Generation and characterization of highly constitutive active histamine H3 receptors

Constitutive activation of G-protein-coupled receptors is a well recognized phenomenon, and G-protein-coupled receptor antagonists have been found to possess inverse agonist activity. Constitutive activation of histamine H3 receptor is recently documented in in vivo as well as in recombinant receptor systems in vitro. Several H3 antagonists have been shown to act as inverse agonists and such profiles of H3 antagonists have been implicated in their pharmacological functions. Here we report the construction and characterization of a highly constitutive active H3 receptor (MT6), in which the 357 alanine residue was converted to lysine (A357K). We generated a series of mutated H3 receptors and their functions were examined in human embryonic kidney (HEK) 293 cells. Among them, induced mutation at the amino acid 357 position (A357K) showed a dramatically enhanced response to thioperamide-induced cAMP accumulation compared with the cells expressing wild-type (WT) H3 receptors, suggesting that the mutation rendered receptors to high constitutive activity. We further characterized by ligand binding assays using membrane fractions, and Ki values of imetit (agonist) and proxyfan (partial agonist) against the MT6 receptors were lower compared with those observed in WT H3 receptors. In contrast, H3 antagonists (thioperamide, ciproxifan, and GT2016) with inverse agonism displayed increased Ki values against the MT6 receptors (2.5- to 5.8-fold), demonstrating more a prominent effect of inverse agonists to the constitutive active receptor. Taken together, these data suggested that A357K mutation in the H3 receptor increased the population of active state receptors that preferably binds to agonists than inverse agonists, which could be termed as a constitutively active mutant of H3 receptor.     

Cloning of rat histamine H(3) receptor reveals distinct species pharmacological profiles

The recent cloning and characterization of the human histamine H(3) receptor cDNA marked a significant step toward a greater understanding of the role of this receptor in the central nervous system. We now report the cloning of the rat histamine H(3) receptor cDNA and demonstrate distinct pharmacological species differences. The rat cDNA clone encodes a putative 445-amino acid protein with 93% identity to the human H(3) receptor. Northern blot analysis revealed a major single entity of 2.7-kb in length expressed only in brain. Transfection of the rat receptor cDNA into SK-N-MC cells conferred an ability to inhibit forskolin-stimulated cAMP formation in response to histamine and other H(3) agonists. N-[(3)H]methylhistamine saturably bound to transfected cells with high affinity (K(d) = 0.8 nM). All agonists tested had low or subnanomolar K(i) values similar to that for the human recombinant receptor. The antagonists thioperamide and clobenpropit also bound with high affinity (K(i) = 4 and 0.4 nM, respectively). This is in contrast to the antagonist profile obtained for the human recombinant receptor that showed K(i) values of 58 and 0.6 nM for thioperamide and clobenpropit, respectively. These data suggest that the low affinity of thioperamide for the human H(3) receptor represents a species difference in pharmacology and not a unique pharmacological subtype. It also was found that chloroproxyfan behaved as a full agonist at the rat recombinant receptor. These findings highlight the significance of validating the pharmacology of experimental compounds at both the rat and human H(3) receptors.     

Different effects of opioid antagonists on mu-, delta-, and kappa-opioid receptors with and without agonist pretreatment

Opioid receptors display basal signaling (constitutive, agonist-independent activity), which seems to be regulated by agonist exposure. Whereas agonist pretreatment desensitizes receptors to subsequent agonist stimulation, basal signaling of mu-opioid receptor (MOR) was shown to increase. Moreover, agonist pretreatment converts the neutral antagonists naloxone and naltrexone into inverse agonists, suppressing basal signaling, whereas analogs with reduced C6-position, e.g., 6beta-naltrexol, remain neutral antagonists at MOR under any condition. This study compares the regulation of basal signaling of MOR, delta-(DOR), and kappa-(KOR) opioid receptors after pretreatment with morphine or receptor-selective agonists, in transfected human embryonic kidney 293 cell membranes. Moreover, naloxone, naltrexone, and related antagonists were compared for binding potency and effect on basal and agonist-stimulated receptor signaling, measuring guanosine 5'-O-(3-[35S]thio)triphosphate binding. The results demonstrate basal activity for each opioid receptor, which is modulated by pretreatment with agonists. Even closely related opioid antagonists display distinct patterns of neutral and inverse effects before and after agonist pretreatment, including distinct efficacies between naloxone and naltrexone at agonist-pretreated DOR and KOR. Pretreatment with different agonists has varying effects on inverse and neutral activities of some analogs tested. These results demonstrate that antagonist efficacy is context-dependent, possibly accounting for paradoxical pharmacological effects. Activity profiles at the three opioid receptors under different conditions could lead to antagonists with optimal clinical properties in treatment of addiction and adverse opioid effects.     

