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Biol Cell (I 996) 86,3Y43 @ Elsevier. Paris

Original article

Inhibition of trichocyst exocytosis and calcium influx in Paramecium by amiloride and divalent cations Daniel Kerbceuf, Jean Cohen * Centre de Gen&ique Moleculaire, assock? 6 1’Universite Pierre et Marie Curie, Centre National de la Recherche Scientifique, Avenue de la Terrasse, 91198 Gif-sur-Yvette cedex, France (Received5 February1996; accepted15 April 1996) Summary - Regulated exocytosis of defensive secretory organelles, the trichocysts, as well as a transient Ca*+-influx can be induced in Paramecium by aminoethyldextran (Kerbceuf and Cohen, J Cell Biot (1990) 11 I, 2527). Knoll et al (Febs Lett (1992) 304, 265) reported that veratridine was also a secretagogue for Paramecium. Here we show that, like aminoethyldextran, veratridine induces a transient Ca*+-influx. Both aminoethyldextran- and veratridine-induced exocytosis and associated Ca*+-influx were: i) blocked in the &Z2 thermosensitive mutant at the non-permissive temperature; and ii) inhibited by amiloride and four divalent cations, Ba*+, Mg*+, Sr*+ and Co*+. This suggests that, although of different chemical nature, arninoethyldextran and veratridine act through the same physiological pathway. In addition, the inhibitory doses are comparable to the ones found to inhibit a hyperpolarization-sensitive Ca*+-current described in Paramecium (Preston et a2 (1992) J Gen Physiol 100, 233). The possibility that the activation of this Ca*+-current by the secretagogue represents an early step in the regulation of trichoeyst exocytosis is discussed.* exocytosis

/ calcium

inflax

/ Paramecium/

amiloride

/ veratridine

Introduction Stimulus-secretion coupling, first described by Douglas [6], most often involves a rise in cytosolic Ca2+, either through the opening of Ca2+-channels of the plasma membrane, generally in excitable cells, or via the release from internal stores, generally in non-excitable cells [20, 361. In the unicell Paramecium, Ca2+ seems to play a key role in signal transduction, since the application of Caz+-ionophores [16, 271 or direct microinjection of Ca2+ into cells induce trichocyst exocytosis [5, lo]. In addition, exocytosis triggered by aminoethyldextran (AED) [22] is associated with Ca2+ movements visualized as a Ca2+-influx [9, 121 and a transient backward swimming [9, 231, a behavior known to arise from an increase in intracellular free Ca2+ in the ciliary area. The isolation and genetic analysis of mutants defective for exocytosis are possible in this cell type [3, 241 and one of the exocytosis mutants, nd12, displays correlated thermosensitive defects in exocytosis and in the associated Ca2+ movements, confirming the tight relationship between these two phenomena [9]. The origin of the Ca2+ involved in trichocyst exocytosis is still not clear. Early reports favored Ca2+ flowing into the cell as the trigger [9, 16, 23, 271, whereas more recent experiments argue in favor of the release of Ca2+ from internal stores as determinant [7, 11, 131. In this paper, we show that veratridine, a drug previously found to be a secretagogue in Paramecium [12], was active on the reference strain but not on the ndl2 mutant. Like AED, veratridine was able to induce a massive Ca2+-influx associated with exocytosis. We also show that amiloride and four divalent

* Correspondence

and reprints

cations inhibit AED- and veratridine-induced exocytosis as well as the associated Ca2+-movements at the same concentrations as those found to inhibit a hyperpolarization-sensitive Ca2+-current of Paramecium [26]. These observations lead us to relate trichocyst exocytosis with the hyperpolarization-sensitive Ca2+-cutrent, suggesting that a Ca2+-influx could play a critical role in the early steps of stimulation.

Materials and methods Strains and culture conditions The reference strain used in this study, d4-500, is a derivative of Paramecium tetraurelia [31] carrying a mutation in the gene pwA which inactivates a ciliary voltage-operated Caz+-channel [28] and thus prevents the avoiding response

