Instrumentation & Reagents for Ion/2nd Messenger Imaging

In this grant, we seek to fully implement one aspect of our overall long term goal of developingadvanced instrumentation and reagents for ion and second messenger imaging and analysis. Ourinstrumentation development efforts have-proceeded quite well in the area of hardware and softwareto support any combination of dual excitation and or dual emission fluorescent ratio imaging. We willapply our unique capabilities to simultaneous ration imaging of calcium ion and cyclic AMP in single livingcells. For calcium we will use the fluorescent dual wavelength excitation-single wavelength emissiondye fura-2, and for cyclic AMP the single wavelength excitation-dual wavelength emission fluorescenceenergy transfer dye "FICRhR". We own the exclusive world-wide rights to this technology for smallmolecules developed at UC San Diego. In this phase I application we will first develop the reagent forcyclic AMP imaging in production quantities, establish methods of stabilization for its distribution anddetermine the proper hardware, software and calibration procedures to accurately utilize it for thedynamic measurement of cyclic AMP in cells. We will then develop techniques for the simultaneousmeasurement of cyclic AMP and calcium in single living cells using the fluorescent probes, "FICRhR" andfura-2. Success at simultaneous measurement of these two major cell second messengers will beimportant in providing the tools to elucidate a myriad of cell functions which are interregulated by cyclicAMP and calcium. Furthermore, success in this project will confirm the applicability of this generalapproach to the dynamic analysis of other substances of intracellular regulatory importance, potentiallyimplicated in a wide variety of diseases. Cancer, heart and certain neurological disease are the mostprominent pathological states in which more detailed knowledge of the dynamics of cyclic AMP andcalcium could help in an understanding of the basic cell biological changes associated with thesediseases.