Effect of local anesthetics on Ca++ binding to lipid-extracted RBC membrane
[영문]It has been shown that local anesthetics and Ca**++ reduce membrane permeabilities to various substances in excitable tissue(Taylor, 1959; Inoue and Frank, 1962; Aceves and Machne, 1963; Feinstein, 1964; Blaustein and Goldman, 1966; Blaustein, 1967; Kuperman et al., 1968; wilcox and Fuchs, 1969; Papahadjopoulos, 1970; Papahadjopoulos, 1972). Also, it was observed that local anesthetics compete with Ca**++ for binding sites in the membrane(Blaustein and Goldman, 1966; Feinstein and Paimre, 1967; Kwant and Weeman, 1960; Suarez-Kurtz et al., 1970;
Forstner and Manery, 1971; Seeman, 1972). Feinstein and other investigators showed that local anesthetics interact with phospholipids and lipids extracted form nerve and muscle (Feinstein, 1963; Feinstein and Paimre, 1964: Blaustein, 1967;
Papahadjopoulos, 1970). In subsequent studies, Feinstein and his coworker showed that local anesthetics react with Ca**++ presumably at the phosphodiester phosphate group of acidic phospholipids(Feinstein and Paimre, 1966).
However, no studies have been reported on whether local anesthetics react with membrane protein and if these local anesthetics still compete with Ca**++ for the binding sites of membrane protein.
In the present study, experiments were carried out to investigate this aspect and the results show that Ca**++ binding to the membrane protein of RBC and egg albumin were inhibited competitively by local anesthetics.
(1) Preparation of RBC ghost membrane fragments: Fresh human blood was obtained from the blood bank and the membrane fragments were isolated by the method of Dodge et al.(1963). After ghosts were obtained, the cell suspension was sonified by a cell disruptor(Model W 1850, Branson sonic Power Co.) for 10∼20 seconds at 60 watts.
(2) Ca**++ binding study: An aliquot of membrane fragment(about 0.5mg protein) transferred to a cover glass was dried in a desiccator in a cold room. The membrane fragments were incubated and washed in a manner to be described later. When the membrane protein was used for the calcium binding study the membrane fragments dried on the cover glasses were immersed to extract lipid from the membrane fragments in a series of solvents in large volumes. The organic solvents used were chloroform-methanol(2:1, v/v), acetone and ethyl ether.