Evidence for the involvement of PI-signaling and diacylglycerol second messengers in the initiation of metamorphosis in the hydroid Hydractinia echinata Fleming

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121,82-89 (1987)

Evidence for the Involvement of PI-Signaling and Diacylglycerol Second Messengers in the Initiation of Metamorphosis in the Hydroid Hydractinia echinata Fleming THOMASLEITZANDWERNER A. MULLER Zoologisches Institut

der Universitiit Heidelberg, Fachrichtung Physiologic, Im Neuenheimer D-6900 Heidelberg 1, Federal Republic of Germany

Received September 2.3, 1986; accepted in revised form November

Feld 230,

12, 1986

Metamorphosis of the planula larvae into polyps does not occur spontaneously but depends on the reception of external trigger stimuli. Artificially, metamorphosis can be initiated by a pulse-type application of Cs+ or tumor-promoting phorbol esters (W. A. Miiller (1985) D#krextiation 29,216~222). In the present study we examined the putative involvement of the phosphatidylinositol system in signal transduction. Planulae of Hydractinia echinata were preincubated with [3H]-inositol. Upon exposure of the larvae to Cs+ the label in inositol trisphosphate (InsPa) increased twofold as early as 15 set after addition of Cs+. Within the first 60 set the levels of inositol monophosphate (InsPi) and inositol bisphosphate (InsPz) were also elevated compared to the values in nonstimulated larvae. After 1 and 3 hr, respectively, of incubation with Cs+, only the label in InsPa was increased. When applied to saponin-permeabilized larvae, InsPa did not induce metamorphosis. But 1,2-dioctanoyl-rat-glycerol (diQ was effective in inducing metamorphosis with a half-maximal effective concentration of 9 @M. The percentage of metamorphosed animals after the application of 5 PM diCs (30 mM Cs’) was increased by the simultaneous application of 1 PM (0.1 GM) of the diacylglycerol kinase inhibitor R 59022. The results are interpreted as evidence for the involvement of the PI-signaling/diacylglycerol transduction system in the initiation of metamorphosis of planula larvae of H. echinata. 0 1987 Academic Press, 1nc.

being the most active compound. Freeman and Ridgway (1987), by studying Ca2+-sensitive photoproteins in the Metamorphosis of the planula larvae of at least some hydroid Phialidium gregarium and several other species, hydrozoan species is induced by contact with marine found bursts of calcium transients in the planula larvae bacteria (Mtiller, 1969, 1973; Freeman and Ridgway, occurring during induction of metamorphosis by Cs+ or 1987). Artificial induction of metamorphosis is achieved bacteria. by pulse-type application of monovalent cations (Cs’, The results of these studies prompted us to examine Li+, K+, Rb+) with Cs+ being the most potent inducer whether a signal transduction pathway found recently (Spindler and Mtiller, 1972; Mtiller and Buchal, 19’73; in vertebrate cells is also involved in initiation of metaBerking, 1984; Freeman and Ridgway, 1987). Although morphosis. This signal transduction mechanism is an involvement of Na+/K+-ATPase has been suspected thought to occur upon reception of a stimulus on the (May and Miiller, 1975; Mtiller and Buchal, 1973) the outer surface of the cell membrane (Berridge and Irvine, mechanism of action of the inducers was unknown. 1984; Fain, 1984; Hokin, 1985; Majerus et al., 1985; BerTwo recent studies shed further light on this problem. ridge, 1986a). The receptor is coupled possibly by means Mtiller (1985) was able to induce metamorphosis of Hy- of a GTP-binding protein (e.g., Joseph, 1985; Taylor and dructinia echinata by application of potent tumor pro- Merritt, 1986) to a phosphodiesterase (phospholipase C) moters with phorbol 12-myristate 13-acetate (PMA)’ which liberates inositol phosphates from phosphatidylinositol (phosphates) (Berridge and Irvine, 1984; Fisher et al, 1984; Hirasawa and Nishizuka, 1985, Berridge, i Abbreviations used: CMP, cytidine monophosphate; diCs, dioctan1986a). Inositol trisphosphate (InsP,) which is generated oylglycerol; DMSO, dimethyl sulfoxide; GroPIns, glycerophosphoinofrom phosphatidylinositol bisphosphate (PtdInsP2) sitol; InsPi, inositol monophosphate; InsP,, inositol bisphosphate; inositol; InsPa, inositol trisphosphate; InsP -3, polyphosphorylated functions as a second messenger by liberating Ca2+from OAG, 1-oleoyl-2-acetyl-sn-glycerol; pH1, intracellular pH, Pi, inorganic intracellular stores (Berridge, 1984a, 1985, Berridge and phosphate; PI-cycle, phosphatidylinositol (phosphates)-cycle; PMA, Irvine, 1984; Exton, 1985). phorbol12-myristate 13-acetate; PtdIns, phosphatidylinositol; PtdInsP, Other products of the action of phospholipase C are phosphatidylinositol monophosphate; PtdInsPa, phosphatidylinositol compounds of the diacylglycerol type which function as bisphosphate. INTRODUCTION

