Immunochemical visualization and quantitation of cyclic AMP in single cells.
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 visualize cAMP within cells. We describe here the fixation and immobilization of cAMP within cells and the production of specific, high titer polyclonal antibodies that recognize cAMP. Relative levels of cAMP immunofluorescence were quantitated in glial cells (oligodendrocytes, astrocytes, Schwann cells, and glioma cells) that were either untreated or treated with activators of endogenous adenylyl cyclase to raise cAMP levels. In treated cells, cAMP immunofluorescence is strongly localized in the perinuclear cytoplasm.
Wiemelt
A P
AP
The Wistar Institute, Philadelphia, Pennsylvania 19104, USA.
Engleka
M J
MJ
Skorupa
A F
AF
McMorris
F A
FA
D000171:Acrolein; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001253:Astrocytes / Q000737:chemistry; D000242:Cyclic AMP / Q000032:analysis* / Q000276:immunology; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D019085:Fluorescent Antibody Technique, Direct; D005910:Glioma / Q000737:chemistry; D007202:Indicators and Reagents; D009457:Neuroglia / Q000737:chemistry; D011817:Rabbits; D012583:Schwann Cells / Q000737:chemistry
D000906:Antibodies; D007202:Indicators and Reagents; D000171:Acrolein; D000242:Cyclic AMP; D000262:Adenylyl Cyclases