fer contained 50 mM HEPES, 250 mM sucrose and 40 PM Car&. In order to disrupt the membranes and give accessto the crystalline contents, purified trichocysts were submitted to an osmotic shock by a two-fold dilution in the same buffer without sucrose. This treatment permits full matrix stretching into 2@-3Opm long needles, due to the presence of external CaC12,as monitored under 100 x phase contrast microscopy. To be sure that the divalent cations used @aa+, Co2+, Mg2+, Mn2+, Sra+)have no inhibitory effect on this matrix stretching, the chloride salts of these ions were added to the dilution buffer to a final concentration of 5 mM and the stretching monitored as above. Test of exocytotic ability Exocytotic stimulation was performed by adding 9 ~1 of a cell suspension to 1 fl of either 30 @I AED or various concentrations of veratridine (Sigma). Routinely, the incubations were performed in 1 mM HEPES (pH 7.0), 40 @vf Cat&, since they were done together with calcium uptake measurements(see below). ln some cases,when divalent cations w-ereused as inhibitors, 1 to 2.5 mM CaC12 was used. Exocytotic ability was determined under 10 x dark field microscopy by the observation of released trichocysts. A semi-quantitative estimation is possible by this method and four cIasses were distinguished: no (-), weak (+)+ intermediate (++) and maximal (+++) exocytosis. The inhibitory effects of divalent cations were determined by brief incubation of the cells in the presence of the ions for 5-10 s prior to stimulation. The inhibitory effect of amiloride (Sigma) was monitored as follows: to avoid surface tension problems which led some cells to stick to the surface of the droplet and release trichocysts by lysis, cells were put on a silicone-coated slide and the secretagogue containing amiloride on a coverslip, which was then lowered onto the cells. The discrepancy between the inhibitory effect documented here in the Rest& and the observations noted by Erxleben and Plattner [7] that amiloride does not inhibit exocytosis could be due to the difficulty of handling amiloride and avoiding artefactual lysis and trichocyst release.

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Fig 1. Veratridine-induced 4sCa2+-uptakeand inhibition by amiloride and Ba2+. I mM veratridine triggers a strong innease of initial Ca2+-uptake (open squares), compared to non-stimulated cells (open circles). 1 mM amiloride (black-diamonds) or 5 mM II&l2 (black triangles) strongly inhibit the veratridine-induced Caa+-influx. Note that the apparent incomplete inhibition of the veratridine-induced Caz+-influx by amiioride corresponds m fact to the basal incorporation in the presence af amiloride done which is higher than the basal incorporation without amiloride (see fig 2, black circles). Each point is the mean of two meastue.ments in one typical experiment.

Calcium uptake measure~ments Caz+-uptakewas measured according to Ker&f and Cohen [9j The cells were harvested in log phase by a 5..min centrifugatior at 30 g, to avoid unwanted exocytosis before the experiment washed once in I mM HEPES (pH 7.0), 40 m CaC12(a caleiun concentration low enough to prevent non-specific bind&g an6 giving a good spe&ic radioactivity of dsCa+, and concemramc to at least to lOOil ceil/ml. After 30 mm at 26T in this incuba. tion buffer, 4tCa (used at a final specific activity of 1 #X/ml; uptake experiments were performed. Par each point, after 2,4 oJ 6 s of labelling in the presence or absenceof secretagoguesant inhibitors, as specified, a 0.5ml sample of cells was quickly diluted in 4 ml of 1 mM HEPES (pH 7.0), 5 mM CaClz at O’C vacuum filtered over 0.45~pm pore size filters (MilIipore Conti nental Water System, Bedford, MA), and washed three Eimes with 4 ml of the same buffer. Each filter was then-counted by~liq uid scintillation. Under our experimental conditions, any ch-angs in the initial rate of uptake provoked by the secretagogueswith or without inhibitors wilt directly reflect the modification of the membrane permeability to Ca2+, thus of the influx of Ca2*, ;ts discussedby Mauger er al [ 171. Other methods to quantify the calcium movements were not used here due to technical probJems peculiar to the cell type used. Indeed, the direct visualization of calcium concentration changes using fluorescent calcium sensors [36] is not easily applicable in Paramecium since this organism cannot esterify me dye (observed~at least forPura%AM; &andchamp and Let-tier, personal communication), a step necessaryfor trapping into the cytosol Results Veratridine stimulates exocytosis and its associated calcium influx Veratridine, known to be an agonist of voltage-operated Na+-chatmels [4, 141, was shown to induce exocytosis~in Paramecium j 121. We carried out 4sCa2+-uptake measurements and observed a~strong Ca**-infktx~ in the presence of I mM veratridine~ (fig l), similar in kinetics andamp@tude to the AEDGnduced Ca*+-influx [9]. AED does not elicit exocytosis nor Ca2+ influx in the mutamnd12, indicating that this mutations affects a very early step in the process, the entry of Ca*+ itself or an upstream event. When veratridine was applied to PI&ndl2 cells grown at 3S’C (see Materi& and methuds) neither exocytosis nor backward swimming could be detected, strortgly suggesting that Vera&dine and AED act on a common pathway. In addition, the absence of stimulation of ndZ2 cells by veratridine shows t&t the @ion of this secretapgue on wild type ceIIs is~physioi@cal and not due to artefactual permeabilisation of plasma rnembrane. Amiloride and divaietqcutions its associated Ca2+ influ

