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 Ca(2+)-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 microM, 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 BAPTA-AM (100 microM) inhibits RVD but not hepoxilin A3 formation, as tested by the RVD reconstitution assay; (iv) RVD is inhibited by the G-protein inhibitors, GDP beta S (1 microM) 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 beta 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.
Margalit
A
A
Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Livne
A A
AA
Funder
J
J
Granot
Y
Y
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000097:blood; D000328:Adult; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000097:blood; D048429:Cell Size / Q000502:physiology; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D007038:Hypotonic Solutions; D066298:In Vitro Techniques; D008465:Mechanoreceptors / Q000502:physiology; D008954:Models, Biological; D009997:Osmotic Pressure; D037342:Pertussis Toxin; D010741:Phospholipases A / Q000097:blood*; D054467:Phospholipases A2; D047428:Protein Kinase Inhibitors; D012969:Sodium Fluoride / Q000494:pharmacology; D013314:Stress, Mechanical; D013873:Thionucleotides / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D007038:Hypotonic Solutions; D047428:Protein Kinase Inhibitors; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; C023427:guanosine 5'-O-(2-thiodiphosphate); C038243:8-hydroxy-11,12-epoxyeicosa-5,9,14-trienoic acid; D012969:Sodium Fluoride; D037342:Pertussis Toxin; D010741:Phospholipases A; D054467:Phospholipases A2; D019204:GTP-Binding Proteins; D015126:8,11,14-Eicosatrienoic Acid; D002118:Calcium