Effect of the ionic strength and prostaglandin E2 on the free Ca2+ concentration and the Ca2+ influx in human red blood cells

Human red blood cells (RBCs) were loaded with the Ca2+-sensitive fluorescent dye fura-2 to investigate the effects of media ionic strength and prostaglandin E2 (PGE2) on the intracellular free Ca2+ concentration ([Ca2+]i). [Ca2+]i of intact RBCs in a Ca2+-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 Ca2+-containing low ionic strength (LIS) solutions, [Ca2+]i was significantly lower than in the Ca2+-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 [Ca2+]i was seen after 1 h. In Ca2+-free (0 Ca2+ plus 15 μM EGTA) media, [Ca2+]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 PGE2 on passive Ca2+ influx was investigated on ATP-depleted RBCs. Ca2+ 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/(lcells×h) in HIS, and from 36.7±5.3 to 8.6±1.2 μmol/(lcells×h) in LIS. Prostaglandin E2 (PGE2; 10−7–10−11 M), dissolved in deionised water or in ethanol, did not affect [Ca2+]i in either normal or in ATP-depleted RBCs suspended in Ca2+-containing HIS medium. Finally, the addition of carbachol (100 μM) did not affect [Ca2+]i. The present findings suggest that stimulation of the Ca2+-activated K+ channel by PGE2, reported in [J. Biol. Chem. 271 (1996) 18651], cannot be mediated via increased [Ca2+]i.