0012-1606/87 $3.00 Copyright All rights

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

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second messengers by stimulating protein kinase C (e.g., Nishizuka, 1984; Ashendel, 1985; Bell, 1986). Tumor-promoting phorbol esters bind to protein kinase C in a manner similar to diacylglycerols (Brockerhoff, 1986; Jeffrey and Liskamp, 1986) and are potent activators of this enzyme. The involvement of the phosphatidylinositol (phosphates) breakdown and protein kinase C in cell proliferation and differentiation has been observed often (Michell, 1982; Berridge, 1984b; Berridge et ah, 1985; Vicentini and Villereal, 19136)and combined with the results of Miiller (1985) and Freeman and Ridgway (1987) it is reasonable to assume that these cellular signal pathways are also of significance in hydrozoan metamorphosis. To obtain further insight into these mechanisms we investigated the liberation of inositol phosphates by larvae of Hydractinia echinata which had been incubated with labeled i:nositol and induced thereafter to undergo metamorphosis by pulse-type application of cs+. We further tried to induce metamorphosis with a synthetic endogenous “phor’bol ester,” namely, dioctanoylglycerol (diCs), and we used the diacylglycerol kinase inhibitor R 59022 (de Chaffoy de Courcelles et al., 1985) with the aim of potentiating the effect of Cs+ or diacylglycerol on induction of metamorphosis. Additionally, InsPs was applied to larvae which were permeabilized with saponin. MATERIAL9

AND

METHODS

Animals. Wild colonies of H. echinata are maintained in our laboratory and planula larvae are routinely raised as described elsewhere (Mtiller, 1984). Chemicals. Myo-[1,2-3H(N)]inositol (sp act 1.74 TBq/ mmole) was purchased from NEN (Dreieich). Prepacked columns of AG l-X8 resin (formate form, 200-400 mesh) were from Bio-Rad. The diacylglycerol kinase inhibitor R 59022 was obtained from Janssen Chimica and 1,2dioctanoyl-rat-glycerol (Idi&), saponin, and myoinositol trisphosphate were obtained from Sigma. Quickszint 1 liquid scintillator (Zinsser) was used for liquid scintillation counting. All otlher chemicals from different sources (Baker, Fluka, Merck, and Serva) were of analytical grade. Incubation of larvae. Otnehundred larvae (4 days old) were incubated with approximately 185 kBq (corresponding to 4.8 X 10’ cpm) [3H]inositol for 16 hr in 1 ml of sterile seawater in lo-ml disposable PPN tubes (Greiner). Subsequently the larvae were centrifuged at 8009 for 1 min and washed su’ccessively with 2 X 4 and 1 X 3 ml of sterile seawater. After the last wash the weight of seawater + larvae was adjusted to 400 pg, and 100 ~1

in Hydractinia

83

of 580 mM CsCl (or 100 ~1of sterile seawater for controls) was added to induce 100% metamorphosis (standard procedure used in our laboratory to induce metamorphosis in virtually all larvae after 3 hr of incubation; see Berking, 1984; Mtiller, 1985). The incubations were performed without Li+ and stopped by adding 1.5 ml methanol-chloroform (2:1, v/v) after 15 set, 1 min, or 3 min for one batch of animals and after 1 or 3 hr for another batch. Analysis of metabolites. The samples were homogenized on ice with a Branson Sonifier Cl5 equipped with a microtip. Chloroform (2x 250 ~1) was used to wash the microtip and added to the sample. HCl (0.5 ml, 2.4 M) was added and the tubes were vortexed and centrifuged thereafter to facilitate separation of the phases. The organic phase was removed and the aqueous phase was washed with 1 ml of chloroform. The combined organic phase was then washed with 2 ml of methanol-l M HCl (l:l, v/v) and the washing was added to the aqueous phase. The combined aqueous phase was evaporated in a Speed Vat. with heating. Inositol phosphates and glycerophosphoinositol (GroPIns) were separated according to the method described by Berridge et al., (1983). The dried sample was dissolved in 1 ml of 6.25 mMNa,B,07 and loaded onto 0.6 ml of the ion exchanger AG l-X8. Two additional 0.5-ml portions of 6.25 mMNazB407 were used to wash the tube and passed through the column. The 2-ml eluate (containing inositol) was mixed with 6 ml of Quickszint 1 and counted as a gel. The columns were washed with 10 ml of 5 mM inositol. GroPIns was eluted with 5 ml of 5 mM sodium tetraborate/60 mM sodium formate, inositol monophosphate (InsPi) was eluted with 5 ml of 0.1 M formic acid/O.2 M ammonium formate, inositol bisphosphate (InsPz) was eluted with 5 ml of 0.1 M formic acid/O.4 M ammonium formate, and InsP3 was eluted with 5 ml of 0.1 M formic acid/O.8 M ammonium formate (Batty et ah, 1985) directly into 20-ml scintillation vials. To regenerate the ion exchanger and to elute the remaining inositol polyphosphates (InsP,,3) 5 ml of 4 M formic acid was passed through. All eluates were mixed with 7.5 ml of scintillator and counted as a gel for 10 min or until the counting error (2~) was less than 2%. Quench correction was not done. All samples with the same eluate were quenched to the same degree and were therefore directly comparable. Metamorphosis assay. The compounds to be tested were added to the larvae (100-200 per 3.5 cm 4 dish) in 6 ml of sterile seawater to give the concentrations indicated in the figures. R 59022 was predissolved in methanol and diCs in dimethyl sulfoxide (DMSO). The concentrations of methanol and DMSO, respectively, never exceeded 0.5 and 0.3%) respectively. With these concentrations the

