induce MOR internalization. We examined whether this interaction occurred in mature mammalian neurons at the level of receptor desensitization. Coapplication of low concentrations of DAMGO did not increase morphine-induced desensitization in LC neurons but caused a lesser degree of desensitization than DAMGO alone. We also failed to observe an enhancement by DAMGO of morphine-induced desensitization in the electrically stimulated guinea pig ileum myenteric plexus-longitudinal muscle preparation. In HEK293-MOR1 cells, low concentrations of DAMGO did not convert morphine into a receptor-internalizing agent. The data presented here fail to support the theory that low concentrations of DAMGO can increase morphine-induced MOR desensitization or internalization.

Differential effects of agonists on adenylyl cyclase superactivation mediated by the kappa opioid receptors: adenylyl cyclase superactivation is independent of agonist-induced phosphorylation, desensitization, internalization, and down-regulation.

Prolonged activation of opioid receptors followed by agonist removal leads to adenylyl cyclase (AC) superactivation. In this study, we examined in CHO cells stably expressing the human or rat kappa opioid receptor (hkor or rkor) whether agonists had differential abilities to induce AC superactivation and whether the hkor and rkor exhibited differential AC superactivation. Pretreatment of the hkor with (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate (U50,488H) induced AC superactivation in a time- and dose-dependent manner, reaching a plateau at 4 h and 0.1 microM. The extents of AC superactivation after a 4-h pretreatment of the hkor with saturating concentrations of agonists were in the order of the full agonists U50,488H, dynorphin A(1-17), (+/-)-ethylketocyclazocine, etorphine, and U69,593 > the high-efficacy partial agonist nalorphine > the low-efficacy partial agonists nalbuphine, morphine, and pentazocine. Interestingly, the full agonist levorphanol caused much lower AC superactivation than other full agonists and reduced the AC superactivation induced by U50,488H and dynorphin A(1-17) in a dose-dependent manner. The order of relative efficacies of agonists in causing AC superactivation mediated by the rkor was similar to that mediated by the hkor and the extents of AC superactivation were slightly lower. Because the rkor does not undergo U50,488H (1 microM)-induced phosphorylation, desensitization, internalization, and down-regulation in these cells, the degree of AC superactivation is independent of these processes. This is among the first reports to demonstrate that relative efficacies of agonists in causing AC superactivation generally correlated with those in activating G proteins and a full agonist reduced AC superactivation induced by another full agonist.

Influence of agonist efficacy and receptor phosphorylation on antagonist affinity measurements: differences between second messenger and reporter gene responses.

The ability of an antagonist to bind to a receptor is an innate property of that ligand-receptor chemical interaction. Provided no change in the antagonist or receptor chemical nature occurs, this affinity should remain constant for a given antagonist-receptor interaction, regardless of the agonists used. This fundamental assumption underpins the classification of receptors. Here, measurements of beta2-adrenoceptor-mediated cAMP accumulation and cAMP response-element (CRE)-mediated reporter-gene transcription revealed differences in antagonist affinity that depended upon agonist incubation time and the efficacy of the competing agonist. In cAMP accumulation studies (10-min agonist incubation), antagonist affinities were the same regardless of the agonist used. The CRE-reporter gene assay (5 h of incubation) antagonist affinities were 10-fold lower in the presence of isoprenaline and adrenaline than when salbutamol or terbutaline were present (e.g., log KD propranolol -8.65 +/- 0.08, n = 22, and -9.68 +/- 0.07, n = 17, for isoprenaline and salbutamol-induced responses, respectively). Isoprenaline and adrenaline were more efficacious in functional studies, and their ability to internalize GFP-tagged human beta2-adrenoceptors. Longer-term cAMP studies also showed significant differences in KD values moving toward that seen with gene transcription. Agonist-dependent differences in antagonist affinity were reduced for reporter-gene responses when a phosphorylation-deficient mutant of the beta2-adrenoceptor was used. This study suggests that high-efficacy agonists induce a chemical modification in beta2-adrenoceptors (via phosphorylation) that reduces antagonist affinities. Because reporter-gene assays are used for high-throughput screening in drug discovery, less efficacious or partial agonists may be more reliable than highly efficacious agonists when reporter-gene techniques are used to estimate antagonist affinity.

