In a recent publication, researchers from the University of Miami Miller School of Medicine (USA) describe that Serotonin released by human beta cell inhibits glucagon secretion by alpha cells. They demonstrated that this paracrine loop was mediated via the cAMP pathway. To do so, they captured in live human pancreatic islet cells cAMP signals using a specific fluorescent biosensor.
Cell-based calcium assays have been a mainstay of GPCR (G protein coupled receptor) drug discovery and signal transduction research since the mid 1990s. However, a calcium readout alone might be ambiguous, because many cellular processes produce calcium. Furthermore, it does not differentiate between signalling pathways routed through the different types of heterotrimeric G proteins. While effects on GPCRs which exhibit effects on Gs proteins lead to an elevated level of the second messaeger cAMP by activating the activity of adenylate cyclase, signalling through Gi proteins inhibits adenylate cyclase and thus leads to a decrease of cAMP. Finally signalling through Gq proteins leads to phospolipase C activation which cleaves Phosphatidylinositol 4,5-bisphosphate (PIP2) to Diacetlyglycerol (DAG) and Inositol trisphosphate (IP3) which leads to an increase in Ca2+ level (for an overview see reference 1).
Dye based methods to measure Ca2+ are very well established, whereas¬†there are no dyes available for most of the other second messengers in these signalling pathways.
Genetically encoded fluorescent biosensors for live cell discovery
That’s why Montana Molecular¬ developed genetically encoded fluorescent biosensors for second messengers such as cAMP, DAG, PIP2, and Ca2+, references 2, 3). [Read more…]
In drug discovery screening campaigns as well as in fundamental research activities, cellular parameters have to be measured and monitored in living cells regularly. Often fluorescent dyes are used to detect and follow some of the parameters. These methods are powerful, especially in screening large numbers of compounds, but have their limitations e.g. when it comes to measuring more than one parameter in the same living cell, or when specific locations in the cell shall be targeted.
To overcome these limitations and to provide tools to especially look into GPCR related signalling in the most comprehensive and detailed way currently possible, Montana Molecular¬†have developed genetically encoded fluorescent biosensors to measure parameters such as cAMP, DAG, PIP2, Ca2+and voltage changes in living cells. [Read more…]