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Taste Receptor Expression and Calcium Signaling Optimization in Lung Cells

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Taste Receptor Expression and Calcium Signaling Optimization in Lung Cells Taste Receptor Expression and Calcium Signaling Optimization in Lung Cells Andrew Kennedy, A.S. – Biology (B.S.) Major SMART-Transfer Program Faculty Advisor: Robert Tarran, PhD – UNC School of Medicine Department of Cell Biology and Physiology Research Goals Taste Receptors in the Lungs Optimizing Protocol for Cell Bitter or Sweet Agonist Cell Exterior Signaling Assay 7 Transmembrane Domains • Taste Receptors (TRs) NH3 are a type of G protein coupled • This project will attempt to optimize an assay TM5 TM6 TM7 TM3 TM4 receptor (GPCR) found for calcium signaling in cells that do not TM1 TM2 TrpM5 endogenously express TRs COOH in human oral taste [3] • Results will assist in developing protocols for a G G buds GTP future study in transfecting the non- G • Sweet and bitter taste GDP endogenous TRs for use in a screening of receptor (T1R1-3 and compounds for activation of Ca2+ signaling T2R1-60 respectively) • When a florescent calcium indicator (Fluo-4) is The heterotrimeric G protein G dissociates from (G + G ) “Gustducin” are expressed in the given to a cell, light is emitted by the indicator airways and when it binds to calcium. This emitted light can gastrointestinal tract[3] be picked up by microscopes, which shows Key Movement • To date, studies of TRs that the cell is using calcium for signaling Endoplasmic Reticulum Cell Interior Activation in nasal and lung epithelia show TG Figure 1. Diagram of G protein (Gustducin) downstream calcium TG Ca2+ Ca2+ Ca2+ during agonism signaling pathways, Ca2+ 2+ 2+ Ca Overall'increase'in'cytoplasmic'Ca ' Ca2+ leading to various 2+ Ca Ca2+ 2+ Cell Exterior 2+ Ca Ca Bitter or Sweet Agonist 2+ Ca Ca2+ + immune responses Ca2+ TG Na 2+ + Ca Na Ca2+ Ca2+ + 2+ NH Ca Na 2+ 3 2+ Ca Inhibits Pump such as antimicrobial Ca Ca2+ 2+ 2+ TG Ca2+ Ca Pumps Ca 2+ Ca into the ER Ca2+ leaks SERCA Pump into the SERCA Pump cytosol peptide secretion and Ca2+ Ca2+ 2+ 2+ 2+ Ca 2+ Ca Ca Ca 2+ Ca 2+ 2+ TM7 2+ Ca Ca TM5 TM6 [3, 4] Ca Ca2+ TM3 TM4 inflammation TM1 TM2 Endoplasmic Endoplasmic Ca2+ TrpM5 Reticulim Reticulim • 2+ Ca COOH While over 60 TR + genes have been G Na PLC 2 discovered, the G G Figure 3. Diagram of Thapsigargin (TG) Ca2+ Ca2+ distribution of TRs 2+ 2+ Ca2+ mechanism for increasing intracellular Ca Ca2+ Ca IP 2+ throughout all airway 3 Ca2+ Ca cell types has not concentrations. (Left) Thapsigargin enters ATP PDE been well the cell while ER functions normally. (Right) cAMP 3 characterized Thapsigargin inhibits the SERCA (sarco/ R Key 3 2+ Movement IPR • A process for showing endoplasmic reticulum Ca ATPase) pump Endoplasmic Reticulum Cell Interior 2+ Activation which genes are resulting in a net decrease in ER [Ca ] expressed, called Figure 2. Diagram of taste receptor signaling pathways after the Gustducin subunit α (Gα) dissociates from both the GPCR and qPCR, will be used for Gβ/Gγ lung cells Results Taste Receptors in the Lungs Optimizing Protocol for Cell Fold Change of Genes Relative to GAPDH and CFTR Signaling Assay 1.0 Calu3 10µm 0.5 HBEC 10µm HASMC 0.06 0.04 0.02 3T3 5µM Fluo-4 Pre-TG 10µm 10µm Fold Change Fold 0.004 0.002 HEK293 3µM Pre-TG HEK293 3µM Post-TG 0.000 10µm 10µm H2O CFTR GNAT3 TAS1R1TAS1R2TAS1R3TAS2R1 TAS2R3 TAS2R4 TAS2R5 TAS2R7TAS2R8TAS2R9TRPM5TRPM8 TAS2R10TAS2R13TAS2R14TAS2R16TAS2R19TAS2R20TAS2R30TAS2R31TAS2R38TAS2R42TAS2R43TAS2R45TAS2R46TAS2R50TAS2R60 Gene Name Figure 5. Different airway cells show varied expression of TR genes. qPCR results graphed as the fold change, where fold change of a gene = 2^(-∆∆Ct) HEK293 5µM Pre-TG HEK293 5µM Post-TG Figure 4. 2 cell lines are able to load Fluo4 and show appropriate Significance Ca2+ response with positive control (TG). Cell lines (3T3 fibroblasts, HEK293) were plated onto collagen-coated coverslips and loaded • Results of the qPCR show varied lung TR gene expression (Figure 5). No clear pattern of TR with 3 or 5uM fluo4 for 40 min. Coverslips were transferred to metal chambers for imaging on epifluorescent microscope (Nikon Eclipse expression was shown from these data, and it will require more samples to be tested Ti). Cells were submerged in Ringer’s solution for duration of • Cells show suitable loading of Fluo-4 (Figure 4). HEK-293 cells exposed to 3µM Fluo-4 imaging. Images were taken before adding TG (pre-TG) and after showed similar florescence to the 5µM group, meaning future imaging for this cell type can adding TG (post-TG). (A) image of 3T3 with 5uM Fluo4 pre-TG (B) be carried out using a smaller amount (3µM) of Fluo-4. image of 3T3 with 5uM Fluo4 post-TG (C) image of HEK293 with 3uM Fluo4 pre-TG (D) image of HEK293 with 3uM Fluo4 post-TG (E) image of HEK293 with 5uM Fluo4 pre-TG (F) image of HEK293 with 3uM Fluo4 post-TG References 1. Callahan-Lyon, P., Electronic cigarettes: human health effects. Tob Control, 2014. 23 Suppl 2: p. ii36-40. 2. Hahn, J., et al., Electronic cigarettes: overview of chemical composition and exposure estimation. Tob Induc Dis, 2014. 12(1): p. 23. 3. Kinnamon, S.C., Taste receptor signalling - from tongues to lungs. Acta Physiol (Oxf), 2012. 204(2): p. 158-68. 4. Lee, R.J., et al., Bitter and sweet taste receptors regulate human upper respiratory innate immunity. J Clin Invest, 2014. 124(3): p. 1393-405. 5. Greene, T.A., et al., Probenecid inhibits the human bitter taste receptor TAS2R16 and suppresses bitter perception of salicin. PLoS One, 2011. 6(5): p. e20123. .
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