a4

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solvents did not induce any observable effects in control experiments. The incubations were performed for 3 hr, after which the larvae were washed three times with sterile seawater. The percentage of metamorphosed animals was scored the day after. For the rationale for scoring metamorphosed animals, see Mtiller (1969,1985). For permeabilization 200-300 larvae in 0.5 ml of sterile seawater were mixed with 25 ~1 of a solution of saponin in sterile seawater (final concentration, 170 pugsaponin/ ml). Incubation was performed for 10 min at room temperature with an additional 25 ~1of InsPS in sterile 1020 mosmol sucrose (final concentration, 10 p&f) or 25 ~1 of 1020 mosmol sucrose (control). The larvae were washed three times with sterile seawater and suspended in 0.5 ml of seawater supplemented with InsPS (final concentration, 10 PM). After 2 hr the larvae were washed and transferred to petri dishes (3.5 cm 4) containing 6 ml of seawater. Permeabilization was demonstrated in initial experiments by the uptake of a normally impermeant dye (Procion brilliant yellow). The cells of the larvae were permeable for 14 to 2 hr since after that time the dye was no longer able to enter the cells. Larvae appeared

21000

16 060

15000 3 700 E ,”

f .> .: .z -n d

3 050

2 400 650

T

I 1 1

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to be normal after saponin treatment. They underwent normal metamorphosis when induced with 116 mMCsC1. RESULTS

Inositol phosphate production, The formation of inositol phosphates after initiating metamorphosis by means of Cs+ is shown in Figs. 1 and 2. It is evident that 15 set after addition of Cs+, InsPa rises to approximately 200% of the control value. After 1 min of stimulation InsPs production reaches 215%. After 3 min production is slowed down but nevertheless significantly higher than in the control. The formation of InsPI and InsP2 is also stimulated after addition of Cs+. Percentage elevation, compared to control, is not as high as in the case of InsPs but nevertheless it is significant. With GroPIns and highly phosphorylated inositol the results are inconclusive due to the high variability in the values and the low amount of incorporated radioactivity. The short-term incubations were repeated several times and the results were always similar (data not shown). With longer incubations the results are somewhat different (Fig. 2). Only the values for InsPz and inositol

970 lnositol 750

530 450

315

160 105

90

460

3

270

1;

l&l

sb Incubation

FIG. 1. Formation of inositol phosphates during the initiation quadruplicates. 0, Induction with 116 mM Cs+; n , control (control

15

60

75

160

time (s)

of metamorphosis (short-term incubations). Shown are means f SEM of values did not change significantly over a lo-min period (data not shown)).

LEITZ AND MULLER

PI-Signaling

in

I

lnosilol

6roP Ins

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15000~

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HydraAinia

1500

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600

400

Incubation FIG. 2. Formation of inositol phosphates during the initiation triplicates. 0, Control; W, induction with 116 mM Cs+.

time(hr)

of metamorphosis

polyphosphates (InsP& were different from control values after such long-term incubations. InsPz was elevated nearly twofold after a 3-hr stimulation with Cs+, and InsP,,3 values were lowered to approximately 45% of the control. Values for InsPI were lower in stimulated samples but this difference was not significant. With GroPIns and InsPs no considerable differences were obtained between control and experimental values. Control and experimental values for inositol decreased with increasing incubation time, suggesting a further incorporation from intracellular pools.

(long-term

incubations).

