Mutations of Cys-17 and Ala-271 in the human histamine H2 receptor determine the species selectivity of guanidine-type agonists and increase constitutive activity

In a steady-state GTPase activity assay, N-[3-(1H-imidazol-4-yl)propyl)]guanidines and N(G)-acylated derivatives are more potent and efficacious at fusion proteins of guinea pig (gpH(2)R-G(salphaS)) than human (hH(2)R-G(salphaS)) histamine H(2) receptor, coupled to the short splice variant of G(salpha), G(salphaS). Whereas Ala-271 (hH(2)R) and Asp-271 (gpH(2)R) in transmembrane domain 7 were identified to determine the potency differences of guanidine-type agonists, the molecular basis for the efficacy differences remains to be elucidated. A homology model of the gpH(2)R suggested that an H-bond between Tyr-17 and Asp-271 stabilizes an active receptor conformation of the gpH(2)R. In the present study, we generated a mutant hH(2)R-G(salphaS) with Cys-17--> Tyr-17/Ala-271--> Asp-271 exchanges (hH(2)R-->gpH(2)R) that exhibited an enhanced level of constitutive GTPase activity and adenylyl cyclase activity compared with wild-type hH(2)R-G(salphaS) and gpH(2)R-G(salphaS). Potencies and efficacies of guanidines and N(G)-acylguanidines were increased at this mutant receptor compared with hH(2)R-G(salphaS), but they were still lower than at gpH(2)R-G(salphaS), suggesting that aside from Tyr-17 and Asp-271 additional amino acids contribute to the distinct pharmacological profiles of both species isoforms. Another hH(2)R-G(salphaS) mutant with a Cys-17--> Tyr-17 exchange showed inefficient coupling to G(salphaS) as revealed by reduced agonist-stimulated GTPase and basal adenylyl cyclase activities. Collectively, our present pharmacological study confirms the existence of an H-bond between Tyr-17 and Asp-271 favoring the stabilization of an active receptor conformation. Distinct potencies and efficacies of agonists and inverse agonists further support the concept of ligand-specific conformations in wild-type and mutant H(2)R-G(salphaS) fusion proteins.     

Probing ligand-specific histamine H1- and H2-receptor conformations with NG-acylated Imidazolylpropylguanidines

Impromidine (IMP) and arpromidine (ARP)-derived guanidines are more potent and efficacious guinea pig (gp) histamine H(2)-receptor (gpH(2)R) than human (h) H(2)R agonists and histamine H(1)-receptor (H(1)R) antagonists with preference for hH(1)R relative to gpH(1)R. We examined N(G)-acylated imidazolylpropylguanidines (AIPGs), which are less basic than guanidines, at hH(2)R, gpH(2)R, rat H(2)R (rH(2)R), hH(1)R, and gpH(1)R expressed in Sf9 cells as probes for ligand-specific receptor conformations. AIPGs were similarly potent H(2)R agonists as the corresponding guanidines IMP and ARP, respectively. Exchange of pyridyl in ARP against phenyl increased AIPG potency 10-fold, yielding the most potent agonists at the hH(2)R-G(salpha) fusion protein and gpH(2)R-G(salpha) identified so far. Some AIPGs were similarly potent and efficacious at hH(2)R-G(salpha) and gpH(2)R-G(salpha). AIPGs stabilized the ternary complex in hH(2)R-G(salpha) and gpH(2)R-G(salpha) differently than the corresponding guanidines. Guanidines, AIPGs, and small H(2)R agonists exhibited distinct agonist properties at hH(2)R, gpH(2)R, and rH(2)R measuring adenylyl cyclase activity. In contrast to ARP and IMP, AIPGs were partial H(1)R agonists exhibiting higher efficacies at hH(1)R than at gpH(1)R. This is remarkable because, so far, all bulky H(1)R agonists exhibited higher efficacies at gpH(1)R than at hH(1)R. Collectively, our data suggest that AIPGs stabilize different active conformations in hH(2)R, gpH(2)R, and rH(2)R than guanidines and that, in contrast to guanidines, AIPGs are capable of stabilizing a partially active state of hH(1)R.     

