Graduation Year

2018

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Thomas Taylor-Clark, Ph.D.

Committee Member

Craig Doupnik, Ph.D.

Committee Member

Sami Noujaim, Ph.D.

Committee Member

Javier Cuevas, Ph.D.

Committee Member

Srinivas Tipparaju, Ph.D.

Keywords

ion channel, pulmonary disease, reactive oxygen species, sensory nerves

Abstract

Sensory nerves detect conditions in the external and internal environment and permit behavioral and physiological responses to maintain homeostasis and ensure survival. As such, sensory nerves detect a wide variety of stimuli including heat, touch, pH, and vibration. The precise nature of these responses is dependent upon the initiating stimulus and site of activation. Potentially threatening stimuli (noxious heat, cellular damage, chemical irritants) activate nociceptive sensory nerves through the gating of ion channels expressed at sensory nerve terminals, thereby evoking defensive reflexes such as cough, watering eyes and limb withdrawal as well as sensations of pain or discomfort. While these responses are typically protective, inflammation alters peripheral neuronal activity resulting in unpleasant sensations, and aberrant reflexes which are the hallmark symptoms of pulmonary disease(Costello et al., 1999).

Unfortunately, current treatments which focus on specific symptoms such as bronchospasm and inflammation are not effective for all individuals and fail to address underlying sensory nerve activity(Barnes, 2012). Therefore, understanding mechanisms underlying the increased activity of ion channels expressed at sensory nerve terminals such as the nonselective cation channels transient receptor potential channels ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) may lead to the development of novel therapeutics for the treatment of inflammatory pulmonary diseases. As inflammation induces mitochondrial dysfunction through inhibition of the electron transport chain (mETC), we investigated the impact of mitochondrial dysfunction on the activation of TRPA1 and TRPV1 using dissociated vagal neurons. Using live cell Ca2+ imaging to measure channel activation, we found that mitochondrial dysfunction induced with antimycin A (complex III inhibitor), carbonyl cyanide m-chlorophenyl hydrazone (CCCP, mitochondrial uncoupling agent), and rotenone (complex I inhibitor) activates a portion of vagal neurons which express TRPA1 and/or TRPV1.

Using antimycin A, we discovered that across all neurons were Ca2+ responses were diminished (averaged across all neurons) with knockout/inhibition of either TRPA1 or TRPV1 and largely abolished with dual knockout/inhibition of both channels. Interestingly, the diminished response with TRPA1 knockout/inhibition was due to the decrease in the magnitude of the Ca2+ flux in neurons responding while decreased responses with TRPV1 knockout/inhibition were due to the decrease in percentage of neurons which responded, not Ca2+ flux magnitude. Mitochondrial dysfunction results in the increased reproduction of ROS and mitochondrial depolarization. Therefore, we evaluated the relationship between mitochondrial depolarization (using JC-1) and ROS production (using mitoSOX Red) and channel activation (Ca2+ imaging with FURA-2AM). Although only a portion of TRPA1/TRPV1 expressing neurons responded to mitochondrial inhibition with CCCP, rotenone or antimycin A that there was no difference in ROS production or mitochondrial depolarization between responding and non-responding neurons.

We also investigated the impact of these ROS on the activation of TRPA1 and TRPV1. We found that the activation of TRPA1, but not TRPV1, is likely downstream of mitochondrial ROS production as 1) antimycin A induced Ca2+ fluxes were diminished by co-treatment with ROS scavengers in transfected HEK293 cells and dissociated vagal neurons, 2) ROS production (measured with mitoSOX Red simultaneously with Ca2+ imaging) was correlated with Ca2+ fluxes in TRPA1, but not TRPV1, expressing neurons, and 3) the ROS-unresponsive TRPA1 K620A mutant did not respond to antimycin A. As studies regarding ROS production are hindered by the lack of suitable methods to measure ROS production due to caveats associated with the use of ROS sensitive dyes and the lack of sensitivity offered by redox sensitive proteins, we also sought to increase the sensitivity of roGFP1, a redox sensitive variant of GFP by replacing one of the reactive cysteines with the more reactive selenocysteine. We successfully produced a redox sensing protein with increased sensitivity. However, this protein exhibited low expression levels and a greatly diminished dynamic range.

Download

Included in

Neurosciences Commons

Share

COinS