Influence of pH on the Binding of Diphenylmethylenepiperidines by 5-HT2B Receptors in Rat Stomach Fundus.

Abstract

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...