Interaction of Serotonin_1AReceptors from Bovine Hippocampus with Tertiary Amine Local Anesthetics

Abstract

SUMMARY1. We have examined the interaction of tertiary amine local anesthetics with the bovine hippocampal serotonin 1A (5-HT 1A ) receptor, an important member of the G-proteincoupled receptor superfamily.2. The local anesthetics inhibit specific agonist and antagonist binding to the 5-HT 1A receptor at a clinically relevant concentration range of the anesthetics. This is accompanied by a concomitant reduction in the binding affinity of the 5-HT 1A receptor to the agonist. Interestingly, the extent of G-protein coupling of the receptor is reduced in the presence of the local anesthetics.3. Fluorescence polarization measurements using depth-dependent fluorescent probes show that procaine and lidocaine do not show any significant change in membrane fluidity. On the other hand, tetracaine and dibucaine were found to alter fluidity of the membrane as indicated by a fluorescent probe which monitors the headgroup region of the membrane.4. The local anesthetics showed inhibition of agonist binding to the 5-HT 1A receptor in membranes depleted of cholesterol more or less to the same extent as that of control membranes in all cases. This suggests that the inhibition in ligand binding to the 5-HT 1A receptor brought about by local anesthetics is independent of the membrane cholesterol content.5. Our results on the effects of the local anesthetics on the ligand binding and G-protein coupling of the 5-HT 1A receptor support the possibility that G-protein-coupled receptors could be involved in the action of local anesthetics.

Ligand Binding Characteristics of the Human Serotonin1A Receptor Heterologously Expressed in CHO Cells

“…The data shown in the table represent the means±SEM of three independent experiments. See Materials and Methods for other details.in excellent agreement with the K d value reported earlier by us for the native bovine hippocampal 5-HT 1A receptor[33,35,[37][38][39]. Moreover, this is also in agreement with the K d values for recombinant 5-HT 1A receptors reported by other groups[57,58].Table 1also shows that the 5-HT 1A receptors expressed in CHO-5-HT 1A R cells…”

“…In addition, the 5-HT 1A receptor antagonists represent a major class of molecules with potential therapeutic effects in anxiety-or stress-related disorders [30]. We have earlier solubilized and partially purified the 5-HT 1A receptor from bovine hippocampus [31,32] and have shown modulation of ligand binding to the bovine hippocampal 5-HT 1A receptor by metal ions, guanine nucleotides, alcohols, local anesthetics, membrane cholesterol, and covalent modifications of the disulfides and sulfhydryl groups [33][34][35][36][37][38][39][40].…”

G-Protein-Dependent Cell Surface Dynamics of the Human Serotonin1A Receptor Tagged to Yellow Fluorescent Protein

“…In addition, 5-HT 1A receptor antagonists represent a major class of molecules with potential therapeutic effects in anxiety-or stress-related disorders (25). We have earlier shown modulation of ligand binding activity of the 5-HT 1A receptor isolated from the bovine hippocampus with agents that perturb G-proteins (26,27), local anesthetics (28), and membrane cholesterol (29,30). In addition, we have solubilized and partially purified the 5-HT 1A receptor from both the native bovine hippocampus (17) and heterologously expressed human 5-HT 1A receptors from Chinese hamster ovary cells (31), where the receptor was found to possess very similar pharmacological characteristics as in the native system (32).…”

Anestésicos locais: interação com membranas biológicas e com o canal de sódio voltagem-dependente

“…12 Trabalhos na literatura mostram várias evidências da interação de AL com outras proteínas além do canal de sódio voltagem dependente, dentre elas, as sinalizadoras como: calmodulina, canais de potássio, receptores de acetilcolina, ATPases microssomais e mitocondriais, citocromo oxidase, proteína G, proteína EnvZ -que age na transdução de sinais por ativação da porina e proteína quinase Cα, receptores de serotonina 1A, proteína tirosina kinase e receptores de opióides. [13][14][15][16][17][18] Desde os trabalhos pioneiros de Hodgkin e Huxley, 19 em 1953, sabe-se que os anestésicos locais têm efeito direto nos canais de sódio e que interagem com diferentes graus de afinidade com essas proteínas, dependendo de seu estado funcional (ativado, inativado, em repouso). Inicialmente postulou-se que essa afinidade diferencial estaria relacionada à presença das formas carregada e neutra dos anestésicos em pH fisiológico.…”