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Protistology 6 (2), 103-110 (2009)
Protistology
Role of Ca2+ and cAMP in a cell signaling pathway for resting cyst formation of ciliated protozoan Colpoda cucullus
Tatsuomi Matsuoka, Asuka Kondoh, Kunihisa Sabashi, Nobuaki Nagano, Takahiko Akematsu, Akemi Kida and Ryota Iino
Institute of Biological Science, Faculty ofScience, Kochi University, Kochi, Japan
Summary
Resting cyst formation (encystment) of Colpoda cucullus is caused by an increase in an external Ca2+ concentration or overpopulation of Colpoda vegetative cells. The Ca2+-mediated or overpopulation-mediated encystment was suppressed by Ca2+ channel blockers (Cd2+, La3+, Ni2+), Ca2+-chelating reagents (EGTA, BAPTA), calmodulin antagonists (W-7, trifluoperazine), ^p-cAMPS (an cAMP analog antagonist) and 2’-deoxyadenosine (a P-site inhibitor of adenylate cyclase). On the other hand, by the addition of Ca2+ ionophore A23187, IBMX (an inhibitor of phosphodiesterase), cAMP or its membrane-permeable derivative, the encystment was prominently induced even in the Ca2+-free medium. These results suggest that Colpoda encystment may be mediated by an increase in cAMP concentration through the activation of adenylate cyclase whose activity is possibly regulated by Ca2+/calmodulin
Key words: cAMP, Ca2+/calmodulin, cyst formation, Colpoda
Introduction
As already described by Gutiérrez et al. (2001), Matsusaka (2006) and perhaps other protozoologists, resting cysts of protozoa is one of the cryptobiotic forms, the process of whose formation involves a gene-regulated cyto-differentiation (Matsusaka, 1979; Matsusaka et al., 1989; Martín-González et al., 1991; Gutiérrez et al., 2000; Izquierdo et al., 2000) and cytoplasmic dehydration prior to a stop of metabolic activity. To survive on the soil surface where the puddles temporarily appear and rapidly
dry out, the terrestrial colpodid ciliates transform into resting cysts resistant to drying, freezing and higher temperatures (Taylor and Strickland, 1936; Foissner, 1993; Gutiérrez, 2001; Maeda et al., 2005), and they excyst to proliferate when favorable conditions are regained. The resting cyst formation (encystment) is induced by an increase in mainly external Ca2+ concentration (a signal for coming desiccation) in Colpoda cucullus (Yamaoka et al., 2004) or the overpopulation of vegetative cells in C. duodenaria (Strickland, 1940) and C. cucullus (Maeda et al., 2005). The Ca2+-induced encystment
© 2009 by Russia, Protistology
of Colpoda is, on the other hand, suppressed by components released from bacteria (a signal indicating the existence of food) (Yamasaki et al., 2004) and components contained in plant leaves such as peptides, porphyrins, etc. (Tsutsumi et al., 2004; Yamasaki et al., 2004).
The purpose of the present study is to elucidate the signaling pathway leading to Colpoda encyst-ment which is activated by the addition of Ca2+ in the surrounding medium or a cell-to-cell stimula-tion with Colpoda vegetative cells. We revealed, by means of Ca2+ channel blockers (Kasai and Neher, 1992; Mlinar and Enyeart, 1993; Leech et al., 1994; Lee et al., 1999; Triggle 2006), Ca2+ chelating reagents and Ca2+ ionophore A23187 (Reed and Lardy, 1972), calmodulin antagonists such as W-7 (Hidaka et al., 1981) or trifluoperazine (Vandonselaar et al., 1994), that the signaling pathway leading to encystment might be activated by an Ca2+/calmodulin (Ca2+/ CaM). In addition, we obtained results suggesting that an increase in intracellular cAMP concentration was involved in encystment induction. In the present paper, a partial process of an encystment-inducing signaling pathway including Ca2+/CaM and cAMP will be discussed.
Material and methods
Colpoda cucullus was cultured in 0.05% (w/v) cereal infusion inoculated with bacteria (Entero-bactor aerogenes) as food. The bacteria, which were supplied by the Institute for Fermentation, Osaka, Japan, were cultured on agar plates containing 1.5% agar, 0.5% polypepton, 1% meat extract and 0.5% NaCl. For encystment induction, vegetative cells cultured for 1-2 days were collected by centrifugation (1,500 g, 2 min), rinsed twice in 1 mM Tris-HCl (pH 7.2), and then transferred into 200 ^l of test solutions at cell density of 500-1,000 cells/ml or 5,000~10,000 cells/ml (overpopulation).
