Immunochemical Visualization and Quantitation of Cyclic AMP in Single Cells

Abstract

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.Many signals that regulate the growth, development, and metabolism in cells use cAMP as a second messenger (1, 2). Neurotransmitters, growth factors, and hormones signal to appropriate receptors, causing either the stimulation or inhibition of adenylyl cyclase through intermediate guanyl nucleotide regulatory proteins. Adenylyl cyclase catalyzes the conversion of ATP to cAMP, which acts by binding to protein kinase A (PKA 1 ) and causing the release of activated catalytic subunits, or by binding and activating cAMP-gated ion channels (3-6). Activated PKA regulates the function of pre-existing cellular proteins by selective phosphorylation and also regulates the synthesis of new cellular proteins by phosphorylating and altering the activity of transcription factors (7-9).In the oligodendrocyte, the myelinating cell of the central nervous system, cAMP is thought to play independent roles in the regulation of cell growth and differentiation. Precursor cells, which are mobile and proliferative, exit the cell cycle upon exposure to cAMP analogs (10). In oligodendrocyte-lineage cells that are differentiating, exposure to cAMP analogs or activators of adenylyl cyclase increases the rate of differentiation (11,12). Whereas experimentally induced cAMP can have potent regulatory effects in these processes, the role of cAMP in normal oligodendrocyte development is not well understood. Cyclic AMP has similar effects in the peripheral myelinating glia, Schwann cells (13), and it is thought that axonal contact causes an elevation in cAMP, inducing proliferation and differentiation (14).Many questions remain about how cAMP relays signals, due in part to technical limitations that have prevented determination of the subcellular distribution of cAMP or comparison of cAMP levels between single cells. Homogeneous populations of cells may vary in their cAMP content from cell to cell, and conventional assays of cAMP extracted from homogenates of such a population generate a simple average level for the set (15). These assay procedures also mask any useful information about cell-specific changes in cAMP levels in the heterogeneous environments of tissue or cell culture. Finally, there have been no methods for accurately measuring free cAMP levels within ...

Neuronal Cyclic AMP Controls the Developmental Loss in Ability of Axons to Regenerate

“…1 B), where they persist to adult. Consistent with this measure of absolute cAMP levels of dissociated DRG neurons, when immu-nostained in situ with a cAMP antibody (Wiemelt et al, 1997), there is obviously much more cAMP in P0 than in P5 DRG neurons (Fig. 1C).…”

“…cAM P immunostaining was performed on DRG cryosections with a rabbit polyclonal antiserum according to Wiemelt et al (1997). Isolated, intact DRGs were fixed with acrolein for 1 hr at room temperature (RT), incubated in 20% sucrose until equilibrium, embedded with optimum cutting temperature (OC T) matrix (Tissue-Tek; Miles Inc., Elkhart, I N), and cryosectioned at 5 m. The fixed RG neurons or DRG cryosections were washed in a quenching solution containing 1 mg /ml glycine for 15 min at RT, followed by 1% (w/ v) sodium cyanoborohydride for 15 min.…”

The Orphan G Protein-coupled Receptor GPR17 Negatively Regulates Oligodendrocyte Differentiation via Gαi/o and Its Downstream Effector Molecules

“…Incubation with analogs of cAMP or sustained elevation of endogenous cAMP by either activation of adenylyl cyclase (10,23,26,39,40) or inhibition of the cAMP-hydrolyzing enzyme phosphodiesterase-7 (11) have been reported to increase oligodendrocyte differentiation, demonstrating that regulation of cAMP intracellular abundance is an efficient mechanism to control oligodendrocyte maturation. Accordingly, CNS myelin debris, a known inhibitor of oligodendroglial differentiation, partly exerts its action by attenuating cAMP levels in oligodendrocytes, which can be overcome by treatment with either dibutyryl-cAMP or rolipram, an inhibitor of phosphodiesterase-4 (12).…”

cAMP-induced Auditory Supporting Cell Proliferation is Mediated by ERK MAPK Signaling Pathway

“…Cochlear explants were cultured under conditions identical to those used to measure cAMP levels in the presence or absence of FSK. Cochlear explants were then sectioned and stained with cAMP antibodies to detect the presence of cAMP-like immunoreactivity (cAMP-LI) in FSK-treated and untreated control cochlear explants (Shen et al 1999;Wiemelt et al 1997). FSK-treated cochlear explants showed a marked increase in cAMP-LI staining in the supporting cells, compared with the supporting cells in the untreated control explants (Fig.…”

“…cAMP immunohisto-chemistry was performed with a polyclonal antiserum which has been shown to be highly specific for cAMP in tissues prepared as described above (Wiemelt et al 1997). Cochlea explant sections, 6 μM, were cut in the coronal plane on a cryostat at −17°C, picked up on ProbeOn slides (Fisher) and air dried.…”