title: Selective inhibition of human platelet phospholipase A2 by buffering cytoplasmic calcium with the fluorescent indicator quin 2. Evidence for different calcium sensitivities of phospholipases A2 and C.
abstract: Human platelets labelled with either [14C]arachidonic acid or [32P]orthophosphate were loaded or not with the Ca2+ fluorescent indicator quin 2. They were then incubated in the presence or in the absence of human thrombin (1 U/ml) in a medium where Ca2+ concentration was adjusted near zero or to 1 mM. Under these conditions, phospholipase A2 activity, as detected by the release of [14C]arachidonate and of its metabolites, or by the hydrolysis of [14C]phosphatidylcholine, was severely impaired in quin 2-loaded platelets upon removal of external Ca2+. However, Ca2+ was not required in non-loaded platelets, where a maximal phospholipase A2 activity was detected in the absence of external Ca2+. In contrast, phospholipase C action, as determined from the amounts of [14C]diacylglycerol, [14C]- or [32P]phosphatidic acid formed, appeared to be much less sensitive to the effects of quin 2 loading and of Ca2+ omission. By using various concentrations of quin 2, it was found that the inhibitory effect exerted against phospholipase A2 could be overcome by external Ca2+ only when the intracellular concentration of the calcium chelator did not exceed 2 mM. At higher concentrations averaging 3.5 mM of quin 2, phospholipase A2 activity was fully suppressed even in the presence of external Ca2+, whereas phospholipase C was still active, although partly inhibited. It is concluded that platelet phospholipase A2 requires higher Ca2+ concentrations than phospholipase C to display a maximal activity. By comparing platelet phospholipase A2 activity under various conditions with the values of cytoplasmic free Ca2+ as detected by quin 2 fluorescence, it is proposed that cytoplasmic free Ca2+ in control platelets stimulated with thrombin can attain concentrations above 1 microM, probably close to 5-10 microM, as recently determined with the photoprotein aequorin (Johnson, P.C., Ware, J.A., Cliveden, P.B., Smith, M., Dvorak, A.M. and Salzman, E.W. (1985) J. Biol. Chem. 260, 2069-2076).
authors.forename: M F
authors.initials: MF
authors.lastname: Simon
authors.forename: H
authors.initials: H
authors.lastname: Chap
authors.forename: L
authors.initials: L
authors.lastname: Douste-Blazy
mesh_terms: D000634:Aminoquinolines / Q000494:pharmacology*; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D010740:Phospholipases / Q000037:antagonists & inhibitors*; D010741:Phospholipases A / Q000037:antagonists & inhibitors*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D013929:Thromboxane B2 / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*
chemical_list: D000634:Aminoquinolines; D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D010710:Phosphates; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010743:Phospholipids; D016718:Arachidonic Acid; D013929:Thromboxane B2; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D010738:Type C Phospholipases; D013917:Thrombin; C035739:Quin2; D002118:Calcium