Receptor occupancy regulates Ca2+ entry and intracellular Ca2+ redistribution in activated human platelets.
Fura-2-loaded human platelets were used to study Ca2+ release from intracellular compartments, as well as Ca2+ influx from the extracellular space. We investigated the response towards the endoperoxide/thromboxane-receptor agonist. U46619, and the inhibitor of the endoplasmic-reticulum Ca(2+)-ATPase, thapsigargin. U46619 dose-dependently depleted intracellular Ca2+ stores, followed by active sequestration of released Ca2+. Ca2+ influx induced by U46619 largely relies on receptor occupancy. Removing the thromboxane analogue from its receptor by using the endoperoxide/thromboxane-receptor antagonist BM 13177 largely blunted U46619-mediated Ca2+ influx. The Ca(2+)-ATPase inhibitor thapsigargin evoked a gradual rise in intracellular Ca2+, which was potentiated by a preceding activation of platelets with the receptor agonist U46619. This agonist-sensitizing effect also depends on receptor occupancy. Removing U46619 from its receptor by addition of the endoperoxide/thromboxane-receptor antagonist BM13177 suppressed the sensitizing effect completely. Furthermore, interrupting downstream receptor signalling events by raising intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP) again suppressed the U46619-sensitizing effect on thapsigargin-induced Ca2+ release. This study indicates that the process of Ca2+ release followed by resequestration in response to a platelet agonist by its own is not sufficient to produce the sensitizing effect. Rather, a continuously occupied receptor triggering sustained downstream signalling events seems to be required for sensitization. The presence of a receptor agonist may induce an increased cycling of Ca2+ between the agonist-responsive and the thapsigargin-dischargeable compartment, leading to faster and more intense accumulation of Ca2+ in the cytosolic compartment after inhibition of the Ca(2+) ATPase. Suggestively, receptor occupancy increases the Ca(2+)-releasing potency of thapsigargin by coupling the thapsigargin-sensitive Ca(2+)-storing compartments with an agonist-responsive compartment that exhibits a high leakage rate in stimulated platelets.
Brüne
B
B
University of Konstanz, Faculty of Biology, Federal Republic of Germany.
von Appen
F
F
Ullrich
V
V
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D002118:Calcium / Q000097:blood*; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D011464:Epoprostenol / Q000494:pharmacology; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D009599:Nitroprusside / Q000494:pharmacology; D015539:Platelet Activation / Q000502:physiology*; D011450:Prostaglandin Endoperoxides, Synthetic / Q000378:metabolism / Q000494:pharmacology; D017482:Receptors, Thromboxane / Q000819:agonists / Q000037:antagonists & inhibitors / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013449:Sulfonamides / Q000494:pharmacology; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology
D011450:Prostaglandin Endoperoxides, Synthetic; D017482:Receptors, Thromboxane; D013449:Sulfonamides; D013729:Terpenes; D009599:Nitroprusside; D013928:Thromboxane A2; D019284:Thapsigargin; C040624:sulotroban; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D011464:Epoprostenol; D000242:Cyclic AMP; D006152:Cyclic GMP; D002118:Calcium