Simultaneous analysis of adenosine 3',5'-cyclic monophosphate accumulation and adenosine 5'-triphosphate metabolism in cultured cells preincubated with [2-3H]adenine.
A simple and sensitive method based on metabolic labeling was developed for the simultaneous analysis of cyclic AMP accumulation and ATP metabolism in small numbers of cultured cells. Cells are preincubated overnight with [2-3H]adenine to label the ATP pool to a high specific activity. After cell stimulation the metabolites are extracted in a small volume of aqueous acetic acid and chloroform and separated without further manipulation by one-dimensional thin-layer chromatography and the radioactivity incorporated is determined by liquid scintillation counting. With ATP labeled to about 6 Ci/mmol, the lower limit of cyclic AMP detection is 2 fmol, a sensitivity that is comparable to the radioimmunoassay of acetylated cyclic AMP. In primary neurons and a neural cell line, for example, levels of ATP and its metabolites change when large amounts of cyclic AMP are generated, each with its unique pattern. ATP is also depleted when metabolic energy is consumed concomitantly with stimulation of cyclic AMP production by agonists, probably as a result of an increase in ion pump activity following cation influx. As ATP is utilized for cyclic AMP production and simultaneously for many other processes, an assessment of its metabolism in parallel with that of cyclic AMP is critical. We suggest that the method described here is particularly advantageous over other methods for this purpose.
Suidan
H S
HS
Department of Biochemistry, University of Cambridge, United Kingdom.
Tolkovsky
A M
AM
D000225:Adenine; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D000242:Cyclic AMP / Q000378:metabolism*; D006632:Histamine / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D014316:Tritium
D014316:Tritium; D006632:Histamine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D000225:Adenine; D007545:Isoproterenol