3′:5′-Cyclic AMP-dependent 3′:5′-cyclic GMP accumulation in Dictyostelium discoideum

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Abstract
Suspensions of 3′:5′-cyclic AMP (cAMP)-sensitive cells of Dictyostelium discoideum responded to a cAMP pulse with increased 3′:5′-cyclic GMP (cGMP) levels. Under the assay conditions used (2 × 10 8 cells per ml in 10 mM phosphate buffer, pH 6.0) cAMP (5 × 10 -8 M final concentration) increased cGMP levels from 1 pmol per 10 7 cells to 7 pmol per 10 7 cells in 10 sec and basal levels were recovered in 20-25 sec. cGMP accumulation did not occur when cells were in the cAMP-insensitive stage. cAMP-sensitive cells responded with increased cGMP levels when triggered by 5 × 10 -8 M 5′-CH 2 -cAMP or 10 -5 M adenosine-5′-methylmonophosphate (5′-AMPMe) but not after addition of 5 × 10 -8 M 3′:5′-cyclic IMP (cIMP) or 5 × 10 -8 M 5′-AMP. As agonists of cAMP, 5′-CH 2 -cAMP and 5′-AMPMe have, respectively, more than 10% and 1% the chemotactic activity of cAMP, while cIMP has 0.01% the activity of cAMP and 5′-AMP is inactive up to a concentration of 10 -3 M. cAMP-mediated cGMP formation was dependent upon cAMP concentration, with a half-maximal cAMP concentration of about 10 -8 M. This cAMP concentration agrees closely with that necessary for half-maximal receptor occupation. cAMP-mediated cGMP formation was independent of the presence of extracellular Ca 2+ ; cell aggregation and chemotaxis were also independent of the presence of external Ca 2+ . Therefore, cAMP action does not depend on stimulation of the Ca 2+ influx. cAMP was found to mediate desensitization of cAMP-dependent cGMP formation. Addition of 5 × 10 -8 M cAMP to sensitive cells induced a desensitization period that lasted 1-5 min. Desensitization was dependent on the cAMP concentration. Finally, we propose that the translation of a chemotactic signal from the cell surface to pseudopod formation in Dictyostelium involves changes in the levels of cGMP.