Entrapping of the spin label tempocholine into human erythrocytes by resealing after hyposmolar stress. Comparison with haemolysis. The effects of some membrane-active substances.

Human erythrocytes were subjected to a sudden hyposmolar stress by suspension in solutions of varying salt concentrations in the presence of the spin label tempocholine. The enlarged pores in the erythrocyte membranes produced by the influx of water, followed by stretching, allowed the passage of the spin label, so that a certain amount of tempocholine was entrapped when the erythrocytes spontaneously resealed with closing of the pores. The excess of spin label in the external solution was then reduced to a diamagnetic species by the addition of ascorbic acid. The positively charged tempocholine and the ascorbic acid did not penetrate properly resealed erythrocytes, so that the electron spin resonance (ESR) signal from the entrapped spin label constituted a measure of the effective resealing of the pores and rifts in the membrane subsequent to hyposmolar stress. Some drug substances were found to influence the entrapping curves obtained when the amount of entrapped spin label was plotted against the osmolarity. Chlorpromazine, trifluoperazine, nicardipine, amperozide and haloperidol gave rise to a dose-dependent decrease of the entrapping of tempocholine, especially at low osmolarities. The exclusion of Ca2+ and Mg2+ ions from the solutions increased the action of chlorpromazine. The protective action against haemolysis brought about by a number of membrane-active substances at low concentrations [2] had its counterpart in the entrapping curve observed with chlorpromazine at 0.1 mM. It is suggested that the substances in this series exerted their action on the resealing process by interaction with the calmodulin system.