Skip to Main Content
 

Global Search Box

 
 
 
 

Files

File List


pritam dissert final 070816.pdf (19.74 MB)

ETD Abstract Container

Abstract Header

Cross Talk Between TRPA1 and TRPV1 Ion-Channels: Role of Nitric Oxide

Sinharoy, Pritam
http://orcid.org/0000-0002-6322-260X
http://rave.ohiolink.edu/etdc/view?acc_num=kent1467381679

Abstract Details

2016, PHD, Kent State University, College of Arts and Sciences / Department of Biological Sciences.
Abstract: Propofol is an intravenous anesthetic that is widely used during general anesthesia and for a variety of outpatient procedures. However, unwanted side effects associated with the use of propofol include apnea and acute pain upon infusion and vasodilation resulting in hypotension. Therefore, we aimed to investigate the cellular signaling cascade by which propofol enhances pain and regulates vasomotor tone in vitro. Aim 1: Background: We previously demonstrated that the intravenous anesthetic, propofol, restores the sensitivity of transient receptor potential (TRP) vanilloid channel subtype-1 (TRPV1) receptors via a protein kinase C epsilon (PKCe)-dependent and transient receptor potential ankyrin channel subtype-1 (TRPA1)-dependent pathway in sensory neurons. The extent to which the two pathways are directly linked or operating in parallel has not been determined. Using a molecular approach, our objectives of the current study were to confirm that TRPA1 activation directly results in PKCe activation and to elucidate the cellular mechanism by which this occurs. Results: F-11 cells were transfected with complimentary DNA (cDNA) for TRPV1 only or both TRPV1 and TRPA1. Intracellular Ca2+ concentration was measured in individual cells via fluorescence microscopy. An immunoblot analysis of the total and phosphorylated forms of PKCe, nitric oxide synthase (nNOS), and TRPV1 was also performed. In F-11 cells containing TRPV1 only, neither propofol nor AITC induced PKCe or TRPV1 phosphorylation. Moreover, NOS inhibition blocked propofol-and AITC-induced restoration of TRPV1 sensitivity and PKCe phosphorylation, and PKCe inhibition prevented the nitric oxide donor, SNAP, from restoring TRPV1 sensitivity. Also, propofol-and AITC-induced phosphorylation of nNOS and nitric oxide (NO) production were blocked with the TRPA1-antagonist, HC-030031. Conclusions: These data demonstrate that the AITC- and propofol-induced restoration of TRPV1 sensitivity is mediated by a TRPA1-dependent, nitric oxide synthase-dependent activation of PKCe. Aim 2: Background: TRP ion channels have recently emerged as key regulators of vasomotor tone and many general anesthetic agents modulate vasomotor tone both in vivo and in vitro. Previously, our laboratory demonstrated the role of TRPA1 ion channel in propofol-mediated vasodilation in vivo. Moreover, our preliminary in vitro studies have indicated that a unique yet complex interaction between TRPA1 and TRPV1 appears to mediate the depressor response in coronary micro vessels that involves activation of eNOS. However the mechanism by which propofol-induced stimulation of TRPA1 activates eNOS is yet to be defined in mouse coronary artery endothelial cells (MCAECs). Therefore, our objective was to elucidate the cellular signaling pathway(s) by which propofol modulates endothelial NO production via TRPA1-dependent pathway in MCAECs that leads to alterations in vasomotor tone. Results: MCAECs subjected to immunoblot analysis, FACS analysis, immunocytochemical analysis and calcium imaging revealed that functional TRPA1 and TRPV1 are co-expressed in MCAECs, which mediates propofol and capsaicin-induced, increases in intracellular calcium levels. Moreover, our immunoblot analysis and griess assay data suggest that propofol-induced phosphorylation of eNOS and subsequent NO production is mediated via- endothelial TRPA1 and involves Ca2+ -dependent activation of CaMKII and PI3K signaling pathway. Finally, proximity ligation assay (PLA) data analysis suggests that propofol-induced stimulation of TRPA1 leads to molecular interaction between TRPA1 and TRPV1 in MCAECs. Conclusions: These data conclusively demonstrate that propofol activates eNOS via a TRPA1-mediated, Ca2+- dependent activation of CaMKII and PI3K signaling pathway in MCAECs. Moreover, propofol-induced stimulation of the TRPA1 leads to a physical interaction between TRPA1 and TRPV1 in coronary endothelium.
Derek Damron, Ph.D. (Advisor)
Ian Bratz, Ph.D. (Committee Member)
Gary Koski, Ph.D. (Committee Member)
Jennifer Mcdonough, Ph.D. (Committee Member)
Soumitra Basu, Ph.D. (Other)
126 p.
Biomedical Research; Cellular Biology
TRPA1, TRPV1, ion channels, nitric oxide, cross-talk, PKC epsilon

Recommended Citations

Citations

  • Sinharoy, P. (2016). Cross Talk Between TRPA1 and TRPV1 Ion-Channels: Role of Nitric Oxide [Doctoral dissertation, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=kent1467381679

    APA Style (7th edition)

  • Sinharoy, Pritam. Cross Talk Between TRPA1 and TRPV1 Ion-Channels: Role of Nitric Oxide. 2016. Kent State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=kent1467381679.

    MLA Style (8th edition)

  • Sinharoy, Pritam. "Cross Talk Between TRPA1 and TRPV1 Ion-Channels: Role of Nitric Oxide." Doctoral dissertation, Kent State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=kent1467381679

    Chicago Manual of Style (17th edition)

kent1467381679
270
© 2016, all rights reserved.
This open access ETD is published by Kent State University and OhioLINK.