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Abstract

When human platelets are stimulated with thrombin or activators of protein kinase C, cytosolic pH (pH_i) increases due to activation of Na⁺/H⁺ exchange. In order to further elucidate the molecular mechanisms that regulate the exchanger, we used sodium fluoride, which is a known activator of guanine nucleotide-binding proteins (G proteins) in platelets. Although NaF induced the mobilization of Ca²⁺ from intracellular storage sites in fura2-loaded platelets, it failed to raise pH_i as determined from the fluorescence of 2,7-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein-loaded platelets. Furthermore, when thrombin (0.1 unit/ml) or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) had raised pH_i from 7.13 ± 0.05 to 7.35 ± 0.07 (n = 30), addition of NaF (2.5-10 mM) rapidly restored pH_i to values found before stimulation. Conversely, preincubation of platelets with low concentrations of NaF (2.5 mM) completely prevented alkalinization in response to thrombin or TPA. Unlike ethylisopropylamiloride, which completely blocked Na⁺/H⁺ exchange, NaF did not prevent the recovery of pH_i from an artificial acid load. Hence, the inhibitory action of NaF is restricted to receptor-mediated activation of the antiport. In order to investigate whether the NaF effect was attributable to a G protein, platelets were preincubated with N-ethylmaleimide (50 microM), which is known to inhibit the adenylyl cyclase-inhibitory G protein. N-Ethylmaleimide treatment not only prevented inhibition of adenylyl cyclase by epinephrine but also completely reversed the inhibitory effect of NaF on the Na⁺/H⁺ exchanger. Our data suggest the existence of a novel G protein which is activated by fluoride and functions as a negative regulator of the Na⁺/H⁺ exchanger in platelets.