Differential efficacies of somatostatin receptor agonists for G-protein activation and desensitization of somatostatin receptor subtype 4-mediated responses

Both the histamine H1-receptor (H1R) and H2-receptor (H2R) exhibit pronounced species selectivity in their pharmacological properties; i.e., bulky agonists possess higher potencies and efficacies at guinea pig (gp) than at the corresponding human (h) receptor isoforms. In this study, we examined the effects of NG-acylated imidazolylpropylguanidines substituted with a single phenyl or cyclohexyl substituent on H1R and H2R species isoforms expressed in Sf9 insect cells. N1-(3-Cyclohexylbutanoyl)-N2-[3-(1H-imidazol-4-yl)propyl]guanidine (UR-AK57) turned out to be the most potent hH2R agonist identified so far (EC50 of 23 nM in the GTPase assay at the hH2R-Gsalpha fusion protein expressed in Sf9 insect cells). UR-AK57 was almost a full-hH2R agonist and only slightly less potent and efficacious than at gpH2R-Gsalpha. Several NG-acylated imidazolylpropylguanidines showed similar potency at hH2R and gpH2R. Most unexpectedly, UR-AK57 exhibited moderately strong partial hH1R agonism with a potency similar to that of histamine, whereas at gpH1R, UR-AK57 was only a very weak partial agonist. Structure/activity relationship studies revealed that both the alkanoyl chain connecting the aromatic or alicyclic substituent with the guanidine moiety and the nature of the carbocycle (cyclohexyl versus phenyl ring) critically determine the pharmacological properties of this class of compounds. Collectively, our data show that gpH1R and gpH R do not necessarily exhibit preference for bulky agonists (2) compared with hH1R and hH2R, respectively, and that UR-AK57 is a promising starting point for the development of both potent and efficacious hH1R and hH2R agonists.     

Similar apparent constitutive activity of human histamine H(2)-receptor fused to long and short splice variants of G(salpha).

Fusion proteins allow for the analysis of receptor/G protein coupling under defined conditions. The beta(2)-adrenoceptor (beta(2)AR) fused to the long splice variant of G(salpha) (G(salphaL)) exhibits a higher apparent constitutive activity than the beta(2)-adrenoceptor fused to the short splice variant of G(salpha) (G(salphaS)). Experimentally, this results in higher efficacy and potency of partial agonists and in higher efficacy of inverse agonists at the beta(2)AR fused to G(salphaL) relative to the beta(2)AR fused to G(salphaS), indicating that the agonist-free beta(2)AR and the beta(2)AR occupied by partial agonists promote GDP dissociation from G(salphaL) more efficiently than from G(salphaS). In fact, the GDP affinity of G(salphaS) fused to the beta(2)AR is higher than the GDP affinity of G(salphaL) fused to the beta(2)AR. We asked the question whether the histamine H(2)-receptor (H(2)R) exhibits similar coupling to G(salpha) splice variants as the beta(2)AR. To address this question, we studied H(2)R-G(salpha) fusion proteins expressed in Sf9 cells. In contrast to beta(2)AR-G(salpha) fusion proteins, the potencies and efficacies of partial agonists and the efficacies of inverse agonists were similar at the H(2)R fused to G(salphaL) and G(salphaS) as assessed by guanosine-5'-O-(3-thio)triphosphate binding and/or steady-state GTPase activity. However, the time course analysis of guanosine-5'-O-(3-thio)triphosphate binding indicated that G(salphaS) fused to the H(2)R possesses a higher GDP-affinity than G(salphaL) fused to the H(2)R. Our data show that the H(2)R fused to G(salphaL) and G(salphaS) possesses similar constitutive activity and is insensitive to differences in GDP affinity of G(salpha) splice variants. Thus, GDP affinity of G proteins does not generally determine constitutive activity of receptors.     

