The treatment of aspirinated platelets with the endomembrane Ca2+-ATPase inhibitor thapsigargin (Tg) induces a large increase in cytosolic pH (pHi), as measured with the intracellular fluorescent indicator 2´,7´-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. In contrast, Tg induces a decrease in pHi in the presence of the Na+/H+ exchanger inhibitor 5-(N,N-hexamethylene)-amiloride (NHA). Both effects are inhibited if the cytosolic free Ca2+ concentration ([Ca2+]i) is chelated by loading with bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid tetra-acetoxymethyl ester (BAPTA-AM). Without BAPTA, the pH effects are inhibited in the presence of BSA or the phospholipase A2 inhibitor oleoyloxyethylphosphocholine. These observations are consistent with the Tg-induced pH effects being mediated at least in part by the release of arachidonic acid (ArA) on activation of phospholipase A2 by the increased [Ca2+]i. Exogenous ArA promotes a rapid decrease in pHi in platelets suspended in a high-[Na+] medium, and an increase in pHi if platelets are depolarized by suspension in a high-[K+] medium in the presence of valinomycin and the external pH is increased to 7.9. The protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) behaves like ArA, although ArA is not a protonophore. It is concluded that ArA activates a proton conductance across the plasma membrane. The latter is inhibited by La3+. In high-[Na+] media, the pHi previously decreased by ArA recovers rapidly on removal of ArA with BSA. The effect is prevented by NHA. The recovery after BSA is much slower if FCCP rather than ArA is used to decrease pHi, but it is fast again with both ArA and FCCP. Furthermore, pHi previously decreased by ArA also recovers readily on inhibition of the ArA-activated H+ conductance with La3+, and the effect is NHA-sensitive. When pHi is decreased with the K+/H+ ionophore nigericin, a rapid recovery is activated by ArA followed by BSA (but not by BSA alone). The effect is independent of Ca2+ and protein kinase C. It is concluded that ArA, besides activating the H+ conductance, also acts as an activator of the Na+/H+ exchanger.

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