PT - JOURNAL ARTICLE AU - Johnson, Santina C. AU - Uppaluru, Harshvardhan AU - Oswald, William AU - Francis, C. Michael AU - Gong, Na AU - Leavesley, Silas AU - Rich, Thomas TI - Characterization of Agonist-Induced Ca<sup>2+</sup> Signals in Human Airway Smooth Muscle Cells Using Excitation Scanning Hyperspectral Imaging and Image Analysis Approaches AID - 10.1124/jpet.560.101200 DP - 2024 Jun 01 TA - Journal of Pharmacology and Experimental Therapeutics PG - 560 VI - 389 IP - S3 4099 - http://jpet.aspetjournals.org/content/389/S3/560.short 4100 - http://jpet.aspetjournals.org/content/389/S3/560.full SO - J Pharmacol Exp Ther2024 Jun 01; 389 AB - Abstract ID 101200Poster Board 560Calcium signals plays critical roles in the physiology and pathophysiology of the respiratory system. Thus, proteins that regulate Ca2+ signaling pathways are often potential therapeutic targets. However, there are significant gaps in our understanding Ca2+ signaling pathways complicating the use of agents that alter Ca2+ signals as potential pharmacologic therapies. Thus, there is a need to better understand signal specificity and information content within Ca2+ signaling pathways. We propose the use of spectral imaging and dynamic region of interest (ROI) tracking software to better understand the impacts of GqPCR agonists on Ca2+ signals in human airway smooth muscle cells (HASMCs). Unlike traditional approaches in which investigators analyze static ROIs, we identify and track the location of ROIs as a function of time. Characteristics of Ca2+ signals in specific ROIs can then be quantified. This approach allows for a more nuanced and precise representation of agonist-induced Ca2+ signals in HASMCs. We have focused on the response of HASMCs to three distinct GqPCR agonists: two agonists that trigger contraction of HASMCs, carbachol and histamine; and one agonist that triggers relaxation of HASMCs, chloroquine. Utilizing excitation scan-based hyperspectral imaging, we capture and visualize the Ca2+ signals induced by these agonists. This advanced imaging method is complemented by a comprehensive analytical approach that integrates with dynamic ROI tracking software. ROI features (Ca2+ signal characteristics) were then analyzed using principal component analysis (PCA) and subsequent k-means clustering or K nearest neighbor methods. This approach enables us to dynamically track Ca2+ signals and categorize them based on parameters including area, duration, and amplitude. This approach has revealed unique patterns and characteristics of Ca2+ signals distinct to each agonist, offering potential insight into signal specificity. These observations may shed light on the distinct effects of carbachol, histamine, and chloroquine in modulating Ca2+ signals and subsequent regulation of HASMC contractile state. In addition, this overall approach may be applicable to the scientific interrogation of a wide range of intracellular signals.This work was supported by NIH R01HL169522, P01HL066299, R01HL058506, K25HL136869, and TL1TR003106.