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.     

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.     

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.     

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.     

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.     

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.     

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.     

Salvinorin A, an active component of the hallucinogenic sage salvia divinorum is a highly efficacious kappa-opioid receptor agonist: structural and functional considerations

The diterpene salvinorin A from Salvia divinorum has recently been reported to be a high-affinity and selective kappa-opioid receptor agonist (Roth et al., 2002). Salvinorin A and selected derivatives were found to be potent and efficacious agonists in several measures of agonist activity using cloned human kappa-opioid receptors expressed in human embryonic kidney-293 cells. Thus, salvinorin A, salvinorinyl-2-propionate, and salvinorinyl-2-heptanoate were found to be either full (salvinorin A) or partial (2-propionate, 2-heptanoate) agonists for inhibition of forskolin-stimulated cAMP production. Additional studies of agonist potency and efficacy of salvinorin A, performed by cotransfecting either the chimeric G proteins Gaq-i5 or the universal G protein Ga16 and quantification of agonist-evoked intracellular calcium mobilization, affirmed that salvinorin A was a potent and effective kappa-opioid agonist. Results from structure-function studies suggested that the nature of the substituent at the 2-position of salvinorin A was critical for kappa-opioid receptor binding and activation. Because issues of receptor reserve complicate estimates of agonist efficacy and potency, we also examined the agonist actions of salvinorin A by measuring potassium conductance through G protein-gated K(+) channels coexpressed in Xenopus oocytes, a system in which receptor reserve is minimal. Salvinorin A was found to be a full agonist, being significantly more efficacious than (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U50488) or (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U69593) (two standard kappa-opioid agonists) and similar in efficacy to dynorphin A (the naturally occurring peptide ligand for kappa-opioid receptors). Salvinorin A thus represents the first known naturally occurring non-nitrogenous full agonist at kappa-opioid receptors.     

The bulky N6 substituent of cabergoline is responsible for agonism of this drug at 5-hydroxytryptamine 5-HT2A and 5-HT2B receptors and thus is a determinant of valvular heart disease

Fibrotic valvular heart disease (VHD) has been observed in patients with Parkinson's disease treated with dopamine receptor agonists such as pergolide and cabergoline. 5-Hydroxytryptamine(2B) receptor (5-HT(2B)R) agonism is the most likely cause, but other 5-HT receptors may also play a role in VHD. We aimed at characterizing the molecular fragment of cabergoline responsible for agonism at 5-HT(2B)R and 5-HT(2A)R. Cabergoline with an allyl substituent at N(6) behaved as a potent 5-HT(2B)R full agonist in relaxation of porcine pulmonary arteries and as a weaker 5-HT(2A)R partial agonist in contraction of coronary arteries. The same was true for cabergoline derivatives with cyclopropylmethyl, propyl, or ethyl at N(6). However, agonism was converted into antagonism, when the N(6) substituent was methyl. 6-Methylcabergoline retained agonism compared with cabergoline at human dopamine D(2LONG) and human dopamine D(2SHORT) receptors as determined by guanosine 5'-O-(3-[(35)S]thio)triphosphate binding. In porcine aortic valve cusps, 5-HT-induced contractions were inhibited by ketanserin (5-HT(2A/2C)R antagonist) but not by N-(1-methyl-1H-5-indolyl)-N'-(3-methyl-5-isothiazolyl)urea (SB204741) (5-HT(2B)R antagonist). In porcine valvular interstitial cells, cabergoline-induced activation of extracellular signal-regulated kinase (ERK) 1/2, an initiator of cellular proliferation and activity, was blocked by (R)-(+)-4-(1-hydroxy-1-(2,3-dimethoxyphenyl)methy1)-N-2-(4-fluorophenylethyl)piperidine (MDL100907) (5-HT(2A)R antagonist) and N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-1,1'-biphenyl-4-carboxamide (GR127935) (5-HT(1B)R antagonist), whereas the stimulatory effect on [(3)H]proline and [(3)H]glucosamine incorporations (indices of extracellular matrix collagen and glycosaminoglycan) was blocked by MDL100907. We conclude that the bulky N(6) substituent of cabergoline is responsible for 5-HT(2A)R and 5-HT(2B)R agonism. The increased ERK1/2 phosphorylation and production of extracellular matrix by cabergoline are mediated by 5-HT(2A)Rs. However, the moderate potency of cabergoline at native 5-HT(2A)Rs suggests that these are not the preferential target in VHD in vivo.     

