Calcium- and lead-activated morphological changes in human erythrocytes: a spin label study of the cytoplasm

Stimulation of the human red blood cell (RBC; erythrocyte) by Ca2+ or Pb2+ ions induced potassium efflux (the so-called Gárdos effect). Time-dependent changes in the volume and shape of the RBCs were monitored on the basis of mean cell volume and light scattering. Changes in the RBC cytoplasm were followed using electron paramagnetic resonance (EPR), after labeling of the cytoplasmic glutathione with 2,2,6,6-tetramethyl-4-maleimidopiperidine-N-oxyl. The RBC could still be activated in vitro with Ca2+ (plus ionophore A23187) or Pb2+ ions. Pb2+ retained its stimulatory-inhibitory activity. No linear relationship between the reciprocal of cell volume and the mobility (correlation time) of the probe was found over the morphological range covered upon Ca2+ activation. The response of the probe is considered to be mainly governed by the microviscosity due to hemoglobin and any influence by cytoskeleton products could not be discerned. Pretreatment of the RBC with N-ethylmaleimide (1 mM for 30 min) at room temperature prevented spin labeling. However, the RBC still responded to the ion activation, whereas this effect was eliminated after pretreatment at 37 degrees C. EPR of spin-labeled cells, before and after reaching the sphero-echinocytic state, resulted in similar final spectra. Spectra were also observed from RBCs fixed in various states with glutaraldehyde.