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.     

Pharmacological properties of ABT-239 [4-(2-{2-[(2R)-2-Methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile]: I. Potent and selective histamine H3 receptor antagonist with drug-like properties

Histamine H3 receptor antagonists are being developed to treat a variety of neurological and cognitive disorders that may be ameliorated by enhancement of central neurotransmitter release. Here, we present the in vitro pharmacological and in vivo pharmacokinetic profiles for the nonimidazole, benzofuran ligand ABT-239 [4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl)benzonitrile] and compare it with several previously described imidazole and nonimidazole H3 receptor antagonists. ABT-239 binds to recombinant human and rat H3 receptors with high affinity, with pK(i) values of 9.4 and 8.9, respectively, and is over 1000-fold selective versus human H1, H2, and H4 histamine receptors. ABT-239 is a potent H3 receptor antagonist at recombinant human and rat receptors, reversing agonist-induced changes in cAMP formation (pK(b) = 7.9 and 7.6, respectively), guanosine 5'-O-(3-[35S]thio) triphosphate ([35S]GTPgammaS) binding (pK(b) = 9.0 and 8.3, respectively), and calcium mobilization (human pK(b) = 7.9). ABT-239 also competitively reversed histamine-mediated inhibition of [3H]histamine release from rat brain cortical synaptosomes (pK(b) = 7.7) and agonist-induced inhibition of contractile responses in electric field stimulated guinea pig ileal segments (pA2 = 8.7). Additionally, ABT-239 is a potent inverse agonist, inhibiting constitutive [35S]GTPgammaS binding at both rat and human H3 receptors with respective pEC50 values of 8.9 and 8.2. ABT-239 demonstrates good pharmacokinetic characteristics in rat, dog, and monkey with t1/2 values ranging from 4 to 29 h, corresponding with clearance values and metabolic turnover in liver microsomes from these species, and good oral bioavailability ranging from 52 to 89%. Thus, ABT-239 is a selective, nonimidazole H3 receptor antagonist/inverse agonist with similar high potency in both human and rat and favorable drug-like properties.     

The role of transmembrane helix 5 in agonist binding to the human H3 receptor

We have used alanine scanning mutagenesis to identify residues in transmembrane domain 5 of the histamine H3 receptor that are important for agonist binding. All of the mutants generated were functionally expressed as demonstrated by their ability to bind [(125)I]iodoproxyfan with comparable affinity to the wild-type receptor and their ability to inhibit forskolin-stimulated cAMP formation when activated by histamine. Many mutations produced small changes in the potency of histamine, but the most pronounced reduction in potency and affinity of the agonists, histamine, R-alpha-methylhistamine, imetit, and impentamine, was seen with mutation of glutamate 206. Our modeling suggests that this residue plays a key role in ligand binding by interacting with the imidazole ring of histamine. Interestingly, L199A greatly reduced agonist potency in functional assays but had only minor effects on agonist affinity, implicating a role for this residue in the mechanism of receptor activation. We also studied the functional effects of the mutations by linking the receptor to calcium signaling using a chimeric G protein. A comparison of the two functional assays demonstrated contrasting effects on agonist activity. Histamine, imetit, and impentamine were full agonists in the cAMP assay, but imetit exhibited only partial agonist activity through the chimeric G protein. Furthermore, impentamine, another potent agonist in the cAMP assay, was only able to activate the E206A mutant in the calcium assay despite being inactive at the wild-type receptor. These observations suggest that the agonist receptor complexes formed by these three different H3 agonists are not conformationally equivalent.     