Identification of A1 and A2 adenosine receptors in the rat spinal cord

The adenosine receptors in membranes prepared from rat ventral and dorsal lumbar spinal cord were characterized by comparing the binding characteristics of [3H]5'-N-ethylcarboxamide adenosine ([3H]NECA), an agonist with nearly equal affinities at the A1 and A2 adenosine receptor subtypes, with those of [3H]N6-[(R)-1-methyl-2-phenylethyl]adenosine ([3H]R-PIA), an A1-selective agonist. Saturation isotherms of the ventral and dorsal spinal cord yielded Kd values 1.9 to 2.3 nM for [3H]R-PIA and 18.1 to 19.5 nM for [3H]NECA. The Bmax for [3H]NeCA was approximately twice the Bmax for [3H]R-PIA in ventral and dorsal halves (267 vs. 128 fmol/mg of protein and 402 vs. 206 fmol/mg of protein, respectively). Displacement of specific [3H]NECA binding by the A2-selective agonist, 2-(phenylamino)adenosine, the relatively nonselective antagonist, theophylline and six A1-selective agonists, R-PIA, S-PIA, N6-(cyclohexyl)adenosine, N6-(cyclopentyl)adenosine, N6-(m-aminophenyl)adenosine and N6-(m-iodophenyl)adenosine, revealed two [3H]NECA binding components with the characteristics of A1 and A2 receptors. All curves best fit a two-site model when analyzed by the computer program LIGAND. R-PIA, N6-(cyclohexyl)adenosine and N6-(cyclopentyl)adenosine were the most potent displacers at the first site (Ki = 0.6-1.4 nM). All A1-selective agonists were poor displacers of [3H]NECA at the second site (Ki = 0.6-18.6 microM). The A2-selective agonist, 2-(phenylamino)adenosine, was as potent as R-PIA in displacing [3H]NECA from this site with a Ki value 0.57 microM. Finally, the A1 and A2 adenosine receptor-mediated inhibition and stimulation of adenylate cyclase were demonstrated directly in synaptic membranes prepared from the spinal cord.     

Effects of histamine, H1- and H2-receptor antagonists on the activity of serotonergic neurons in the dorsal raphe nucleus

We investigated the effects of histamine applied by microiontophoresis onto serotonin-containing (serotonergic) cells recorded extracellularly in the dorsal raphe nucleus of the rat. Application of histamine at low iontophoretic currents (1-5 nA) produced a rapid depression of the firing of all serotonergic neurons tested. The H1-receptor antagonists mepyramine and diphenhydramine were unable to attenuate the histamine-induced response. Antagonism of the effect of histamine by the iontophoretic application of the H2-receptor antagonists cimetidine and metiamide was not possible to evaluate since both were found to exert potent inhibitory effects by themselves. In contrast, the nonimidazole-derived H2-receptor antagonist ranitidine, which had no effect by itself, selectively antagonized the histamine-induced depression of neuronal activity. Histidine, 3-methylhistamine and a variety of histamine agonists selective for H1- or H2-receptors were unable to mimic the effect of histamine in dorsal raphe. Histamine's effects may, in part, be mediated at a gamma-aminobutyric acid receptor complex as the gamma-aminobutyric acid antagonists bicuculline and picrotoxin rapidly and reversibly antagonized both the histamine- and the cimetidine-induced depression of serotonin cell firing; the glycine antagonist strychnine selectively blocked the inhibitory effect of glycine without altering the histamine-induced response. These data show an inhibitory effect of histamine on serotonin-containing neurons in the dorsal raphe; this effect may be partially mediated at a subtype of H2-receptor. These data further indicate that the inhibitory effects of histamine and cimetidine observed in the dorsal raphe nucleus may result, in part, from an action directly or indirectly at a gamma-aminobutyric acid receptor complex.     

