Pharmacological characterization of serotonin-stimulated phosphoinositide turnover in brain regions of the immature rat

The effects of serotonin (5-HT) and related agonists and antagonists on phosphoinositide turnover have been investigated in several brain regions of the immature rat. In the presence of LiCl, 5-HT caused a marked increase in total [3H]inositol phosphate levels in cortical (maximal effect + 420%, EC50 = 7 microM) and to a lesser extent in hippocampal and striatal slices prepared from the immature (8-day-old) rat; the cortical 5-HT-induced phosphoinositide response was tetrodotoxin resistant. The magnitude of the increase in the cortical phosphoinositide response caused by 5-HT was maximal at 1 day postnatal and progressively declined to reach 6% of this maximal response in the adult. After incubation of immature (8-day-old) rat cortical slices for 2.5 min with 5-HT (in the absence of LiCl), inositol 1-phosphate, inositol 1,4-bisphosphate, inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate levels increased about 2-fold. A variety of 5-HT2 or mixed 5-HT1/5-HT2 agonists stimulated total [3H]inositol phosphate formation in the immature rat cortex and hippocampus with a rank order of potency [alpha(+)-methyl-5-HT greater than quipazine greater than MK 212 greater than 5-HT] which resembles their potencies at the 5-HT2 binding site. In contrast, the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin, the 5-HT1B agonists 1-(m-trifluoromethylphenyl)piperazine and 1-(m-chlorophenyl)-piperazine and the 5-HT3 agonist 2-methyl-5-HT were inactive.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of peptidic and nonpeptidic delta-opioid agonists on guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding in brain slices from Sprague-Dawley rats

Previous studies have demonstrated that peptidic and nonpeptidic delta-opioid receptor agonists have different effects depending on the measure. For example, nonpeptidic delta-opioid agonists, but not peptidic agonists, produce convulsions in rats, and in vitro studies suggested that peptidic and nonpeptidic delta-opioid agonists might have differential mechanisms of receptor downregulation. The present study evaluated potential differences between peptidic and nonpeptidic delta-opioid agonists in their ability to activate G proteins using guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) autoradiography experiments in rat brain slices. The peptidic agonist [d-Pen(2),d-Pen(5)]-enkephalin and the nonpeptidic agonist (+)BW373U86 [(+)-4-[alpha(R)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-hydroxyphenyl)methyl]-N,N-diethylbenzamide] demonstrated concentration-dependent increases in [(35)S]GTPgammaS binding that were attenuated by the delta-opioid antagonist naltrindole. (+)BW373U86 was more potent and efficacious than the peptidic agonist, and this difference remained consistent across brain regions where significant stimulation was observed. In addition, multiple delta-opioid compounds were evaluated for their agonist activity in this assay. These data suggested that differences between peptidic and nonpeptidic delta-opioid agonists in behavioral studies were most likely caused by differences in agonist efficacy. Finally, these data also revealed that [(35)S]GTPgammaS autoradiography could be used to compare efficacy differences among agonists across various brain regions in rat brain slices.     

Positron emission tomography imaging using an inverse agonist radioligand to assess cannabinoid CB1 receptors in rodents

[11C]MePPEP is an inverse agonist and a radioligand developed to image cannabinoid CB1 receptors with positron emission tomography (PET). It provides reversible, high specific signal in monkey brain. We assessed [11C]MePPEP in rodent brain with regard to receptor selectivity, susceptibility to transport by P-glycoprotein (P-gp), sensitivity to displacement by agonists, and accumulation of radiometabolites. We used CB1 receptor knockout mice and P-gp knockout mice to assess receptor selectivity and sensitivity to efflux transport, respectively. Using serial measurements of PET brain activity and plasma concentrations of [11C]MePPEP, we estimated CB1 receptor density in rat brain as distribution volume. CB1 knockout mice showed only nonspecific brain uptake, and [11C]MePPEP was not a substrate for P-gp. Direct acting agonists anandamide (10 mg/kg), methanandamide (10 mg/kg), CP 55,940 (1 mg/kg), and indirect agonist URB597 (0.3 and 0.6 mg/kg) failed to displace [11C]MePPEP, while the inverse agonist rimonabant (3 and 10 mg/kg) displaced >65% of [11C]MePPEP. Radiometabolites represented ~13% of total radioactivity in brain between 30 and 120 min. [11C]MePPEP was selective for the CB1 receptor, was not a substrate for P-gp, and was more potently displaced by inverse agonists than agonists. The low potency of agonists suggests either a large receptor reserve or non-overlapping binding sites for agonists and inverse agonists. Radiometabolites of [11C]MePPEP in brain caused distribution volume to be overestimated by approximately 13%.     

