Prostaglandin E2 Stimulates a Ca2+-dependent K+ Channel in Human Erythrocytes and Alters Cell Volume and Filterability

Qing Li,Volker Jungmann,Anatoly Kiyatkin,Philip S Low

doi:10.1074/jbc.271.31.18651

Qing Li, Volker Jungmann + Show 2 more

Open Access

https://doi.org/10.1074/jbc.271.31.18651

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Abstract

To understand the mechanism by which human red blood cells (RBCs) contribute to hemostasis and thrombosis, we have examined the effects of metabolites released by activated platelets on intact RBCs. Prostaglandin E2 (PGE2), a signal molecule produced by activated platelets, was observed to lower the filterability of human erythrocytes by approximately 30% at 10(-10) M. PGE2 also caused a reduction in mean cell volume of approximately 10%. The shrinkage of red cells after PGE2 treatment was confirmed by documenting a decrease in osmotic fragility and an increase in cell density following exposure to the hormone. Careful analysis, however, revealed that only approximately 15% of the erythrocytes responded to stimulation with PGE2. Examination of the cause of cell shrinkage showed that induction of a PGE2-stimulated K+ efflux pathway leading to rapid loss of cellular K+ was responsible. The PGE2-stimulated K+ loss was also observed to be Ca2+-dependent, suggesting the possible involvement of the Gardos channel. Gardos channel participation was supported by the observation that two Gardos channel inhibitors, charybdotoxin and clotrimazole, independently blocked the PGE2-stimulated K+ efflux. Further evidence for Gardos channel activation came from experiments aimed at characterizing the efflux pathway followed by the obligatory counterion. Thus, K+ efflux was readily stimulated even when NO3- was substituted for Cl-, suggesting that neither KCl cotransport nor Na/K/2Cl cotransport plays a prominent role in the PGE2-induced cell shrinkage. Further, the anion transporter band 3 was implicated as the counterion efflux route, since DIDS inhibited the PGE2-stimulated cell volume change without blocking the change in membrane potential. Taken together, we propose that release of PGE2 by activated platelets constitutes part of a mechanism by which activated platelets may recruit adjacent erythrocytes to assist in clot formation.

Highlights

Red blood cells (RBCs)1 comprise 99% of the blood cells in circulation
We report here that prostaglandin E2 (PGE2) activates the Gardos channel in human erythrocytes and thereby induces Kϩ efflux, cell shrinkage, and reduced cell filterability
We have demonstrated that PGE2 reduces human erythrocyte filterability by stimulating Kϩ efflux leading to a loss of osmotic water and cell shrinkage

Summary

EXPERIMENTAL PROCEDURES

Materials—PGE2, clotrimazole, and Drabkins solution for hemoglobin measurements were purchased from Sigma. The washed erythrocytes at 10% hematocrit in buffer A were treated with 0.001 volumes of PGE2 dissolved in ethanol and incubated for the desired times. The filtration time of the erythrocyte suspension was normalized to the filtration time of the bovine serum albumin solution. After incubating at 4 °C for 4 min, cell suspensions were centrifuged (1,000 ϫ g, 5 min) and 100 ␮l of supernatant was mixed with 10 volumes of Drabkins solution. In the Gardos channel inhibition studies, the RBC suspension (10% hematocrit) was preincubated with charybdotoxin (ChTX) at room temperature for 100 min or in clotrimazole for 20 min prior to initiation of the Kϩ efflux assays. The cell suspension was added to 3 ml of a 2 ␮M diS-C3-(5) solution in buffer A, and after 5 min of equilibration, the suspension was centrifuged at 1,000 ϫ g for 5 min. The fluorescence of the supernatant was measured and used to calculate the cells’ membrane potential according to the method of Hladky and Rink [23], assuming a resting potential in fresh cells of Ϫ9 mV [23]

RESULTS

TABLE I

Estimated membrane potentiala

DISCUSSION