inhibit both exocytosis and

Amiloride, an inhibitor of cation transport in many eel1 types [2, 8, 35], has been shown to inhibit a new type of calcium channel in Paramecium, a hyperpolarization-sensitive Ca2+-channel [26]. When am&ride was introduced in the stimulation medium, a dose-dependent inhibitory effeet was~observed both on AED- and veratridine-triggered exocytosis. For AED-triggered exocytosis; the inhibition progressively appeared at an&ride concentrations from 125 PM to I m&l and beyond, where exocytosis was completely mhibited (see-the top part of fig 3). Exocytosis triggered by 0.S mM veratridine was also inhibited-bye

Inhibition

of trichocyst exocytosis

1 n&l amiloride. The inhibitory effect of 1 mM amiloride was fully reversible after at least 1 min of incubation, when cells were washed in the same buffer without amiloride. This suggests that the highest ainiloride concentrations used here do not affect the cell viability over the duration of the experiments. To test the effect of Ba2+, COG+,Mg2+, Mn2+ and St-z+on exocytosis, we first checked that these ions had no effect on trichocyst stretching itself, since our test is based on the observation of presence/absence of decondensed trichocysts. Using the irz vitru assay described in Muter-i& and methods, we found that only Mn2+ was inhibitory for trichocyst stretching. Therefore, only the four other divalent cations were used to test AED- and veratridine-triggered exocytosis, in the buffer containing 40 /LM Ca2+. Ba2+ and Co2+ down to 0.5 mM gave a full inhibition of exocytosis elicited by both secretagogues, whereas Mg2+ and Sr2+ gave the same inhibition only at 5 mM. In the presence of 2.5 mM Ca2+, a complete inhibition by Ba2+ was obtained at 10 mM and half inhibition at 1 mM. When introduced in the stimulation medium, 1 mM amiloride inhibited the veratridine-induced (fig 1) and the AED-induced (fig 2) increase of 4sCa2+-uptake. Varying the amiloride concentration from 125 PM to 2 mM showed that this inhibitory effect is concentration-dependent and fairly well correlated with the inhibition of exocytosis (fig 3). The amiloride ICs,, for exocytosis and Caz+-uptake is cu 0.25 mM. The effect of Ba2+, the ion with the most inhibitory effect on exocytosis, was studied on the veratridineinduced (fig 1) and the AED-induced (fig 4) Ca2+-influx. The uptake of 4sCa2+ by both stimulated and non-stimulated cells was severly reduced in the presence of 5 mM Ba2+. The same effect was observed in the presence of 5 mM Mg2+ or Sr2+ (data not shown). By varying Ba2+ concentrations in 4sCa2+-uptake experiments, we could obtain dose-response curves reasonably correlated to the inhibitory effect on exocytosis (fig 5). The Ba*+ ICs,, was cu4op.M.

41

stimulation sequence, between the reception of the stimulus itself and the calcium entry [9]. As this mutation blocks a step downstream from both AED and veratridine actions, there should be a point of convergence in the stimulation pathway upstream from the site of action of ndl2; iii) AEDand veratridine-triggered exocytosis and Ca2+-influx have the same sensitivity to amiloride and divalent cations. The inhibition of veratridine effects by Ba2+ indicates that this agonist does not act on the depolarization-sensitive ciliary Ca2+-channel, as initially proposed by Schultz and Schade [29], since this channel should be stimulated rather

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time (s)

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Fig 2. Effect of amiloride on the ABD-induced %a2+-uptake. Initial 4XJa2+-uptakeof non-stimulated (circles) and AED-stimulated (squares) cells measured in the absence (open symbols) or the presence (black symbols) of 1 mM amiloride. The basal Ca2+-influx was bigher in the presence of amiloride than in its absence. However, in the presence of amiloride, no stimulation was observed with ABD. Each point is the mean of two measurements in one typical experiment.

Jliscussion Using two different secretagogues, AED and veratridine, and two kinds of inhibitors of exocytosis, amiloride and divalent cations, we investigated the relationships between the stimulation and the calcium movements associated with exocytosis. We showed that both secretagogues seemed to act through the same pathway, and that this pathway is inhibited by the same substances that inhibit the hyperpolarization-sensitive Ca2+-current of Paramecium.

