title: Rat platelet arachidonate metabolism in the presence of Ca2+, Sr2+ and Ba2+: studies using intact platelets and semi-purified phospholipase A2.
abstract: To document further the involvement of external Ca2+ in the platelet-induced activation process, we have studied the arachidonate metabolism of intact washed rat platelets in the presence of different concentrations of Ca2+, Sr2+ or Ba2+. The thrombin-induced mobilization of radiolabeled arachidonate preincorporated into platelet phospholipids was followed as well as the subsequent formation of labeled cyclooxygenase and lipoxygenase products. Results indicate that upon thrombin stimulation (0.2 U/ml), the release of endogenous arachidonate and the formation of its metabolites are reduced by 50-90% only by omission of Ca2+ as compared to 1 mM Ca2+ in the suspending medium. At higher Ca2+ concentrations (5 mM), the arachidonate mobilization and metabolite formation are inhibited and the data are thus close to those obtained in the absence of Ca2+. In the presence of Sr2+ or Ba2+, the results indicate that these cations can substitute for Ca2+. As for Ca2+, an optimum concentration is found for Sr2+ and Ba2+ (3-5 mM), and higher concentrations inhibit the metabolism of arachidonic acid. As the above data might be compatible with the possible entry of Sr2+ and Ba2+ into platelets upon stimulation, we also studied the activity of a semi-purified preparation of phospholipase A2 from rat platelets. This activity was assayed (pH 9.2) using heat-denatured [3H]arachidonate-prelabeled phospholipids as substrate. The results show that this phospholipase A2 activity was strongly Ca2+-dependent. In addition, we found that unlike Mg2+, Sr2+ and Ba2+ are able to greatly enhance this activity. Relative efficiency (Vmax) was in the order Ca2+ greater than Sr2+ greater than Ba2+. Taken together, these findings suggest that external Ca2+ may play a major role in the regulation of rat platelet activity. Our interpretation is in line with the view that Sr2+ or Ba2+ could enter the platelet through a mechanism common to Ca2+ (a Ca2+ channel). Although direct evidence is awaited from the results of further studies which are in progress, it can reasonably be considered that Sr2+ or Ba2+ might cause platelet-induced activation mimicking a rise in the cytosolic Ca2+ and subsequent activation of Ca2+-dependent enzymes.
mesh_terms: D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000097:blood*; D001464:Barium / Q000494:pharmacology*; D001792:Blood Platelets / Q000201:enzymology / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D007473:Ion Channels / Q000378:metabolism; D010740:Phospholipases / Q000097:blood*; D010741:Phospholipases A / Q000097:blood*; D054467:Phospholipases A2; D010972:Platelet Activating Factor / Q000097:blood; D051381:Rats; D012643:Selenium / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology
chemical_list: D001095:Arachidonic Acids; D007473:Ion Channels; D010972:Platelet Activating Factor; D001464:Barium; D016718:Arachidonic Acid; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2; D013917:Thrombin; D012643:Selenium; D002118:Calcium