Mutation in a protein kinase C phosphorylation site of the 5-HT 1A receptor preferentially attenuates Ca 2+ responses to partial as opposed to higher-efficacy 5-HT 1A agonists

The Thr 149Ala mutation in a putative protein kinase C phosphorylation site of the 5-HT 1A receptor’s second intracellular loop has been shown to affect the closing of Ca 2+ channels and Ca 2+ mobilisation without interfering with the inhibitory cAMP pathway (Mol Pharmacol 52 (1997) 164). Here, the Ca 2+ responses for a series of 5-HT 1A agonists were compared between the wild-type (wt) and mutant Thr 149Ala 5-HT 1A receptor as part of a fusion protein containing a G α 15 protein. Neither the mutation nor the fusion process modified the [ 3H]WAY 100635-based ligand binding profile of the fusion proteins as compared to the wt 5-HT 1A receptor protein. Whereas at the wt 5-HT 1A receptor, 5-HT induced a Ca 2+ response in CHO-K1 cells via endogenous G i/o proteins, the Ca 2+ response to 5-HT at the mutant Thr 149Ala 5-HT 1A receptor was fully dependent on either the co-expression or the fusion to a recombinant G α 15 protein. Buspirone, flesinoxan and 8-OH-DPAT produced a graded partial response (26 to 62%) at the wt 5-HT 1A:G α 15 fusion protein; F 13640, 5-CT and F 14679 behaved as higher-efficacy agonists with maximal Ca 2+ responses similar to 5-HT. The maximal Ca 2+ responses at the mutant Thr 149Ala 5-HT 1A:G α 15 fusion protein were significantly attenuated for flesinoxan and 8-OH-DPAT (−45 and −36%, respectively); the response to the other 5-HT agonists was not significantly affected. A similar effect was observed upon treatment with phorbol 12-myristate 13-acetate at the Thr 149Ala 5-HT 1A:G α 15 fusion protein. In conclusion, the amplitude of the Ca 2+ responses induced by partial, but not that to fuller 5-HT 1A receptor agonists, is affected by the Thr 149Ala mutation of the 5-HT 1A:G α 15 fusion protein.

Chronic Morphine Treatment Inhibits Opioid Receptor Desensitization and Internalization

Chronic opioid receptor (OR) activation by morphine causes distinct cellular adaptations responsible for the development of tolerance. The present study examines the effect of chronic morphine exposure on the ability of high-efficacy agonists to mediate delta-OR (DOR) and mu-OR (MOR) uncoupling and internalization, two regulatory mechanisms contributing to rapid desensitization of OR function. Chronic morphine treatment (1 microm; 72 hr) of DOR carrying neuroblastoma x glioma (NG108-15) hybrid cells, a prototypical model system frequently used to study cellular aspects of opioid tolerance, completely blocked the capacity of [d-Ala2, d-Leu5]enkephalin (DADLE) and etorphine to desensitize opioid-stimulated [35S]GTPgammaS binding and to mediate DOR internalization. Similar findings were obtained on stably DOR- and MOR-transfected human embryonic kidney (HEK) 293 cells. Chronic morphine treatment also heterologously impaired agonist regulation of non-opioid G-protein-coupled receptors, such as the m(4)-muscarinic acetylcholine receptor and the brain-type cannabinoid receptor. As a possible underlying mechanism, we found that chronic morphine treatment completely blocked agonist-induced redistribution of beta-arrestin1 in both NG108-15 and stably MOR-transfected HEK293 cells. Moreover, attenuation of beta-arrestin1 function appears to depend on persistent stimulation of MAP kinase activity during the course of chronic morphine treatment, because coincubation of the cells together with the MAP kinase blocker PD98059 fully restored beta-arrestin1 translocation and receptor internalization. These results demonstrate that chronic morphine treatment produces adaptational changes at the beta-arrestin1 level, which in turn attenuates agonist-mediated desensitization and internalization of G-protein-coupled receptors.

