Effect of the ionic strength and prostaglandin E 2 on the free Ca 2+ concentration and the Ca 2+ influx in human red blood cells

Abstract

Human red blood cells (RBCs) were loaded with the Ca 2+-sensitive fluorescent dye fura-2 to investigate the effects of media ionic strength and prostaglandin E 2 (PGE 2) on the intracellular free Ca 2+ concentration ([Ca 2+] i). [Ca 2+] i of intact RBCs in a Ca 2+-containing physiological (high) ionic strength (HIS) solution was 75.1±8.3 nM after 5 min incubation, increasing to 114.9±9.6 nM after 1 h. In Ca 2+-containing low ionic strength (LIS) solutions, [Ca 2+] i was significantly lower than in the Ca 2+-containing HIS solution ( p=0.041 or 0.0385 for LIS solutions containing 200 or 250 mM sucrose, respectively), but, as in HIS solution, an increase of [Ca 2+] i was seen after 1 h. In Ca 2+-free (0 Ca 2+ plus 15 μM EGTA) media, [Ca 2+] i decreased (ranging from 15 to 21 nM), but were not significantly different in HIS or LIS, and did not change following 1 h incubation. The effect of the ionic strength and PGE 2 on passive Ca 2+ influx was investigated on ATP-depleted RBCs. Ca 2+ influx was faster during the initial 10 min in comparison with the subsequent time period (10−45 min), both in HIS and LIS media, decreasing from 20.3±1.9 to 12.9±1.3 μmol/(l cells×h) in HIS, and from 36.7±5.3 to 8.6±1.2 μmol/(l cells×h) in LIS. Prostaglandin E 2 (PGE 2; 10 −7–10 −11 M), dissolved in deionised water or in ethanol, did not affect [Ca 2+] i in either normal or in ATP-depleted RBCs suspended in Ca 2+-containing HIS medium. Finally, the addition of carbachol (100 μM) did not affect [Ca 2+] i. The present findings suggest that stimulation of the Ca 2+-activated K + channel by PGE 2, reported in [J. Biol. Chem. 271 (1996) 18651], cannot be mediated via increased [Ca 2+] i.