Detection of cAMP Accumulation Assays versus Real Time Monitoring

Traditionally, detection of cAMP is based on accumulation assays. A radioimmu-noassay-based method to asses directly cAMP concentration from tissue and cell preparations was introduced in the late 1960s (Steiner et al. 1969). The general principle behind such types of accumulation assays is that changes in intracellular cAMP are detected by the competition between cAMP in the sample and a labeled form of cAMP for binding to an anti-cAMP antibody. With several variations, this method is still the most widely used to date. Several protocols are now available based on this simple approach, exploiting a whole plethora of technologies, from radiometric to enzymatic and with application for high-throughput screening (Williams 2004). Although such methods can be highly sensitive (Golla and

Seethala 2002) and remain essential research tools, their limitation derives from the necessity of fixing and/or breaking apart the sample and extracting the second messenger from the cells. In doing so, any information relating to the spatial organization of the cAMP signal is lost. In addition, cAMP accumulation assays are restricted to assessing the second messenger concentration in steady-state conditions or changes in a timescale of minutes. Therefore, their time resolution is rather poor. Moreover, only average information relative to total cAMP changes in a cell population is attainable, whereas the concentration of free cAMP in the individual cell is more physiologically relevant.

Thus, the conventional way of assessing cAMP concentration is clearly inadequate to respond to the request of high spatial and temporal resolution necessary to investigate compartmentalized signaling. An important step forward in the study of the spatial and temporal aspects of cAMP signaling came from the development of real-time detection approaches. Such methodologies allow to measure accurately quantitative and dynamic parameters of the cAMP signaling networks. In addition, they provide unprecedented resolution both in time and in space, reporting cAMP changes as they happen in the complex intracellular environment.

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