The number of encysted cells were counted in 80-120 randomly chosen per measurement, and were expressed as a percentage of the total number of cells (80-120 cells). Columns (points) and attached bars correspond to the means of 5-7 identical measurements (the number of measurements shown in the figures) and standard errors. All test solutions contained at least 1 mM Tris-HCl (pH 7.2) (Figs 1, 2, 3B, 4, 5, 6A) or both 1 mM Tris-HCl (pH 7.2) and 0.1% DMSO (Figs 3A, 3C, 6B). Other components contained in the test solutions were labeled in the figures. The rates of encystment (%) were measured at 7-8 hr after the onset of induction. Asterisks (*) and double asterisks (**) in figures show significant
differences among columns at p < 0.05 and p < 0.01, respectively (Mann-Whitney test).
N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride (W-7 hydrochloride), verapamil hydrochloride, diltiazem hydrochloride, 3-isobutyl-1-methylxanthine (IBMX), 2’-deoxy-adenosine monohydrate (2’-dA), forskolin and adenosine 3’, 5’-cyclic monophosphothioate were purchased from Wako Pure Chemical Industries, A23187 (free acid), ethylenebis (oxy-2, 1-phenylenenitrilo) tetraacetic acid tetra (acetoxy-methyl) ester (BAPTA-AM) from Calbiochem, N6, 2’-O-dibutyryladenosine 3’, 5’-cyclic monophosphate sodium salt (db-cAMP), N2, 2’-O-dibutyrylguanosine 3’, 5’-cyclic monophosphate sodium salt (db-cGMP), adenosine 3’, 5’-cyclic monophosphate sodium salt monohydrate (cAMP), guanosine 3’, 5’-cyclic monophosphate sodium salt (cGMP) from Sigma-Aldrich, Inc., ^p-isomer (^p-cAMPS) from Biolog Life Sci. Inst. and trifluoperazine dihydrochloride (TFP) from MP Biomedicals, LLC. BAPTA-AM, forskolin and A23187 were dissolved in dimethyl sulfoxide (DMSO) to give 10 mM, 10 mM and 1 mM stock solutions, respectively, and 10 ^l of each stock solution was added into 10 ml of 1 mM Tris-HCl (pH 7.2) to produce final concentrations of test solutions at 10 ^M, 10 ^M and 1 ^M, respectively (final concentration of DMSO at 0.1%).
Results and Discussion
Ca2+-mediated encystment induction and effects of Ca2+ channel blockers
It has been previously reported that 1 mM Ca2+ could induce encystment of C. cucullus (Yamaoka et al., 2004), but the minimum Ca2+ concentration for inducing encystment is still unknown. In the present study, first, the encystment-inducing effects of several salts (CaCl2, NaCl, KCl, MgCl2) in the surrounding medium were investigated at concentrations raging from 1 ^M to 1 mM. At concentrations less than 1 mM, the external NaCl, KCl or MgCl2 hardly showed an encystment-inducing activity, while much lower concentrations of CaCl2 had marked encystment-inducing activity (even 10 ^M CaCl2 was effective for encystment induction) (Fig. 1). As previously suggested (Yamaoka et al., 2004), the results also indicate that the effect of CaCl2 may not be due to Cl-, but to Ca2+ at least in these concentrations. In order to examine whether or not the increase in external Ca2+ concentration may cause inflow of Ca2+ through Ca2+ channels,
Fig. 1. Effects ofvarious salts (ions) on the induction of Colpoda encystment. Every solution contained 1 mM Tris-HCl (pH 7.2) and various concentrations of salts.
the effects of Ca2+ channel blockers such as divalent or trivalent cations (Mn2+, Cd2+, La3+, Ni2+, La3+), verapamil and diltiazem on encystment induction were examined (Fig. 2). As shown in Fig. 2, Ca2+-mediated encystment was significantly suppressed by the addition of Cd2+, La3+ or Ni2+ into the surrounding medium, while Mn2+, verapamil and diltiazem did not affect encystment. The inhibitory effects of divalent and trivalent metal ions against several types of Ca2+ channels are not so specific (Kasai and Neher, 1992; Mlinar and Enyeart, 1993; Leech et al., 1994; Lee et al., 1999), while verapamil and diltiazem are specific blockers against a subclass of voltage-gated Ca2+ channels, L-type channels (Triggle, 2006). In consequence, the Ca2+ channels involved in the induction of encystment of Colpoda may not be L-type Ca2+ channels.
