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

Dictyostelium discoideum:

Regulation of Calcium homeostasis by cGMP.

Die Rolle von cGMP für die Entwicklung der sozialen Amöbe Dictyostelium discoideum.

Untersuchung zur Regulation der Kalziumhomöostase durch cGMP.

Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften

Universität Konstanz Fachbereich Biologie

Daniel Felix Lusche Konstanz, Januar 2004

To my family

Publications:

Lusche D.F., Malchow D.

Developmental control of cAMP-induced Ca²⁺-influx by cGMP: Influx is delayed and reduced in a cGMP-phosphodiesterase D deficient mutant of Dictyostelium discoideum. Cell Calcium 2004, accepted June 2004

Lusche D.F., Kaneko H., Malchow D.,

cGMP-phosphodiesterase antagonists inhibit Ca²⁺ -influx in Dictyostelium discoideum and bovine cyclic-nucleotide-gated-channel.

Eur.J.Pharmacol.2004, submitted Malchow D., Lusche D.F., Schlatterer C.,

A link of Ca²⁺ to cAMP oscillations in Dictyostelium: the calmodulin antagonist W 7 potentiates cAMP relay and transiently inhibits the acidic Ca²⁺-store. BMC Developmental Biology 2004, May 17;4(1):7

Schlatterer C., Happle K., Lusche D.F., Sonnemann J.,

Cytosolic [Ca²⁺]-transients in Dictyostelium discoideum depend on the filling state of internal stores and on an active SERCA Ca²⁺-pump. J. Biol. Chem.

2004 Apr.30;279(18):18407-14

Marcus Tullius Cicero Aeguam memento rebus in arduis servare mentem

Table of contents

General Introduction ______________________________________________ 1 Cyclic AMP-Signaltransduction _________________________________________ 2 Cyclic GMP is an important second messenger in D. discoideum and other

eukaryotes.__________________________________________________________ 4 Ca²⁺ a second messenger required for chemotaxis and differentiation_________ 5 Aim of this work______________________________________________________ 7 General Abbreviations ________________________________________________ 8 Cyclic GMP-phosphodiesterase antagonists inhibit Ca²⁺ -influx in

Dictyostelium discoideum and bovine cyclic-nucleotide-gated-channel ___ 9 Abstract ____________________________________________________________ 9 Introduction _________________________________________________________ 9 Material and Methods ________________________________________________ 11 Results ____________________________________________________________ 15 Discussion _________________________________________________________ 24 References _________________________________________________________ 28 Developmental control of cAMP-induced Ca²⁺-influx by cGMP: Influx is

delayed and reduced in a cGMP-phosphodiesterase D deficient mutant of Dictyostelium discoideum ________________________________________ 31

Abstract ___________________________________________________________ 31 Introduction ________________________________________________________ 31 Material and Methods ________________________________________________ 33 Results ____________________________________________________________ 35 Discussion _________________________________________________________ 46 References _________________________________________________________ 49 Identification of Ca²⁺-channels in D. discoideum. _____________________ 51

Introduction ________________________________________________________ 51 Material and Methods ________________________________________________ 51 Results ____________________________________________________________ 58 Discussion _________________________________________________________ 65 Outlook ____________________________________________________________ 66 References _________________________________________________________ 67 cGMP is involved in regulation of light-scattering oscillations. __________ 69

Results ____________________________________________________________ 69 A link of Ca²⁺ to cAMP oscillations in Dictyostelium: the calmodulin

antagonist W 7 potentiates cAMP relay and transiently inhibits the acidic Ca²⁺-store ______________________________________________________ 71

Abstract ___________________________________________________________ 71

Introduction ________________________________________________________ 71 Materials and Methods _______________________________________________ 72 Results ____________________________________________________________ 75 Discussion _________________________________________________________ 83 References _________________________________________________________ 86 General discussion ______________________________________________ 89

Future work ________________________________________________________ 91 Summary_______________________________________________________ 92 Zusammenfassung ______________________________________________ 94 References _________________________________________________________ 96

General Introduction

Members of the family Dictyostelidae are eukaryotic cells that are grouped separately within the animal kingdom (Baldauf et al., 1997). Together with Physarum and Planoptotostelium they are clustered together in the monophyletic crown group Mycetozoa. In addition to their unique evolutionary and taxonomic position, these cells possess an unusual lifecycle. Besides a sexual period cells undergo a phase of asexual development initiated by nutrient depletion when growing. The main feature of this phase is a transition from unicellular cells to a multicellular structure (Figure 1) first called a mound, which proceeds to a slug and will finally culminate to a fruiting body consisting of two differentiated cell types, dead stalk cells and viable spores.

Figure 1: Life cycle of D. discoideum. Photograph was obtained from Prof. Dr. R. Mutzel.

This structure is composed of approximately 10⁵ cells. The setup of this structure requires that individual cells aggregate to a centre which is made possible by their chemotactic behaviour using cAMP as the most potent chemoattractant. The cells are thought to be driven to aggregation and multicellularity by an altruistic behaviour caused by a “green beard effect” of certain genes whereby the cells are able to recognize their allel’s presence, “the green beard”, in other cells (Queller et al., 2003).

Biological and medically relevant processes, such as phagocytosis, chemotaxis, apoptosis, morphogenesis, terminal differentiation occurring either during vegetative stages or later in the asexual lifecycle can be studied in the laboratory.

Here, axenic cells of the species Dictyostelium discoideum can grow in liquid culture or with bacteria on agar plates that will be phagocytosed by the cells. The development of the cells can be launched by removal of the media or by lack of bacteria in the vicinity of the cells.

Cyclic AMP-Signaltransduction

Cyclic AMP requirement during early development

Cyclic AMP- signal transduction is essential for progress of development after nutrient depletion [reviewed in (Kimmel et al., 2003; Saran et al., 2002)]. Cyclic AMP acts outside and inside the cells. During chemotaxis cells are capable of detecting and relaying the cAMP signal to neighbouring cells such that a spatio- temporal cAMP gradient can be established. This system causes the occurrence of cAMP oscillations. They can be visualized by optical density waves obtained from cells streaming to aggregation centres on agar plates that correlate with waves of secreted cAMP proceeding from the centre to the outermost cells (Kriebel et al., 2003; Patel et al., 2000) or by measurements of cells in suspension where cAMP oscillates in parallel to spike-shaped light-scattering changes induced by morphological changes as a consequence of cAMP binding to single cells (Wurster et al., 1989). Even in the slug cAMP concentrations and downstream targets oscillate as observed by monitoring GFP-tagged cAMP-signalling components (Dormann et al., 2001b; Dormann et al., 2002). The participating effector molecules of cAMP, however, and its induced signalling pathway change during development. This is reflected by the expression patterns of the 4 known cAMP-receptors cAR1-cAR4. At the early stages in development cAR1 is the major receptor, while the remaining receptors are predominantly expressed during different parts of late development albeit with lower affinity for cAMP [references in (Kimmel et al., 2003)]. cAR1 like the other receptor forms belongs to the G-protein coupled receptors and is responsible for the cAMP relay. In Figure 2 the proposed cAMP-relay after receptor activation is shown (Laub et al., 1998; Saran et al., 2002). Upon cAMP stimulation the Gβγ-subunit dissociates from Gα2 and activates the adenylyl-cyclase A (ACA) that synthesizes cAMP which will be subsequently

secreted by the cells and can then act paracrine. The activation is very complex and also involves a cytosolic activator of adenylyl-cyclase (CRAC), RasC, a Ras interacting protein (Rip3), a nucleotide exchange factor (RasGEF), a novel factor pianissimo and a cyclase activating protein (CAP) that is similar to the known bacterial protein (Noegel et al., 2003; Saran et al., 2002).