Pharmacological profile of histaprodifens at four recombinant histamine H1 receptor species isoforms

There are differences in the pharmacological properties of phenylhistamines and histaprodifens between guinea pig histamine H(1) receptor (gpH(1)R) and human histamine H(1) receptor (hH(1)R). The aim of this study was to analyze species differences in more detail, focusing on histaprodifen derivatives and including the bovine histamine H(1) receptor (bH(1)R) and rat histamine H(1) receptor (rH(1)R). H(1)R species isoforms were coexpressed with the regulator of G protein signaling RGS4 in Sf9 insect cells. We performed [(3)H]mepyramine binding assays and steady-state GTPase assays. For a novel class of histaprodifens, the chiral histaprodifens, unique species differences between hH(1)R, bH(1)R, rH(1)R, and gpH(1)R were observed. The chiral histaprodifens 8R and 8S were both partial agonists at gpH(1)R, but only 8R was a partial agonist at the other H(1)R species isoforms. An additional phenyl group in chiral histaprodifens 10R and 10S, respectively, resulted in a switch from agonism at gpH(1)Rto antagonism at hH(1)R, bH(1)R, and rH(1)R. In general, histaprodifens showed the order of potency hH(1)R < bH(1)R < rH(1)R < gpH(1)R. An active-state model of gpH(1)R was generated with molecular dynamics simulations. Dimeric histaprodifen was docked into the binding pocket of gpH(1)R. Hydrogen bonds and electrostatic interactions were detected between dimeric histaprodifen and Asp-116, Ser-120, Lys-187, Glu-190, and Tyr-432. We conclude the following: 1) chiral histaprodifens interact differentially with H(1)R species isoforms; 2) gpH(1)R and rH(1)R, on one hand, and hH(1)R and bH(1)R, on the other hand, resemble each other structurally and pharmacologically; and 3) histaprodifens interact with H(1)R at multiple sites.     

Multiple differences in agonist and antagonist pharmacology between human and guinea pig histamine H1-receptor

Species isoforms of histamine H2-, H3-, and H4-receptors differ in their pharmacological properties. The study aim was to dissect differences between the human H1R (hH1R) and guinea pig H1R (ghH1R). We coexpressed hH1R and gpH1R with regulators of G-protein signaling in Sf9 insect cells and analyzed the GTPase activity of Gq-proteins. Small H1R agonists showed similar effects at hH1R and gpH1R, whereas bulkier 2-phenylhistamines and histaprodifens were up to approximately 10-fold more potent at gpH1R than at hH1R. Most 2-phenylhistamines and histaprodifens were more efficacious at gpH1R than at hH1R. Several first-generation H1R antagonists were approximately 2-fold, and arpromidine-type H1R antagonists up to approximately 10-fold more potent at gpH1R than at hH1R. [3H]Mepyramine competition binding studies confirmed the potency differences of the GTPase studies. Phe-153-->Leu-153 or Ile-433-->Val-433 exchange in hH1R (hH1R-->gpH1R) resulted in poor receptor expression, low [3H]mepyramine affinity, and functional inactivity. The Phe-153-->Leu-153/Ile-433-->Val-433 double mutant expressed excellently but only partially changed the pharmacological properties of hH1R. Small H1R agonists and 2-phenylhistamines interacted differentially with human and guinea pig H2R in terms of potency and efficacy, respectively. Our data show the following: 1) there are differences in agonist- and antagonist-pharmacology of hH1R and gpH1R encompassing diverse classes of bulky ligands. These differences may be explained by higher conformational flexibility of gpH1R relative to hH1R; 2) Phe-153 and Ile-433 are critical for proper folding and expression of hH1R; and 3) H2R species isoforms distinguish between H1R agonists.     

