Initiation of RVD response in human platelets: Mechanical-biochemical transduction involves pertussis-toxin-sensitive G protein and phospholipase A₂

Platelets revert hypotonic-induced swelling by the process of regulatory volume decrease (RVD). We have recently shown that this process is under the control of endogenous hepoxilin A₃. In this work, we investigated the mechanical-biochemical transduction that leads to hepoxilin A₃ formation. We demonstrate that this process is mediated by pertussis-toxin-sensitive G protein, which activates Ca²⁺-insensitive phospholipase A₂, and the sequential release of arachidonic acid. This conclusion is supported by the following observations: (i) RVD response is blocked selectively by the phospholipase A₂ inhibitors manoalide and bromophenacyl-bromide (0.2 and 5 μm, respectively) but not by phospholipase C inhibitors. The addition of arachidonic acid overcame this inhibition; (ii) extracellular Ca²⁺ depletion by EGTA (up to 10 m m) does not affect RVD; (iii) intracellular Ca²⁺ depletion by BAPTAAM (100 μm) inhibits RVD but not hepoxilin A₃ formation, as tested by the RVD reconstitution assay; (iv) RVD is inhibited by the G-protein inhibitors, GDP_βS (1 μm) and pertussis toxin (1 ng/ml). This inhibition is overcome by addition of arachidonic acid or hypotonic cell-free eluate that contains hepoxilin A₃; (v) NaF, 1 m m, induces hepoxilin A₃ formation, tested by the RVD reconstitution assay; and (vii) GDP_βS inhibits hepoxilin A₃ formation associated with flow. Therefore, it seems that G proteins are involved in the initial step of the mechanical-biochemical transduction leading to hepoxilin A₃ formation in human platelets.