Na+/H+ exchange modulates Ca2+ mobilization in human platelets stimulated by ADP and the thromboxane mimetic U 46619.
According to recent observations ADP stimulates platelets via activation of Na+/H+ exchange which increases cytosolic pH (pHi). This event initiates formation of thromboxane A2 (via phospholipase A2) and, thereafter, inositol 1,4,5-trisphosphate (via phospholipase C) which is known to mobilize Ca2+ from intracellular storage sites. We investigated changes in pHi and cytosolic free Ca2+, [Ca2+]i, activating platelets with ADP and the thromboxane mimetic U 46619. We found that ADP (5 microM) increased pHi from 7.15 +/- 0.08 to 7.35 +/- 0.04 (n = 8) in 2'-7'-bis-(carboxyethyl)-5,6-carboxyfluorescein-loaded platelets, whereas thromboxane A2 formation was inhibited by indomethacin. ADP also induced a dose-dependent Ca2+ mobilization in fura2-loaded platelets which again was not affected by indomethacin. [Ca2+]i increased by 54 +/- 10 nM (n = 8) at 1 microM and by 170 +/- 40 nM (n = 7) at 10 microM ADP above the resting value of 76 +/- 12 nM (n = 47). Inhibition of Na+/H+ exchange by ethylisopropylamiloride (EIPA) reduced ADP-induced Ca2+ mobilization by more than 65% in indomethacin-treated platelets. This inhibition could be completely overcome by artificially raising pHi using either NH4Cl or the Na+/H+ ionophore monensin. We found that U 46619 increased pHi by 0.18 +/- 0.05 at 0.1 microM and by 0.29 +/- 0.07 (n = 7) at 1.0 microM above the resting value via an EIPA-sensitive mechanism. In conflict with the proposed role of the Na+/H+ exchange we found that U 46619 raised [Ca2+]i via a mechanism that for more than 50% depended on intact Na+/H+ exchange. Again, artificially elevating pHi restored U 46619-induced Ca2+ mobilization despite the presence of EIPA. Thus, our data show that Na+/H+ exchange is a common step in platelet activation by prostaglandin endoperoxides/thromboxane A2 and ADP and enhances Ca2+ mobilization independently of phospholipase A2 activity.
Siffert
W
W
Institut für Physiologie, Ruhr-Universität, Bochum, Federal Republic of Germany.
Siffert
G
G
Scheid
P
P
Akkerman
J W
JW
D001572:Benzofurans; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007213:Indomethacin / Q000494:pharmacology; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology*; D017923:Sodium-Hydrogen Exchangers; D013050:Spectrometry, Fluorescence
D001572:Benzofurans; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D011450:Prostaglandin Endoperoxides, Synthetic; D017923:Sodium-Hydrogen Exchangers; D002118:Calcium; D016257:Fura-2; D007213:Indomethacin