Figure 2: The cAMP-relay. A model for cAMP relay of a single cell according to Saran et al., 2002 is shown.

The cAMP produced will be degraded within the cell by RegA, a specific intracellular cAMP phosphodiesterase and extracellularly by a secreted cAMP- phosphodiesterase PdsA and a spliced form of PdsA that is membrane bound ensuring the termination of the cAMP response. The synthesis and degradation of cAMP is controlled by feedback loops involving a cAMP-dependent protein kinase (PKA) and an extracellular regulated protein kinase (ERK 2). The mechanism for the release of cAMP is still unknown, although the required apparatus seems to be located at the rear end of the cells (Kriebel et al., 2003). Adaptation of the receptors to the cAMP-stimulus by a mechanism not completely understood supports the detection of the spatio-temporal gradients. The high affinity of cAR1 assures a high frequency of wave initiations and therefore efficient aggregation, which should be important in the natural habitat, the soil (Dormann et al., 2001a).

Cyclic AMP requirement during multicellularity in late development The slug can be subdivided in different zones containing the differentiating prespore cells and several subtypes of prestalk cells emerging already at the mound. The cell subtypes can be identified with specific genetic markers. The sorting of the cells with prestalk cells at the front and prespore cells at the rear end

of the slug is a consequence of the cAMP waves, whereby the tip of the slug acts as an organising centre for the cAMP waves [(Dormann et al., 2001a; Dormann et al., 2001b,) and references therein]. The autocatalytic cAMP waves show specific spiral shaped patterns within the cell mass, but the speed of the waves is slower than during aggregation due to the differently expressed cAR subtypes, that do have a lower affinity for cAMP. Another control system for the extracellular cAMP concentration is provided by the developmentally regulated secretion of a phosphodiesterase inhibitor (PDI) that controls the activity of PdsA (Riedel et al., 1971). Thus, the extracellular cAMP concentrations are adjusted during development. Cell movement within the mound and the slug are still controlled by the cAMP, since a gradient is established by the prestalk cells that secrete more PdsA than prespore cells. The cell movement and sorting during these stages resemble processes during gastrulation. For terminal differentiation resulting in the completion of the fruitingbody, PKA induced gene expression is a key step of the cAMP response [(Kay, 2002; Saran et al., 2002) and references]. Cells that show higher PKA activity show precocious sporulation. Therefore a pathway including PKA contributes to cell fate. This has recently been confirmed by the discovery of an transcription factor downstream of PKA, SrfA (Escalante et al., 2002).

Cyclic GMP is an important second messenger in D. discoideum and other eukaryotes.

There is a second oscillatory component albeit mainly within the cell, cGMP. The information on cGMP is scarce compared to cAMP. Cyclic GMP is generally synthesized by guanylyl-cyclases and degraded by specific and non specific phosphodiesterases (Bosgraaf et al., 2002c). In the tool box for cGMP pathways of eukaryotes cGMP-dependent protein kinases, CNras, IRAGs, cGMP-regulated Ca^2-channels and K⁺-channels are generally present, but other previously unsuspected proteins are likely to be added (Kim et al., 2003). In D. discoideum some components of the cGMP metabolism and potential effector molecules have been cloned recently (Bosgraaf et al., 2002c). These enzymes are activated but not exclusively after cAMP stimulation. Like in mammalian cells there are several forms of guanyly-cylcases and phosphodiesterases present. The details of the proteins involved in D. discoideum are addressed in the introductions of the following sections. With respect to the ultimate function(s) of cGMP little is known

for this organism. This is reflected in the weak phenotypes resulting from knock out strains or overexpressors. Several groups have presented evidence by the use of these KO strains that cGMP alters chemotaxis and translocation of myosin II to the cortex of the cells after cAMP stimulation and supported older findings obtained from a chemically generated mutant (streamer F) that reacts towards cAMP stimulation with an elevated intracellular cGMP level accompanied by a prolonged phosphorylation and association of myosin II with the cytoskeleton (Bosgraaf et al., 2002a; Liu et al., 1993). Chemotaxis is altered in this strain and the cell streams during aggregation are broader than in the wildtype. cGMP is also involved in the osmotic stress response but the specificity of this function remains to be elucidated since the cAMP signalling pathway also contributes to this response (Ott et al., 2000; Oyama, 1996). Phototaxis and thermotaxis of the slug also seem to be regulated by cGMP like it is the case in Caenorhabditis elegans (Komatsu et al., 1996).

Ca²⁺ is a second messenger required for chemotaxis and differentiation

Role of Ca²⁺ during early development

Convincing evidence that cAMP mediated chemotaxis during early development depends on proper regulation of Ca²⁺-homeostasis is derived from experiments done by Unterweger et al. [(Unterweger et al., 1995) and reviewed in (Malchow et al., 1996a)]. Introduction of Ca²⁺-buffers such as EGTA and its derivative BAPTA into the cytosol by scrape loading causes the cells to round up and reduces the ability of the cells to chemotax. In addition, cells chemotax to a capillary filled with a Ca²⁺ ionophore, A23187 (Malchow et al., 1982). The basal [Ca²⁺]i concentration of 50 nM of unstimulated cells and a [Ca²⁺]i transient upon addition of cAMP is a consequence of a regulation that involves Ca²⁺-stores and Ca²⁺-fluxes across the plasma membrane and the presences of Ca²⁺ -binding proteins as described also for other eukaryotic cells [reviewed in (Malchow et al., 1996a; Newell, 1995a)].

Stimulation with cAMP induces a Ca²⁺-influx and a rise of [Ca²⁺]i that are triggered by emptying of Ins(1,4,5)P3 sensitive and fatty acid sensitive Ca²⁺-stores [(Malchow et al., 1996a; Schaloske et al., 2000; Schaloske et al., 1998;

Sonnemann et al., 1997) and references therein]. Ins(1,4,5)P3 can be generated by phospholipase C (PLC) (Drayer et al., 1992) or by hydrolysis of Ins(1,3,4,5,6)P5

(Drayer et al., 1994). The different pathways as sources for the generation of Ins(1,4,5)P3 seem to be important for initiation of Ca²⁺- entry supported by the finding that PLC is activated after cAMP stimulation by Ga2.Moreover, half of the Ca²⁺-influx was shown to be independent of Gα2. Likewise [Ca²⁺]i did not dependent on Gα2 but partly on Gβγ (Sonnemann et al., 1998). Thus, PLC alone is not sufficient for activation of the Ins(1,4,5)P3 pathway. Fatty acids generated by phospholipase A2 can substitute for the cAMP induced Ca²⁺ -release in a Gα2 and Gβγ dependent manner (Schaloske et al., 1997). The Ca²⁺-influx and the rise of [Ca²⁺]i after stimulation with cAMP are severely affected but not abolished in a mutant HM1049 that lacks a gene homologous to InsP3 receptors (iplA). Thus, one storage compartment should be altered. The cells of this strain show normal chemotaxis, but no rise of [Ca²⁺]i under standard conditions (Traynor et al., 2000).

Chemotaxis, however, is altered under conditions where external Ca²⁺ was varied (Schlatterer, manuscript in preparation). In addition, a small rise of [Ca²⁺]i might still occur in the mutant, but this Ca²⁺ might be readily taken up, since pumps are very active (Schlatterer et al. 2003, submitted).

Downstream reactions that might explain the contribution of Ca²⁺ to the oscillating chemotactic behaviour reflected in light-scattering and optical density wave recordings include alterations in the formation of the actin filaments [(Furukawa et al., 2003) and references therein], in myosin regulation (Newell, 1995a), in the status of EF-hand containing proteins and other proteins that directly bind Ca²⁺

[(Gauthier et al., 2001; Myre et al., 2002; Newell, 1995a) and references therein]

and in adenylyl-cyclase activation (Schaap et al., 1995) in vitro, in guanylyl-cyclase activation in vitro (Roelofs et al., 2002).