AED and veratridine stimulate the same pathway AED and veratridine are very different molecules. AED is a highly charged polyamine (cu 40 charges per molecule) whose mode of action is still hypothetical [5] and veratridine is a well known agonist of vertebrate Na+-channels [4, 141 whose target in Paramecium is not known. Therefore, the mode of stimulation of AED and veratridine are expected to be different as well as the physiological pathways they stimulate. However, as already proposed by Knoll et ul [12], we showed here that the action of both secretagogues converge to a point where a common mechanism is stimulated: i) veratridine and AED induce the same associated events, exocytosis, backward swimming and Ca2+-influx: ii) the ndl2 mutation was shown to block a very early step in the

' ' "'I*'1 0

I 0.1

I IO

[amiloride] in k,l

Fig 3. Amiloride dose-response curves. 4QZaz+-uptalce of AEDstimulated cells in the presence of amiloride expressedas its ratio over the maximal uptake without amiloride, measured at 2 s and

---

.---~-

0 0

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2

tirnz (s)

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J?ig 4. Effect of Ba*+ on the AED-induced 4Xa*+-uptake. Initial 4sCaz+-uptakeof non-stimulated (circles) and AED-stimulated (squares) cells were measured in the absence (open sye~bols)or the presence (black symbuls) of 5 n&f Bat& Each point is the mean of two measurements in one typical experiment.

Fig 5. Ba2+dose-responsecurves. 45CaZ+-uptakeof AED-stimulated cells in presence of BaC& expressed as its ratio over tbe maximal uptake without BaC&, measured at 2 s and pletted against the Ba*+ concentration. Each point is the mean of four measurements. The symbols +++ to - in the upper bar represent the amount of trichocysts released upon stimulation in the presence of Ba2+(seeMaterials and methods).

than inhibited by Ba2+. I-Iowever, the doses at which amiIoride and divalent cations act are consistent with the K.o

given by Preston et uZ [26] fork the hyperpolarization-sensltive (%+-current (0.08 mM, 0.67 mM, 2 2 mM, 2 2 mM and cu 0.39 mM for Ba2+, CG+, MgZ+, Sr*+ and amiloride respectively), although they were measured in different conditions. This suggests that the hyperpolarization-sensi.~ tive Ca2+-current could be related to exocytosis in some way. The fute of calcium during stimulation of exocytcwis We already proposed that a sharp hyperpolarization could be a stimulus for trichoeyst exocytosis [5]. Indeed, electric

shocks given from outside elicit preferential exocytoGs toward the positive electrode 119, 32, 371, where the mem . brane is hyperpolarized: low concentrations ~of cationic detergents induce exocytosis [16], most likely by preferential insertion into the external face of the plasma membr8nethus increasing the transmembrane potential [ 181. Concern. ing highly charged polycationic secretagogues, such a:< AED, we proposed [5] that they act through a mechanism of surface negative charge screening, also known to incEase the transmembrane potential [ 181. From the results presented here: we propose that the tar. get of the hypeqolarizingzlike effect of AED could be the activation of the hyperpolarization-sensitive Ca2+-current. Veratridine would act as an agonist of this chaimel, rather than by changing the poiarization of the membrane. Both secretagogues would therefore activate the same-paxhway through different mechanisms. It has- been shown recently that a Ca** release from subplasmalemmal stores [33, 34] would be the major sonree of Ca*+ necessary for exocytotic membrane fusion [7, 1I, -131. The authors also SuggeSEd that the massive Cap+-influx observed during exocytosis [9, 12] occurred only after cotipletion of membrane fusion and would serve as store-refilling. Our present data suggest however that at least a minor part of the Ca2+-influx should precede exocytosis, suggesting the occurrence of a mechanism of Ca*+-induced Ca2* pleases Of interest is the work of tinge et ul [13] showing that the cortical Ca2+ stores of Paramecium present strong structuraj and pharmacological homology with muscle sarcoqlasmic reticulum where a mechanism of Caz+-induced Ca2+ relate has long been shown to be critical for muscle contiaction. ln agreement with all the data now available, we can pm. pose a model for the stimulation of trichocyst exocyto$s. The critical step induced by the stimulation is the opening of a hyperpolarization-sensitive Ca*+-charnel. All agents giving a sharp hyperpolarization, as discussed above9 would trigger this channel, as will a&o other agonists through direct binding. The opening of the charmel would per& some calcium to flow in and to trigger a kind of WCinduced Ca2+ release, followed by a massive Ca2+-influx RJ refill the stores. The calcium rise in the cytosol would if% turn trigger exocytotic membrane fusion and have &XI side effects, such as ciliary reversal. The opening of the chanfiel per se depolarizes the membrane, since it increases the Ca.conductance of the membrane, and therefore provokes its own inactivatiofi. This is in good agreement with the observation that local trigger of exocytosis does not propagate over the cell surface [22]. Electrophysiologicai studies of secretagogue 3timu&ion of wild type and nd12 cells tit the non-permissivi temperature have to be undertaken to further understands the pro” cess. However, recent advances in molecular~cloning b> functiona compiementation of nd mutants (Skouri a& Cohen, unpublished) will allow the cloning and sequencing of the nd12 gene, so that its possible relationship with C$*channels can be esiabiished. Acknowledgments This work was supported by the Centre National de la Recherchc Scientifique. the lnstitut National de la San@?et de la Recherche Vlkdicale (grant 910503), and a fellowship from the Ligue Nationale Franqaise contre le Cancer to DK. V&Zthank And& Adoutte (Orsay. France), B Gomperts (London, [JK), I. Madeddu (Gif-sur-Yvette-. France) and R Preston (PhiladcJphia,USA) t&r Fruitful discussionsand C Creutz (Charlottesville, USA). for crit ical reading of the manuscript.

