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Localização de anestesicos locais do tipo amino-amida em lipossomas uni e multilamelares

by Fraceto, Leonardo Fernandes

Abstract (Summary)
Literature carries many theories about the mechanism of action of local anesthetics (LA). We can highlight those that focus on the direct effect of LA on the sodium channel protein and the ones that consider the interaction of anesthetic molecules with the lipid membrane phase. The direct correlation between LA hydrophobicity and anesthetic potency is well known. So, it is reasonable to consider that the interaction of these amphiphilic compounds with the membrane could be of great importance, as well as the neutral (uncharged) forro interactio_ since its affinity for the membrane is higher than the protonated LA species. To better understand the LA - lipid membrane phase interactio_ we have chosen to study the neutral forro of four amino-amide local anesthetics (lidocaine, etidocaine, mepivacaine and bupivacaine) in unilamellar liposomes of phosphatidylcholine (EPC). The choice was made because these LA are clinically used and there are just a few studies involving the neutral species in membrane systems. These anesthetics belong to a homologous series but show quite different physicochemical properties such as hydrophobicity and steric parameters (mainly the cyclic vs. the non-cyclic). We have employed different spectroscopic techniques such as Electron Paramagnetic Resonance (EPR), Fluorescence, Infta-Red and Nuclear Magnetic Resonance (NMR) of hydrogen and phosphorus to obtain information about the specific interaction of LA with the membrane. In EPR and fluorescence measurements membrane probes inserted to different depths of the lipid acyl chain revealed that ali the four anesthetics led to a decrease in membrane organizatio_ but with quantitative differences related to the preferential positioning of each molecule inside the bilayer. IR and phosphorus-NMR measurements have clearly shown that partition of the LA molecule favors the access of water molecules up to the phosphate and glycerol moieties. These results are quite compatible with the decrease in membrane organization and can be explained by the spacement created by the LA insertion in-between the lipid molecules. Hydrogen NMR eH-NMR) techniques evidenced LA partition inside the membrane, as shown by the broadening of the anesthetics lH peaks when they move from the water to the membrane phase. Longitudinal relaxation time (TI) measurements were quite useful in the investigation of the preferential positioning of LA in the bilayer: just the more hydrophobic anesthetics (etidocaine and bupivacaine) were able to disturb the mobility of lipid hydrogens in the acyl chain core region. Nuclear Overhauser effect (ROESY) experiments revealed LA:lipid cross-peaks consistent with the preferential insertion of these molecules. Considering that LA crosses the membranes in a fast way and that its location inside the bilayer is best represented by a distribution function (against the bilayer normal), we have brought together clear evidences of regions with greater probabilities of finding the LA, i.e., where the LA stay most of the time. A model for the LA location inside the bilayer is presented. We show here that local anesthetics partition causes a greater change in membrane organization in unilamellar than in multilamellar EPC vesicles, previously studied in our laboratory. Membrane perturbation is not proportional to the LA hydrophobicity, even when equal LA:lipid molar ratios are present inside the membrane. Each LA seems to have a preferential positioning inside the bilayer, determined by its chemical properties, i.e., the more hydrophobic (etidocaine and bupivacaine) prefer deeper regions than the more hydrophilic (lidocaine and mepivacaine), which aromatic rings would lye in the glycerol moiety. We believe that the deeper insertion would favor anesthetic potency by facilitating the access ofthe molecule to hydrophobic sites ofthe sodium channel protein
This document abstract is also available in Portuguese.
Bibliographical Information:

Advisor:Eneida de Paula; Eneida de Paula [Orientador]; Maria Helena Andrade Santana; Fred Yukio Fujiwara

School:Universidade Estadual de Campinas

School Location:Brazil

Source Type:Master's Thesis

Keywords:anestesia local fluorescencia ressonancia magnetica nuclear membranas biologia

ISBN:

Date of Publication:03/28/2000

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