Measurement of ionized calcium in blood platelets with a new generation calcium indicator.
Fura 2, a new generation calcium indicator, has a 30 fold brighter fluorescence than Quin 2, shows wavelength shifts upon calcium binding and has a relatively low buffering capacity for free calcium. Quin 2, the most widely used fluorophore, on the other hand, shows no wavelength shifts and has a very high affinity for free calcium. Therefore, we have compared the relative merits of these two fluorophores for monitoring agonist induced alterations in platelet cytosolic calcium. Platelets loaded with Fura 2 showed a significant rise in cytosolic calcium when stirred with agonists such as epinephrine, arachidonate and thrombin, whereas Quin 2 loaded platelets demonstrated a rise in cytosolic calcium only with thrombin stimulation. A rise in agonist induced calcium in Fura 2 loaded platelets was prevented when the cells were exposed first to antagonists such as aspirin or prostaglandin E1. Arachidonate refractory platelets, upon stirring with a single agonist, did not show a significant elevation in cytosolic calcium. However, when refractory platelets were first exposed to epinephrine and then challenged with arachidonate, they revealed a significant elevation in cytosolic calcium. Unlike Quin 2, Fura 2 at the highest concentration tested did not inhibit platelet function. Improved properties of Fura 2 suggest that it may be a useful agent to study agonist induced alterations in cytosolic calcium levels in blood platelets.
D000634:Aminoquinolines / Q000032:analysis; D001095:Arachidonic Acids / Q000494:pharmacology; D001572:Benzofurans / Q000032:analysis*; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000032:analysis; D004837:Epinephrine / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000032:analysis*; D016257:Fura-2; D013917:Thrombin / Q000494:pharmacology
D000634:Aminoquinolines; D001095:Arachidonic Acids; D001572:Benzofurans; D005456:Fluorescent Dyes; D013917:Thrombin; C035739:Quin2; D002118:Calcium; D016257:Fura-2; D004837:Epinephrine