Ligand-specific contribution of the N terminus and E2-loop to pharmacological properties of the histamine H1-receptor

There are species differences between human histamine H(1) receptor (hH(1)R) and guinea pig (gp) histamine H(1) receptor (gpH(1)R) for phenylhistamines and histaprodifens. Several studies showed participation of the second extracellular loop (E2-loop) in ligand binding for some G protein-coupled receptors (GPCRs). Because there are large species differences in the amino acid sequence between hH(1)R and gpH(1)R for the N terminus and E2-loop, we generated chimeric hH(1)Rs with gp E2-loop (h(gpE2)H(1)R) and gp N terminus and gp E2-loop (h(gpNgpE2)H(1)R). hH(1)R, gpH(1)R, and chimeras were expressed in Sf9 insect cells. [(3)H]Mepyramine binding assays and steady-state GTPase assays were performed. In the series hH(1)R > h(gpE2)H(1)R > h(gpNgpE2)H(1)R, we observed a significant decrease in potency of histamine 1 in the GTPase assay. For phenoprodifen 5 and the chiral phenoprodifens 6R and 6S, a significant decrease in affinity and potency was found in the series hH(1)R > h(gpE2)H(1)R > h(gpNgpE2)H(1)R. In addition, we constructed new active-state H(1)R models based on the crystal structure of the human beta(2)-adrenergic receptor (hbeta(2)AR). Compared with the H(1)R active-state models based on the crystal structure of bovine rhodopsin, the E2-loop differs in its contact to the ligand bound in the binding pocket. In the bovine rhodopsin-based model, the backbone carbonyl of Lys187 (gpH(1)R) interacts with large histaprodifens in the binding pocket, but in the hbeta(2)AR-based model, Lys187 (gpH(1)R) is located distantly from the binding pocket. In conclusion, the differences in N terminus and E2-loop between hH(1)R and gpH(1)R exert an influence on affinity and/or potency for histamine and phenoprodifens 5, 6R, and 6S.     

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.     

Functional differences between full and partial agonists: evidence for ligand-specific receptor conformations

The interaction of an agonist-bound G-protein-coupled receptor (GPCR) with its cognate G-protein initiates a sequence of experimentally quantifiable changes in both the GPCR and G-protein. These include the release of GDP from G(alpha), the formation of a ternary complex between the nucleotide-free G-protein and the GPCR, which has a high affinity for agonist, followed by the binding of GTP to G(alpha), the dissociation of the GPCR/G-protein complex, and the hydrolysis of GTP. The efficacy of an agonist is a measure of its ability to activate this cascade. It has been proposed that efficacy reflects the ability of the agonist to stabilize the active state of the GPCR. We examined a series of beta(2)-adrenoceptor (beta(2)AR) agonists (weak partial agonists to full agonists) for their efficacy at promoting two different steps of the G-protein activation/deactivation cycle: stabilizing the ternary complex (high-affinity, GTP-sensitive agonist binding), and steady-state GTPase activity. We obtained results for the wild-type beta(2)AR and a constitutively active mutant of the beta(2)AR (beta(2)AR(CAM)) using fusion proteins between the GPCRs and G(salpha) to facilitate GPCR/G-protein interactions. There was no correlation between efficacy of ligands in activating GTPase and their ability to stabilize the ternary complex at beta(2)AR(CAM). Our results suggest that the GPCR state that optimally promotes the GDP release and GTP binding is different from the GPCR state that stabilizes the ternary complex. By strongly stabilizing the ternary complex, certain partial agonists may reduce the rate of G-protein turnover relative to a full agonist.     

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.     