Thus, it is important to identify further components involved in Ca²⁺-influx and in the generation of [Ca²⁺]i increase to elucidate the different pathways

Ca²⁺ as a regulator of determination, differentiation and morphogenesis

Development of D. discoideum can be influenced by the ion composition of the media surrounding the cells (Maeda, 1970). Already during growth the relative Ca²⁺ content in the cytosol and the stores of the cells contributes to the determination of the cell fate of the individual cell during development (Azhar et al., 2001; Azhar et al., 1998). Cells with high [Ca²⁺]i are preferentially in S and early

G2 phase and tend to become prestalk cells, while cells that are in a later stage of the cell cycle tend to have low [Ca²⁺]i and become prespores. However, the correlation is not absolute and other factors also contribute to the determination.

The presence of Ca²⁺ influences aggregation and terminal differentiation. Excess Ca²⁺ during growth leads to smaller fruiting bodies, while during differentiation this effect is less pronounced but abnormally long slugs and a stalky phenotype are observed. Low Ca²⁺ shows adverse effects. The latter effect might be a consequence of Ca²⁺ affecting aggregation territory numbers with high Ca²⁺

resulting in smaller numbers, an observation that lacks a definite explanation yet, but might be related to a factor called conditioned medium factor (CMF) that senses cell density and whose signal transduction pathway involves Ca²⁺ -influx (Yuen et al., 1995). In the slug, the anterior localized prestalk cells (see introduction) show significantly higher [Ca²⁺]i than the prespore cells although the latter sequester Ca²⁺ more efficiently [(Baskar et al., 2000; Cubitt et al., 1995;

Maeda et al., 1973) references therein, (Schlatterer et al., 2001)]. This resulting gradient of [Ca²⁺]i steepens with high Ca²⁺ treatment of the slugs and conversely flattens for slugs treated with low Ca²⁺. It’s suggested that the Ca²⁺ gradient along the different cells rather than the Ca²⁺ content of a single cell contributes to determination of the cell type proportion of a slug. Moreover, a differentiation inducing factor, DIF, produced by prespore cells induces a rise in [Ca²⁺]i and prestalk cell differentiation, but suppresses prespore differentiation [(Azhar et al., 1997), reviewed in (Kay, 2002)]. Thus, it appears that in cells developing under high external Ca²⁺ conditions the prespore pathway is favoured.

Notably, there are hints that Ca²⁺ might still be involved in the regulation of the cAMP response. Degradation of PdsA and PdsA inhibitor which will alter the cAMP available is regulated by the Ca²⁺-content of the ER, with depletion of the ER promoting the degradation (Coukell et al., 1990; Coukell et al., 1992). Finally, Ca²⁺

has been shown to be involved in spore germination. During activation and swelling of the spores Ca²⁺-efflux occurs and once amoeba emerge Ca²⁺ is taken up [(Lydan et al., 1995) and references].

Aim of this work

The goal of this study has been to elucidate the role of cGMP in Ca²⁺-homeostasis whereby the regulation of Ca²⁺-influx was emphasized. Initial experiments using a

chemically mutagenized mutant, streamer F (see above), that showed a prolonged and elevated rise in cGMP and Ca²⁺-influx led to the conclusion that cGMP might positively regulate Ca²⁺-influx. Since the genes involved in cGMP metabolism were not cloned at the beginning of this study and the proteins physically unstable, we used pharmacological and molecular and bioinformatical approaches to investigate a cGMP / Ca²⁺ link, focusing on the discovery of (a) Ca²⁺ -channel(s).

With the rapid discovery of unusual cGMP-binding proteins and their corresponding genes by a Dutch and a Scottish group, mutants became available and we were able to obtain a decisive mutant lacking a cGMP-phosphodiesterase to investigate the involvement of cGMP in Ca²⁺-influx. This was done in the first part. A second question that we asked was the contribution of Ca²⁺ and specific Ca²⁺-stores to cAMP and cGMP oscillations that accompany light-scattering oscillations caused by morphological changes of cells in suspension. We measured light-scattering oscillations, cAMP oscillations of cells in suspension and used the calmodulin antagonist W7 that releases Ca²⁺ from internal stores to investigate this question.

General Abbreviations

Ax2, axenic strain 2; ACA, adenylyl cyclase A; CRAC, cytosolic activator of adenylyl-cyclase; cAR1, cAMP receptor 1; CaM, calmodulin; CNG, cyclic- nucleotide-gated; [Ca²⁺]e, extracellular Ca²⁺ concentration; [Ca²⁺]i, intracellular Ca²⁺-concentration; ePDE, extracellular phosphodiesterase; EDTA,

Ethylendinitrilotetraacetic acid; EGTA, Ethylenglycol-O,O’-bis(2-aminoethyl)- ,N,N,N’,N’ tetraacetic acid; Gbp, cGMP-binding protein; GFP, green fluorescent protein; CaMKII, calmodulin kinase II, CMA, concanamycin A; DTT, Dithiothreitol;

EIA, enzyme immuno assay; HEK, 293 Human embryonic kidney cells; IBMX, 3- isobutyl-1-methylxanthine; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; kb, kilo base;

kDa, kilo Dalton; MEQ, 4-{[3,4-(methylenedioxy)benzyl]amino}-6-

methoxyquinazoline; PLC, Phospholipase C; ORF, open reading frame; wt, wildtype; PDE, phosphodiesterase; KO, knock out; PKG, cGMP-dependent kinases; SDS, sodiumdodecylsulfat; Rip A, Ras-interacting protein; Tween 20;

Polyoxyethylen-Sorbitan –Monolaurat; XeC, Xestospongin C

Cyclic GMP-phosphodiesterase antagonists inhibit Ca

- influx in Dictyostelium discoideum and bovine cyclic- nucleotide-gated channel

Abstract

1 We used antagonists of cGMP-phosphodiesterases to examine the role of cGMP for light-scattering oscillations and cAMP-induced Ca²⁺-influx in Dictyostelium discoideum, however, SCH 51866 (SP) and Sildenafil citrate were poor inhibitors of cGMP-hydrolysis.

2 Instead, SP (IC50=16 µM) and Sildenafil blocked chemoattractant (cAMP) - induced Ca²⁺ -influx as determined with a Ca²⁺-specific electrode.

3 SP (150 µM) affected neither spontaneous cGMP transients during light- scattering-oscillations nor cAMP-mediated K⁺-efflux.

4 SP and Sildenafil are competitive inhibitors of cGMP phosphodiesterases.

However, the activity of cGMP-dependent protein kinase (PKGIα) was not altered by SP (150 µM). By contrast, patch-clamp measurements of bovine cone cGMP- gated channels (CNGA3), stably expressed in HEK 293 cells, revealed reversible, competitive and dose-dependent inhibition of sodium currents by SP (IC50=25 µM) and Sildenafil (30 µM), but not by another inhibitor of cGMP-phosphodiesterases, UK 114,542.

5 The possibility that D. discoideum cells also express a cGMP-regulated channel is supported by our finding that LY 83583 (35 µM), known to inhibit cyclic nucleotide-gated channels as well as guanylyl-cyclases, reduced cAMP-induced Ca²⁺-influx in D. discoideum, but did not affect cAMP-induced cGMP accumulation.