Lorcaserin, a novel selective human 5-hydroxytryptamine2C agonist: in vitro and in vivo pharmacological characterization

5-Hydroxytryptamine (5-HT)(2C) receptor agonists hold promise for the treatment of obesity. In this study, we describe the in vitro and in vivo characteristics of lorcaserin [(1R)-8-chloro-2,3,4,5-tetrahydro-1-methyl-1H-3 benzazepine], a selective, high affinity 5-HT(2C) full agonist. Lorcaserin bound to human and rat 5-HT(2C) receptors with high affinity (K(i) = 15 +/- 1 nM, 29 +/- 7 nM, respectively), and it was a full agonist for the human 5-HT(2C) receptor in a functional inositol phosphate accumulation assay, with 18- and 104-fold selectivity over 5-HT(2A) and 5-HT(2B) receptors, respectively. Lorcaserin was also highly selective for human 5-HT(2C) over other human 5-HT receptors (5-HT(1A), 5-HT(3), 5-HT(4C), 5-HT5(5A), 5-HT(6), and 5-HT(7)), in addition to a panel of 67 other G protein-coupled receptors and ion channels. Lorcaserin did not compete for binding of ligands to serotonin, dopamine, and norepinephrine transporters, and it did not alter their function in vitro. Behavioral observations indicated that unlike the 5-HT(2A) agonist (+/-)-1-(2,5-dimethoxy-4-phenyl)-2-aminopropane, lorcaserin did not induce behavioral changes indicative of functional 5-HT(2A) agonist activity. Acutely, lorcaserin reduced food intake in rats, an effect that was reversed by pretreatment with the 5-HT(2C)-selective antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-yl-carbamoyl]indoline (SB242,084) but not the 5-HT(2A) antagonist (R)-(+)-alpha-(2,3-dimethoxyphenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidine-methanol (MDL 100,907), demonstrating mediation by the 5-HT(2C) receptor. Chronic daily treatment with lorcaserin to rats maintained on a high fat diet produced dose-dependent reductions in food intake and body weight gain that were maintained during the 4-week study. Upon discontinuation, body weight returned to control levels. These data demonstrate lorcaserin to be a potent, selective, and efficacious agonist of the 5-HT(2C) receptor, with potential for the treatment of obesity.     

Inverse agonism at heptahelical receptors: concept, experimental approach and therapeutic potential

Inverse agonists (negative antagonists) are ligands that stabilize the inactive conformation (R) of receptors according to the two-state receptor model. The active conformation (R*) of heptahelical receptors, i.e. G protein-coupled receptors, has high affinity for G proteins. According to ternary complex models of receptor activation, the R*G complex is in equilibrium with R + G, with spontaneous activity in the absence of agonist. Inverse agonists, having a higher affinity for R, shift R*G towards R + G, decreasing the spontaneous activity of receptors. Agonists have the opposite effect, with a higher affinity for R*. Neutral antagonists have the same affinity for R and R* and compete for both agonists and inverse agonists. Inverse agonists have been recently proposed for a variety of heptahelical receptors. Methods to detect inverse agonists among antagonists are based on the determination of ligand affinity at R and R* with binding experiments, and on the modulation of G protein activity (GTP binding and hydrolysis) or of effector activity. Receptor inverse agonists, but also G protein antagonists and GTPase inhibitors, decrease spontaneous G protein activity corresponding to R*G. Receptor agonists, G protein agonists and GTPase inhibitors increase effector basal activity, but receptor inverse agonists decrease it. The therapeutic potential of inverse agonists is proposed in human diseases ascribed to constitutively active mutant receptors and may be extended to diseases related to wild-type receptor over-expression leading to the increase of R*. Some of the therapeutic effects of presently used receptor antagonists may be related to their inverse agonist properties. Inverse agonists lead to receptor upregulation, offering new approaches to tolerance and dependence to drugs.     