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.     

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.     

Constitutive activity and inverse agonism at the M2 muscarinic acetylcholine receptor

Introduction of a single-point mutation (Asn to Tyr) at position 410 at the junction between transmembrane domain 6 and the third extracellular loop of the human M(2) muscarinic acetylcholine (mACh) receptor generated a mutant receptor (N410Y) that possesses many of the hallmark features of a constitutively active mutant receptor. These included enhanced agonist binding affinity and potency, in addition to agonist-independent accumulation of [(3)H]inositol phosphates in cells coexpressing the chimeric Galpha(qi5) protein and the N410Y mutant M(2) mACh receptor. Constitutive activity was sensitive to inhibition by a range of muscarinic ligands, including those used clinically in the management of overactive bladder (oxybutynin, tolterodine, and darifenacin), indicating that these ligands behave as inverse agonists at the M(2) mACh receptor. Long-term (24-h) treatment of Chinese hamster ovary cells expressing the N410Y mutant M(2) mACh receptor with certain mACh receptor inverse agonists (atropine, darifenacin, and pirenzepine) elicited a concentration-dependent up-regulation of cell surface receptor expression. However, not all ligands possessing negative efficacy in the [(3)H]inositol phosphate accumulation assays were capable of significantly up-regulating receptor expression, perhaps indicating a spectrum of negative efficacies among ligands traditionally defined as mACh receptor antagonists. Finally, structurally distinct agonists exhibited differences in their relative potencies for the activation of Galpha(i/o) versus Galpha(s), consistent with agonist-directed trafficking of signaling at the N410Y mutant, but not at the wild-type M(2) mACh receptor. This indicates that the N410Y mutation of the M(2) mACh receptor alters receptor-G-protein coupling in an agonist-dependent manner, in addition to generating a constitutively active receptor phenotype.     

G protein-dependent pharmacology of histamine H3 receptor ligands: evidence for heterogeneous active state receptor conformations

Previously reported pharmacological studies using the imidazole-containing histamine H3 receptor ligands GT-2331 (Cipralisant) and proxyfan resulted in a range of classifications (antagonist, agonist, and protean) for these compounds. We examined the role that the signaling system, with particular emphasis on the type of G protein, had on the pharmacology observed for H3 ligands. Ligands were assessed using assays measuring neurotransmitter release, cAMP, and guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding. Whereas clobenpropit and ciproxifan were consistently antagonists, GT-2331, proxyfan, and imetit exhibited differential activity. Although GT-2331 and proxyfan exhibited little agonist activity in neurotransmitter release assays, both demonstrated full agonism relative to (R)-alpha-methylhistamine in cAMP assays. In [35S]GTPgammaS binding assays, GT-2331 and proxyfan demonstrated partial agonism. Imetit showed full agonism in most assays, but it was slightly less efficacious in a neurotransmitter release assay and in [35S]GTPgammaS binding at the human H3 receptor. To further examine these ligands, we coexpressed G alpha16 or chimeric G alpha q/i5 in human embryonic kidney cells expressing the human H3 receptor and assayed intracellular calcium and cAMP levels. GT-2331, proxyfan, and imetit demonstrated full agonism in all assays of cAMP activity. However, in cells expressing G alpha16, they exhibited minimal agonism in calcium mobilization assays, whereas imetit showed partial agonism. When G alpha q/i5 was used, the activity of both GT-2331 and proxyfan increased, whereas imetit became a full agonist. These results demonstrate that GT-2331 and proxyfan's differential pharmacology at the H3 receptor depends on the type of G protein used and provide indirect evidence for differential ligand-bound active states that mediate signaling by the H3 receptor.     

