Detection of multiple H3 receptor affinity states utilizing [3H]A-349821, a novel, selective, non-imidazole histamine H3 receptor inverse agonist radioligand.

1. A-349821 is a selective histamine H3 receptor antagonist/inverse agonist. Herein, binding of the novel non-imidazole H3 receptor radioligand [3H]A-349821 to membranes expressing native or recombinant H3 receptors from rat or human sources was characterized and compared with the binding of the agonist [3H]N--methylhistamine ([3H]NMH). 2. [3H]A-349821 bound with high affinity and specificity to an apparent single class of saturable sites and recognized human H3 receptors with 10-fold higher affinity compared to rat H3 receptors. [3H]A-349821 detected larger populations of receptors compared to [3H]NMH. 3. Displacement of [3H]A-349821 binding by H3 receptor antagonists/inverse agonists was monophasic, suggesting recognition of a single binding site, while that of H3 receptor agonists was biphasic, suggesting recognition of both high- and low-affinity H3 receptor sites. 4. pKi values of high-affinity binding sites for H3 receptor competitors utilizing [3H]A-349821 were highly correlated with pKi values obtained with [3H]NalphaMH, consistent with labelling of H3 receptors by [3H]A-349821. 5. Unlike assays utilizing [3H]NMH, addition of GDP had no effect on saturation parameters measured with [3H]A-349821, while displacement of [3H]A-349821 binding by the H3 receptor agonist histamine was sensitive to GDP. 6. In conclusion, [3H]A-349821 labels interconvertible high- and low-affinity states of the H3 receptor, and displays improved selectivity over imidazole-containing H3 receptor antagonist radioligands. [3H]A-349821 competition studies showed significant differences in the proportions and potencies of high- and low-affinity sites across species, providing new information about the fundamental pharmacological nature of H3 receptors.

Cloning and pharmacological characterization of the monkey histamine H3 receptor.

Species differences have been described previously for histamine H(3) receptor pharmacology. Rat selective histamine H(3) receptor ligands such as ciproxifan and A-304121 (2-amino-1-[4-[3-(4-cyclopropanecarbonyl-phenoxy)-propyl]-piperazin-1-yl]-propan-1-one) show over 100-fold selectivity for the rat receptor compared to the human receptor. To date, however, the pharmacology of the cloned monkey histamine H(3) receptor has not been examined. In this study, we cloned the monkey histamine H(3) receptor gene (H(3)R) and evaluated the receptor pharmacology in binding and functional assays. The monkey histamine H(3) receptor is highly homologous to the human receptor with 438 identities in their 445 amino acid sequences, but less homologous to the rat receptor. However, unlike the human or rat, we found no evidence for additional splicing for the monkey H(3)R. Pharmacological analysis indicated that the monkey receptor exhibited similar pharmacological profiles to those of the human receptor, providing critical information for characterizing histamine H(3) receptor ligands in monkey behavioral models.

Species comparison and pharmacological characterization of rat and human CB2 cannabinoid receptors.

Pharmacological effects of cannabinoid ligands are thought to be mediated through cannabinoid CB1 and CB2 receptor subtypes. Sequence analysis revealed that rat and human cannabinoid CB2 receptors are divergent and share 81% amino acid homology. Pharmacological analysis of the possible species differences between rat and human cannabinoid CB2 receptors was performed using radioligand binding and functional assays. Pronounced species selectivity at the rat cannabinoid CB2 receptor (50- to 140-fold) was observed with AM-1710 (3-(1,1-Dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo[c]chromen-6-one) and AM-1714 (3-(1,1-Dimethyl-heptyl)-1-9-dihydroxy-benzo[c]chromen-6-one). In contrast, JWH-015 ((2-Methyl-1-propyl-1H-indol-3-yl)-napthalen-1-yl-methanone) was 3- to 10-fold selective at the human cannabinoid CB2 receptor. Endocannabinoid ligands were more human receptor selective. Cannabinoid CB2 receptor antagonist, AM-630 ((6-Iodo-2-methyl-1-(2-morpholin-4-yl-ethyl)-1H-indol-3-yl)-(4-methoxy-phenyl)-methanone) was more potent at the rat receptor in radioligand binding and functional assays than that of the human receptor. The findings of the pharmacological differences between the human and rat cannabinoid CB2 receptors in this study provide critical information for characterizing cannabinoid ligands in in vivo rodent models for drug discovery purpose.

Genetic and pharmacological aspects of histamine H3 receptor heterogeneity.

Histaminergic H3 receptors modulate the release of neurotransmitters within the CNS and periphery. Ligands for these receptors have potential clinical utility in a variety of disease states. However, the pharmacological characteristics of these receptors have been enigmatic for more than a decade because of the diversity of pharmacological effects observed with the limited number of heretofore-available compounds. Recent cloning of the H3 receptor has revealed interspecies differences in the protein sequences in key regions, the existence of splice variants that differ in composition between species, and potential differences in signal transduction processes between either different tissues and/or species. This review attempts to summarize these findings within the context of the molecular biological and pharmacological data accumulated to date. Also, we suggest a nomenclature strategy to reduce potential confusion that has arisen from different naming systems used by various investigators. While some facets of this genetic and pharmacological diversity help to rationalize various aspects of H3 receptor heterogeneity, there remains an insufficient repertoire of selective ligands, assays, or other measures to completely resolve all components of this diversity. The promise of newly available tools to further explore H3 receptor function may provide the insight to bring the promised clinical potential of H3 receptor ligands to realization.

Pharmacological and molecular characterization of a dorsal root ganglion cell line expressing cannabinoid CB(1) and CB(2) receptors.

The behavioral effects evoked by cannabinoids are primarily mediated by the CB(1) and CB(2) cannabinoid receptor subtypes. In vitro pharmacology of cannabinoid receptors has been elucidated using recombinant expression systems expressing either CB(1) or CB(2) receptors, with limited characterization in native cell lines endogenously expressing both CB(1) and CB(2) receptors. In the current study, we report the molecular and pharmacological characterization of the F-11 cell line, a hybridoma of rat dorsal root ganglion neurons and mouse neuroblastoma (N18TG2) cells, reported to endogenously express both cannabinoid receptors. The present study revealed that both receptors are of mouse origin in F-11 cells, and describes the relative gene expression levels between the two receptors. Pharmacological characterization of the F-11 cell line using cannabinoid agonists and antagonists indicated that the functional responses to these cannabinoid ligands are mainly mediated by CB(1) receptors. The non-selective cannabinoid ligands CP 55,940 and WIN 55212-2 are potent agonists and their efficacies in adenylate cyclase and MAPK assays are inhibited by the CB(1) selective antagonist SR141716A (SR1), but not by the CB(2) selective antagonist SR144528 (SR2). The endocannabinoid ligand 2AG, although not active in adenylate cyclase assays, was a potent activator of MAPK signaling in F-11 cells. The analysis of CB(1) and CB(2) receptor gene expression and the characterization of cannabinoid receptor pharmacology in the F-11 cell line demonstrate that it can be used as a tool for interrogating the endogenous signal transduction of cannabinoid receptor subtypes.