The membrane concentraton of a local anesthetic (Chlorpromazine)

Abstract 1. 1. The classical partition theory of anesthesia proposed by Meyer 1 , Baum 2 , Overton 3 and Meyer and Hemmi 4 predicted that the concentration of anesthetic in the membrane should be of the order of 0.03–0.06 mole anesthetic per 1 of membrane lipid. In order to measure the membrane concentration of a local anesthetic directly, the adsorption of chlorpromazine to hemoglobin-free erythrocyte ghost membranes was measured at concentrations known to be membrane stabilizing for both nerve fibers and erythrocytes. 2. 2. In the chlorpromazne concentration region for membrane stabilization and anesthesia, namely 2·10 −6 –2·10 −5 M, the adsorption of chlorpromazine by erythrocyte ghost membranes was associated with a single set of binding sites. When these sites were 50% occupied (50% membrane stabilization) the membrane concentration of the anesthetic was about 0.03 mole drug per 1 wet membrane. When the sites were fully occupied (at around 2·10 −5 M) the membrane concentration was 0.066 mole anesthetic per 1 wet membrane. The range of 0.03–0.066 M approximately agrees with the values predicted by the partition theory. 3. 3. At chlorpromazine concentrations higher than 3·10 −6 M, which corresponds to lytic concentrations of this surface-active anesthetic, the membrane concentrations increased very steeply. The membrane concentration of these “lytic sites” is much higher than 0.066 mole/l wet membrane. 4. 4. The maximum membrane concentration of 0.006 M for the “stablization sites” did not vary with the temperature. 5. 5. The affinity constant for the adsorption of chlorpromazine was 165 000 M −1 at 22°. The affinity constant varies inversely with the temperature. 6. 6. The effect of pH on the membrane concentration of chlorpromazine was also examined. At the extracellular concentration of 2.5·10 −6 M the membrane/buffer partition coefficient was 4300 at pH 6.8 and 3500 at pH 6.2. 7. 7. The free energy of adsorption was −9400 cal/mole. The enthalpy of adsorption was negative at 14° (−500 cal/mole), and became more negative (−3000 cal/mole) at higher temperature. These findings, according to the theory of Nemethy and Scheraga 52,53 , suggest that the chlorpromazine—membrane interaction is hydrophobic in nature. 8. 8. The bulk volume of 0.066 mole chlorpromazine in 1 l wet membrane would occupy between 12 and 25 cm 3 or 1.2–2.5% of the wet membrane volume. The bulk volume of the molecules cannot completely account for the 4.5% expansion of the erythrocyte membrane area that is known to occur in the presence of 2·10 −5 M chlorpromazine.