IMMUNOCHEMICAL VISUALIZATION AND QUANTITATION OF CYCLIC-AMP IN SINGLECELLS

Adenosine 3':5'-cyclic monophosphate (cAMP) is a key second messenger in signaling pathways governing many cellular processes. To define the subcellular localization and relative abundance of cAMP, we developed a novel immunochemical approach based on acrolein fixation to visuali ze cAMP within cells. We describe here the fixation and immobilization of cAMP within cells and the production of specific, high titer polyc lonal antibodies that recognize cAMP. Relative levels of cAMP immunofl uorescence were quantitated in glial cells (oligodendrocytes, astrocyt es, Schwann cells, and glioma cells) that were either untreated or tre ated with activators of endogenous adenylyl cyclase to raise cAMP leve ls, In treated cells, cAMP immunofluorescence is strongly localized in the perinuclear cytoplasm.