cGMP accumulation induced by hypertonic stress in Dictyostelium discoideum

The change in extracellular osmolarity from 0.07 osm to 0.38 osm caused rapid cell shrinkage and loss of pseudopodes in Dictyostelium discoideum amoebae and induced elevation of total (cellular + extracellular) cGMP with a 2.5-min lag. cGMP accumulation reached a peak at 10-15 min after the change, and then the total cGMP gradually decreased. cGMP first accumulated intracellularly and was then secreted. A roughly identical osmotic concentration was required for the accumulation when the effect of KCl and glucose was tested. The non-osmolytes, formamide and ethanol, did not induce the accumulation. We concluded that hypertonic stress induces cGMP accumulation in D. discoideum amoebae. The hypertonic stress-induced accumulation of cGMP was observed in a streamer F mutant (NP368) that lacks cGMP-specific phosphodiesterase. While Dictyostelium cells also have nonspecific phosphodiesterases that degrade both cGMP and cAMP, hypertonic stress induced only a small increase in cAMP in wild type and streamer F cells. These results suggest that hypertonic stress-induced accumulation of cGMP is due to the activation of guanylate cyclase rather than the inhibition of phosphodiesterases. Binding of folic acid to the specific receptors on the cell surface induces a rapid transient accumulation of cGMP that reaches a peak at 10 s. When cells were stimulated by folic acid after the addition of 0.31 M glucose, rapid transient cGMP accumulation was observed immediately after the stimulation by folic acid and prolonged cGMP accumulation was induced 2-3 min after the addition of glucose irrespective of the timing of folic acid stimulation. These results suggest that the hypertonic stress-induced and the receptor-mediated accumulation proceed independently of one another. 2,3-Dimercapto-1-propanol, a thiol-reducing reagent, induces prolonged cGMP accumulation similar to hypertonic stress. However, the hypertonic stress-induced cGMP accumulation was enhanced by EDTA and was not suppressed by folic acid and cAMP. These characteristics are distinct from the reducing reagent-induced accumulation that is suppressed by EDTA, folic acid, and cAMP. These findings show that hypertonic stress has a unique effect on the activation of guanylate cyclase.