Enzyme immunoassays of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate using acetylcholinesterase

Pradelles, Philippe; Grassi, Jacques; Chabardès, Danielle; Guiso, Nicole

doi:10.1021/ac00180a014

Cited by 151 publications

(84 citation statements)

References 18 publications

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“…Indeed, numerous biochemical ELISA kits for cAMP have been developed based on this technique and the antibodies used recognize acetyl-or succinyl-derivatized cAMP with high sensitivity. However, antibody recognition of unmodified cAMP such as naturally occurring cAMP in cells is dramatically lower (42).…”

Section: Resultsmentioning

confidence: 99%

Immunochemical Visualization and Quantitation of Cyclic AMP in Single Cells

Wiemelt

Engleka

Skorupa

et al. 1997

Journal of Biological Chemistry

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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 ...

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Section: Resultsmentioning

confidence: 99%

Immunochemical Visualization and Quantitation of Cyclic AMP in Single Cells

Wiemelt

Engleka

Skorupa

et al. 1997

Journal of Biological Chemistry

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“…Adenine nucleotides were also determined by 31 P NMR as indicated above. cAMP was measured as described (27) using a cAMP EIA kit (Cayman Chemical Co.).…”

Section: Gc-ms and Nmr Methodology-gc-ms Measurements Of 13 C Ormentioning

confidence: 99%

3-Isobutylmethylxanthine Inhibits Hepatic Urea Synthesis

Nissim

Horyn²,

Nissim

et al. 2008

Journal of Biological Chemistry

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“…HTE cells were scraped from wells with a rubber policeman, homogenized, and boiled for 3 min to denature the protein in the samples and to release cAMP. The cAMP concentration was then determined by enzyme immunoassay (Cayman Chemical) (38).…”

Section: Methodsmentioning

confidence: 99%

Role of Adhesins and Toxins in Invasion of Human Tracheal Epithelial Cells by Bordetella pertussis

Bassinet

Gueirard²,

Maître

et al. 2000

Infect Immun

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Bordetella pertussis, the agent of whooping cough, can invade and survive in several types of eukaryotic cell, including CHO, HeLa 229, and HEp-2 cells and macrophages. In this study, we analyzed bacterial invasiveness in nonrespiratory human HeLa epithelial cells and human HTE and HAE0 tracheal epithelial cells. Invasion assays and transmission electron microscopy analysis showed that B. pertussis strains invaded and survived, without multiplying, in HTE or HAE0 cells. This phenomenon was bvg regulated, but invasive properties differed between B. pertussis strains and isolates and the B. pertussis reference strain. Studies with B. pertussis mutant strains demonstrated that filamentous hemagglutinin, the major adhesin, was involved in the invasion of human tracheal epithelial cells by bacteria but not in that of HeLa cells. Fimbriae and pertussis toxin were not found to be involved. However, we found that the production of adenylate cyclase-hemolysin prevents the invasion of HeLa and HTE cells by B. pertussis because an adenylate cyclase-hemolysin-deficient mutant was found to be more invasive than the parental strain. The effect of adenylate cyclase-hemolysin was mediated by an increase in the cyclic AMP concentration in the cells. Pertactin (PRN), an adhesin, significantly inhibited the invasion of HTE cells by bacteria, probably via its interaction with adenylate cyclase-hemolysin. Isolates producing different PRNs were taken up similarly, indicating that the differences in the sequences of the PRNs produced by these isolates do not affect invasion. We concluded that filamentous hemagglutinin production favored invasion of human tracheal cells but that adenylate cyclase-hemolysin and PRN production significantly inhibited this process.

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Enzyme immunoassays of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate using acetylcholinesterase

Cited by 151 publications

References 18 publications

Immunochemical Visualization and Quantitation of Cyclic AMP in Single Cells

Immunochemical Visualization and Quantitation of Cyclic AMP in Single Cells

3-Isobutylmethylxanthine Inhibits Hepatic Urea Synthesis

Role of Adhesins and Toxins in Invasion of Human Tracheal Epithelial Cells by Bordetella pertussis

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