Inhibition

ot’ trichocyst

References 1 Adoutte A (1988) Exocytosis: biogenesis, transport and secretion of trichocysts. In: Paramecium (Gortz HD, ed) Springer Verlag, Berlin, 325-362 2 Benos DJ (1982) Amiloride: a molecular probe of sodium transport in tissues and cells. Am J Physio1242, Cl3 1-C 145 3 Bonnemain H, Gulik-Krzywicki T, Grandchamp C, Cohen J (1992) Interactions between genes involved in exocytotic membrane fusion in Paramecium. Genetics 130,461-470 4 Catteral WA (1975) Activation of the action potential Na+ ionophore of cultured neuroblastoma cells by veratridine and batrachotoxm J Biol Chem 250,4053-4059 5 Cohen J, Kerbceuf D (1993) Calcium and trichocyst exocytosis in Paramecium: genetic and physiological studies. In: h4embrune traffic in protozoa (Plattner H, ed) JAI PressInc, Greenwich Conn, 61-81 6 Douglas WW (1974) Involvement of calcium in exocytosis and the exocytosis vesiculisation sequence. Biochem Sot Symp 39, l-28 7 Erxleben C, Plattner H (1994) Ca*+ release from suplasmalemma1 stores as a primary event during exocytosis in Paramecium cells. J Cell Biol 127,935-945 8 Garcia ML, King VF, Shevell JL, Slaughter RS, SuarezKurtz G, Wincluist RJ, Kaczorowski GJ (1990) Amiloride analogs inhibit L-type calcium channels and display calcium entry blocker activity. J i3iol Chem 265, 3763-3771 9 Kerbceuf D, Cohen J (1990) A Ca2+influx associated with exocytosis is specifically abolished in a Paramecium exocytotic mutant. J Cell Bioi 1I L 2527-2535 10 Klauke N, Plattner H (1993) Mobilization of intracellular calcium for exocytosis in Paramecium cells. Eur J Cell Biol 60 (supp 37), 142 (Abstr) 11 Knoll G, Grasslet A, Braun C, Probst W, Hohne-Zell B, Plattner H (1993) A calcium influx is neither strictly associated with nor necessatyfor exocytotic membrane fusion in Paramecium-cells. Cell Calcium 14, 173-183 12 Knoll G, Kerbmuf D, Plattner H (1992) A rapid calcium influx during exocytosis in Paramecium cells is followed by a rise in cyclic GMP within 1s. FEBS L&t 304,265-268 13 Lange S, Klauke N, Plattner H (1995) Subplasmalemmal Ca*+ stores of probable relevance for exocytosis in Paramecium. Alveolar sacs share some but not all characteristics with sarcoplasmic reticulum. Cell Calcium 17,335-344 14 Leibowitz MD, Sutro JB, Hille B (1986) Voltage-dependent gating of veratridine-modified Na+ channels. J Gen Physiol 87,2546 15 Lima 0, Gulik-Kizywicki T, Sperling L (1989) Puramecium trichocysts isolated with their membranes are stable in the presence of millimolar Ca*+.J Cell Sci 93,557-564 16 Matt H, Bihnski M, Plattner H (1978) Adenosine triphosphate, calcium and temperature requirements for the final steps of exocytosis in Paramecium cells. J Cell Sci 32,67-86 17 Mauger JP, Poggioli J, Guesdon F, Claret M (1984) Noradrenaline, M

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