Cyclic GMP in the development of the social amoeba Dictyostelium discoideum : Regulation of Calcium homeostasis by cGMP.

Abstract

In the work presented here it was shown that cyclic GMP is involved in the signalling pathway of the chemoattractant cAMP. Utilizing a strain (PdeD-KO) deficient in the cGMP-specific phosphodiesterase that shows a prolonged and elevated cAMP-induced cGMP transient it was found that cGMP negatively and developmental time-dependently regulates cAMP-induced Ca2+-influx. Cyclic GMP also delayed Ca2+-influx. Intracellular Ca2+-stores and Ca2+-pumps were not targeted by cGMP. By contrast, Ca2+-channels in the plasma membrane are likely to be the target of cGMP regulation. The inhibition by cGMP was confirmed by application of the cGMP-analogue 8-pCPT-cGMP in wildtype cells. Moreover, inhibition was also achieved by known mammalian inhibitors of cGMP phosphodiesterases, although surprisingly there was no prolonged and elevated cGMP transient. I showed that two of these substances investigated, SCH 51866 (SP) and Sildenafil Zitrat (Viagra®) directly compete with cGMP for a cyclic nucleotide regulated and Ca2+-permeable channel (CNGA3). This effect was specific, since a Sildenafil derivative, UK 114,542 did not inhibit the channel and since a cAMP-induced K+-channel was not inhibited by SP. This was also true for the cGMP-binding site, since there was no effect of SP on the activity of a cGMPdependent proteinkinase. Another convincing evidence for a Ca2+-channel at the plasma membrane to be a target of cGMP was the lack of SP-inhibition of Ca2+- influx in PdeD-KO cells. By searching the available databases I identified the sequences of three Ca2+- channels, whereby two of them could be targeted by cGMP: A Ca2+-channel similar to Ins(1,4,5)P3-receptors of the endoplasmatic reticulum, a mechanosensitive channel (Ms-channel) and a two-pore channel (TPC). The analysis of the TPC was launched. DdTPC is present with a single copy within the genome of D. discoideum. Northern blotting and RT-PCR revealed that its mRNA is expressed late in development. Thus, DdTPC seems to be necessary for late development, possibly for prestalk and prespore differentiation. The channel responsible for early development might be the Ms-channel. In the last part of this work cGMP was established as a regulator of light-scattering oscillations besides cAMP. PdeD-KO cells displayed oscillations with elevated amplitude and shorter period lengths. By contrast, a mutant, CAP-KO, with a Summary reduced cGMP transient, showed reduced amplitudes. The mechanism of the oscillations was furthermore revealed by the aid of the calmodulin antagonist W7 showing that cAMP synthesis is coupled to Ca2+. The link is based on release of Ca2+ from acidic Ca2+-stores. W7 transiently inhibits the proton-pump causing additional Ca2+-release. Ca2+ activates Ca2+-dependent phospholipase C, which is a key enzyme in the cAMP-elevating pathway. The light-scattering oscillations are therefore the result of concerted action of cAMP, cGMP and Ca2+. Note added in proof: shortly before the final version of this work, the remaining fragment of DdTPC was cloned.