L. Cavallini et al., ARACHIDONIC-ACID ACTIVATES A PROTON CONDUCTANCE PATHWAY AND THE NA+ H+ EXCHANGER IN PLATELETS/, Biochemical journal, 319, 1996, pp. 567-574
The treatment of aspirinated platelets with the endomembrane Ca2+-ATPa
se inhibitor thapsigargin (Tg) induces a large increase in cytosolic p
H (pH(i)), as measured with the intracellular fluorescent indicator ',
7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. In contrast, Tg induc
es a decrease in pH(i), in the presence of the Na+/H+ exchanger inhibi
tor 5-(N,N-hexamethylene)-amiloride (NHA). Both effects are inhibited
if the cytosolic free Ca2+ concentration ([Ca2+](i)) is chelated by lo
ading with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetr
a-acetoxymethyl ester (BAPTA-AM). Without BAPTA, the pH effects are in
hibited in the presence of BSA or the phospholipase A(2) inhibitor ole
oyloxyethylphosphocholine. These observations are consistent with the
Tg-induced pH effects being mediated at least in part by the release o
f arachidonic acid (ArA) on activation of phospholipase A(2) by the in
creased [Ca2+](i). Exogenous ArA promotes a rapid decrease in pH(i) in
platelets suspended in a high-[Na+] medium, and an increase in pH(i)
if platelets are depolarized by suspension in a high-[K+] medium in th
e presence of valinomycin and the external pH is increased to 7.9. The
protonophore carbonyl cyanide p-trifluoromerhoxyphenylhytirazone (FCC
P) behaves like ArA, although ArA is not a protonophore. It is conclud
ed that ArA activates a proton conductance across the plasma membrane.
The latter is inhibited by La3+. In high-[Na+] media, the pH(i) previ
ously decreased by ArA recovers rapidly on removal of ArA with BSA. Th
e effect is prevented by NHA. The recovery after BSA is much slower if
FCCP rather than ArA is used to decrease pH(i), but it is fast again
with both ArA and FCCP. Furthermore, ppH(i) previously decreased by Ar
A also recovers readily on inhibition of the ArA-activated H+ conducta
nce with La3+, and the effect is NHA-sensitive. When pH(i) is decrease
d 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. II is concluded that ArA, besides activ
ating the H+ conductance, also acts as an activator of the Na+/H+ exch
anger.