G-protein sensitivity of ligand binding to human dopamine D(2) and D(3) receptors expressed in Escherichia coli: clues for a constrained D(3) receptor structure

Human dopamine D(2) and D(3) receptors were expressed in Chinese hamster ovary (CHO) and Escherichia coli cells to compare their ligand binding properties in the presence or absence of G-proteins and to analyze their ability to interact with G(i/o)-proteins. Binding affinities of agonists (dopamine, 7-OH-DPAT, PD128907, lisuride) and antagonists/inverse agonists (haloperidol, risperidone, domperidone, spiperone, raclopride, nemonapride), measured using [(125)I]iodosulpride and [(3)H]7-OH-DPAT, were similar for hD(3) receptors in E. coli and CHO cell membranes. Both agonists and antagonists showed 2- to 25-fold lower binding affinities at hD(2) receptors in E. coli versus CHO cell membranes (measured with [(3)H]spiperone), but the rank order of potencies remained similar. Purported inverse agonists did not display higher affinities for G-protein-free receptors. In CHO membranes, GppNHp decreased high affinity agonist ([(3)H]7-OH-DPAT) binding at hD(2) receptors but not at hD(3) receptors. Also, [(3)H]7-OH-DPAT (nanomolar concentration range) binding was undetectable at hD(2) but clearly measurable at hD(3) receptors in E. coli membranes. Addition of a G(i/o)-protein mix to E. coli membranes increased high affinity [(3)H]7-OH-DPAT binding in a concentration-dependent manner at hD(2) and hD(3) receptors; this effect was reversed by addition of GppNHp. The potency of the G(i/o)-protein mix to reconstitute high affinity binding was similar for hD(2) and hD(3) receptors. Thus, agonist binding to D(3) receptors is only slightly affected by G-protein uncoupling, pointing to a rigid receptor structure. Furthermore, we propose that the generally reported lower signaling capacity of D(3) receptors (versus D(2) receptors) is not due to its lower affinity for G-proteins but attributed to its lower capacity to activate these G-proteins.     

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.     

5-hydroxytryptamine2A receptor inverse agonists as antipsychotics

We have used a cell-based functional assay to define the pharmacological profiles of a wide range of central nervous system active compounds as agonists, competitive antagonists, and inverse agonists at almost all known monoaminergic G-protein-coupled receptor (GPCR) subtypes. Detailed profiling of 40 antipsychotics confirmed that as expected, most of these agents are potent competitive antagonists of the dopamine D2 receptor. Surprisingly, this analysis also revealed that most are potent and fully efficacious 5-hydroxytryptamine (5-HT)2A receptor inverse agonists. No other molecular property was shared as universally by this class of compounds. Furthermore, comparisons of receptor potencies revealed that antipsychotics with the highest extrapyramidal side effects (EPS) liability are significantly more potent at D2 receptors, the EPS-sparing atypical agents had relatively higher potencies at 5-HT2A receptors, while three were significantly more potent at 5-HT2A receptors. Functional high-throughput screening of a diverse chemical library identified 530 ligands with inverse agonist activity at 5-HT2A receptors, including several series of compounds related to known antipsychotics, as well as a number of novel chemistries. An analog of one of the novel chemical series, AC-90179, was pharmacologically profiled against the remaining monoaminergic GPCRs and found to be a highly selective 5-HT2A receptor inverse agonist. The behavioral pharmacology of AC-90179 is characteristic of an atypical antipsychotic agent.     

Inverse agonism and neutral antagonism at wild-type and constitutively active mutant delta opioid receptors

The delta opioid receptor modulates nociceptive and emotional behaviors. This receptor has been shown to exhibit measurable spontaneous activity. Progress in understanding the biological relevance of this activity has been slow, partly due to limited characterization of compounds with intrinsic negative activity. Here, we have used constitutively active mutant (CAM) delta receptors in two different functional assays, guanosine 5'-O-(3-thio)triphosphate binding and a reporter gene assay, to test potential inverse agonism of 15 delta opioid compounds, originally described as antagonists. These include the classical antagonists naloxone, naltrindole, 7-benzylidene-naltrexone, and naltriben, a new set of naltrindole derivatives, H-Tyr-Tic-Phe-Phe-OH (TIPP) and H-Tyr-TicPsi[CH2N]Cha-Phe-OH [TICP(Psi)], as well as three 2',6'-dimethyltyrosine-1,2,3,4-tetrahydroquinoline-3-carboxylate (Dmt-Tic) peptides. A reference agonist, SNC 80 [(+)-4-[(alphaR)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide], and inverse agonist, ICI 174864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu), were also included. In a screen using wild-type and CAM M262T delta receptors, naltrindole (NTI) and close derivatives were mostly inactive, and TIPP behaved as an agonist, whereas Dmt-Tic-OH and N,N(CH3)2-Dmt-Tic-NH2 showed inverse agonism. The two latter compounds showed negative activity across 27 CAM receptors, suggesting that this activity was independent from the activation mechanism. These two compounds also exhibited nanomolar potencies in dose-response experiments performed on wild-type, M262T, Y308H, and C328R CAM receptors. TICP(Psi) exhibited strong inverse agonism at the Y308H receptor. We conclude that the stable N,N(CH3)2-Dmt-Tic-NH2 compound represents a useful tool to explore the spontaneous activity of delta receptors, and NTI and novel derivatives behave as neutral antagonists.     

