In this initial Phase I grant application it is proposed to fully implement one aspect of our overall long term goal of developing advanced instrumentation and reagents for ion and second messenger imaging and analysis. Our instrumentation development efforts have-proceeded quite well in the area of hardware and software to support any combination of dual excitation and or dual emission fluorescent ratio imaging. We will apply our unique capabilities to simultaneous ration imaging of calcium ion and cyclic AMP in single living cells. For calcium we will use the fluorescent dual wavelength excitation- single wavelength emission dye fura-2, and for cyclic AMP the single wavelength excitation- dual wavelength emission fluorescence energy transfer dye """"""""FICRhR"""""""". Atto Instruments, Inc. owns the exclusive world-wide rights to this technology for small molecules developed at UC San Diego. In this phase I application we will first develop the reagent for cyclic AMP imaging in production quantities, establish methods of stabilization for its distribution and determine the proper hardware, software and calibration procedures to accurately utilize it for the dynamic measurement of cyclic AMP in cells. We will then develop techniques for the simultaneous measurement of cyclic AMP and calcium in single living cells using the fluorescent probes, """"""""FICRhR"""""""" and fura-2. Success at simultaneous measurement of these two major cell second messengers will be important in providing the tools to elucidate a myriad of cell functions which are interregulated by cyclic AMP and calcium. Furthermore, success in this project will confirm the applicability of this general approach to the dynamic analysis of other substances of intracellular regulatory importance, potentially implicated in a wide variety of diseases. Cancer, heart and certain neurological disease are the most prominent pathological states in which more detailed knowledge of the dynamics of cyclic AMP and calcium could help in an understanding of the basic cell biological changes associated with these diseases.
| DeBernardi, M A; Brooker, G (1998) Simultaneous fluorescence ratio imaging of cyclic AMP and calcium kinetics in single living cells. Adv Second Messenger Phosphoprotein Res 32:195-213 |
| DeBernardi, M A; Brooker, G (1996) Single cell Ca2+/cAMP cross-talk monitored by simultaneous Ca2+/cAMP fluorescence ratio imaging. Proc Natl Acad Sci U S A 93:4577-82 |
