In the work presented we established cGMP as an important regulator of Ca2+ -influx stimulated by cAMP. A detailed discussion of the individual experiments has been provided in the designated sections. Here, I will discuss the implications of the results for the general background.
The regulatory role of Ca2+ for chemotaxis and light-scattering oscillations is currently a major issue for Dictyostelium research. Light-scattering oscillations and chemotaxis, as recently confirmed, depend on the production of cAMP after cAMP-receptor stimulation [Lusche, Malchow unpublished, (Zhang et al., 2003), reviewed in (Saran et al., 2002)]. A regulation by Ca2+ replaces a relatively simple by a more complex model for the cAMP signal relay involving Ca2+ fluxes across the plasma membrane, within the cytosol and in Ca2+ sequestering organelles.
The calmodulin antagonist W7 proved to be a useful tool to investigate and confirm a link between alterations in Ca2+-homeostasis and cAMP oscillations. A release of Ca2+ from internal stores here induced by W7 causes an increase of cAMP oscillations. It became evident that changes in [Ca2+]i and in the state of a protein(s) containing a calmodulin binding site alter light-scattering oscillations and consequently cause morphological alterations that are mirrored by light-scattering spikes (Wurster et al., 1989). These alterations were connected to the morphological changes during chemotaxis, which depend on correct alterations of the cytoskeleton (Yumura, 1993). Several experimental data exist that show that Ca2+ is involved in the regulation of the cytoskeleton and chemotaxis: Mutants in Ca2+-dependent actin binding proteins display unusual chemotactic behaviour (Furukawa et al., 2003; Lee et al., 1998; Newell, 1995b; Rivero et al., 1996);
Buffering or blocking of the increase of [Ca2+]i abolishes chemotaxis and cells also chemotax to an ionophore that releases Ca2+ from internal Ca2+ stores (Malchow et al., 1982; Schaloske et al., 2000; Unterweger et al., 1995). Additions of Ca2+
during light-scattering oscillations severely alter the pattern of the spike-shaped oscillations (Gerisch et al., 1979; Malchow et al., 1996b). Moreover, addition of Ca2+ to EGTA preincubated cells, a pretreatment that prevents spike-shaped oscillations, induces light-scattering oscillations (Lusche, Malchow unpublished observation). Some of the observations might be explained by the indirect activation of the cAMP transient by Ca2+. The experiments done with W7 not only
reveal the link, but also attribute an important role to internal Ca2+-stores for cAMP oscillations. Thus, experimental evidence exists and indicates that chemotaxis and light-scattering oscillations depend both on cAMP and Ca2+-concentration. What is the contribution of the cGMP oscillations?
cGMP oscillations are slightly advanced compared to cAMP oscillations and therefore are not in phase with light-scattering spikes in wildtype cells (Wurster B., 1977). Since the absence of 78% of cGMP-hydrolysis in PdeD KO cells caused a prolonged and elevated cGMP transient after cAMP stimulation (Bosgraaf et al., 2002b), the observed elevated and faster light-scattering oscillations are a consequence of a higher concentration of cGMP. In addition, it has been shown that cGMP is crucial for chemotaxis (Bosgraaf et al., 2002a). An elevated cGMP concentration in a PdeD KO strain promotes efficient suppression of lateral pseudopods and more efficient chemotaxis. It is interesting that this is reflected by more efficient light-scattering oscillations. Are, therefore, cGMP and cAMP ultimately linked or does an indirect connection exist? The answer can not be given satisfactorily. However, preliminary data showing that cGMP does not decrease during CMA and Caffeine treatment as shown for W7 induced increase of cAMP transients, indicate that cGMP oscillations can be uncoupled at least temporarily from cAMP oscillations (Lusche, Malchow unpublished data). It seems to be more likely that both oscillations are linked indirectly by Ca2+ oscillations. It was shown here that cGMP negatively couples to Ca2+-influx but does not act at the stores. Cyclic GMP therefore regulates the [Ca2+]i via Ca2+-influx and possibly cAMP production, a question that is under current investigation. Since cGMP production itself can be regulated by Ca2+ (probably via a small guanylyl-cyclase binding protein identified in the database by us and others, clone SSC670 from the D. discoideum cDNA project, Tsukuba, Japan.), Ca2+ also provides a negative feedback for the cGMP transient and thus termination of the response necessary for cGMP oscillations.
How is Ca2+-influx regulated by cGMP? As discussed before, cGMP either targets a Ca2+-channel directly or indirectly. Thus, we had to identify a Ca2+-channel.
Information from the Dictyostelium sequencing consortium indicates the existence of three Ca2+-channels, one of them being a putative Ryanodine / Ins(1,4,5)P3
receptor (Traynor et al., 2000). The remaining two are a putative mechanosensitive and a putative two-pore Ca2+channel (TPC). The Dictyostelium
TPC shows 24% homology to AtTPC and contains the typical domain architecture of other TPC (Furuichi et al., 2001; Ishibashi et al., 2000). It is interesting that like the plant channels but unlike the mammalian channel putative EF-hand regions exist between the two ion channel domains. This might indicate that the channel itself is regulated by Ca2+. We investigated the expression profile of DdTPC present as a single gene copy in D. discoideum. The channel is not expressed during the time when Ca2+-influx occurs first but during early culmination.
Mechanosensitive channels are known to transport Ca2+ and are regulated by cGMP (Yao et al., 2002). It is intriguing that this is the channel that regulates Ca2+ -influx after 4 hours of development. Both channels could contribute to elucidate the regulatory mechanism of Ca2+-entry by cGMP.
