The effect of human rhinovirus-16 on thermo-TRP channels, hypotonic activation and ATP release

Atkinson, Samantha Kimberley

Abstract

Human rhinovirus (RV-16) infections are major cause of upper respiratory tract infections (URTI) and are associated with acute exacerbations of asthma and COPD. URTIs are associated with excessive production of mucus within the airway, particularly in sufferers of chronic respiratory conditions. This mucus may lead to altered osmolarity within the airway and contribute to hypotonic stimulation of the airway epithelium and airway hypersensitivity leading to cough. Hypotonic stimulation has previously been shown to activate transient receptor potential (TRP) vanilloid 4 (TRPV4) ultimately leading to ATP release via pannexin-1. RV-16 has been previously shown to cause upregulation of TRP channel expression in the airway, however, the mechanism of common cold cough is unknown. We hypothesise that infection with RV-16 causes increased ATP release in the airway leading to airway hypersensitivity and increased cough frequency.

Infection of airway epithelial (A549) and transfected astrocytoma (1321N1) cell lines with synthetic viral stimulus poly(I:C) failed to cause any change in expression or function of TRPA1, V1, V4 or P2X3 when monitored using RT-PCR, western blot analysis or intracellular calcium signalling. However, infection with RV-16 led to reduced function of thermo-TRP channels without concurrent reduction in expression although function was restored to basal levels by late infection.

The role of hypotonic stimulation on A549 cells was investigated with the aim of further delineating the mechanism of ATP release following hypotonic stimulation. Calcium-calmodulin kinase was identified to play an integral role in downstream ATP release, and that blockade of calcium-calmodulin kinase or pannexin-1 significantly reduced ATP release following hypotonic stimulation. Furthermore, immunofluorescent analysis identified a distinct distribution pattern of pannexin-1 prior to and post hypotonic stimulation, which could be effectively abolished through inhibition of pannexin-1, or significantly reduced following inhibition of calcium-calmodulin kinase, TRPV4 or MLC phosphorylation.

Finally, RV-16 infection led to increased ATP release from 72 hours through 168 hours post infection, independent of further stimulus at a time point which coincides with the development of viral cough in vivo. Furthermore, RV-16 infection led to a glycolytic shift in cell metabolism without any increase in cell death.

In summary, RV-16 induced ATP release, mediated by calcium-calmodulin kinase, and through TRPV4 and pannexin-1 activation, may underpin the development of a viral cough during an upper respiratory tract infection.

Citation

Atkinson, S. K. (2019). The effect of human rhinovirus-16 on thermo-TRP channels, hypotonic activation and ATP release. (Thesis). Hull York Medical School, the University of Hull and the University of York. Retrieved from https://hull-repository.worktribe.com/output/4221658

• Thesis Type: Thesis
• Deposit Date: Jun 26, 2019
• Publicly Available Date: Feb 23, 2023
• Keywords: Medicine
• Public URL: https://hull-repository.worktribe.com/output/4221658
• Additional Information: Hull York Medical School, The University of Hull and The University of York
• Award Date: Jan 1, 2019