Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in models of cardiovascular disease. We previously suggested sphingosine-1-phosphate (S1P) activates volume-sensitive Cl- current (ICl,swell) by ROS-dependent signaling. S1P and its analog, FTY720 (fingolimod), primarily act via G-protein coupled receptors (S1PR; S1PR1-3 in heart), but several intracellular S1P ligands are known. We investigated how these agents regulate ICl,swell. ICl,swell was elicited by bath S1P (500 nM), FTY720 (S1PR1,3 agonist; 10 μM), and SEW2871 (S1PR1 agonist; 10 μM) and was fully inhibited by DCPIB, a specific blocker. These data suggested role of S1PR in activation of ICl,swell. Surprisingly, neither CAY10444 (S1PR3 antagonist; 10 μM) nor VPC23019 (S1PR1,3 antagonist; 13 μM) blocked FTY720-induced ICl,swell. Also, gallein a pan Gbeta-gamma inhibitor, failed to block the S1P-induced current. Moreover, 100 nM FTY720 applied via the pipette evoked a larger, faster activating current than 10 μM bath FTY720. Similarly, 500 nM S1P gave larger, faster activating ICl,swell when added to the pipette than when added in the bath. In contrast to FTY720, bath S1P-induced ICl,swell was blocked by CAY10444, but a 3-fold higher concentration failed to eliminate the response to pipette S1P, and VPC23019 failed to suppress bath and pipette S1P-induced currents. Taken together, inconsistencies in the responses to S1PR agents and the greater sensitivity to pipette than bath S1P and FTY720 support the notion that intracellular ligands rather than sarcolemmal S1PR activated ICl,swell. Next we tested if S1P and FTY720, like osmotic swelling, require both NADPH oxidase and mitochondrial ROS production to evoke ICl,swell. S1P- and FTY720-induced ICl,swell were blocked by rotenone but were insensitive to gp91ds-tat, suggesting only mitochondrial ROS production was needed. One possibility is that S1P and FTY720 elicit ICl,swell by binding to mitochondrial prohibitin-2, an S1P ligand whose knockdown augments mitochondrial ROS productions. These data suggest ICl,swell may be activated by S1P accumulation in ischemia-reperfusion and CHF. Understanding S1P-signaling that elicits ICl,swell may provide insight into electrophysiological mechanisms of cardiac pathology and help identify novel targets for therapy.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1460 |
| Date | 01 January 2013 |
| Creators | Desai, Pooja |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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