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 x 10(8) cells per ml in 10 mM phosphate buffer, pH 6.0) cAMP (5 x 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 x 10(-8) M 5'-CH(2)-cAMP or 10(-5) M adenosine-5'-methylmonophosphate (5'-AMPMe) but not after addition of 5 x 10(-8) M 3':5'-cyclic IMP (cIMP) or 5 x 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 x 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.