Identification and specific blockade of two receptors for histamine in the cardiovascular system.

Histamine caused a fall in blood pressure in anesthetized dogs and cats which was only partially attenuated by mepyramine (pyrilamine), a histamine type H1-receptor antagonist. Further treatment with burimide or metiamide, type H2-receptor antagonists, caused nearly complete attenuation of the response to histamine. Burimamide alone had no effect on vasodilatation produced by histamine in the dog gracilis muscle whereas mepyramine alone caused a partial attenuation. An H2-receptor agonist, 4-methylhistamine and an H1-receptor agonist, 2-(2-pyridyl)ethylamine, both produced vasodilatation which was blocked by metiamide and mepyramine, respectively. Constriction of the saphenous vein produced by histamine was found to involve interaction with H1-receptors only. In the intact dog, histamine increased heart rate and decreased left ventricular dp/dt through direct effects. Mepyramine prevented the increase in heart rate but did not affect the chronotropic actions of isoproterenol and glyceryl trinitrate. H1-receptor blockade did not alter inotropic effects whereas subsequent H2-receptor blockade prevented the negative inotropic effect of histamine. It is concluded that both peripheral vascular and cardiac responses to histamine are mediated through activation of H1- and H2-histamine receptors.     

Characterization of histamine receptors in isolated rabbit veins

Veins were isolated from 16 sites of the rabbit venous tree and responses to histamine and histamine receptor agonists were studied to characterize the histamine receptors. Isometric contraction and relaxation of ring segment preparations were recorded. Histamine produced concentration-dependent contractions in all veins in the resting state. Both the maximum response and pD2 value varied remarkably from vein to vein and regional differences in sensitivities to histamine varied considerably from previous findings in dog veins. Also in the precontracted state with a vasoconstricting agent, histamine predominantly contracted the veins. The contractile responses to histamine, in both resting and precontracted states, were antagonized competitively by the histamine H1-receptor antagonist, mepyramine. On the other hand, histamine relaxed the precontracted veins, in the presence of mepyramine. Selective H2-receptor agonists, dimaprit and impromidine, relaxed the precontracted veins even in the absence of mepyramine. These responses to histamine were antagonized competitively by the H2-receptor antagonist, cimetidine or ranitidine. The present study provides quantitative and systematic data regarding histamine receptors in rabbit veins. We propose that: 1) there are both vasoconstrictor H1-receptors and vasodilator H2-receptors, 2) histamine generally contracts rabbit veins through predominant H1-receptors and that 3) the H2-receptor-mediated relaxation does not depend on the presence of the endothelium.     

2-Methoxymethyl-salvinorin B is a potent kappa opioid receptor agonist with longer lasting action in vivo than salvinorin A

Salvinorin (Sal) A is a naturally occurring, selective kappa opioid receptor (KOPR) agonist with a short duration of action in vivo. Pharmacological properties of a C(2) derivative, 2-methoxymethyl (MOM)-Sal B, were characterized. MOM-Sal B bound to KOPR with high selectivity and displayed approximately 3-fold higher affinity than U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate] and Sal A. It acted as a full agonist at KOPR in guanosine 5'-O-(3-[(35)S]thio)triphosphate binding and was approximately 5- and approximately 7-fold more potent than U50,488H and Sal A, respectively. In Chinese hamster ovary cells stably expressing KOPR, all three kappa agonists internalized or down-regulated KOPR to similar extents, with MOM-Sal B being the most potent. In mice, MOM-Sal B (0.05-1 mg/kg s.c.) caused immediate and dose-dependent immobility lasting approximately 3 h, which was blocked by norbinaltorphimine. In contrast, ambulation in a Y-maze was increased when rats received MOM-Sal B (1-5 mg/kg s.c.). In addition, MOM-Sal B (0.5-5 mg/kg i.p.) produced antinociception (hot-plate test) and hypothermia in a dose-dependent manner in rats. MOM-Sal B was more potent than U50,488H in both tests and more efficacious than U50,488H in the hot-plate test. These latter two in vivo effects were blocked by norbinaltorphimine, indicating KOPR-mediated actions. Sal A at 10 mg/kg elicited neither antinociception nor hypothermia 30 min after administration to rats. In summary, MOM-Sal B is a potent and efficacious KOPR agonist with longer lasting in vivo effects than Sal A.     