Inverse agonist activity of selected ligands of platelet-activating factor receptor

The receptor for platelet-activating factor (PAFR) is a member of the G protein-coupled receptor (GPCR) family. According to the allosteric ternary complex model, GPCRs exist in an equilibrium between different conformations. Agonist binding promotes and stabilizes the receptor in an active conformation. On the other hand, ligands that stabilize the inactive conformation are known as inverse agonists. Due to the association of platelet-activating factor (PAF) with diverse physiological and pathological processes, considerable efforts have been invested in the development of antagonists to PAFR. A large number of these molecules has been shown to specifically interact with PAFR but, surprisingly, little is known about their impact on the conformation of the receptor and its activity. By using a constitutively active mutant (L231R) of the human PAFR and by transiently coexpressing the wild-type (WT) receptor with the G(alpha)q subunit of the trimeric G protein, we were able to address this issue with ligands of diverse structures such as phospholipids, benzodiazepines, furans, and others. We demonstrated that some of these molecules are potent inverse agonists. For example, when cells (WT PAFR + G(alpha)q) were exposed to WEB2086, SM10661, or alprazolam, the basal inositol phosphate production was reduced by 53 +/- 6, 44 +/- 3, and 54 +/- 4%, respectively. The decrease in basal inositol phosphate production by WEB2086 was significantly inhibited by a more neutral antagonist BN52021, confirming the specificity of the reaction. We demonstrate here that WEB2086 and other known ligands previously considered as antagonists can act as inverse agonists on the human PAF receptor.     

Constitutive coupling of a chimeric dopamine D2/alpha 1B receptor to the phospholipase C pathway: inverse agonism to silent antagonism by neuroleptic drugs

Neuroleptic drugs have been suggested to act as inverse agonists at the dopamine D2 receptor, but no link between therapeutic efficacy and ligand's intrinsic activity could be determined. Since the resolving capacity to monitor inverse agonism at dopamine D2 receptors is limited, we speculated that receptor constitutive activation could be enhanced by constructing chimeric D2/alpha 1B receptors. Marked inverse agonist responses with a series of dopamine antagonists were obtained by: 1) exchange of the D 2short receptor's 3ICL by that of the alpha 1B-adrenoceptor, 2) incorporation of an activating mutation (Ala 279 Glu) in the distal portion of its 3ICL, and 3) coexpression with a G alpha11 protein. This chimeric D2/alpha 1B receptor construct displayed a ligand binding profile comparable to that of the wild-type (wt) D 2short receptor and an effector activation profile close to that of the wt alpha 1B-adrenoceptor. Most of the dopamine antagonists attenuated by -54 to -59% basal inositol phosphates (IP) formation, thus clearly acting as inverse agonists. Ziprasidone behaved as a silent antagonist (+5% versus basal IP level) and antagonized both dopamine-mediated (pK B, 7.61) and tropapride-mediated (pK B, 8.52) IP responses. Clozapine, olanzapine, and raclopride displayed partial inverse agonist properties (-31, -67, and -71% versus tropapride, respectively), whereas bromerguride (+63%) and cis-(+)-5-methoxy-1-methyl-2-(di-n-propylamino tetralin) [(+)-UH 232] (+88%) demonstrated positive agonism. In conclusion, analyses with the chimeric D2/alpha 1B Ala 279 Glu 3ICL receptor construct suggest that neuroleptic drugs can be differentiated on the basis of their intrinsic activity, as they can either activate, inhibit, or be silent at this receptor construct.     

Intrinsic efficacy of antipsychotics at human D2, D3, and D4 dopamine receptors: identification of the clozapine metabolite N-desmethylclozapine as a D2/D3 partial agonist