Activation of alpha-2 adrenergic receptors augments neurotransmitter-stimulated cyclic AMP accumulation in rat brain cerebral cortical slices

Norepinephrine-stimulated cyclic AMP (cAMP) accumulation in brain is mediated by both alpha and beta adrenergic receptors. The functional activity of these receptors can be differentiated by examining the response to isoproterenol alone and isoproterenol + 6-fluoronorepinephrine, and alpha adrenergic agonist. The present study was undertaken to define the pharmacological characteristics of the alpha adrenergic component associated with cAMP accumulation in brain. Using a prelabeling technique it was found that norepinephrine- or isoproterenol plus 6-fluoronorepinephrine-stimulated cAMP accumulation was only inhibited partially by an alpha-1 adrenergic receptor antagonist. In contrast, alpha-2 adrenergic receptor antagonists inhibited completely that portion of the response exceeding that produced by isoproterenol alone (alpha adrenergic augmentation). Furthermore, selective alpha-1 adrenergic agonists were incapable of enhancing the cAMP response to isoproterenol, whereas alpha-2 adrenergic agonists were active in this regard. Partial agonists for the alpha-2 adrenergic receptor were ineffective as augmentors. The data suggest that the alpha adrenergic component of the norepinephrine response in rat brain slices has characteristics of both alpha-1 and alpha-2 receptors, with the alpha-2 adrenergic component being a major contributor in this regard.     

3H]CGS 21680, a selective A2 adenosine receptor agonist directly labels A2 receptors in rat brain

Characterization of the adenosine A2 receptor has been limited due to the lack of available ligands which have high affinity and selectivity for this adenosine receptor subtype. In the present study, the binding of a highly A2-selective agonist radioligand, [3H]CGS 21680 (2-[p-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamido adenosine) is described. [3H]CGS 21680 specific binding to rat striatal membranes was saturable, reversible and dependent upon protein concentration. Saturation studies revealed that [3H]CGS 21680 bound with high affinity (Kd = 15.5 nM) and limited capacity (apparent Bmax = 375 fmol/mg of protein) to a single class of recognition sites. Estimates of ligand affinity (16 nM) determined from association and dissociation kinetic experiments were in close agreement with the results from the saturation studies. [3H]CGS 21680 binding was greatest in striatal membranes with negligible specific binding obtained in rat cortical membranes. Adenosine agonists ligands competed for the binding of 5 nM [3H]CGS 21680 to striatal membranes with the following order of activity; CGS 21680 = 5'-N-ethylcarboxamidoadenosine greater than 2-phenylaminoadenosine (CV-1808) = 5'-N-methylcarboxamidoadenosine = 2-chloroadenosine greater than R-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than N6cyclopentyltheophylline greater than S-phenylisopropyladenosine. The nonxanthine adenosine antagonist, CGS 15943A, was the most active compound in inhibiting the binding of [3H]CGS 21680. Other adenosine antagonists inhibited binding in the following order; xanthine amine congener = (1,3-dipropyl-8-(2-amino-4-chloro)phenylxanthine greater than 1,3-dipropyl-8-cyclopentylxanthine greater than 1,3-diethyl-8-phenylxanthine greater than 8-phenyltheophylline greater than 8-cyclopentyltheophylline = xanthine carboxylic acid congener greater than 8-parasulfophenyltheophylline greater than theophylline greater than caffeine. The pharmacological profile of both adenosine agonist and antagonist compounds to compete for the binding of [3H]CGS 21680 was consistent with a selective interaction at the high affinity adenosine A2 receptor. A high positive correlation (r = 0.98, P less than .01) was observed between the pharmacological profile of adenosine ligands to inhibit the binding of [3H]CGS 21680 and the selective binding of [3H]NECA (+50 nM CPA) to high affinity A2 receptors. However, some differences between these assays were found for compounds which have moderate affinity and nonselective actions at both the A1 and A2 adenosine receptor subtypes. Unlike data obtained with nonselective adenosine ligands, the present results indicate that [3H]CGS 21680 directly labels the high affinity A2 receptor in rat brain without the need to block binding activity at the A1 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