6 Utilizing a PdeD null strain that exhibits a prolonged and elevated cGMP transient following receptor activation, we found that the inhibition of Ca²⁺-influx by SP in the wildtype was absent in the mutant

7 Our results show that SP and Sildenafil are antagonists of a Ca²⁺-permeable channel (CNGA3) and that both compete with cGMP for a regulatory site of Ca²⁺- influx in D. discoideum.

Introduction

The social amoeba Dictyostelium discoideum is a unicellular eukaryote that enters multicellularity and differentiates when being subjected to nutrient depletion.

Between 10⁴ to 5*10⁶ cells aggregate to form differently sized slugs and, within 24 h, a fruiting body that carries the spore mass (Bonner J.T. 2002 http://dictybase.org/). Chemotaxis leading to aggregation and differentiation requires complex processing of environmental information by the individual cell. As in many cell types and tissues, several second messengers, among them cAMP, cGMP and Ca²⁺, are recognized to be part of these processes. Although progress has been made in understanding their contribution to chemotaxis and some aspects of development, the signalling pathways and their interaction remain to be clarified.

In D.discoideum, cAMP functions as a secretable first messenger and chemotactic agent to induce aggregation and to activate distinct signalling pathways within the individual amoebae. In addition, cAMP acts as a second messenger for signal relay, gene expression, terminal stalk cell differentiation, and spore germination (reference in Brown et al., 1999; Brzostowski et al., 2001; Dormann et al., 2001;

Firtel et al., 2000). By binding to a serpentine receptor, cAR1, cAMP not only evokes an increase of intracellular cAMP but also a transient ten-fold increase in intracellular cGMP concentration. In contrast to cAMP, cGMP is mainly detected within the cells and only a small amount was found to be secreted (Van Haastert et al., 1997; VanHaastert P.J.M., 1983).

Cyclic AMP and cGMP are implicated in the regulation of Ca²⁺ -homeostasis. Upon binding of cAMP to its receptor, Ca²⁺ is released from both acidic and Ins(1,4,5)P3- sensitive Ca²⁺ stores leading to a capacitative Ca²⁺ -influx across the plasma membrane and an increase in cytosolic Ca²⁺ concentration (reviewed in Newell et al., 1995; Schaloske et al., 1997). Evidence for an involvement of cGMP in the regulation of Ca²⁺ -influx comes from experiments with a mutant “streamer F“and more recently with a “pdeD-KO” strain that show a prolonged and elevated cGMP response after cAMP stimulation resulting in an altered chemotactic movement and abnormally extended aggregation streams (Meima et al., 2002; Menz et al., 1991; Ross et al., 1981). Strikingly, Ca²⁺ -influx in PdeD-KO cells is inhibited while cGMP is elevated (Lusche et al. manuscript in preparation). In addition, ⁴⁵Ca²⁺ - flux studies done with a series of mutants defective in the cGMP pathway strengthened the notion that cGMP contributes to regulation of cAMP-induced Ca²⁺ -influx (Kuwayama et al., 1998).

Recently, progress has been made in understanding the metabolism of cGMP in D. discoideum (reviewed in Bosgraaf et al., 2002). Synthesis is mediated by two guanylyl cyclases, a membrane bound and a soluble form, DdGCA and DdsGC, respectively (Roelofs et al., 2001; Roelofs et al., 2002). Two cGMP-specific phosphodiesterases responsible for degradation of cGMP have been cloned (Bosgraaf et al., 2002). D. discoideum PDE3 accounts for 20% of the total cGMP- hydrolysing activity of the amoebae. The remaining 78% of cGMP-hydrolysing activity in lysates originates from an enzyme encoded by PdeD that is activated by low cGMP concentrations and to a small extent from another protein PdeE (Kuwayama et al., 2001; Saran et al., 2002). The streamer F mutant and PdeD-KO cells lack 80% of cGMP-hydrolysing activity but retained the 20 % activity corresponding to D. discoideum PDE3. In addition, genes encoding two phosphodiesterases with different substrate preferences have been sequenced.

PDE1 / PdsA is an enzyme with a higher specificity for cAMP than for cGMP. It exists as a soluble, secreted form and as a membrane-bound form reviewed in (Franke et al., 1992). In contrast, D. discoideum PDE2 / Reg A is an intracellular enzyme that specifically degrades intracellular cAMP reviewed in (Saran et al., 2002).

To study the role of cGMP in D. discoideum, we utilized known mammalian PDE inhibitors that affect either hydrolysis of cGMP or its regulation by cGMP.

Surprisingly, we observed only slight inhibition of cGMP-hydrolysis by these drugs.

Although the type V phosphodiesterase inhibitors employed in this study, act competitively with cGMP we found that these compounds altered cAMP-induced Ca²⁺ -influx. Therefore, we analysed whether they regulate other cGMP-binding proteins. Bovine PKGIα was not affected. However, the PDE5 / PDE1 inhibitors SCH 51866 (SP) and Sildenafil citrate reversibly inhibited the cGMP-induced current of a bovine cyclic nucleotide gated channel (CNGA3). This finding indicates that the Ca²⁺-channel present in D. discoideum might be directly or indirectly regulated by cGMP.

Material and Methods

Guanosine, DMSO and DTT were purchased from Sigma, St. Louis, (USA); 3’.5’,- cGMP, 3’,5’-cAMP, 5’AMP, 5’GMP, and adenosine were obtained from Boehringer, Mannheim (Germany). [8,5-³H]cGMP was obtained from Amersham,

Braunschweig and [2,8-³H]cAMP from ICN, Irvine, Ca (USA). Dipyridamol, MY 5445, Vinpocetine and Zaprinast were from Biomol, Hamburg (Germany) and MEQ, Trequinsin were from Calbiochem/Novabiochem, DMPPO was kindly provided by H. Coste, Glaxo Wellcome (France), E 4021 by M. Takeuchi and T.

Saeki, Eisai Co, Tsukuba-shi, Ibaraki (Japan). SCH 51866 by R.Watkins, Schering-Plough Research Inst. Kenilworth, NJ, (USA), SKF 96231 by S.Trowbridge, Smith Kline Beecham(UK), Sildenafil citrate by E. Bischoff, Bayer AG, Wuppertal, (Germany) and UK 114,542 by Mrs.S.D. Srodzinski, Pfizer, Sandwich, Kent (UK); PKGIα and VASPtide were kindly given by Dr. E. Butt and Prof. Dr. U.Walter, Medizinische Universitaetsklinik, Wuerzburg (Germany)

Cultivation of D. discoideum cells

Cells were grown at 23°C in axenic medium as described (Schaloske et al., 1997).

Growing PdeD-KO cells were supplemented with 10 µg/ml blasticidin.

Differentiation was induced by washing the cells two times with ice-cold 17 mM K⁺ / Na⁺ phosphate buffer. Cells were incubated at a cell density of 2*10⁷ cells/ml.

Light-scattering recordings of cells in suspension

Light-scattering measurements of cells were done as described by Gerisch and Hess (Gerisch et al., 1974). The extinction of a cell suspension (2*10⁷ cells/ml) aerated in a cuvette was monitored at 500 nm in a Zeiss PM6 spectrophotometer.

cAMP-pulses (1 µM) were applied to a cell suspension (2*10⁷ cells/ml) every 6 min. while monitoring light-scattering changes. Inhibitor was added after at least three control pulses 1-2 h before the onset of spike-shaped oscillations. For every pulse, the magnitude of the first light scattering change was determined.

Measurement of cAMP-induced Ca²⁺ -influx and K⁺-efflux

Net Ca²⁺ and K⁺-fluxes were measured after cAMP stimulation by means of an electrode sensitive for either K⁺ or Ca²⁺ in a cell suspension at 23°C as described previously (Aeckerle et al., 1985; Bumann et al., 1984). In brief, 5*10⁷ cells (Ca²⁺ - electrode) or 2.5*10⁸ cells/ml (K⁺-electrode) were incubated in nominally Ca²⁺ free buffer (Tricine pH 7.0, supplemented with 5 mM KCl for Ca²⁺ -flux studies).

