Neuropharmacology

Biography

Biography: Thomas Rich

Abstract

Cyclic nucleotides are ubiquitous second messengers known to differentially regulate many cellular functions over a wide range of timescales. Several lines of evidence suggest that intracellular distributions of cAMP and cGMP are not uniform, and that compartmentalization is largely responsible for signaling specificity within the these signaling pathways. However, real time measurements of cAMP and cGMP signals have been hampered by the low signal-to-noise ratio of fluorescence and FRET probes, as well as the inability to simultaneously measure multiple signals at discrete subcellular localtions within the same cell. In addition, measurements are typically performed in two spatial dimensions (2D), further limiting insight into second messenger signaling systems. Here we present novel hyperspectral imaging approaches that increase signal-to-noise ratios of fluorescence and FRET probes and allow real time, multiplexed measurements of intracellular signaling pathways in three spatial dimensions (3D). We have used hyperspectral approaches to visualize cyclic nucleotide gradients in several cell types, including pulmonary endothelial cells. Interestingly, in endothelial cells gradients primarily form along the apical to basolateral axis. These gradients would not be discerned by traditional imaging approaches. These data suggest that 2D imaging studies of cyclic nucleotide compartmentalization may lead to erroneous conclusions about the existence of second messenger gradients, and that 3D studies are required to assess mechanisms of signaling specificity. Results also demonstrate that novel hyperspectral imaging technologies are powerful tools for measuring biochemical processes in discrete subcellular domains. This work was supported by NIH grants P01HL066299, S10RR027535, and the Abraham Mitchell Cancer Research Fund.