5-HT1A receptor activation and anti-cataleptic effects: high-efficacy agonists maximally inhibit haloperidol-induced catalepsy

Studies have shown that 5-HT1A receptor ligands modulate antipsychotic-induced catalepsy. Here, we further examined the role of intrinsic activity at 5-HT1A receptors in these effects. The anti-cataleptic effects of 5-HT1A receptor ligands with positive intrinsic activity [from high to low: 3-chloro-4-fluorophenyl-(4-fluoro-4-{[(5-methyl-6-methylamino-pyridin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl-methanone fumaric acid salt (F 13714), eptapirone, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), 2-[4-[4-(7-methoxy-1-naphtyl) piperazino]butyl]-4-methyl-2H,4H-1,2,4-triazin-3,5-dione maleic acid salt (F 11461), buspirone, 2-[4-[4-(7-benzofuranyl)piperazino]butyl]-4-methyl-2H,4H-1,2,4-triazin-3,5-dione (F 12826), ipsapirone, and (s)-N-tert-butyl-3-(4-(2-methoxyphenyl)piperazine-1-yl)-2-phenylpropanamide hydrochloride (WAY 100135)] and negative intrinsic activity [N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexanecarboxamide dihydrochloride (WAY 100635)] were examined. Catalepsy was induced by the classical antipsychotic haloperidol (0.63 mg/kg) and measured in the cross-legged position test and in the bar test. All 5-HT1A receptor agonists, except WAY 100135, significantly attenuated the effects of haloperidol in the cross-legged position test. All agonists had similar effects in the bar test, except ipsapirone, which failed to attenuate haloperidol-induced catalepsy. In contrast to the effects observed with the agonists, the inverse agonist WAY 100635 appeared to enhance haloperidol-induced catalepsy in both tests, in agreement with earlier findings. The maximal effects of the 5-HT1A receptor ligands to attenuate catalepsy correlated positively with the rank order of their intrinsic activity at 5-HT1A receptors (either catalepsy test: rS=0.92, P<0.001). F 13714, which had the highest intrinsic activity, maximally inhibited haloperidol-induced catalepsy in the cross-legged position and bar tests (100% and 99% inhibition, respectively). Because the magnitude of the anti-cataleptic effects of 5-HT1A receptor ligands correlates positively with their intrinsic activity, it is likely that F 13714 has marked anti-cataleptic effects because of its high intrinsic activity at 5-HT1A receptors.

Antibody Capture Assay Reveals Bell-Shaped Concentration-Response Isotherms for h5-HT1AReceptor-Mediated Gαi3Activation: Conformational Selection by High-Efficacy Agonists, and Relationship to Trafficking of Receptor Signaling

Although serotonin 5-HT(1A) receptors couple to several Gi/o G-protein subtypes, little is known concerning their differential activation patterns. In this study, in membranes of Chinese hamster ovary cells expressing h5-hydroxytryptamine(1A) receptors (CHO-h5-HT(1A)), isotherms of 5-HT-stimulated guanosine-5'-O-(3-[(35)S]thio)-triphosphate ([(35)S]GTPgammaS) binding were biphasic, suggesting coupling to multiple G-protein subtypes. The high potency component was abolished by preincubation with an antibody recognizing Galpha(i3) subunits and was resistant to induction of [(35)S]GTPgammaS dissociation by unlabeled GTPgammaS, thus yielding a bell-shaped concentration-response isotherm. To directly investigate Galpha(i3) activation, we adopted an antibody-capture/scintillation proximity assay. 5-HT and other high-efficacy agonists yielded bell-shaped [(35)S]GTPgammaS binding isotherms, with peaks at nanomolar concentrations. As drug concentrations increased, Galpha(i3) stimulation progressively returned to basal values. In contrast, the partial agonists (-)-pindolol and 4-(benzodioxan-5-yl)1-(indan-2-yl)piperazine (S15535) displayed sigmoidal stimulation isotherms, whereas spiperone and other inverse agonists sigmoidally inhibited [(35)S]GTPgammaS binding. Agonist-induced stimulation and inverse agonist-induced inhibition of Galpha(i3) activation were i) abolished by pretreatment of CHO-h5-HT(1A) cells with pertussis toxin; ii) reversed by the selective 5-HT(1A) antagonist (N-[2-[4-(2-methoxy-phenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)-cyclohexane-carboxamide) fumarate (WAY100,635), and iii) absent in nontransfected CHO cell membranes. 5-HT isotherms could be modified by altering sodium concentration; only stimulatory actions were observed at 300mM NaCl, whereas only inhibitory actions were seen at 10 mM NaCl. Furthermore, bell-shaped isotherms were not detected at short incubation times, suggesting time-dependent changes in receptor/Galpha(i3) coupling. Taken together, these data show that low but not high concentrations of high-efficacy 5-HT(1A) agonists direct receptor signaling to Galpha(i3). In contrast, partial agonists favor h5-HT(1A) receptor signaling to Galpha(i3) over a wide concentration range, whereas inverse agonists inhibit constitutive Galpha(i3) activation.

