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A5968 - Lowered Expression of Soluble Adenylate Cyclase (sAC) and Cellular Redox Enzymes in Human Airway Smooth Muscle Cells Derived from Asthma
Author Block: A. Ghosh1, R. A. Panettieri2, D. J. Stuehr1; 1Pathobiology, Cleveland Clinic, Lerner Research Institute, Cleveland, OH, United States, 2Institute for Translational Medicine and Science, Rutgers University, New Brunswick, NJ, United States.
Rationale Our earlier study (ref.) found that lung soluble guanylate cyclase (sGC) is dysfunctional in asthma and impedes the Nitric oxide (NO)-based bronchodilation. The molecular hallmarks of sGC dysfunction revealed sGC to become heme-free and NO unresponsive, but this was overcome by pharmacologic sGC agonists (BAY 41-2272 and BAY 60-2270). In order to further understand molecular mechanisms causing inefficient bronchodilation in asthma, we performed intensive expression profiling in human airway smooth muscle cells (HASMCs), of key regulators involved in the β-adrenergic-sAC-cAMP and NO-sGC-cGMP bronchodilation signal cascades. Methods The methods of analysis involve western blotting for protein expression and sGC activity assay by cGMP estimations on ELISA. Results While the protein expression of β-adrenergic receptor (β2-AR) was unaltered from both control (n=16) and asthma (n=17), we found significantly low expression of soluble adenylate cyclase in the HASMCs derived from asthma relative to healthy controls. In contrast, the expression of sGC (β1 subunit) was variable in the asthma samples, and in certain cases seemed regulated by the expression of chaperone, hsp90. The expression of phosphodiesterases (PDEs, specifically PDE2A and PDE5A), remained unequivocal with no distinct pattern of lowering or elevation in the analyzed HASMCs. Analyzing for cellular redox enzymes, we found low levels of catalase and thioredoxin-1 (Trx1) in a small cohort of HASMCs and in corresponding lung tissue samples from asthma. Conclusion These data indicate that the dysregulation in the two signal pathways in asthma may essentially be upstream at the level of the two soluble cyclases, (sGC and sAC) with a lesser dependence on the downstream regulators like PDEs. Low levels of the vital redox enzymes can increase oxidant stress which may cause lung sGC to become heme-free and may represent one of the possible mechanisms of heme-free sGC accumulation in asthma. Our present study is poised to reveal distinct molecular pathways causing hindered bronchodilation and mechanisms triggering generation of heme-free sGC in asthma. Reference Ghosh A, Koziol-White CJ, Asosingh K, Cheng G, Ruple L, Groneberg D, Friebe A, Comhair SA, Stasch JP, Panettieri RA Jr, Aronica MA, Erzurum SC, Stuehr DJ (2016) Soluble guanylate cyclase as an alternative target for bronchodilator therapy in asthma. Proc Natl Acad Sci U S A 113(17):E2355-E2362.