Megakaryocyte purinergic receptor electrophysiology, calcium dynamics and the effects of acetylsalicylic acid
Young, Jose Pompilio.
Thesis (Ph.D.)--Georgetown University, 2010.; Includes bibliographical references.; Text (Electronic thesis) in PDF format. Purinergic receptor activation increases [Ca2+]i in a fluctuating fashion, triggering oscillatory outward Ca2+ activated K+ currents in rat megakaryocytes (MKs). Whole cell and nystatin perforated patch clamp techniques were used to analyze changes in ionic conductance in MK with acetylsalicylic acid (ASA), an antithrombotic agent and COX-1 inhibitor. MKs are a model for platelet reactivity, particularly in ASA treatment failure (ASA resistance). Freshly isolated MKs were incubated 30 min in the absence or presence of 1mM ASA. Using a K+ rich internal solution, outward currents were recorded in response to 10 ℗æM ATP, 10 ℗æM ADP and 5 ℗æM 2MeSADP in the voltage clamp mode. Agonist-induced currents decreased in amplitude over time, but this decline was attenuated by ASA in both continuous and repeated agonist challenge, indicating increased MK reactivity with ASA treatment. In separate experiments heterologous desensitization was observed when MKs were stimulated with ADP after exposure to a thromboxane receptor agonist (U46619) indicating cross-talk between thromboxane-purinergic pathways. Different cells, treated with ASA or MRS2179 (P2Y1 receptor antagonist), were stimulated with 2MeSADP. The dose response curve was shifted to the left in both cases, suggesting increased MK reactivity. ASA also caused an increased interval between currents (delay). ASA attenuated desensitization of purinergic receptors and increased delay. In Ca2+ imaging studies of primary rat MKs, ASA enhanced [Ca2+]i elevation associated with , a central part of platelet activation, in two major pathways: inositol 1,4,5-triphosphate (IP3)-mediated Ca2+ mobilization from intracellular stores and store-operated Ca2+ entry (SOCE) via STIM1-Orai1 interaction. Ca2+-imaging studies indicated ASA-treated MKs had higher 2MeSADP-evoked (a specific agonist for the P2Y1 and P2Y12 ADP receptors) [Ca2+]i, as well as enhanced store-operated Ca2+ entry through the plasma membrane following emptying of intracellular Ca2+ stores with thapsigargin. Immunohistochemistry revealed STIM1 puncta formation and subplasma membrane localization, which are required for SOCE, and are more prominent in ASA-treated MKs.; ASA treatment may potentiate Ca2+ release from internal stores through IP3Rs, which may also enhance internal Ca2+ store depletion to trigger greater STIM1-Orai1-mediated SOCE. These findings may be relevant to ASA resistance, because individual variations in sensitivity to the multiple effects of ASA on signaling pathways could result in insensitivity to its antiplatelet effects in some patients.
MetadataShow full item record
Showing items related by title, author, creator and subject.
Calcium Signaling in Cardiac Myocytes: Contributions from Mitochondria, Sodium-Calcium Exchanger, and Ryanodine Receptor Haviland, Sarah Suzanne (Georgetown University, 2013)This dissertation explores three critical players in the cardiac Ca2+ signaling pathway. First, I examined the role of mitochondria in Ca2+ signaling by generating a Ca2+ probe, mitycamE31Q, with 1.5μM affinity for ...
Divekar, Shailaja Dilip (Georgetown University, 2008)ABSTRACT Growth factors are thought to activate estrogen receptor-alpha ERα by phosphorylating the serines in the N-terminus of the receptor. However, this mechanism does not account for the conformational changes that ...
Regulation of MicroRNAs Targeting the Angiogenic Switch Molecule Fibroblast Growth Factor Binding Protein 1 by Retinoic Acid Receptor Activation Baker, Tabari (Georgetown University, 2014)This dissertation examines the role of retinoic acid receptor activation in the post-transcriptional regulation of a fibroblast growth factor binding protein. Previous work showed that all-trans retinoic acid (ATRA) reduces ...