Portal de Periódicos da CAPES

The displacement of membrane calcium by a local anesthetic (chlorpromazine)

1969; Elsevier BV; Volume: 193; Issue: 2 Linguagem: Inglês

10.1016/0005-2736(69)90194-1

1879-2642

W.O. Kwant, Philip Seeman,

Biochemical Analysis and Sensing Techniques

1. The effect of chlorpromazine on the membrane concentration of Ca2+ was examined. Erythrocyte ghosts, which were free of hemoglobin and Ca2+, were mixed with 45Ca2+ in the presence of different concentrations of Ca2+ and chlorpromazine. The amount of Ca2+ adsorbed to the membrane was determined from the amount of 45Ca2+ remaining in the supernatant. 2. It was found that [3H]sorbitol rapidly equilibrated over the entire water space of the ghost suspension. [14C]Inulin, however, completely permeated only about 80% of the ghosts. The water space for 45Ca2+ was found to be similar to that for inulin; this was obtained by saturating the membrane sites for Ca2+ (which occurred between 10 and 50 mM Ca2+), measuring the radioactivity in the supernatant, and comparing it with the control value. 3. The membrane concentration of Ca2+ was determined as a function of the extracellular Ca2+ concentration. At free concentrations of Ca2+ below 1 mM the adsorption isotherm indicated one set of binding sites with a saturation capacity of 0.081 mole Ca2+ per 1 of solid (and 30% hydrated) membrane, and with an affinity constant of 1940 l membrane solid per mole Ca2+. 4. The addition of saponin (2 mg/mg dry ghost) greatly increased the inulin space and the adsorption of Ca2+ to the membranes. Rehemolysis of the ghosts also increased the binding of Ca2+ to the mebranes. Saponin and rehemolysis presumably exposed intracellular sites by disrupting the membrane. 5. Chlorpromazine, at free concentrations of 1–10 μM (or total concentrations 0f 0.1–1 mM) competitively displaced the membrane-bound Ca2+. The maximum amount of membrane Ca2+ displaced when the membrane was saturated with chlorpromazine (0.066 mole chlorpromazine per 1 solid membrane) was 0.021 mole Ca2+ per 1 membrane solid. It was concluded that 1 Ca2+ was displaced from the membrane by about 2 chlorpromazine molecules.