3′:5′-Cyclic AMP-dependent 3′:5′-cyclic GMP accumulation in Dictyostelium discoideum
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 × 108 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 107 cells to 7 pmol per 107 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′-CH2-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′-CH2-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 Ca2+; cell aggregation and chemotaxis were also independent of the presence of external Ca2+. Therefore, cAMP action does not depend on stimulation of the Ca2+ 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.