Electrode potentials were recorded with a pH meter (Metrohm, Herisau, Switzerland). Ca²⁺-flux was analysed 4 -6 h and K⁺-efflux 8 -9 h after induction of starvation. cAMP was applied 5 min after the addition of SP and subsequently

cAMP pulses were given every 5 min. Inhibitors were applied in DMSO (0.1%), a concentration that neither effect basal Ca²⁺ concentration nor induced Ca²⁺-fluxes.

Extracellular cAMP-hydrolysis assay

[2,8-³H]cAMP hydrolysis was measured during spike-shaped light-scattering oscillations in the presence or absence of the inhibitor. At the maximum of a light- scattering spike 3 µCi / ml of [2,8-³H]cAMP were applied to the cells and samples were taken every 30 s. Hydrolysis was stopped by the addition of a solution containing 0.1 mM EGTA, 5 mM cAMP, 5 mM 5’AMP and 5 mM adenosine and samples were kept on ice. After centrifugation at 12100g at 4°C for 2 min. the supernatant was applied to PEI-cellulose F (Merck, Darmstadt, Germany) sheets and chromatographed in 3.5 M LiCl. Spots were visualized under UV-illumination, excised and counted in a Beckman scintillation counter.

Preparation of cell extracts

Cells differentiated for 2 h were washed three times in ice-cold Hepes buffer (20 mM, pH 7.2) and resuspended at a density of 1.5*10⁸ cells/ml. Cells were lyzed by passage through a nuclepore filter (5 µm) and collected in buffer containing 3% (w / v) sucrose, 50 mM KCl, 1 mM MgCl2, 20 µg/ml leupeptin, 1 µg/ml aprotinin and 2.5 mM DTT. Extracts were handled as described previously (Schaloske et al., 1998).

Phosphodiesterase assay

The assay was modified after Thompson et al. and Gulyassy et al. (Gulyassy et al., 1976; Thompson et al., 1974). The cGMP-hydrolysing activity in extracts of D.

discoideum is due to cGMP-specific and unspecific phosphodiesterase activities.

Unspecific phosphodiesterases utilize both cAMP and cGMP as substrate.

Unspecific activity is reduced to 5% by preincubation of extracts with 10 mM DTT (Bulgakov et al., 1983). The remaining cAMP-hydrolysing activity presumably corresponds to the cAMP-specific enzyme Reg A. Cytosolic extracts were preincubated for 10 min either with or without 10 mM DTT. The reaction-buffer contained 20 mM HEPES-buffer pH 7.2, 50 mM KCl, 1 mM MgCl2, 500 nM cGMP and 3.3 µCi/ml [8,5-³H]cGMP in a total volume of 150 µl. Reactions were started by the addition of the extract and inhibitors or DMSO as a vehicle, respectively.

Extracts were diluted until cGMP-hydrolysis of the control was linear. Samples

were taken in duplicate after 5 min of incubation at 25°C. The reaction was terminated by the addition of a stop solution containing 0.1 M EDTA, 5 mM cGMP, 5 mM 5’GMP and 5 mM guanosine. Samples were subsequently treated as described under extracellular cAMP-hydrolysis assay. The running buffer was 75 mM KCl.

Measurement of PKGIα activity

Activity of bovine PKGIα was measured according to Butt et al. (Butt et al., 1994).

Mixtures contained 20 mM Tris/HCl, 10 mM MgCl2, 5 mM 2-Mercaptoethanol, 0.01% (w/v) BSA, 50 ng PKGIα, 5 µM cGMP. Incorporation of phosphate into the specific substrate VASPtide was started by the addition of 50 µM [γ-³²P]ATP (100 cpm/pMol). Samples were incubated for 5 min at 30°C. Addition of 0.3M EDTA (pH 7.0) and application of the sample onto P-81 cellulose (Whatman) terminated the reaction. After washing with 75 mM phosphoric acid the samples were air-dried and counted. Measurements were performed either in the presence of the inhibitor or the vehicle DMSO.

Determination of total cGMP

Total cGMP concentrations of developing cells were determined using an enzyme immuno assay (Amersham Pharmacia, Freiburg, Germany). Roughly 4 h after the onset of starvation spike-shaped oscillations can be recorded by measuring light- scattering of cells in suspension. These oscillations are accompanied by spike- shaped changes in intracellular cGMP concentrations. Samples were collected during light-scattering oscillations (2*10⁷ cells / ml) before and after the addition of the inhibitor and quenched with HClO4 (1 M final concentration). This was followed by neutralization with 3 M KHCO3 (Wurster et al., 1977). Subsequently, the amount of cGMP was determined according to the manufacturer’s instructions.

Patch-clamp recordings

Currents in excised patches of HEK 293 cells stably expressing bovine cone CNGA3 channels were recorded as described (Weyand et al., 1994). In brief, patches were exposed to a solution containing 140 mM NaCl, 10 mM Hepes, 10 mM EGTA. The same solution was used in the pipette. For channel opening and inhibitor studies the appropriate cGMP concentration was added with or without inhibitor. Leak currents in cGMP free buffer solution were subtracted.

Results

Effects of SP on total cGMP concentration and on cGMP-hydrolysis In D. discoideum, light-scattering oscillations in cell suspension can be measured after about 4 hours of differentiation. Light-scattering oscillations mirror transient morphological changes of the cells accompanied by cAMP and cGMP oscillations (Wurster et al., 1989). Cyclic GMP concentrations oscillate slightly in advance of cAMP oscillations (Wurster et al., 1977). To address the question whether inhibition of cGMP-phosphodiesterases in wildtype Ax 2 cells would lead to a prolonged and elevated increase of cGMP similar to the streamer F mutants, we applied a series of known PDE inhibitors. One compound, SP, a potent PDE1/PDE5 inhibitor, caused strong alterations in spike-shaped light-scattering oscillations (data not shown). Therefore we were interested whether the cGMP concentration was also affected. In three experiments, the change in total cGMP concentration in the presence of 300 µM SP was 105 ± 39% of control. In cell extracts cGMP-hydrolysis was inhibited by only 17± 9 % using 200 µM SP and could not be further increased by 600 µM SP. SP specifically inhibited cGMP- hydrolysis as indicated by a specificity ratio of 2.5 ( Table 1). We conclude that the inhibition of cGMP-hydrolysis was not sufficient to further raise the transient increase of cGMP concentration.

Effect of different phosphodiesterase inhibitors on cGMP-hydrolysis Since SP did not inhibit cGMP-hydrolysis in D. discoideum to a similar extent as in mammals, we attempted to identify a more potent compound. Currently there are 11 families of phosphodiesterases classified by their pharmacological and kinetic profiles, substrate specificity and cellular as well as subcellular distribution (Rascon et al., 2002) and references therein.

Table 1 summarizes the results obtained for 12 different compounds tested for their potential to inhibit cGMP-hydrolysis in D. discoideum extracts.

Even at rather high concentrations, the inhibitors only modestly affected cGMP- hydrolysing activity.

The most potent drugs were Zaprinast and HL 724, yet their specificity was low.

Zaprinast inhibited cAMP-hydrolysis with equal potency (data not shown). Except for DMPPO, HL 724 and MY 5445, the majority of the drugs including Sildenafil

citrate, the most frequently used drug for PDEV inhibition, were specific for cGMP- hydrolysis.

