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A7173 - Mapping Spatial Distributions of cAMP Signals in Human Airway Smooth Muscle Cells
Author Block: T. C. Rich1, N. Annamdevula1, A. Britain1, A. Westbrook1, D. A. Deshpande2, R. B. Penn2, S. J. Leavesley1; 1Ctr for Lung Biology, Univ of South Alabama, Mobile, AL, United States, 2Medicine, Thomas Jefferson University, Philadelphia, PA, United States.
Rationale: β2-adrenoreceptor (β2AR) agonists are an effective therapy for reversing the acute bronchoconstriction associated with an asthma attack. However, chronic use of beta-agonists results in diminished effectiveness, and long-acting beta-agonists may trigger severe exacerbation of asthma symptoms. In order to understand the effects of transient and prolonged activation of β2ARs, we need to better understand the spatial distribution and kinetics of β2AR agonist-induced cAMP signals and the resultant distribution of PKA activity in human airway smooth muscle cells (HASMs). Most single cell approaches to measure cAMP are based upon Förster resonance energy transfer (FRET) between donor and acceptor fluorophores attached to a cAMP binding protein. Binding of cAMP triggers a conformational change that reduces FRET efficiency. Unfortunately, FRET probes have notoriously low signal-to-noise ratios, prohibiting accurate assessment of the distribution of cAMP (FRET efficiency) in 3D (x,y,z). Thus, the vast majority of FRET imaging experiments are conducted in 2D. We have developed hyperspectral imaging and analysis approaches to allow FRET measurement in 5D: three spatial dimensions (x,y,z), wavelength (λ), and time (t). This has allowed us to map the kinetics and spatial distribution of agonist-induced cAMP signals and resultant changes in PKA activity in HASMs.
Methods: FRET-based cAMP (H188) and PKA/phosphatase (AKAR4) activity probes were transiently expressed in HASMs. Emission scan-based hyperspectral imaging approaches were used to measure cAMP-mediated changes in FRET signals in three spatial dimensions using a Nikon A1R confocal microscope. Custom image analysis scripts to calculate FRET signals were written in the matlab programming environment.
Results: Using the 5D hyperspectral imaging approaches described above, we observed that 1 µM isoproterenol triggered substantial cAMP gradients in HASMs. cAMP levels were high at the apical face and low at the basolateral face of cells. In addition, punctate cAMP signals were observed at distal regions of HASMs. In contrast, we observed that 1 µM PGE1 triggered cAMP signals that appeared to originate from the nuclear/perinuclear space. We also observed gradients in the balance between PKA and phosphatase activities in response to 1 µM isoproterenol.
Conclusions: These results demonstrate that hyperspectral imaging and image analysis approaches offer powerful tools to study the dynamics and spatial distribution of cAMP signals within HASMs. The results also indicate the potential of these approaches to map cAMP signals and resultant changes in balance between PKA and phosphatase activities to specific cellular responses. (NIH R01HL058506, P01HL066299, S10RR027535, S10OD020149, and AHA 16PRE27130004)