Bidirectional allosteric effects of agonists and GTP at alpha(2A/D)-adrenoceptors

Agonists and GTP exert reciprocal effects on the stability of the G protein-coupled receptor/G protein complex, implying bidirectional control over the receptor/G protein interface. To investigate this relationship, we compared the ability of a series of hydroxyl-substituted phenethylamine and imidazoline agonists to stimulate [(35)S]guanosine 5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS) binding in membranes from alpha(2A/D)-adrenergic receptor-transfected PC12 cells with the magnitude of the GTP-induced reduction in agonist affinity in [(3)H]rauwolscine-binding studies. Agents previously described as full and partial agonists in functional studies showed similar relative efficacies in promoting GTP binding (r = 0.97) as well as similar relative potencies (r = 0.94). Efficacy among agonists for promotion of [(35)S]GTPgammaS binding was closely correlated with the relative influence of GTPgammaS on agonist binding (r = 0.97), consistent with a bidirectional allosteric influence by agonists and GTP on receptor/G protein complexation. In an additional series of tolazoline derivatives, a range in efficacy from full agonism to strong inverse agonism was observed, depending on the presence or absence of hydroxyl substituents. Together these results suggest that agonist-induced repositioning of transmembrane helices via their hydroxyl interactions is a critical determinant of the stability of the receptor/G protein complex and therefore of agonist efficacy.     

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.     

An 80-amino acid deletion in the third intracellular loop of a naturally occurring human histamine H3 isoform confers pharmacological differences and constitutive activity

In this article, we pharmacologically characterized two naturally occurring human histamine H3 receptor (hH3R) isoforms, hH3R(445) and hH3R(365). These abundantly expressed splice variants differ by a deletion of 80 amino acids in the intracellular loop 3. In this report, we show that the hH3R(365) is differentially expressed compared with the hH3R(445) and has a higher affinity and potency for H3R agonists and conversely a lower potency and affinity for H3R inverse agonists. Furthermore, we show a higher constitutive signaling of the hH3R(365) compared with the hH3R(445) in both guanosine-5'-O-(3-[35S]thio) triphosphate binding and cAMP assays, likely explaining the observed differences in hH3R pharmacology of the two isoforms. Because H3R ligands are beneficial in animal models of obesity, epilepsy, and cognitive diseases such as Alzheimer's disease and attention deficit hyperactivity disorder and currently entered clinical trails, these differences in H3R pharmacology of these two isoforms are of great importance for a detailed understanding of the action of H3R ligands.     

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.     

Distinct interactions of human beta1- and beta2-adrenoceptors with isoproterenol, epinephrine, norepinephrine, and dopamine

Fluorescence studies with purified human beta(2)-adrenoceptor (beta(2)AR) revealed that the endogenous catecholamines, (-)-epinephrine (EPI), (-)-norepinephrine (NE), and dopamine (DOP), stabilize distinct active receptor conformations. However, the functional relevance of these ligand-specific conformations is as yet poorly understood. We addressed this question by studying fusion proteins of the beta(1)-adrenoceptor (beta(1)AR) and beta(2)AR with the short and long splice variants of G(s)alpha (G(s)alpha(S) and G(s)alpha(L)), respectively. Fusion proteins ensure efficient receptor/G-protein coupling and defined stoichiometry of the coupling partners. EPI, NE, DOP, and the prototypical synthetic betaAR agonist, (-)-isoproterenol (ISO), showed marked differences in their efficacies at stabilizing the high-affinity ternary complex at beta(1)AR-G(s)alpha and beta(2)AR-G(s)alpha fusion proteins. Ternary complex formation was more sensitive to disruption by GTP with the beta(2)AR than with the beta(1)AR. Generally, in steady-state GTPase assays, ISO, EPI, and NE were full agonists, and DOP was a partial agonist. Exceptionally, at beta(1)AR-G(s)alpha(L), NE was only a partial agonist. Generally, in adenylyl cyclase assays, ISO, EPI, and NE were full agonists, and DOP was a partial agonist. At beta(2)AR-G(s)alpha(L), NE was only a partial agonist. There was no correlation between efficacy at stabilizing the ternary complex and activating GTPase, and there were also dissociations between K(i) values for high-affinity agonist binding and EC(50) values for GTPase activation. In contrast to synthetic partial agonists, DOP did not exhibit increased efficacy at betaAR-G(s)alpha(L) versus betaAR-G(s)alpha(S) fusion proteins. In conclusion, our data with betaAR-G(s)alpha fusion proteins show that endogenous catecholamines and ISO stabilize distinct conformations in the beta(1)AR and beta(2)AR.     

