INITIATION OF RVD RESPONSE IN HUMAN PLATELETS - MECHANICAL-BIOCHEMICAL TRANSDUCTION INVOLVES PERTUSSIS-TOXIN-SENSITIVE G-PROTEIN AND PHOSPHOLIPASE A(2)

Platelets revert hypotonic-induced swelling by the process of regulato ry volume decrease (RVD). We have recently shown that this process is under the control of endogenous hepoxilin A(3). In this work, we inves tigated the mechanical-biochemical transduction that leads to hepoxili n A(3) formation. We demonstrate that this process is mediated by pert ussis-toxin-sensitive G protein, which activates Ca2+-insensitive phos pholipase A(2), and the sequential release of arachidonic acid. This c onclusion is supported by the following observations: (i) RVD response is blocked selectively by the phospholipase A(2) inhibitors manoalide and bromophenacyl-bromide (0.2 and 5 mu M, respectively) but not by p hospholipase C inhibitors. The addition of arachidonic acid overcame t his inhibition; (ii) extracellular Ca2+ depletion by EGTA (up to 10 mM ) does not affect RVD; (iii) intracellular Ca2+ depletion by BAPTA-AM (100 mu M) inhibits RVD but not hepoxilin A(3) formation, as tested by the RVD reconstitution assay; (iv) RVD is inhibited by the G-protein inhibitors, GDP(beta)S (1 mu M) and pertussis toxin (1 ng/ml). This in hibition is overcome by addition of arachidonic acid or hypotonic cell -free eluate that contains hepoxilin A(3); (v) NaF, 1 mM, induces hepo xilin A(3) formation, tested by the RVD reconstitution assay; and (vii ) GDP(beta)S inhibits hepoxilin A(3) formation associated with flow. T herefore, it seems that G proteins are involved in the initial step of the mechanical-biochemical transduction leading to hepoxilin A(3) for mation in human platelets.