Regulation by histamine of cyclic nucleotide levels in sympathetic ganglia.

An investigation has been carried out of the role of histamine H1- and H2-receptors in the control of cyclic guanosine 3':5'-monophosphate (cGMP) and cyclic adenosine 3':5'-monophosphate (cAMP) levels in blocks of bovine superior cervical ganglion. The data suggest that activation of H1-receptors is associated with cGMP accumulation and that activation of H2-receptors is associated with cGMP accumulation. Histamine increased both cGMP and cGMP levels with similar time course and concentration-response relationships. Low concentrations of the H1-receptor agonist 2-(2-aminoethyl)thiazole increased cGMP but not cAMP levels. Conversely low concentrations of the H2-receptor agonist 4-methylhistamine increased cAMP but not cGMP levels. H1-receptor antagonists blocked the histamine-induced increase in cGMP at low concentrations but blocked the cAMP increase only at substantially higher concentrations. Conversely, H2-receptor antagonists blocked the histamine-induced increased in cAMP but not cGMP. The effects of histamine on cyclic nucleotide levels did not appear to be mediated via the release of an endogenous neurotransmitter. The histamine-induced increase in cGMP appeared to be mediated through calcium: the increase in cGMP required the presence of calcium in the extracellular medium, and the calcium ionophore A23187 caused a calcium-dependent increase in cGMP. When considered with previous electrophysiological and biochemical findings in sympathetic ganglia, a correspondence can be seen: both histamine (at H1-receptors) and acetylcholine (at muscarinic receptors) raise cGMP levels and are associated with excitatory actions; both histamine (at H2- receptors) and dopamine raise cAMP levels and are associated with inhibitory actions.     

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.     

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.     

Isolated parietal cells: histamine response and pharmacology

Isolated, partially purified or enriched rat gastric mucosal parietal cells were shown to respond to histamine and other histaminic H2-receptor agonists as measured by an increased accumulation of [14C]aminopyrine. The response was temperature-dependent, related to parietal cell purity and inhibited selectively and reversibly by H2-receptor antagonists. H1-receptor antagonists noncompetitively inhibited histamine, carbamylcholine and dibutyryl cyclic AMP-stimulated aminopyrine accumulation. The affinity constants calculated for the H2-receptor agonists and antagonists were similar to those previously determined in the studies of activation and inhibition of adenylyl cyclase in enriched parietal cell preparations. The results strongly suggest that a correlation exists between the ability of histamine and its analogs to stimulate isolated rat parietal cell function and their ability to stimulate adenylyl cyclase activity. Potentiation of aminopyrine accumulation in the presence of histamine and carbamylcholine is due to specific receptor effects of each secretagogue.     

In vitro pharmacology of clinically used central nervous system-active drugs as inverse H(1) receptor agonists

The human histamine H(1) receptor (H(1)R) is a prototypical G protein-coupled receptor and an important, well characterized target for the development of antagonists to treat allergic conditions. Many neuropsychiatric drugs are also known to potently antagonize this receptor, underlying aspects of their side effect profiles. We have used the cell-based receptor selection and amplification technology assay to further define the clinical pharmacology of the human H(1)R by evaluating >130 therapeutic and reference drugs for functional receptor activity. Based on this screen, we have reported on the identification of 8R-lisuride as a potent stereospecific partial H(1)R agonist (Mol Pharmacol 65:538-549, 2004). In contrast, herein we report on a large number of varied clinical and chemical classes of drugs that are active in the central nervous system that display potent H(1)R inverse agonist activity. Absolute and rank order of functional potency of these clinically relevant brain-penetrating drugs may possibly be used to predict aspects of their clinical profiles, including propensity for sedation.