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Ouabain's Influence on TRPV4 Channels of Epithelial Cells: An Exploration of TRPV4 Activity, Expression, and
Arturo Ponce1, Isabel Larre2,3, Lidia Jimenez1
1Department of Physiology, Biophysics and Neurosciences, CINVESTAV-IPN, Mexico City 07360, Mexico.
Abstract:
Ouabain, a substance originally obtained from plants, is now classified as a hormone because it is produced endogenously in certain animals, including humans. However, its precise effects on the body remain largely unknown. Previous studies have shown that ouabain can influence the phenotype of epithelial cells by affecting the expression of cell-cell molecular components and voltage-gated potassium channels. In this study, we conducted whole-cell clamp assays to determine whether ouabain affects the activity and/or expression of TRPV4 channels. Our findings indicate that ouabain has a statistically significant effect on the density of TRPV4 currents (dITRPV4), with an EC50 of 1.89 nM. Regarding treatment duration, dITRPV4 reaches its peak at around 1 h, followed by a subsequent decline and then a resurgence after 6 h, suggesting a short-term modulatory effect related to on TRPV4 channel activity and a long-term effect related to the promotion of synthesis of new TRPV4 channel units. The enhancement of dITRPV4 induced by ouabain was significantly lower in cells seeded at low density than in cells in a confluent monolayer, indicating that the action of ouabain depends on intercellular contacts. Furthermore, the fact that U73122 and neomycin suppress the effect caused by ouabain in the short term suggests that the short-term induced enhancement of dITRPV4 is due to the depletion of PIP2 stores. In contrast, the fact that the long-term effect is inhibited by PP2, wortmannin, PD, FR18, and IKK16 suggests that cSrc, PI3K, Erk1/2, and NF-kB are among the components included in the signaling pathways.
Insights
Ouabain significantly affects TRPV4 channel activity and expression, with effects dependent on cell density and involving complex signaling pathways. This hormone influences cellular function through both short-term modulation and long-term synthesis of TRPV4 channels.
Area of Science:
- Physiology
- Molecular Biology
- Cell Biology
Background:
- Ouabain, an endogenous hormone, has largely unknown physiological effects.
- Previous research links ouabain to epithelial cell phenotype modulation.
- Ouabain's impact on voltage-gated potassium channels is documented.
Purpose of the Study:
- To investigate ouabain's effect on Transient Receptor Potential Vanilloid 4 (TRPV4) channel activity and expression.
- To determine the concentration-dependent effect (EC50) of ouabain on TRPV4 currents.
- To elucidate the temporal dynamics and underlying mechanisms of ouabain's action on TRPV4 channels.
Main Methods:
- Whole-cell patch clamp electrophysiology was employed to measure TRPV4 currents.
- Cell density variations (low vs. confluent) were used to assess the role of intercellular contacts.
- Pharmacological inhibitors were used to probe signaling pathways involved in ouabain's effects.
Main Results:
- Ouabain significantly modulated TRPV4 current density (dITRPV4) with an EC50 of 1.89 nM.
- A biphasic effect was observed: a peak at 1 hour followed by a decline and resurgence at 6 hours.
- Ouabain's effect was diminished in low-density cultures, highlighting dependence on cell-cell interactions.
- Short-term effects were linked to Phosphatidylinositol 4,5-bisphosphate (PIP2) depletion, while long-term effects involved cSrc, PI3K, Erk1/2, and NF-kB signaling pathways.
Conclusions:
- Ouabain exerts both short-term modulatory and long-term synthesis-promoting effects on TRPV4 channels.
- Intercellular contacts are crucial for ouabain's action on TRPV4 channels.
- Ouabain's effects are mediated by distinct signaling cascades, involving PIP2 metabolism and kinase pathways, for short-term and long-term actions, respectively.
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