Role of Trp Channels In Mediating The Calcium Signaling Response of Brain Endothelial Cells to Mechanical Stretch

<p>Traumatic brain injury (TBI) often results in disruption of the blood brain barrier (BBB), which is an integral component to maintaining the central nervous system homeostasis. Recently cytosolic calcium levels ([Ca²⁺]i), observed to elevate following TBI, have been shown to influence endothelial barrier integrity. However, the mechanism by which TBI-induced calcium signaling alters the endothelial barrier remains unknown. In the present study, an <em>in vitro</em> BBB model was utilized to address this issue. Exposure of cells to biaxial mechanical stretch, in the range expected for TBI, resulted in a rapid cytosolic calcium increase. Modulation of intracellular and extracellular Ca²⁺ reservoirs indicated that Ca²⁺ influx is the major contributor for the [Ca²⁺]i elevation. Application of pharmacological inhibitors was used to identify the calcium-permeable channels involved in the stretch-induced Ca²⁺ influx. Antagonist of transient receptor potential (TRP) channel subfamilies, TRPC and TRPP, demonstrated a reduction of the stretch-induced Ca²⁺ influx. RNA silencing directed at individual TRP channel subtypes revealed that TRPC1 and TRPP2 largely mediate the stretch-induced Ca²⁺ response. In addition, we found that nitric oxide (NO) levels increased as a result of mechanical stretch, and that inhibition of TRPC1 and TRPP2 abolished the elevated NO synthesis. Further, as myosin light chain (MLC) phosphorylation and actin cytoskeleton rearrangement are correlated with endothelial barrier disruption, we investigated the effect mechanical stretch had on the myosin-actin cytoskeleton. We found that phosphorylated MLC was increased significantly by 10 minutes post-stretch, and that inhibition of TRP channel activity or NO synthesis both abolished this effect. In addition, actin stress fibers formation significantly increased 2 minutes post-stretch, and was abolished by treatment with TRP channel inhibitors. These results suggest that, in brain endothelial cells, TRPC1 and TRPP2 are activated by TBI-mechanical stress and initiate actin-myosin contraction, which may lead to disruption of the BBB.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation (PhD)

Year dc:date.available

2012

Author dc:creator

• Berrout, Jonathan

Contributors dc:contributor

• Roger G. O’Neil
• Edgar T. Walters
• Pramod Dash

• BBB
• TRP
• calcium
• TBI
• mechanical stress
• Fura 2
• ion channel
• endothelial barrier
• Cell Biology
• Cellular and Molecular Physiology
• Medicine and Health Sciences
• Other Neuroscience and Neurobiology

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/256

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-1292