Drugs that antagonize D2-like receptors are effective antipsychotics, but the debilitating movement disorder side effects associated with these drugs cannot be dissociated from dopamine receptor blockade. The "atypical" antipsychotics have a lower propensity to cause extrapyramidal symptoms (EPS), but the molecular basis for this is not fully understood nor is the impact of inverse agonism upon their clinical properties. Using a cell-based functional assay, we demonstrate that overexpression of Galphao induces constitutive activity in the human D2-like receptors (D2, D3, and D4). A large collection of typical and atypical antipsychotics was profiled for activity at these receptors. Virtually all were D2 and D3 inverse agonists, whereas none was D4 inverse agonist, although many were potent D4 antagonists. The inverse agonist activity of haloperidol at D2 and D3 receptors could be reversed by mesoridazine demonstrating that there were significant differences in the degrees of inverse agonism among the compounds tested. Aripiprazole and the principle active metabolite of clozapine NDMC [8-chloro-11-(1-piperazinyl)-5H-dibenzo [b,e] [1,4] diazepine] were identified as partial agonists at D2 and D3 receptors, although clozapine itself was an inverse agonist at these receptors. NDMC-induced functional responses could be reversed by clozapine. It is proposed that the low incidence of EPS associated with clozapine and aripiprazole used may be due, in part, to these partial agonist properties of NDMC and aripiprazole and that bypassing clozapine blockade through direct administration of NDMC to patients may provide superior antipsychotic efficacy.     

Evaluation of histamine H1-, H2-, and H3-receptor ligands at the human histamine H4 receptor: identification of 4-methylhistamine as the first potent and selective H4 receptor agonist

The histamine H(4) receptor (H(4)R) is involved in the chemotaxis of leukocytes and mast cells to sites of inflammation and is suggested to be a potential drug target for asthma and allergy. So far, selective H(4)R agonists have not been identified. In the present study, we therefore evaluated the human H(4)R (hH(4)R) for its interaction with various known histaminergic ligands. Almost all of the tested H(1)R and H(2)R antagonists, including several important therapeutics, displaced less than 30% of specific [(3)H]histamine binding to the hH(4)R at concentrations up to 10 microM. Most of the tested H(2)R agonists and imidazole-based H(3)R ligands show micromolar-to-nanomolar range hH(4)R affinity, and these ligands exert different intrinsic hH(4)R activities, ranging from full agonists to inverse agonists. Interestingly, we identified 4-methylhistamine as a high-affinity H(4)R ligand (K(i) = 50 nM) that has a >100-fold selectivity for the hH(4)R over the other histamine receptor subtypes. Moreover, 4-methylhistamine potently activated the hH(4)R (pEC(50) = 7.4 +/- 0.1; alpha = 1), and this response was competitively antagonized by the selective H(4)R antagonist JNJ 7777120 [1-[(5-chloro-1H-indol-2-yl)-carbonyl]-4-methylpiperazine] (pA(2) = 7.8). The identification of 4-methylhistamine as a potent H(4)R agonist is of major importance for future studies to unravel the physiological roles of the H(4)R.     

Developmental changes in muscarinic receptor-stimulated phosphoinositide metabolism in rat brain

Muscarinic receptor-stimulated phosphoinositide hydrolysis was investigated in rat brain during ontogeny by measuring the accumulation of [3H]inositol phosphates ([3H]InsPs) in cerebral cortex slices at various ages. Experiments with carbachol and acetylcholine showed that [3H]InsPs accumulation was maximal in 7-day-old rats (1477 +/- 98% of basal) and lowest in adult (75 days) rats (428 +/- 24% of basal). No differences were found in the EC50 values for both cholinergic agonists. This effect appeared to be mediated by the M1-muscarinic receptor subtype as it was blocked by pirenzepine with Ki = 29.1 +/- 7.1 nM (adults) and 87.9 +/- 18.2 nM (7-day-old rats). Incorporation of [3H]inositol into phospholipid decreased from day 3 to adulthood; however, when data of [3H]InsPs release were corrected for the incorporation at a given age, the highest stimulation by cholinergic agonists was still observed in 7-day-old rats. Among the other neurotransmitters tested (norepinephrine, histamine and serotonin), all known to stimulate phosphoinositide metabolism, none had the same developmental profile of [3H]InsPs accumulation as cholinergic agonists. In contrast to carbachol- and acetylcholine-stimulated phosphoinositide hydrolysis, the density of muscarinic binding sites, measured by [3H]quinuclidinyl benzilate binding, increased from day 3 to day 75. Acetylcholinesterase activity also increased during development. The dissociation of receptor binding sites from receptor-stimulated phosphoinositide metabolism suggests the presence of a more effective receptor-effector coupling at specific times of neonatal development, particularly 1 week. Furthermore, the fact that maximal stimulation of phosphoinositide hydrolysis coincides with the period of brain growth spurt in the rats suggests that this system in the cerebral cortex might be involved in the processes of cell division and differentiation.     