Constitutive activity of histamine h(3) receptors stably expressed in SK-N-MC cells: display of agonism and inverse agonism by H(3) antagonists.

Agonist-independent activity of G-protein-coupled receptor, also referred to as constitutive activity, is a well-documented phenomenon and has been reported recently for both the histamine H(1) and H(2) receptors. Using SK-N-MC cell lines stably expressing the human and rat H(3) receptors at physiological receptor densities (500-600 fmol/mg of protein), we show that both the rat and human H(3) receptors show a high degree of constitutive activity. The forskolin-mediated cAMP production in SK-N-MC cells is inhibited strongly upon expression of the G(i)-coupled H(3) receptor. The cAMP production can be further inhibited upon agonist stimulation of the H(3) receptor and can be enhanced by a variety of H(3) antagonists acting as inverse agonists at the H(3) receptor. Thioperamide, clobenpropit, and iodophenpropit raise the cAMP levels in SK-N-MC cells with potencies that match their receptor binding affinities. Surprisingly, impentamine and burimamide act as effective H(3) agonists. Modification of the amine group of impentamine dramatically affected the pharmacological activity of the ligand. Receptor affinity was reduced slightly for most impentamine analogs, but the functional activity of the ligands varied from agonist to neutral antagonist and inverse agonist, indicating that subtle changes in the chemical structures of impentamine analogs have major impact on the (de)activation steps of the H(3) receptor. In conclusion, upon stable expression of the rat and human H(3) receptor in SK-N-MC cells constitutive receptor activity is detected. In this experimental system, H(3) receptors ligands, previously identified as H(3) antagonists, cover the whole spectrum of pharmacological activities, ranging from full inverse agonists to agonists.     

Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. III. Agonist and antagonist properties at serotonin, 5-HT(1) and 5-HT(2), receptor subtypes

Although certain antiparkinson agents interact with serotonin (5-HT) receptors, little information is available concerning functional actions. Herein, we characterized efficacies of apomorphine, bromocriptine, cabergoline, lisuride, piribedil, pergolide, roxindole, and terguride at human (h)5-HT(1A), h5-HT(1B), and h5-HT(1D) receptors [guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding], and at h5-HT(2A), h5-HT(2B), and h5-HT(2C) receptors (depletion of membrane-bound [(3)H]phosphatydilinositol). All drugs stimulated h5-HT(1A) receptors with efficacies (compared with 5-HT, 100%) ranging from modest (apomorphine, 35%) to high (cabergoline, 93%). At h5-HT(1B) receptors, efficacies varied from mild (terguride, 37%) to marked (cabergoline, 102%) and potencies were modest (pEC(50) values of 5.8-7.6): h5-HT(1D) sites were activated with a similar range of efficacies and greater potency (7.1-8.5). Piribedil and apomorphine were inactive at h5-HT(1B) and h5-HT(1D) receptors. At h5-HT(2A) receptors, terguride, lisuride, bromocriptine, cabergoline, and pergolide displayed potent (7.6-8.8) agonist properties (49-103%), whereas apomorphine and roxindole were antagonists and piribedil was inactive. Only pergolide (113%/8.2) and cabergoline (123%/8.6) displayed pronounced agonist properties at h5-HT(2B) receptors. At 5-HT(2C) receptors, lisuride, bromocriptine, pergolide, and cabergoline were efficacious (75-96%) agonists, apomorphine and terguride were antagonists, and piribedil was inactive. MDL100,907 and SB242,084, selective antagonists at 5-HT(2A) and 5-HT(2C) receptors, respectively, abolished these actions of pergolide, cabergoline, and bromocriptine. In conclusion, antiparkinson agents display markedly different patterns of agonist and antagonist properties at multiple 5-HT receptor subtypes. Although all show modest (agonist) activity at 5-HT(1A) sites, their contrasting actions at 5-HT(2A) and 5-HT(2C) sites may be of particular significance to their functional profiles in vivo.     