Future work
The biochemical and molecular characterization of DdTPC and Ms-channel are of utmost importance for the investigation of their regulation by cGMP. The knock out cells of these channels and the GFP-tagged proteins will help to clarify the channels role for Ca2+ homeostasis and the localisation of the channels within single cells and within the multicellular structure, respectively. The use of single KO-strains defective in genes of the cAMP and cGMP signalling pathway should clarify the link between Ca2+, cAMP and cGMP. With the help of similar mutants the pathway induced by W7 could be elucidated. In addition, it will be interesting to investigate whether chemotactic cells respond to W7 filled into a capillary like A23187 and induce a chemotaxis response without receptor stimulation. It will be interesting to investigate whether extracellular Ca2+-oscillations and intracellular cAMP oscillations can be directly linked or even visualized to a possible oscillating activation of Ca2+-stores. A model where PLC / Ins(1,4,5)P3-dependent Ca2+ -oscillations supplemented by the regulation through cGMP coordinate chemotactic oscillations and development (Goldbeter et al., 2000; Hess, 2000) might be established in the future.
Summary
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 binding site, since there was no effect of SP on the activity of a cGMP-dependent 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
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.
Zusammenfassung
In der hier vorgelegten Arbeit wurde gezeigt, dass zyklisches GMP als „second Messenger“ in dem Signalweg des chemotaktischen Botenstoffs cAMP eingreift.
Mittels einer Zelllinie (PdeD-KO), der die cGMP-spezifische Phosphodiesterase fehlt und die sich durch einen erhöhten und verlängerten cAMP-induzierten cGMP Transienten auszeichnet, wurde gefunden, dass cGMP den cAMP-induzierten Ca2+-Einstrom entwicklungsabhängig negativ reguliert und den Ca2+-Einstrom zeitlich verzögert. Intrazelluläre Ca2+-Speicher und dort befindliche Ca2+-Pumpen konnten als Wirkungsorte für cGMP ausgeschlossen werden. Im Gegensatz dazu sind Ca2+-Kanäle in der Plasmamembran die wahrscheinlichen Angriffsorte der cGMP Regulation. Die Hemmung des Ca2+-Einstromes wurde durch Einsatz des membran-permeablen cGMP-Analogons 8-pCPT-cGMP im Wildtyp bestätigt. Es gelang ebenfalls, den Ca2+-Einstrom in Wildtyp Zellen mit bekannten Inhibitoren cGMP-spezifischer Phosphodiesterasen von Säugerenzymen zu hemmen, obwohl überraschenderweise keine Erhöhung und Verlängerung des cGMP-Niveaus auftrat. Ich fand, dass zwei näher untersuchte Substanzen, SCH 51866 (SP) und Sildenafil Zitrat (Viagra®) direkt mit cGMP um einen zyklisch Nukleotid regulierten Ca2+- Kanal (CNGA3) kompetieren. Diese Wirkung ist spezifisch, da ein Derivat von Sildenafil, UK 114,452,den Kanal nicht zu hemmen vermochte und der cAMP-induzierte K+-Kanal durch SP nicht hemmbar war. Dies gilt auch für die Bindestelle, da SP und Sildenafil keinen Einfluss auf die Aktivität einer cGMP-abhängigen Proteinkinase hatten. Ein weiterer überzeugender Hinweis, der für einen an der Plasmamembran befindlichen Ca2+-Kanal als direkten oder indirekten Angriffsort für das cGMP spricht, wurde durch das Ausbleiben der Hemmung des cAMP-induzierten Ca2+-Einstromes durch SP in pdeD KO Zellen erhalten.
Durch eine Untersuchung vorhandener Sequenzdatenbanken wurden drei Ca2+ -Kanäle identifiziert, von denen zwei als Ziel für cGMP in Frage kommen: Der Ins(1,4,5)P3-Rezeptor ähnliche Ca2+-Kanal des endoplasmatischen Retikulums, ein mechanosensitiver Kanal (Ms-Kanal) und ein Zwei-Poren Kanal („two-pore channel, TPC). Mit der Analyse des TPC-Kanals wurde begonnen. DdTPC ist in einer Kopie im Genom von D. discoideum vorhanden. Northern Blot Analyse und RT-PCR ergaben, dass seine mRNA in späten Entwicklungsstadien von D.
discoideum exprimiert wird. Der TPC scheint also in einer späten Phase der
Entwicklung von D. discoideum gebraucht zu werden, möglicherweise für die Differenzierung von Prästielzellen und/oder Präsporenzellen. Der Kanal, der für Ca2+-Einstrom in frühen Entwicklungsstadien verantwortlich ist, könnte vielleicht der Ms-Kanal sein.
Im letzten Teil dieser Arbeit wurde cGMP neben cAMP als Regulator von Lichtstreuungsoszillationen etabliert. PdeD-KO Zellen zeigten erhöhte Amplituden und eine geringere Phasenlänge dieser Oszillationen. Die Amplitude war dagegen reduziert in einer Mutante, CAP-KO, mit reduziertem cGMP-Transienten. Der Mechanismus der Oszillation wurde mit Hilfe des Calmodulin Antagonisten W7 weiterhin um eine Kopplung an eine Ca2+-induzierte cAMP Synthese weiter aufgeklärt. Die Kopplung wird über die Freisetzung von Ca2+ aus sauren Speichern erreicht. W7 hemmt vorübergehend die Protonenpumpe, was zu einer erhöhten Freisetzung von Ca2+ führt.
Ca2+ aktiviert dann die Ca2+-abhängige Phospholipase C, die ein wichtiger Bestandteil des zur Erhöhung des cAMP führenden Signalweges ist. Die Lichtstreuungsoszillationen sind somit ein Resultat des Zusammenwirkens von cAMP, cGMP und Ca2+.
Anmerkung: Kurz vor Abgabe dieser Arbeit wurde die genomische Sequenz des DdTPC durch Klonierung des fehlenden Stückes komplettiert.
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