(2S,3R) beta-methyl-2',6'-dimethyltyrosine-L-tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] is a potent,selective delta-opioid receptor antagonist in mouse brain

The constrained opioid peptide (2S,3R)beta-methyl-2',6'-dimethyltyrosine-L-tetrahydroisoquinoline-3-carboxylic acid [(2S,3R)TMT-L-Tic-OH] exhibits high affinity and selectivity for the delta-opioid receptors (). In the present study, we examined the pharmacological properties of (2S,3R)TMT-L-Tic-OH in mouse brain. A 5'-O-(3-[(35)S]thiotriphosphate) ([(35)S]GTP gamma S) binding assay was used to determine the effect of (2S,3R)TMT-L-Tic-OH on G protein activity in vitro, in mouse brain membranes. delta- (SNC80; (+)-4-[(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N,N-diethyl-benzamide) or mu- (DAMGO; [D-Ala(2), Me-Phe(4),Gly(ol)(5)]enkephalin) selective opioid full agonists stimulated [(35)S]GTP gamma S binding in mouse brain membranes 150 +/- 4.5% and 152 +/- 5.7% over the basal level, respectively. (2S,3R)TMT-L-Tic-OH did not influence basal [(35)S]GTP gamma S binding in mouse brain membranes but dose dependently shifted the dose-response curve of SNC80 to the right, with a K(e) value of 3.6 +/- 0.7 nM. In contrast, (2S,3R)TMT-L-Tic-OH had no effect on the dose-response curve of the mu-selective opioid agonist, DAMGO. Warm water (55 degrees C) tail-flick and radiant heat paw-withdrawal tests were used to determine the in vivo nociceptive properties of (2S,3R)TMT-L-Tic-OH in the mouse. Intracerebroventricular injection of (2S,3R)TMT-L-Tic-OH had no significant effect on withdrawal latencies in either nociceptive tests. (2S,3R)TMT-L-Tic-OH (30 nmol/mouse) attenuated deltorphin II- but not DAMGO-mediated antinociception (40 +/- 13 and 100% of maximal possible effect, respectively) when administered intracerebroventricularly 10 min before the agonist. Taken together these results suggest that (2S,3R)TMT-L-Tic-OH is a potent highly selective neutral delta-opioid antagonist in mouse brain.     

Selectivity and potency of agonists for the three subtypes of cloned human beta-adrenoceptors expressed in Chinese hamster ovary cells

The selectivities, potencies and efficacies of beta3-adrenoceptor (beta3-AR) agonists on human three beta-AR subtypes expressed in Chinese hamster ovary (CHO) cells were investigated using radioligand binding assay and cyclic AMP (cAMP) accumulation assay. The three beta-AR subtypes showed the nature of G protein-coupled receptors with the constitutive activity. BRL37344, CL-316,243 and a newly synthesized beta3-AR agonist N-5984, 6-[2-(R)-[[2-(R)-(3-chlorophenyl)-2-hydroxyethyl]amino]propyl]-2,3-dihydro-1,4-benzodioxine-2-(R)-carboxylic acid, were compared for the potency and selectivity for the beta3-AR. In the radioligand binding assay, the affinity of N-5984 for beta3-ARs was 14, 70 and 220 times more potent than those of BRL37344, isoproterenol and CL-316,243, respectively. N-5984 had higher selectivity than BRL37344 for human beta3-ARs compared with either for beta1-ARs or beta2-ARs. N-5984 showed higher potency and intrinsic activity of cAMP production than BRL37344 in CHO cells expressing the beta3-ARs. CL-316,243 had almost no activity of cAMP production in CHO cells expressing any subtype of beta-ARs. These results indicate that N-5984 is the most potent and selective agonist for human beta3-ARs than any other agonists tested.     

