Initiation of RVD response in human platelets: Mechanical-biochemical transduction involves pertussis-toxin-sensitive G protein and phospholipase A2 |
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Authors: | Alon Margalit Avinoam A Livne Jørgen Funder Yosef Granot |
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Institution: | (1) Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel;(2) Department of Medical Physiology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark |
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Abstract: | 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
m, respectively) but not by phospholipase C inhibitors. The addition of arachidonic acid overcame this inhibition; (ii) extracellular Ca2+ depletion by EGTA (up to 10 mm) does not affect RVD; (iii) intracellular Ca2+ depletion by BAPTAAM (100
m) inhibits RVD but not hepoxilin A3 formation, as tested by the RVD reconstitution assay; (iv) RVD is inhibited by the G-protein inhibitors, GDP
S (1
m) 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 mm, induces hepoxilin A3 formation, tested by the RVD reconstitution assay; and (vii) GDP
S 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.DeceasedThis work is dedicated to the memory of Prof. A.A. Livne. It was carried out at the Amelia (Mimi) Rose Laboratory for Cellular Signal Transduction at the Department of Life Sciences, Ben-Gurion University of the Negev. We thank A. Dannon for helpful discussion. |
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Keywords: | Human platelets Regulatory volume decrease Hepoxilin A3 Phospholipase A2 Gproteins Mechanically induced activation |
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