Effects of chelating reagents or ionophore for Ca2+ on encystment induction
Fig. 2. Effects of Ca2+ channel blockers on the induction of Colpoda encystment. A - divalent (100 ^M Mn2+, 1 ^M Cd2+, 1 ^M Ni2+) and trivalent cations (1 ^M La3+); B - specific blockers (10 ^M diltiazem and 10 ^M verapamil) for L-type Ca2+ channels. The drug concentrations used in this assay are critical for the survival of the cells.
The hydrophobic compound BAPTA-AM permeates into the cell interior, and rapidly splits by means of esterases contained in the cells to form the hydrophilic Ca2+ chelator BAPTA which is trapped by the cells. Loading cells with BAPTA-AM, therefore, is expected to suppress a rise in intracellular Ca2+ concentration. If the intracellular Ca2+ concentration is assumed to be less than 5 X 10-6 M, the presence of 10 ^M BAPTA in the cytoplasm will reduce the free Ca2+ concentration to less than 2 x 10-7 M. As shown in Fig. 3A, Ca2+-mediated or overpopulation-mediated (Fig. 3A, right two columns) encystments were significantly
suppressed by the addition of 10 BAPTA-AM in the surrounding medium. If the concentration of contaminating Ca2+ in the surrounding medium is assumed to be 10-6 M, the addition of 10 ^M EGTA (final concentration) reduces the free Ca2+ concentration to about 10-8 M. The addition of 10 ^M (final concentration) EGTA in the external medium also significantly suppressed overpopulation-mediated encystment (Fig. 3B). These results suggest that overpopulation with Colpoda vegetative cells may cause encystment through an increase in intracellular Ca2+ concentration that is resulted from an inflow of Ca2+ across the plasma membrane.
Fig. 3. Effects of intracellular (BAPTA), extracellular Ca2+ chelator (EGTA) or Ca2+ ionophore A23187 on the induction of Colpoda encystment. A - BAPTA-AM (1G ^M); B - EGTA (1G ^M); C - Ca2+ ionophore A23187 (1 ^M).
Some of the Colpoda encysted even in Ca2+-free Tris-HCl buffer (pH 7.2) (Figs 3A, B; leftmost columns labeled ‘None’). Such spontaneous en-cystment was significantly suppressed by BAPTA or EGTA (Figs 3A, B, leftmost two columns), implying that the spontaneous encystment is possibly mediated by a few Ca2+ contaminating the surrounding medium. When Colpoda vegetative cells were suspended in a Ca2+-free Tris-HCl buffer (pH 7.2), encystment induction was promoted by the addition ofionophore A23187 (Fig. 3C). On the other hand, encystment induced by the addition of
G.1 mM or 1 mM Ca2+ was not further promoted (Fig. 3C). Presumably, in the presence of G.1 mM or 1 mM Ca2+, enough Ca2+ may be supplied
into the cytoplasm for the maximum encystment induction.
Effects of calmodulin antagonists on Ca2+
-MEDIATED ENCYSTMENT INDUCTION
The effects of calmodulin antagonists W-7 and trifluoperazine (TFP) on Ca2+-mediated encystment were examined. As shown in Fig. 4, the encystment-suppression effect of W-7 was rather prominent, although TFP significantly suppressed Ca2+-mediated encystment. These results (Figs 1-4) suggest that a rise in free Ca2+ concentration in an external medium elicits an inflow of Ca2+ through Ca2+ channels which may activate Ca2+-binding
Fig. 4. Effects of 1 ^M W-7 (A) and 0.5 ^M trifluoperazine (B) on the induction of Colpoda encystment. The drug concentrations used in this assay are critical for the survival of the cells.
proteins such as calmodulin. It has been reported that the induction of the encystment of Entamoeba is Ca2+/calmodulin (Ca2+/CaM) dependent (Makioka et al., 2GG1). It is presumed that a signaling pathway leading to the encystment of protozoans may involve a common Ca2+/CaM dependent process.