Selectivity of agonists for the active state of M1 to M4 muscarinic receptor subtypes

We measured the intrinsic relative activity (RA(i)) of muscarinic agonists to detect possible selectivity for receptor subtypes and signaling pathways. RA(i) is a relative measure of the microscopic affinity constant of an agonist for the active state of a GPCR expressed relative to that of a standard agonist. First, we estimated RA(i) values for a panel of agonists acting at the M(4) muscarinic receptor coupled to three distinct G-protein pathways: G(i) inhibition of cAMP accumulation, G(s) stimulation of cAMP accumulation, and G alpha(15) stimulation of phosphoinositide hydrolysis. Our results show similar RA(i) values for each agonist, suggesting that the same active state of the M(4) receptor triggers the activation of the three G proteins. We also estimated RA(i) values for agonists across M(1) to M(4) muscarinic subtypes stably transfected in Chinese hamster ovary cells. Our results show selectivity of McN-A-343 [4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride] for the M(1) and M(4) subtypes and selectivity of pilocarpine for the M(1) and M(3) subtypes. The other agonists tested lacked marked selectivity among M(1) to M(4) receptors. Finally, we estimated RA(i) values from published literature on M(1), M(2), and M(3) muscarinic responses and obtained results consistent with our own studies. Our results show that the RA(i) estimate is a useful receptor-dependent measure of agonist activity.     

Ligand dependency of 5-hydroxytryptamine 2C receptor internalization

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a well characterized phenomenon believed to contribute to receptor desensitization. The 5-hydroxytryptamine (5-HT)2C subtype of serotonin receptor is a GPCR that we have shown to internalize upon agonist incubation. In this study, we have examined the effects of 5-HT2C receptor agonists serotonin, Ro 60-0175 [(S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine], and WAY-161503 [(4aR)-8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one]; partial agonists mCPP [1-(m-chlorophenyl)piperazine] and DOI [(+)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane]; inverse agonists SB-206553 [N-3-pyridinyl-3,5-dihydro-5-methylbenzo(1,2-b:4,5-b')dipyrrole-1(2H)carboxamide] and mianserin; and neutral antagonists SB-242084 [6-chloro-5-methyl-1-[[2-[(2-methyl-3-pyridyl)oxy]-5-pyridyl]carbamoyl]-indoline] and 5-methoxygramine on the internalization of a C-terminal green fluorescent protein (GFP)-tagged 5-HT2C receptor (VSV isoform) expressed in transiently transfected human embryonic kidney cells. We detected internalization with an automated, cell-based fluorescence-imaging system (Arrayscan) and monitored function with intracellular Ca2+ measurements (flourometric imaging plate reader). The 5-HT2C-GFP construct exhibited appropriate pharmacology, and we observed that although all three agonists resulted in similar magnitudes of dose-dependent internalization, the partial agonists resulted in approximately 50% less internalization, and the inverse agonists and neutral antagonists failed to induce internalization. These results were confirmed by confocal microscopy. They demonstrate that the 5-HT2C receptor is internalized by incubation with agonists and partial agonists but not with inverse agonists or neutral antagonists.     

G protein coupling and signaling pathway activation by m1 muscarinic 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.     

Facilitation of constitutive alpha(2A)-adrenoceptor activity by both single amino acid mutation (Thr(373)Lys) and g(alphao) protein coexpression: evidence for inverse agonism

The recombinant human alpha(2A)-adrenoceptor (alpha(2A)-AR, RC 2.1. ADR.A2A) can be transformed into a constitutively activated form in CHO-K1 cells by coexpression with a rat G(alphao) protein. Constitutive activity could be enhanced more by both mutation of Thr(373) of the alpha(2A)-AR to a Lys and Cys(351) of the G(alphao) protein by an Ile. The basal [(35)S]GTPgammaS binding response displayed a constitutive alpha(2A)-AR activity that amounted to 21% of the maximal receptor activation as obtained with 10 microM (-)-adrenaline. UK 14304, BHT 920, d-medetomidine, oxymetazoline, and clonidine acted as efficacious agonists. The enhancement of basal activity was entirely blocked (-50 +/- 3%) by ligands that thus appeared to act as inverse agonists (i.e., RX 811059 and its (+)-enantiomer, (+)-RX 821002, RS 15385, and yohimbine); the potencies of the ligands corresponded with their binding affinities for the alpha(2A)-AR. Fluparoxan and WB 4101 displayed partial inverse agonism. Atipamezole and dexefaroxan at 10 microM were virtually free of intrinsic activity and thus acted as neutral antagonists; idazoxan displayed potent partial agonist properties as observed with BRL 44408 and SKF 86466. The inverse agonist activity induced by (+)-RX 811059 could be reversed by atipamezole with a pK(B) value (8.73 +/- 0.07) that was similar to that required for blockade of the UK 14304-mediated response. Constitutive alpha(2A)-AR activation was mainly observed with the G(alphao) Cys(351)Ile protein compared with the pertussis toxin-resistant mutants of the G(alphai) protein subtypes. The observed spectrum of intrinsic activities for the various ligands suggests that pure, neutral antagonists are rather uncommon in this specified alpha(2A)-AR system.     

