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|Title:||Interactions of sodium calcium exchanger (NCX) and sarco/endoplasmic reticulum calcium ATPase (SERCA) in the porcine coronary artery smooth muscle cells|
|Abstract:||<p>Calcium (Ca<sup>2+</sup>) is an important signaling molecule with tightly regulated movements across cell membranes. Ca<sup>2+</sup> transporters play a key role in this regulation. The sarco/endoplasmic reticulum (SER) Ca<sup>2+</sup> pump (SERCA) and plasma membrane Ca2+ pump (PMCA) keep cytosolic Ca<sup>2+</sup> levels [Ca<sup>2+</sup>]<sub>i</sub> low. The sodium calcium exchanger (NCX) is bidirectional and may work to expel Ca<sup>2+</sup> or bring it into the cell depending on the electrochemical gradient. NCX has been proposed to play a role in refilling the SER Ca<sup>2+</sup> pool.</p> <p>The overall objective of this thesis was to determine the effect of SER Ca<sup>2+</sup> depletion on NCX- SERCA interactions in pig coronary artery smooth muscle by monitoring changes in [Ca<sup>2+</sup>]<sub>i</sub> and the co-localization between NCX and SERCA. NCX mediated increase in [Ca<sup>2+</sup>]<sub>i</sub> was observed in Na+ loaded smooth muscle cells. The effect of SER depletion by thapsigargin was examined on the NCX mediated increase in [Ca<sup>2+</sup>]<sub>i</sub>. A decrease in the NCX mediated increase in [Ca<sup>2+</sup>]<sub>i</sub> was observed upon inhibition of SERCA pump with thapsigargin. The effect of SER depletion on proximity of NCX and SERCA proteins was also investigated using immunofluorescence confocal microscopy using anti-NCXl and anti-SERCA2 antibodies and fluorescence labeled secondary antibodies. Na<sup>+</sup> loaded smooth muscle cells in NMG buffer were incubated with or without SERCA inhibitor thapsigargin. Resulting image stacks were analyzed for the co-localization of NCXl and SERCA2 in the areas near the plasma membrane. SER depletion with thapsigargin increased the co-localization between NCXl and SERCA2 near the plasma membrane of smooth muscle cells. Thus, SER Ca<sup>2+</sup> depletion moves plasma membrane protein NCX and SER protein SERCA closer to each other and decreases the NCX mediated Ca<sup>2+</sup> entry in the cell. These interactions may be crucial to smooth muscle Ca<sup>2+</sup> regulation and has the ability to be a potential therapeutic target during oxidative stress. However, the nature of these interactions needs to be explored.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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