Prehemolytic impact of phenothiazine drugs on the attachment of spectrin network in red blood cells

Abstract

Introduction: Chlorpromazine, thioridazine, and trifluoperazine are phenothiazine drugs that cause colloid-osmotic hemolysis of human erythrocytes by unknown mechanism. To clarify this mechanism, the impact of these drugs on the βsp (1.4 MHz) and γ1sp (9 MHz) dielectric relaxations was investigated. Each relaxation was shown to reduce its strength on the severing specific bridge that connects the spectrin network with the lipid membrane. For βsp relaxation, this is the spectrin-actin-glycophorin C bridge while for γ1sp relaxation this is the spectrin-ankyrin-band 3 bridge. Aim: To elucidate the mechanisms of the effects of phenothiazine drugs in prehemolytic concentrations on the red blood cell plasma membrane using scanning temperature-dependent (thermal) differential dielectric spectroscopy. Materials and methods: Erythrocytes were isolated from freshly drawn blood and 100 μl of them were suspended in 1 ml isotonic solution of 10 mM NaCl and mannitol (working medium) containing the indicated concentration of the drug for 10 min at 23°C. The treated erythrocytes were isolated, suspended in working medium, hematocrit 0.55, and heated (heating rate 1.5°C/min) above the denaturation temperature of spectrin (TA≈49.5°C) in order to obtain the differential dielectric spectroscopy data. The complex admittance, Y* = Y’+j.Y”, of the tested suspensions was continuously measured and separated into its real (Y’) and imaginary (Y”) parts using Solartron 1260 Impedance Frequency Analyzer. Results: At pre-hemolytic concentrations, each drug inhibited these two relaxations, predominantly the γ1sp relaxation. The results could be interpreted in terms of a sigmoid effect of the drugs on the spectrin-ankyrin-band 3 bridge severing it at concentration just prior to the start of massive hemolysis. Conclusions: The study points at the possible mechanism of erythrocyte damage after treatment with phenothiazine drugs at prehemolytic concentrations. This is probably due to the disruption of the bridges between the phospholipid bilayer and the submembrane spectrin network.