The comparative pharmacology and up-regulation of rat neuronal nicotinic receptor subtype binding sites stably expressed in transfected mammalian cells

We stably transfected human embryonic kidney cells (HEK 293 cells) with genes encoding rat neuronal nicotinic receptor alpha2, alpha3, or alpha4 subunits in combination with the beta2 or beta4 subunit to generate six cell lines that express defined subunit combinations that represent potential subtypes of rat neuronal nicotinic acetylcholine receptors (nAChRs). These cell lines were designated KXalpha2beta2, KXalpha2beta4, KXalpha3beta2, KXalpha3beta4, KXalpha4beta2, and KXalpha4beta4. The Kd values of [3H](+/-)epibatidine ([3H]EB) binding to membranes from these six cell lines ranged from approximately 0.02 to 0.3 nM. The pharmacological profiles of the agonist binding sites of these putative nAChR subtypes were examined in competition studies in which unlabeled nicotinic ligands, including 10 agonists and two antagonists, competed against [3H]EB. Most nicotinic ligands examined had higher affinity for the receptor subtypes containing the beta2 subunit compared with those containing the beta4 subunit. An excellent correlation (r > 0.99) of the binding affinities of the 10 agonists was observed between receptors from KXalpha4beta2 cells and from rat forebrain tissue, in which [3H]EB binding represents predominantly alpha4beta2 nAChRs. More important, the affinities (Ki values) for the two tissues were nearly identical. The densities of the binding sites of all six cell lines were increased after a 5-day exposure to (-)-nicotine or the quaternary amine agonist carbachol. These data indicate that these cell lines expressing nAChR subunit combinations should be useful models for investigating pharmacological properties and regulation of the binding sites of potential nAChR subtypes, as well as for studying the properties of nicotinic compounds.     

Constitutive activity and ligand selectivity of human, guinea pig, rat, and canine histamine H2 receptors

Previous studies revealed pharmacological differences between human and guinea pig histamine H(2) receptors (H(2)Rs) with respect to the interaction with guanidine-type agonists. Because H(2)R species variants are structurally very similar, comparative studies are suited to relate different properties of H(2)R species isoforms to few molecular determinants. Therefore, we systematically compared H(2)Rs of human (h), guinea pig (gp), rat (r), and canine (c). Fusion proteins of hH(2)R, gpH(2)R, rH(2)R, and cH(2)R, respectively, and the short splice variant of G(salpha), G(salphaS), were expressed in Sf9 insect cells. In the membrane steady-state GTPase activity assay, cH(2)R-G(salphaS) but neither gpH(2)R-G(salphaS) nor rH(2)R-G(salphaS) showed the hallmarks of increased constitutive activity compared with hH(2)R-G(salphaS), i.e., increased efficacies of partial agonists, increased potencies of agonists with the extent of potency increase being correlated with the corresponding efficacies at hH(2)R-G(salphaS), increased inverse agonist efficacies, and decreased potencies of antagonists. Furthermore, in membranes expressing nonfused H(2)Rs without or together with mammalian G(salphaS) or H(2)R-G(salpha) fusion proteins, the highest basal and GTP-dependent increases in adenylyl cyclase activity were observed for cH(2)R. An example of ligand selectivity is given by metiamide, acting as an inverse agonist at hH(2)R-G(salphaS), gpH(2)R-G(salphaS), and rH(2)R-G(salphaS) in the GTPase assay in contrast to being a weak partial agonist with decreased potency at cH(2)R-G(salphaS). In conclusion, the cH(2)R exhibits increased constitutive activity compared with hH(2)R, gpH(2)R, and rH(2)R, and there is evidence for ligand-specific conformations in H(2)R species isoforms.     