Differential activation of Gq/11 and Gi(3) proteins at 5-hydroxytryptamine(2C) receptors revealed by antibody capture assays: influence of receptor reserve and relationship to agonist-directed trafficking.

As determined by a guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding assay, which does not distinguish G protein subtypes, 5-hydroxytryptamine (5-HT) and 2(S)- 1-(6-chloro-5-fluoro-1H-indol-1-yl)-2-propanamine fumarate (Ro600175) behaved as full agonists at human 5-HT(2C) (h5-HT(2C)) receptors (VSV isoform) stably expressed in Chinese hamster ovary (CHO) cells, whereas 1-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), d-lysergic acid diethylamide (LSD), and lisuride exhibited partial agonist properties. After treatment with pertussis toxin to uncouple 5-HT(2C) receptors from Gi/Go but not Gq/11, DOI and LSD were as efficacious as 5-HT and Ro600175 in stimulating [(35)S]GTPgammaS binding, whereas lisuride still exhibited low efficacy (40%). Correspondingly, in a scintillation proximity assay employing specific antibodies against Gq/11, 5-HT, Ro600175, DOI, and LSD behaved as high-efficacy agonists, whereas lisuride showed efficacy of 36%. In contrast, when employing a specific antibody recognizing Gi(3), DOI and LSD were less efficacious (80 and 30%, respectively) than 5-HT and Ro600175, and lisuride was inactive. Agonist actions were specifically mediated by h5-HT(2C) receptors inasmuch as the selective 5-HT(2C) antagonist SB242,084 blocked [(35)S]GTPgammaS binding at both Gq/11 and Gi(3). Agonist potency for stimulation of Gi(3) was ~6- to 8-fold less than for Gq/11, indicating that the latter was preferentially engaged by h5-HT(2C) receptors. Inactivation of h5-HT(2C) receptors with the alkylating agent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline did not modify the efficacy of 5-HT, Ro600175, and DOI at Gq/11, whereas their efficacies were substantially reduced at Gi(3), indicating a greater receptor reserve for the former. Finally, the preferential activation of Gq/11 versus Gi(3) by DOI, LSD, and lisuride was diminished in the presence of lower receptor number. In conclusion, h5-HT(2C) receptors couple to both Gq/11 and Gi(3) in CHO cells, and efficacy for G protein subtype activation is both ligand- and receptor reserve-dependent.

