The poorly characterized G-protein-coupled receptor GPR35 has been suggested as a potential exploratory target for the treatment of both metabolic disorders and hypertension. It has also been indicated to play an important role in immune modulation. A major impediment to validation of these concepts and further study of the role of this receptor has been a paucity of pharmacological tools that interact with GPR35. Using a receptor-β-arrestin-2 interaction assay with both human and rat orthologues of GPR35, we identified a number of compounds possessing agonist activity. These included the previously described ligand zaprinast. Although a number of active compounds, including cromolyn disodium and dicumarol, displayed similar potency at both orthologues of GPR35, a number of ligands, including pamoate and niflumic acid, had detectable activity only at human GPR35 whereas others, including zaprinast and luteolin, were markedly selective for the rat orthologue. Previous studies have demonstrated activation of Gα13 by GPR35. A Saccharomyces cerevisiae-based assay employing a chimaeric Gpa1-Gα13 G-protein confirmed that all of the compounds active at human GPR35 in the β-arrestin-2 interaction assay were also able to promote cell growth via Gα13. Each of these ligands also promoted binding of [35S]GTP[S] (guanosine 5'-[γ-[35S]thio]triphosphate) to an epitope-tagged form of Gα13 in a GPR35-dependent manner. The ligands identified in these studies will be useful in interrogating the biological actions of GPR35, but appreciation of the species selectivity of ligands at this receptor will be vital to correctly attribute function.

If arsenite produces exencephaly by inactivating the Pax3 protein, then the fact that the exencephaly rate was increased in Sp/Sp embryos with no functional Pax3 indicates that arsenite may either induce this defect through additional pathways, or may alter the response via modifier genes. Genetic and environmental factors contributed to the determination of murine sex ratios, with female embryos being more susceptible to loss.

The 5-hydroxytryptamine 2 (5-HT 2 ) receptor class comprises three subtypes 5-HT 2A , 5-HT 2B , 5-HT 2C , which are similar in terms of their sequence, molecular structure, pharmacology and signal transduction pathways.1) All of them belong to the G-protein-coupled-receptors superfamily. They are preferentially coupled to Gq/11 to increase the hydrolysis of inositol phosphates and elevate cytosolic calcium.2)The 5-HT 2B receptor, first found in rat stomach fundus, 3) was one of the latest to be characterised and cloned in mouse, rat and human. [4][5][6][7] As the rest of the 5-HT 2 receptors, 5-HT 2B mediates the release of phosphoinositides, although controversy persists on the possible involvement of other second messengers. [6][7][8][9][10][11][12][13] Interest in the 5-HT 2B receptor has mushroomed as the result of its presence in humans, in both the central nervous system and peripheral tissues. 6,7,14,15) Cyproheptadine (Fig. 1) was chosen as the lead compound for this research project because of its high affinity at 5-HT 2B receptors in rat stomach fundus. In a previous work, 16) we demonstrated the relevance of the amino group in open cyproheptadine analogues. Only compounds having N-H or N-methyl motifs (compounds 1 and 2: 4-diphenylmethylenepiperidine and 4-diphenylmethylene-1-methylpiperidine, respectively), showed significant 5-HT 2B antagonistic activity. This observation supports the previously postulated key role of a cationic amino group in the interaction of ligands at the active site of the aminergic G-coupled protein receptors, by interacting with a conserved anionic aspartate located in the third transmembrane a-helix (TMH) of the receptor. 17)However, a surprising result of our previous study was the total lack of activity of the corresponding quaternary ammonium ion (compound 3: 4-diphenylmethylene-1,1-dimethylpiperidinium iodide). 16)The pH of the biological medium is responsible for the changes in the potency of antagonists at some receptors.18) A question therefore arises whether ligand protonation favours their interaction with 5-HT 2B receptors. To provide new insights on this issue, in this work we studied the influence of increasing the protonation of compounds 2 and 3 (by reducing the pH of the medium) on the antagonism of serotonin in rat stomach fundus preparations. Results and DiscussionDuring the experiments described below, we verified that the pH of the solutions (6, 7.4, 8) remained unchanged over the duration of organ bath experiments (Fig. 2). Cyproheptadine is one of the compounds exhibiting the highest activity at 5-HT 2B receptors. In a previous work we analysed the relevance of the amino group in diphenylmethylenepiperidines (DPMP), which are open cyproheptadine analogues. Only compounds containing N-H or N-methyl motifs, showed significant 5-HT 2B activity. Surprisingly, the corresponding quaternary ammonium salt demonstrated a total lack of activity. Therefore, the question arises whether protonation favours the interaction of these compounds with 5-HT 2B receptors. C...