Role of c AMP in encystment induction
In the presence of 3-isobutyl-1-methylxanthine (IBMX), which is a non-selective inhibitor of phosphodiesterases (Mehats et al., 2GG2), encystment was markedly promoted even in a Ca2+-free medium
Fig. 5. Role of cAMP in Colpoda encystment induction. A - Effects of IBMX (500 ^M); B nucleotides and their derivatives (500 ^M); C - effects of 500 ^M ^p-cAMPS.
effects of cyclic
Fig. 6. Effects of a P-site inhibitor or an activator of adenylate cyclase on Colpoda encystment induction. A - Effects of 1G ^M 2’-deoxyadenosine (2’-dA) which is a P-site inhibitor; B - effects of 1G ^M forskolin (an activator of adenylate cyclase).
(Fig. 5A). This result indicates that a certain cyclic nucleotide may be involved in encystment induction. Therefore, the encystment-inducing effects of cAMP, cGMP or their membranepermeable derivatives (db-cAMP, db-cGMP) were examined. As shown in Fig. 5B, db-cAMP and cAMP remarkably promoted encystment in the Ca2+-free medium. On the other hand, Rp-cAMPS which is a cAMP antagonist (Rothermel et al., 1983, Botelho et al., 1988) tended to suppress the encystment (Fig. 5C). The results suggest that an increase in the intracellular cAMP concentration may occur in a signaling pathway leading to the encystment. Some mammalian adenylate cyclase isoforms are activated by Ca2+/CaM (Sunahara and Taussig, 2GG2). In a signaling pathway for the encystment induction of Colpoda, Ca2+/CaM may also stimulate adenylate cyclase. Actually, as shown in Fig. 6a, the Ca2+-mediated or overpopulationmediated encystment was significantly suppressed by the addition of 2 ’-deoxyadenosine (2 ’ -dA) which is a P-site inhibitor of adenylate cyclase (Fujimori and Pan-Hou, 2GG5). However, encystment was not promoted by the addition of forskolin which is a stimulator of adenylate cyclase (Seamon et al., 1981). One of the mammalian membrane-bound adenylate cyclase isoforms and a soluble adenylate cyclase are not potently stimulated by forskolin (Sunahara and Taussig, 2GG2). The putative adenylate cyclase of Colpoda may be insensitive to forskolin, although it cannot be concluded whether it is of the membrane-bound or soluble type.
Fig. 7 shows a presumably early process of the signaling pathway leading to the encystment
induction of Colpoda, drawn based on the present results. When the external Ca2+ concentration is raised, Ca2+ probably flows into the cell interior through Ca2+ channels. The fact that overpopulation-mediated encystment is suppressed by an intracellular Ca2+ chelator BAPTA (Fig. 3A, right two columns) suggests that cell-to-cell mechanical stimulation (Maeda et al., 2005) with Colpoda cells may increase the intracellular Ca2+ concentration by promoting an inflow of Ca2+ or a release of Ca2+ from intracellular stored vesicles. Intracellular Ca2+ is expected to activate calmodulin through the formation of Ca2+/calmodulin complex (Ca2+/ CaM), which may raise the cAMP concentration by the activation of adenylate cyclase (Sunahara and Taussig, 2002), although the possibility that the Ca2+/CaM suppresses phosphodiesterase activity cannot be eliminated. On the other hand, it is possible that cAMP production and following activation of cAMP-dependent proteins may be located upstream of Ca2+/CaM in the signaling pathway for the encystment induction. That is, the cell-to-cell stimulation due to overpopulation with Colpoda may evoke an elevation of intracellular Ca2+ concentration through proteins regulated by cAMP such as PKA or Epac (Bos, 2003). Therefore, further work will involve determining, by means of in vitro quantitative cAMP assays, changes in the intracellular cAMP concentration caused by the addition of Ca2+ to the external medium. In addition, changes in intracellular Ca2+ concentration must be visualized when the encystment is induced by the addition of Ca2+ to the external medium, by overpopulation with Colpoda cells, or
Fig. 7. A schematic diagram showing an early signaling pathway for encystment induction.
by the addition of membrane-permeable cAMP derivatives.
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Address for correspondence: Tatsuomi Matsuoka.Institute of Biological Science, Faculty of Science, Kochi University, Kochi 780-8520, Japan, e-mail: tmatsuok@cc.kochi-u.ac.jp