Comparison of contractile responses to donitriptan and sumatriptan in the human middle meningeal and coronary arteries

Donitriptan is a potent, high efficacy agonist at 5-HT 1B/1D receptors. We investigated the contractile effects of donitriptan and sumatriptan on human isolated blood vessels of relevance to therapeutic efficacy in migraine (middle meningeal artery) and coronary adverse events (coronary artery). Furthermore, using the concentration–response curves in the middle meningeal artery, we predicted the plasma concentration needed for the therapeutic effect of donitriptan. Both donitriptan and sumatriptan contracted the middle meningeal artery with similar apparent efficacy ( E max: 103±8% and 110±12%, respectively), but the potency of donitriptan (pEC 50: 9.07±0.14) was significantly higher than that of sumatriptan (pEC 50: 7.41±0.08). In the coronary artery, the contraction to donitriptan was biphasic with a significantly higher maximal response ( E max: 29±6%) than sumatriptan ( E max: 14±2%; pEC 50: 5.71±0.16), yielding two distinct pEC 50 values (8.25±0.16 and 5.60±0.24). Incubation with the 5-HT 2 receptor antagonist ketanserin (10 μM) eliminated the low affinity component of the concentration–response curve of donitriptan and the resultant E max and pEC 50 were 9±2% and 7.33±0.21, respectively. Ketanserin was without effect on the sumatriptan-induced contraction. Based on the middle meningeal artery contraction, concentrations ( C max) of donitriptan that may be expected to have a therapeutic efficacy equivalent to that of 50 and 100 mg sumatriptan are predicted to be around 2.5 and 4.3 nM, respectively. Such concentrations are likely to induce only a small coronary artery contraction of 2.9±1.5% and 3.8±2.0%, respectively; these are not different from those by C max concentrations of sumatriptan (1.7±0.4% or 2.2±0.4%). The present results suggest that, like sumatriptan, donitriptan exhibits cranioselectivity and would be effective in aborting migraine attacks with a similar coronary side-effect profile as sumatriptan.

Role of the histidine residue at position 105 in the human alpha 5 containing GABA(A) receptor on the affinity and efficacy of benzodiazepine site ligands.

1. A histidine residue in the N-terminal extracellular region of alpha 1,2,3,5 subunits of the human GABA(A) receptor, which is replaced by an arginine in alpha 4 and alpha 6 subunits, is a major determinant for high affinity binding of classical benzodiazepine (BZ)-site ligands. The effect of mutating this histidine at position 105 in the alpha 5 subunit to an arginine (alpha 5H105R) on BZ-site pharmacology has been investigated using radioligand binding on HEK293 and L(tk-) cells and two electrode voltage clamp recording on Xenopus oocytes in which GABA(A) receptors of subtypes alpha 5, alpha 5H105R, alpha 4 and alpha 6 were co-expressed with beta 3 gamma 2s. 2. The classical BZs, diazepam and flunitrazepam (full agonists on the alpha 5 receptor) showed negligible affinity and therefore negligible efficacy on alpha 5H105R receptors. The beta-carbolines DMCM and beta CCE (inverse agonists on the alpha 5 receptor) retained some affinity but did not exhibit inverse agonist efficacy at alpha 5H105R receptors. Therefore, the alpha 5H105R mutation confers an alpha 4/alpha 6-like pharmacology to the classical BZs and beta-carbolines. 3. Ro15-4513, flumazenil, bretazenil and FG8094, which share a common imidazobenzodiazepine core structure, retained high affinity and were higher efficacy agonists on alpha 5H105R receptors than would be predicted from an alpha 4/alpha 6 pharmacological profile. This effect was antagonized by DMCM, which competes for the BZ-site and therefore is likely to be mediated via the BZ-site. 4. These data indicate that the conserved histidine residue in the alpha subunit is not only a key determinant in the affinity of BZ-site ligands on alpha 5 containing GABA(A) receptors, but also influences ligand efficacy.

Differential signalling of both wild-type and Thr343Arg dopamine D2short receptor by partial agonists in a G-protein-dependent manner