Relationship between rate and extent of G protein activation: comparison between full and partial opioid agonists.

Opioid agonists acting at their receptors alter intracellular events by initiating activation of various types of Gi/Go proteins. This can be measured by the binding of the stable GTP analog [(35)S]guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS). In this study agonist efficacy is defined by the degree to which an opioid stimulates the binding of [(35)S]GTPgammaS. This allows for a definition of full and partial agonists; a full agonist causing a greater stimulation of [(35)S]GTPgammaS binding than a partial agonist. The hypothesis that the rate of agonist-stimulated [(35)S]GTPgammaS binding is dependent upon agonist efficacy was tested using membranes from C6 glioma cells expressing mu- or delta-opioid receptors. At maximal concentrations the rate of agonist-stimulated [(35)S]GTPgammaS binding followed the efficacy of mu-agonists in stimulating [(35)S]GTPgammaS binding, i.e., [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin > morphine > meperidine > butorphanol > nalbuphine. At submaximal concentrations of mu- or delta-full agonists the [(35)S]GTPgammaS association rate was also reduced, such that the rate of [(35)S]GTPgammaS binding correlated with the extent of [(35)S]GTPgammaS bound, whether this binding was stimulated by a full agonist or a partial agonist. Agonists also stimulated [(35)S]GTPgammaS dissociation, showing that binding of this stable nucleotide was reversible. Comparison of the delta-agonists [D-Ser(2),Leu(5)]-enkephalin-Thr and (+/-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxylbenzyl)-N,N-diethylbenzamide, a compound with slow dissociation kinetics, showed the measured rate of G protein activation was not influenced by the agonist switching between receptors. The results are consistent with the idea that the active state(s) of the receptor induced by full or partial agonists is the same, but the number of activated receptors determines the rate of G protein activation.     

Chronic agonist treatment converts antagonists into inverse agonists at delta-opioid receptors

In cellular models, chronic exposure to mu-opioid agonists converts antagonists into inverse agonists at mu-receptors. Such adaptations could contribute to the development of tolerance and/or dependence. To determine whether delta-receptors respond similarly, or whether this adaptation is unique for mu-receptors, this study examined the effects of prolonged agonist exposure on the intrinsic activity of several delta-opioid ligands in GH(3) cells expressing delta-receptors. In opioid naive cells, delta-receptors were constitutively active, and a series of delta-ligands displayed a range of intrinsic activities for G protein activation. Chronic treatment with the full delta-agonist [D-Pen(2,5)]-enkephalin reduced the acute ability of [D-Pen(2,5)]-enkephalin to stimulate and the full inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI-174864) to inhibit G protein activation. In contrast, although naloxone and naltriben exhibited weak partial agonism in opioid naive cells, both ligands acted as full inverse agonists to produce concentration-dependent inhibition of guanosine 5'-O-(3-[(35)S]thio)triphosphate binding after prolonged exposure to [D-Pen(2,5)]-enkephalin or to the partial agonist morphine. This effect was reversed by a neutral delta-antagonist (N,N-bisallyl)-Tyr-Gly-Gly-psi-(CH(2)S)-Phe-Leu-OH (ICI-154129). Finally, as is also characteristic of inverse agonists, naloxone and naltriben demonstrated higher affinities for uncoupled delta-receptors in cells chronically treated with [D-Pen(2,5)]-enkephalin, relative to opioid naive cells. Therefore, this relatively novel adaptation is shared by both mu- and delta-opioid receptors and therefore may serve as an important common mechanism involved the development of tolerance and/or dependence.