Inhibitor Type of mammalian

PDE IC50 Concentration

(µM)

Inhibition of cGMP- hydrolysis

(%)

Specificity ratio

E4021 V 0.004 µM

(Saeki et al., 1995)

600 35 ± 2 1.9

UK114,542 V 0.002 µM

(unpublished) 300 34 ± 3 1.4

DMPPO V 0.003 µM

(Coste et al.,

1995) 300 19 ± 6 0.9

SKF96231 V 1 µM

(Murray et al.,

1991) 100 19 ± 3 4.8

MY5445 V

0.6 µM (Hagiwara et

al., 1984b) 100 5 ± 2 0.2

Sildenafil V

0.0039 µM (Rascon et al.,

2002) 75 28 ± 1 2.0

Zaprinast I,V 10 - 30 µM; 0.3

–1 µM (Silver, 1996)

600 65 ± 6 1.3

MEQ I,V

5.5 µM;

0.36 µM (Takase et al.,

1994)

300 26 ± 13 1.9

SP I,V,IX,X

0.07 µM; 0.063 µM;

1.5 µM; 1 µM (Hetman et al.,

2000;

Vemulapalli et al., 1996)

200 17 ± 9 2.5

Dipyridamol V,VI,VIII,X

0.9 µM 0.38 µM;

4.5 µM 1.1 µM (Rascon et al.,

2002)

300 23 ± 5 1.6

Vinpocetine I

21 µM (Hagiwara et

al., 1984a) 300 32 ± 6 2.6

HL724 III 250 pM

(Ruppert et al., 1982)

300 54 ± 4 1.1

Table 1: Effect of phosphodiesterase inhibitors on cGMP-hydrolysis in D.discoideum The drugs are listed according to their major target in mammalian cells. PDE5, PDE6 and PDE9 specifically hydrolyze cGMP. PDE1 A, PDE1 B and PDE10 also degrade cGMP, however, these enzymes accept also cAMP as their substrate. PDE2 is activated by cGMP and PDE3 is inhibited by cGMP.

Both enzymes preferably hydrolyze cAMP. IC50 values were taken from the literature and

references are indicated by parenthesis. The specificity ratio of cGMP-hydrolysis in the presence and absence of DTT (see material and methods) is presented at the indicated concentration. Data are means of at least three experiments ± SD.

From these experiments we conclude that the commercially available inhibitors of cGMP-specific phosphodiesterases and cGMP-regulated phosphodiesterases are poor inhibitors of D. discoideum cGMP-phosphodiesterases.

SP inhibited cAMP-induced Ca²⁺ -influx

We previously reported that D. discoideum streamer F mutants exhibit a prolonged and elevated Ca²⁺ -influx accompanied by an elevated and prolonged cGMP concentration after cAMP stimulation (Menz et al., 1991). Therefore, we performed recordings of cAMP-induced Ca²⁺-influx from cells in suspension in the presence of SP. SP inhibited cAMP-induced Ca²⁺-influx to more than 50% without altering the kinetics. Figure 3 shows the dose-response curve for the inhibition of cAMP- induced Ca²⁺ -influx by SP. The IC50 for SP was 16 µM and maximum inhibition occurred at 50 µM SP. The incomplete inhibition of cAMP-induced influx might be due to the existence of more than one type of Ca²⁺ -channel (see discussion).

Figure 3: Dose response curve for SP-induced inhibition of cAMP-activated Ca²⁺-

influx.Measurements were done as described in Table 3 (n ≥ 3). Cells were stimulated with 0.1 µM cAMP after preincubation with SP for 5 min. Half maximal inhibition occurred at 16 µM SP.

Since SP is a competitive PDE V inhibitor, we hypothesized that the competition with cGMP was the reason for the reduction of cAMP-induced Ca²⁺ -influx. The possible existence of a cGMP-regulated Ca²⁺ channel was further supported by experiments done with the soluble guanylyl cyclases inhibitor LY 83583. This drug is also known to block cyclic nucleotide gated channels (Leinders-Zufall et al., 1997). The presence of 35 µM LY 83583 reduced cAMP-induced Ca²⁺ -influx by

Concentration (µM)

0 20 40 60 80 100

Ca2+-influx (% control)

0 20 40 60 80 100

34 ± 6% in three experiments without altering cGMP concentration as shown in Figure 4.

Figure 4: LY83583 inhibited cAMP-induced Ca²⁺-influx but did not effect transient cGMP elevations after cAMP-stimulation.One out of three experiments is shown. The addition of cAMP to a cell suspension induced a Ca²⁺ -influx peaking after 30 seconds. LY83583 was applied for 5 minutes before the first cAMP-pulse was given. Samples for determination of total cGMP were taken before and after application of cAMP. The cGMP concentration increased only for 5 ± 9% compared to untreated cells. Experiments were performed and samples processed as described in Material and Methods (n=3).

The inhibitory effect of SP was specific for the cAMP-induced Ca²⁺ -influx, as shown in Table 2.

SP

(µM) Inhibition

(%)

100 -2 ± 28

150 17 ± 6

Table 2: SP did not inhibit cAMP-induced K⁺-efflux. K⁺-efflux was induced by 1 µM cAMP.

Experiments were conducted as described in Material and Methods. K⁺-efflux amounted to 43 ± 15 µM. Data represent means of 3 experiments.

We measured cAMP-induced K⁺ efflux in the presence and absence of the inhibitor. At 100 µM SP, a concentration that maximally inhibited cAMP-induced Ca²⁺ - influx, SP did not change concomitant K⁺-efflux.

Effect of SP on cAMP-induced light-scattering changes, extracellular cAMP-hydrolysis and PKGIα

Given the fact that SP competes with cGMP for its binding site and the lack of an SP-induced cGMP increase, we wondered whether the cause for the inhibition of cAMP-induced Ca²⁺ -influx by SP is mediated by a cGMP-binding protein.

Unspecific binding of SP to the cAMP receptor cAR1 via its guanine base moiety was excluded as the height of the amplitude of the light-scattering responses upon addition of cAMP remained unchanged in the presence of the inhibitor. The height of the amplitude in the presence of 300 µM SP was 99.5 ± 2.5% compared to the control amplitude in four experiments. SP therefore acts either downstream of the receptor or possibly on the extracellular phosphodiesterase.

To test for inhibition of ePDE by SP we measured [2,8-³H]cAMP-hydrolysis during light scattering oscillations. 300 µM SP only weakly affected cAMP-hydrolysis with 18 ± 15% inhibition after 30 s and only 6 ± 10% after 60 s. It is unlikely that this inhibition accounted for the observed changes in the oscillatory pattern, since cAMP was readily degraded within 90 s long before the next spike arose at 420 s.

We then analysed whether SP targeted a PKG. Since the activity of the partially purified D.discoideum PKG is very unstable (Wanner et al., 1990), the activity of bovine PKGIα was measured (Butt et al., 1994).

Concentration (µM)

Phosphorylation (µmol / mg*ml)

Inhibition (%)

Control SP

75 0.8 ± 0.2 0.9 ± 0.2 -12

150 0.5 ± 0.2 0.5 ± 0.2 0

300 0.5 ± 0.2 0.6 ± 0.1 -12.5

Table 3: Measurement of PKGIα activity Activity was tested in the presence and absence of SP or vehicle, respectively. After [γ-³²P]ATP incorporation into VASPtide radioactivity was counted and the activity of PKGIα determined. Data represent means of at least 4 experiments.

Table 3 shows that there was no significant inhibition of the cGMP-activated enzyme at concentrations of SP that already affected light-scattering oscillations.