Cannabinoid agonist signal transduction in rat brain: comparison of cannabinoid agonists in receptor binding, G-protein activation, and adenylyl cyclase inhibition

To investigate differences in agonist affinity, potency, and efficacy across rat brain regions, five representative cannabinoid compounds were investigated in membranes from three different rat brain regions for their ability to maximally stimulate [(35)S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) binding and bind to cannabinoid receptors (measured by inhibition of [(3)H]antagonist binding) under identical assay conditions. In all three brain regions, the rank order of potency for the stimulation of [(35)S]GTPgammaS binding and the inhibition of [(3)H]SR141716A binding for these compounds were identical, with CP55940 approximately levonantradol > WIN55212-2 >/= Delta(9)-tetrahydrocannabinol (Delta(9)-THC) > methanandamide. The rank order of efficacy was not related to potency, and relative maximal agonist effects varied across regions. Receptor binding fit to a three-site model for most agonists, stimulation of [(35)S]GTPgammaS binding fit to a two-site model for all agonists, and high-affinity receptor binding did not appear to produce any stimulation of [(35)S]GTPgammaS binding. WIN55212-2, methanandamide, and Delta(9)-THC also were assayed for the inhibition of adenylyl cyclase in cerebellar membranes. The rank orders of potency and efficacy were similar to those for [(35)S]GTPgammaS binding, but the efficacies and potencies of methanandamide and Delta(9)-THC compared with WIN55212-2 were higher for adenylyl cyclase inhibition, implying receptor/G-protein reserve.     

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.     

Two novel and selective nonimidazole histamine H3 receptor antagonists A-304121 and A-317920: I. In vitro pharmacological effects

Histamine H3 receptor (H3R) antagonists enhance neurotransmitter release and are being developed for the treatment of a variety of neurological and cognitive disorders. Many potent histamine H3R antagonists contain an imidazole moiety that limits receptor selectivity and the tolerability of this class of compounds. Here we present the in vitro pharmacological data for two novel piperazine amide ligands, A-304121 [4-(3-((2R)-2-aminopropanoyl-1-piperazinyl)propoxy)phenyl)cyclopropylmethanone] and A-317920 [N-((1R)-2-(4-(3-(4-(cyclopropylcarbonyl)phenoxy)propyl)-1-piperazinyl)-1-methyl-2-oxo-ethyl-)-2-furamide], and compare them with the imidazole H3R antagonists ciproxifan, clobenpropit, and thioperamide. Both A-304121 and A-317920 bind potently to recombinant full-length rat H3R(pKi values = 8.6 and 9.2, respectively) but have lower potencies for binding the full-length human H3R (pKi values = 6.1 and 7.0, respectively). A-304121 and A-317920 are potent antagonists at rat H3R in reversing R-alpha-methylhistamine [(R)-alpha-MeHA] inhibition of forskolin-stimulated cAMP formation (pKb values = 8.0 and 9.1) but weak antagonists at human H3Rs in cyclase (pKb values = 6.0 and 6.3) and calcium mobilization (pKb values = 6.0 and 7.3) assays in cells co-expressing Galphaqi5-protein. Both compounds potently antagonize native H3Rs by blocking histamine inhibition of potassium-evoked [3H]histamine release from rat brain cortical synaptosomes (pKb values = 8.6 and 9.3) and (R)-alpha-MeHA reversal of electric field-stimulated guinea pig ileum contractions (pA2 values = 7.1 and 8.3). A-304121 and A-317920 are also more efficacious inverse agonists in reversing basal guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding at the human H3R (pEC50 values = 5.7 and 7.0) than are the imidazole antagonists. These novel and selective piperazine amides represent useful leads for the development of H3R antagonist therapeutic agents.