Trinitrobenzenesulfonic acid and fluorodinitrobenzene: probes to study local anesthetic effects in cell membranes.

Abstract

The interaction of local anesthetics with intact erythrocytes was studied by monitoring the extent of reaction of phospholipids with trinitrobenzenesulfonic acid and fluorodinitrobenzene. Incubating erythrocytes with local anesthetics increases the amount of phosphatidylethanolamine and phosphatidylserine available for reaction with trinitrobenzenesulfonic acid and fluorodinitrobenzene. The order of potency of the local anesthetics corresponded to that reported for blocking nerve conduction: dibucaine greater than tetracaine greater than butacaine greater than lidocaine greater than procaine. Treatment of intact erythrocytes with 1 mM tetracaine at 37 degrees C allows 4-5% more of the phosphatidylethanolamine to react with trinitrobenzenesulfonic acid as compared to control cells. Treatment with tetracaine has no effect at 0 degrees C, a temperature at which there is only limited partitioning of the anesthetic into the bilayer. Kinetic analysis of the reaction with trinitrobenzene sulfonic acid showed that the increased number of reactive phosphatidylethanolamine molecules are located mainly on the outer half of the erythrocyte membrane. Tetracaine also increases the number of phosphatidylserine and phosphatidylethanolamine molecules in the erythrocyte membrane which are available to react with the penetrating probe fluorodinitrobenzene. The reaction with PE is increased from 67 to 77% and the reaction of PS is increased from 44 to 57%. Thus tetracaine affects both halves of the lipid bilayer.