SP is an inhibitor of bovine CNGA3

Another target for SP could be a cyclic-nucleotide-gated cation channel. These proteins contain a cyclic nucleotide-binding site at their C-terminus. Moreover, although CNG channels are nonselective cation channels in vitro, the currents recorded from these channels are mainly carried by Ca²⁺ in vivo (Ohyama et al.,

2000). Since no CNG channels were identified in D. discoideum to date, we used bovine CNGA3 stably expressed in HEK 293 cells to record cGMP-induced currents. Indeed, inhibition of the current occurred when SP was applied to the intracellular side of excised patches in the presence of cGMP as shown in Figure 5a,b.

Figure 5: Current recordings of CNGA3 stably expressed in HEK 293 cells. Sodium-currents through CNG-channels in inside-out patches were induced by the addition of cGMP. SP (100 µM) was applied in the presence of 7 µM and 100 µM cGMP as indicated by the beams. Reversibility was tested by washing the patch with buffer solution. One out of 5 experiment over time is shown for a patch clamped at a) positive and b) negative holding potential (-40 mV), respectively. A I-Vm relation of a single cell out of three independent patches is shown in c). Recordings for the SP treated and untreated patch are shown.

Without SP, channels were completely opened by excess cGMP (500 µM). 30 % of the maximal current was recorded at 7 µM cGMP which is close to the km of 8.3 µM measured by Weyand et al. (Weyand et al., 1994). The current at 7 µM was reduced by 100 µM SP applied to the patch. The inhibition in the presence of 100 µM SP was stronger at positive compared to negative potentials (Figure 5a, b).

Inhibition by SP could be reversed by the addition of cGMP in the absence of the inhibitor. Inhibition was generally stronger at a positive than at a negative potential as shown by the I-Vm relation in Figure 5c, indicating that the charge of the SP molecule partly contributes to the inhibitory effect on the channel. Figure 6a shows a dose response curve for the inhibition by SP at +40 mV.

Figure 6: SP competitively and dose-dependently inhibited CNGA3. a) Dose-response curve for SP-mediated inhibition of CNGA3. Ion currents were induced by application of 50 µM cGMP (n ≥ 5). b) Lineweaver-Burke diagram generated for currents induced by variable amounts of cGMP in the presence of 100 µM SP at positive holding potential (n ≥ 5). Data in the linear range of the dose-response curve were calculated using the Hill-equation (Weyand et al., 1994).

Half maximal inhibition of CNGA3 occurred at 50 µM SP. Inhibition was complete at 500 µM SP. Since increasing amounts of cGMP reduced the inhibitory effect of SP, we conclude that both compounds compete for the same binding site. We found that the activity of CNGA3 in the presence of 100 µM SP could be completely restored by the addition of excess cGMP (data not shown). A

SP (µM)

0 100 200 300 400 500 600

Inhibition (%)

0 20 40 60 80 100 120

50 µM cGMP

1 / cGMP (µM-1)

-0,10 -0,05 0,00 0,05 0,10 0,15 0,20 1/ I (pA-1)

0 1 2 3 4 cGMP 5 SP a

b

Lineweaver-Burke plot (Figure 6b) confirmed competition. Maximal currents remained unchanged whereas the km shifted to higher concentrations of cGMP.

Effects of Sildenafil citrate and its derivative on bovine CNGA3 and cAMP-induced Ca²⁺-influx in D. discoideum

Two of the most potent inhibitors of PDEV, Sildenafil citrate and another inhibitor of cGMP-phosphodiesterase, UK 114,542, affected light-scattering oscillations similar to SP in D. discoideum (data not shown). Both drugs like SP inhibited cAMP-induced Ca²⁺-influx in D.discoideum for more than 50 % as shown in Table 4.

Inhibitor Concentration

(µM)

Inhibition (%)

SP 50 54 ± 18

Sildenafil 100 41 ± 6

UK 114,542 150 44 ± 6

Table 4: cAMP-induced Ca²⁺ -influx was inhibited by phosphodiesterase inhibitors. The inhibition by different phosphodiesterase inhibitors was assessed 4 to 6 hours after induction of

development. Cyclic AMP-induced Ca²⁺-influx was measured as described in Material and

Methods. Pulses of 0.1 µM cAMP were applied in the presence or absence of inhibitor at the same extracellular Ca²⁺-concentration. After preincubation with inhibitor for 5 min the first pulse of cAMP was added. Extracellular Ca²⁺ concentration ranged from 1-3 µM. Data represent means of at least 3 experiments (SP n=5).

This finding prompted us to analyse the effects of these compounds on CNGA3.

Sildenafil also proved to inhibit CNGA3, as shown in Figure 5. Channel activity was reduced by 58 ± 14% with 30 µM Sildenafil at +40 mV in three experiments.

Inhibition could be reversed by replacing the bathing solution containing the inhibitor with a mock solution. At –40 mV Sildenafil was less potent (Figure 7a).

The derivative of Sildenafil, UK 114,542, did not inhibit cGMP-induced currents (data not shown), indicating structural requirements for the inhibition of CNGA3 by SP and Sildenafil. These data reveal that the competitive phosphodiesterase inhibitors SP and Sildenafil are novel inhibitors of CNGA3.

Figure 7: Sildenafil-induced inhibition of CNGA3. Sildenafil (30 µM) was applied to the bathing solution in the presence of 10 µM cGMP. One out of three experiments at negative (a) as well as at positive (b) holding potential is shown.

The antagonism of SP on cAMP-induced Ca²⁺-influx is reversed by cGMP

In a PdeD-KO strain cGMP concentrations are higher than in the parental strain and the cGMP transients are prolonged (Meima et al., 2002). Receptor-mediated Ca²⁺-influx, however, was delayed and smaller than in the wildtype (Lusche and Malchow manuscript in preparation). We used this mutant strain to investigate whether the elevated cGMP concentration acts either in concert or competes with SP for a regulatory site of Ca²⁺-influx. Table 5 shows that 25 µM SP, that blocks about 40% of Ca²⁺-influx in the wildtype and is a concentration within the linear range of the dose response curve, did not alter Ca²⁺-influx in the mutant.

Ca²⁺-influx (pmol/10⁷ cells)

Inhibition (%) Control SP

25 ± 3 41 ± 7 -64

45 ± 4 61 ± 3 -35

22 ± 1 16 ± 2 27

32 ± 1 30 ± 4 7

Table 5: SP inhibition was absent in PdeD-KO cells. Ca²⁺ -influx was measured as described for wt cells. 4 independent experiments are shown. Compared to the control the mean influx amounted to 116 ± 35%.

We infer from this experiment that SP and cGMP do not act independently but rather seem to compete for the same site.

Discussion

We demonstrated that two potent cGMP-phosphodiesterases antagonists are also inhibitors of bovine cone CNGA3. SP as well as Sildenafil competed with the physiological ligand cGMP for regulation of CNGA3 in excised patches of HEK 293 cells, albeit the concentration was approximately 3000 times higher for Sildenafil than required for inhibition of mammalian PDE5 (Rascon et al., 2002).

For scrutinizing signalling pathways involving CNG-channels there is a need for specific drugs that alter channel activity. Inhibitors have been described for CNG channels (Kleene, 1994; Leinders-Zufall et al., 1997), yet none of them was found to act competitively. The most widely used inhibitor, L-cis-diltiazem, is a pH, voltage and subtype specific CNG-channel blocker acting at the cytoplasmic site of the channel (Lee et al., 2001) and references therein. Polyamines were also reported to block CNG-channels but competition with the ligand was not tested for (Lu et al., 1999). SP and Sildenafil inhibited CNGA3 at micromolar concentrations.

However, the drugs might also block other CNG-channel subtypes more potently.