G-protein activation and Ca2+ responses by the wild-type D2short receptor and a mutation Thr343Arg, in the distal BBXXB motif of its third intracellular loop, were investigated in CHO-K1 cells in terms of ligand:receptor:G-protein interactions. No evidence was obtained for constitutive, agonist-independent receptor activation, but differences in the ligand-mediated activation profiles of both the wild-type and mutant Thr343Arg D2short receptor were observed. Most of the partial agonists, but not bromocriptine, displayed an enhanced response at the mutant D2short receptor, suggesting that the mutation brings the receptor in a more active state. This enhancement was apparent both at the level of G-protein activation ([35S]GTPγS binding) and at the effector (Ca2+ response) and occurred with different Gα-proteins. Partial agonists were also found to act differently via the wild-type D2short receptor depending on the involved Gα-protein. Compared with higher efficacy agonists, partial agonists displayed Ca2+ responses with slower and dissimilar kinetic properties. Lisuride and in particular bromocriptine produced a more potent response in the co-presence of a Gαo protein instead of a chimeric Gαq/o- or a promiscuous Gα15-protein. S(+)-propylnorapomorphine showed a similar partial response irrespective of the combined Gα-protein. Bromerguride and (+)-UH 232 induced weak (16 to 21% versus dopamine) intrinsic activity in the co-presence of a Gαq/o-protein in contrast to their silent properties with a Gα15- or a GαoCys351Ile-protein. In conclusion, the present data strongly suggest that multiple activation binding sites are involved with these ligands at the D2short receptor, and that their activation may be unravelled by either the mutation or co-expressed Gα-proteins being investigated.

Modulation of cocaine and food self-administration by low- and high-efficacy D1 agonists in squirrel monkeys.

Dopamine D1 ligands have been proposed as candidate medications for cocaine abuse. Previous studies have shown that the ability of D1 ligands to modulate the behavioral effects of cocaine may depend on agonist efficacy. This study investigated the role of agonist efficacy in the ability of D1 ligands to modulate the reinforcing effects of cocaine in monkeys. Squirrel monkeys trained to self-administer cocaine under a second-order schedule of reinforcement were treated daily with D1 agonists varying in efficacy from low to high (SKF 83959 < SKF 77434 < or = SKF 81297 < SKF 82958) and the D1 antagonist SCH 39166. D1 ligands, regardless of efficacy, produced dose-dependent reductions in responding maintained by a maximally effective dose of cocaine. Equivalent doses of each D1 ligand reduced responding for food under a similar second-order schedule, suggesting that the suppression was not specific to cocaine self-administration. When studied in combination with a range of cocaine doses, treatment with the agonists SKF 83959, SKF 77434, SKF 81297, and the antagonist SCH 39166 produced overall rightward and downward shifts in the dose-response function for cocaine self-administration. Treatment with the agonist SKF 82958, however, produced an overall suppression of responding, regardless of the dose of cocaine. In contrast to a high-efficacy agonist, low-efficacy D1 ligands modulated the reinforcing effects of cocaine in a manner consistent with at least a partial antagonism of cocaine self-administration. This delineation of the efficacy-dependent profile of effects for D1 ligands should guide research into their utility as cocaine pharmacotherapies.

The "selective" dopamine D1 receptor antagonist, SCH23390, is a potent and high efficacy agonist at cloned human serotonin2C receptors.

The benzazepine and "selective" dopamine D1 receptor antagonist, SCH23390 [(R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7-ol], shows significant affinity at native serotonin (5-HT)2C receptors. We examined its functional actions at cloned human (h)5-HT2C receptors (VSV isoform) stably expressed in CHO cells. Since 5-HT2C receptors are positively coupled to phospholipase C (PLC), their activation was determined by depletion of membrane-bound pools of pre-labelled [3H]phosphotidylinositol ([3H]PI). SCH23390 showed high affinity (Ki, 9.3 nM) at h5-HT2C sites and depleted [3H]PI with an EC50 of 2.6 nM. Its efficacy was equivalent to that of 5-HT. [3H]PI depletion elicited by SCH23390 was concentration-dependently abolished by the selective 5-HT2C antagonist, SB242,084, with a K(B) of 0.55 nM. Further, in the presence of a fixed concentration of SB242,084 (10 nM), the concentration-response curve for SCH23390 was shifted to the right without loss of maximal effect, yielding a K(B) of 0.57 nM. SCH23390 is a potent and high efficacy agonist at h5-HT2C receptors. Activation of 5-HT2C receptors by SCH23390 may contribute to its functional properties both in animals and in humans.