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

摘要

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 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 Ca~(2+)-containing low ionic strength (LIS) solutions, [Ca2+]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 M 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 mol/(lcells * h) in HIS, and from 36.7 ± 5.3 to 8.6 ± 1.2 mol/(lcells * 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 M) 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.