Although the phosphodiesterase inhibitors share some structural similarities, the drugs do not generally inhibit CNG-channels. Rather, the inhibition required structural prerequisites in addition to the common guanine base moiety. Thus UK 114,542 did not inhibit CNGA3 at all. The specificity of CNG-channel inhibition by Sildenafil and SP with respect to other phosphodiesterase inhibitors is supported by Womack et al. who did not find inhibition of PtdIns(4,5)P2 and ATP regulated currents through rod CNG-channels by Zaprinast and IBMX in oocyte patches (Womack et al., 2000).

Inhibition of phosphodiesterases has been subject to intensive investigations. The design of specific compounds for potential clinical treatments continues to be necessary because of the variety of PDEs present in various tissues. Albeit the specific and effective inhibition of mammalian PDEs, the inhibitors tested failed to potently reduce cGMP-hydrolysis in Dictyostelium discoideum. Either much higher concentrations were required and/or the specificity was low. Taken together, we conclude that the cGMP-activated cGMP-phosphodiesterase activity in D.

discoideum extracts represent unusual enzymes that are different to their mammalian counterparts. Recently genes corresponding to the majority of the cGMP-hydrolysis activity have been cloned (Bosgraaf et al., 2002 and references therein). Their detailed biochemical characterisation remains to be investigated.

However, GpbA / PdeD and GpbB / PdeE are phosphodiesterases with a Zn²⁺ - binding domain. Both of them might be targeted by Zaprinast, a putative Zn²⁺

chelator, and the most potent inhibitor in this study. The lack of inhibition by the available mammalian PDE inhibitors could be due to structural differences in the cGMP-binding sites.

Unexpectedly, although SP caused slight but specific inhibition of cGMP- hydrolysis, there was no additional increase of the cGMP concentration evident during spike-shaped oscillations. An increase in the cGMP concentration was therefore not the cause for the observed SP-induced changes in light-scattering oscillations. This lack of increase in total cGMP is in contrast to mammalian cells, where small amounts of the PDE V inhibitors elevate intracellular cGMP- concentrations in a variety of tissues. For instance, SP competitively inhibited PDE1 and PDE V leading to an increase in intracellular cGMP-concentration in blood platelets. Thus, SP prevented collagen-induced platelet aggregation and proliferation of vascular smooth muscle cells in vitro (Vemulapalli et al., 1996).

Sildenafil inhibited PDE V in smooth muscle of the corpus cavernosum leading to an NO-dependent increase in cGMP, muscle relaxation and subsequent increase in blood flow resulting in penile erection, (reviewed in Maggi et al., 2000).

Nevertheless, the competitive nature of these drugs with respect to cGMP led us to presume that in D. discoideum the putative target (s) responsible for the SP- induced alterations of the light-scattering spikes might contain a cGMP-binding site.

We then found that PDE inhibitors can alter Ca²⁺ -homeostasis in D. discoideum.

SP and also Sildenafil strongly inhibited cAMP-induced Ca²⁺ -influx. This implies the presence of a cGMP-regulated step in the signalling cascade leading to Ca²⁺ - influx. In excised patches of D. discoideum ion channels could not be observed reproducibly due to the difficulties in patch formation (Mueller et al., 1986).

Experimental access to a possible cGMP-binding site of plasma membrane channels is therefore restricted. The Ca²⁺ -influx is triggered by cAMP partly via a G-protein dependent and partly via a G-protein independent pathway. Long chain fatty acids contribute to the regulation of Ca²⁺ -entry by releasing Ca²⁺ from the stores and/or possibly by a regulation of calcineurin A ultimately leading to capacitive Ca²⁺-entry (Kessen et al., 1999). Two types of Ca²⁺ -stores, the acidosomes and Ins(1,4,5)P3 sensitive stores, are involved in the regulation of

Ca²⁺ -influx. Ca²⁺ -influx was observed to be connected to the cGMP metabolism.

Streamer F mutants devoid of 78% of the cGMP-hydrolysing activity exhibited a prolonged cAMP-induced cGMP increase and an elevated and prolonged Ca²⁺ - influx. SP Inhibition of Ca²⁺-influx was maximal with 60% reduction. This indicates that cAMP-induced Ca²⁺ -influx is only partly regulated by cGMP.

As known from other organisms it is possible that cGMP acts either directly or indirectly at a plasma membrane channel or at Ca²⁺ -stores. Indirect effects might be mediated by PKG. PKG mediated regulation of L-type Calcium channels and modulation of Ins(1,4,5)P3-induced Ca²⁺ -release were reported (Biancani, 1998;

Jiang et al., 2000). Given the very small activity changes of bovine PKGIα, we conclude that SP less likely affects D.discodieum PKG. However, the participation of D.discodieum PKG cannot be excluded as the specificity of this enzyme may differ from the mammalian PKG.

In vivo, Ca²⁺ carries considerable fractions of currents through CNG channels in rods and cones (Ohyama et al., 2000). A similar type of Ca²⁺ -permeable channel regulated by cGMP and targeted by SP and Sildenafil might exist in D.

discoideum. In favour of this hypothesis we have shown that LY 83583 also inhibited cAMP-induced Ca²⁺ -influx in D. discoideum in the absence of an increase in the cGMP-concentration. LY 83583 was shown to inhibit CNG- channels directly in salamander olfactory receptor neurons with a Kd of 1,4 µM, presumably by acting inside the channel pore (reference in Leinders-Zufall et al., 1997). A regulation of Ca²⁺ -influx by cGMP is an agreement with the time course of both responses to cAMP. The cGMP rise is maximal after 10 seconds while Ca²⁺ -influx peaks 30 seconds after cAMP stimulation.

Since SP did not affect K⁺-fluxes this also argues against an unspecific binding of the drug to cAR1 that should prevent cAMP-induced Ca²⁺ -influx. Inhibition of extracellular cAMP-hydrolysis and therefore possible inhibitory cAMP accumulation did also not occur in the presence of SP. With respect to the finding that SP and Sildenafil inhibited CNGA3 and that inhibition could be reversed by excess cGMP we propose that cGMP participates in the regulation of Ca²⁺ -influx at the plasma membrane. In support of this we found that inhibition of Ca²⁺-influx by SP in the wildtype was absent in a mutant with elevated cGMP concentrations, indicating that cGMP competes with SP for a regulatory site.

Although the IC50 for inhibition of cGMP-induced currents of the bovine photoreceptor channel by SP and Sildenafil were in the micromolar range, small amounts of SP and Sildenafil at the target site might already interfere with CNG- dependent pathways in vivo. Watkins et al reported an increase of SP concentration of up to 5 µM after 2 hours in blood platelets after injection of 10 mg SP /kg in rats. Sildenafil is reported to cause headache and facial flushing (Cheitlin et al., 1999). It was also shown to affect vision (Laties et al., 2002). Sildenafil is readily taken up and distributed in the tissue. Plasma levels account to 40%

bioavailability and are enhanced in patients older than 65 years and in patients with hepatic impairment. According to this and another report high levels of Sildenafil are bound to plasma proteins (Nichols et al., 2002) and the free plasma concentration calculated to 43nM is low. However, the bound Sildenafil makes up a large reservoir and might also bind to proteins within the membrane. It will also be interesting to elucidate the potency and selectivity of these drugs on different subtypes of CNG-channels as well as on native channels and to perceive the consequences for patients following the application of these drugs. The results of this study should also encourage the search for derivatives of SP and Sildenafil that might act more potently on CNGA3 and presumably on other CNG-channel subtypes.

Acknowledgments

We thank the DFG for financial support and E.Jaworski for expert technical assistance. M.Meima and P.Schaap provided PdeD KO cells, D. F. Lusche received a fellowship from the FAZIT foundation. We thank C.Schlatterer, R.

Schaloske and T.Schmidt-Petri for critically reading of the manuscript