Inverse agonist activity of selected ligands of the cysteinyl-leukotriene receptor 1

Cysteinyl leukotrienes (CysLTs) are associated with several inflammatory processes, including asthma. Due to this association, considerable effort has been invested in the development of antagonists to the CysLT receptors (CysLT(1)R). Many of these molecules have been shown to specifically interact with CysLT(1)R, but little is known about their impact on the conformation of the receptor and its activity. We were especially interested in possible inverse agonist activity of the antagonists. Using a constitutively active mutant (N106A) of the human CysLT(1)R and the wild-type (WT) receptor coexpressed with the G(alphaq) subunit of the trimeric G protein, we were able to address this issue with ligands commonly used in therapy. We demonstrated that some of these molecules are inverse agonists, whereas others act as partial agonists. In cells expressing the CysLT(1)R mutant N106A exposed to Montelukast, Zafirlukast, or 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571), the basal inositol phosphate production was reduced by 53 +/- 6, 44 +/- 3, and 54 +/- 4%, respectively. On the other hand, 6(R)-(4-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E),11(Z),14(Z)-eicosatetraenoic acid (BayU9773) and 1-[2-hydroxy-3-propyl-4-[4-(1H-tetrazole-5-YL)-butoxy]-phenyl ethanone] (LY171883) acted as partial agonists and alpha-pentyl-3-[2-quinolinylmethoxy] benzyl alcohol (REV 5901) as a neutral antagonist. However, in cells expressing CysLT(1)R and G(alphaq), all antagonists used had inverse agonist activity. The decrease in basal inositol phosphate production by ligands with inverse agonist activity could be inhibited by a more neutral antagonist, confirming the specificity of the reaction. We demonstrate here that Montelukast, MK571, and Zafirlukast can act as inverse agonists on the human CysLT(1) receptor.     

Pharmacological and behavioral profile of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl) carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103), a novel 5-hydroxytryptamine(2A) receptor inverse agonist

The in vitro and in vivo pharmacological properties of N-(4-fluorophenylmethyl)-N-(1-methylpiperidin-4-yl)-N'-(4-(2-methylpropyloxy)phenylmethyl)carbamide (2R,3R)-dihydroxybutanedioate (2:1) (ACP-103) are presented. A potent 5-hydroxytryptamine (5-HT)(2A) receptor inverse agonist ACP-103 competitively antagonized the binding of [(3)H]ketanserin to heterologously expressed human 5-HT(2A) receptors with a mean pK(i) of 9.3 in membranes and 9.70 in whole cells. ACP-103 displayed potent inverse agonist activity in the cell-based functional assay receptor selection and amplification technology (R-SAT), with a mean pIC(50) of 8.7. ACP-103 demonstrated lesser affinity (mean pK(i) of 8.80 in membranes and 8.00 in whole cells, as determined by radioligand binding) and potency as an inverse agonist (mean pIC(50) 7.1 in R-SAT) at human 5-HT(2C) receptors, and lacked affinity and functional activity at 5-HT(2B) receptors, dopamine D(2) receptors, and other human monoaminergic receptors. Behaviorally, ACP-103 attenuated head-twitch behavior (3 mg/kg p.o.), and prepulse inhibition deficits (1-10 mg/kg s.c.) induced by the 5-HT(2A) receptor agonist (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride in rats and reduced the hyperactivity induced in mice by the N-methyl-d-aspartate receptor noncompetitive antagonist 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate; MK-801) (0.1 and 0.3 mg/kg s.c.; 3 mg/kg p.o.), consistent with a 5-HT(2A) receptor mechanism of action in vivo and antipsychotic-like efficacy. ACP-103 demonstrated >42.6% oral bioavailability in rats. Thus, ACP-103 is a potent, efficacious, orally active 5-HT(2A) receptor inverse agonist with a behavioral pharmacological profile consistent with utility as an antipsychotic agent.