Downregulation of Guanylate Cyclase Enzyme in Human Asthma model to Investigate NO‐sGc‐ cGMP as a Therapeutic Pathway in Asthma

Abstract

Asthma is characterized by airway smooth muscle hypersensitivity. Common treatments include β‐adrenergic agonist therapy, such as budesonide, to induce airway smooth muscle relaxation via increased cAMP. However, 70% of patients develop resistance to such therapies. Nitric oxide is a locally produced smooth muscle relaxant that operates via stimulation of soluble guanylate cyclase (sGc) to increase cGMP production. In this study, the NO‐sGc‐cGMP pathway was investigated as an alternate pharmacologic pathway for asthma therapy. Previous studies have found that asthma patients are insensitive to NO signaling and have used sGc drugs to directly stimulate the enzyme. The objective of this study was to examine the expression of the two sGc subunits, α and β, within smooth muscle cells isolated from asthma patients in order to understand how NO‐sGC‐cGMP signaling may be disrupted. Further, we examined how budesonide treatment altered sGc expression. RNA sequencing data showed increased mRNA for both sGc subunits in asthmatic patients and that budesonide downregulated the gene expression of both subunits. We hypothesized that this increased gene expression was in part due to a lack of cGMP signaling due to decreased protein levels that is further impaired with steroids such as budesonide. To examine sGc, we measured the protein levels of both the α and β subunits of sGC by western blot in cultured smooth muscle cells. Fatal asthma and non‐asthma donor derived primary airway smooth muscle cells were cultured with and 24 hours of budesonide treatment. Statistical analysis of western blotting was performed by ANOVA. Densitometry of sGC western blots indicated a 2‐fold downregulation of the α (p<0.0001) and 1.5 fold downregulation of the β (p<0.05) subunits in asthmatic compared to normal patients. No significant changes were observed with normal or asthmatic patients with budesonide treatment (p>0.05), indicating no effect of steroids on the NO‐sGc‐cGMP pathway. A downregulation of sGc protein levels in asthmatic patients would lower cGMP production, which could explain the lack of NO responsiveness in these patients and would contribute to increased airway constriction in fatal asthma. These results indicate the NO‐sGc‐cGMP pathway as a potential new therapeutic pathway in asthma, and future work will examine cGMP levels.Support or Funding InformationSupported by R25ES020721 and ASPET SURF Intern ProgramThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.