Shown are means + SEM of

Control values for the inositol phosphates were different when compared between long- and short-term incubations. This difference is possibly due to the heterogeneity of the animal material. Eggs were harvested from mass-cultured Hydractinia and therefore it is uncertain whether different batches originated from the same parents. Induction of metamorphosis As shown in Fig. 3, diCs induced metamorphosis when applied for 3 hr. Effective concentrations were in the range 5 to 100 PM. When 1 PM diCs was used no metamorphoses were found in an-

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DISCUSSION

In this paper we show that generation of InsPs is involved in the initiation of metamorphosis of the planula larvae of H. echinata. The production of InsPs was nearly twofold as early as 15 set after the application of 116 mM Csf, which is known to induce metamorphosis in 100% of larvae when they are treated for 3 hr (Berking, 1984; Miiller, 1985). According to the widely espoused theory InsPs mobilizes Ca2+ from intracellular stores (Berridge, 1984a, 1985, 1986a,b), thereby initiating a cascade of events leading to biological response. We have not yet measured Ca2+mobilization but there is a recent paper by Freeman and Ridgway (1987) dealing with Ca2+ transients during metamorphosis of the hydrozoan Phialidium gregarium. These authors found that Ca2+ transients always accompanied the initiation of meta1K5 54l.” 5al’5 1u4 morphosis induced by Cs+ and bacteria. The Ca2+tranlog concentration diC, (mol. I-‘) sients did not occur when larvae were treated in Ca2+free seawater or in seawater with the Ca2+ channel FIG. 3. Induction of metamorphosis by dioctanoylglycerol (diCs). blocker nifedipine. When treated in such a manner the Shown are means + SEM of triplicates. larvae did not metamorphose, but metamorphosis was initiated immediately after their retransfer into normal other batch of larvae. With 100pMdiC& a metamorphosis seawater. The authors concluded that Ca2+ transients rate of 98% could be achieved. are mediated by Ca2+channels and are necessary for the When applied together with a constant dose of diCs induction of metamorphosis. This is in accordance with (5 PM), 1 pM R 59022 raised the percentage of meta- the observation that La3+ (a potent inhibitor of Ca2+ morphoses from 46.1 (control without R 59022) to 82.7%. entry in rat parotid salivary gland cells (Putney, 1986) With only 10 or 100 nM R 59022 no visible effect was and in A 431 cells (Moolenaar et ah, 1986)) inhibits Cs+noticed (Table 1). On the other hand, 10 pMR 59022 was induced metamorphosis (W. Ludewig, unpublished)). above the optimum as a decrease in the percentage of However, according to Freeman and Ridgway (1987) metamorphosed animals took place (17.7% compared to the Ca2+ transients during the initiation of metamor46.1% of control). phosis are probably not the primary effect of metamorA similar picture emerged after the application of R phic stimulus because the Ca2+ ionophore A23187 in59022with a constant dose of 30 mMCs’, which normally duced large Ca2+ transients but did not trigger metainduces 40-60% metamorphoses after 3 hr of incubation (Table 1). A medium dose of 100 nM R 59022 increased the rate of metamorphoses from 59.8 to 81.6%; all TABLE 1 other concentrations were ineffective. With another INFLUENCE OF THE DIACYLGLYCEROL-KINASE INHIBITOR R 59022 ON batch of larvae the effective dose was 1 pM (data not Cs+- AND di&-INDUCED METAMORPHOSIS OF PLANULA LARVAE OF Hyshown). dructiniu echinata When applied without inducing agents R 59022 (1 nMInduction of metamorphosis by Concentration 1 pM) had no detectable effect on the larvae. With 10 of R 59022 pM R 59022 a sustained contraction of larvae was no5 pM diCs 30 mM Cs+ (M) ticed. After washing the larvae they returned to their normal elongated appearance. At 100 pM, R 59022 was 59.8f 9.3” 46.1 k 11.9 0 (control) lethal to the larvae. n.d.* 1O-9 58.3 f 9.3 When applied to saponin-permeabilized larvae at a lo-* 63.2 f 15.7 56.3 + 11.8 W7 56.7 f 7.5 81.6k 4.3 final concentration of 10 pM, InsPs had no detectable 10-C 82.7 f 5.6 59.2 f 3.9 effect. The larvae remained elongated during the incu1o-5 n.d. 17.7f 8.3 bation period of 2 hr 10 min and also after washing out the InsPs they did not undergo metamorphosis within a Values are percentages of metamorphoses (means f SD of quathe observation period of 24 hr. Subsequent application druplicates (induction with Cs’) or triplicates (diC,)). ’ n.d., not determined. of Cs+ (116 mM) induced normal metamorphosis.

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PI-Signaling

morphosis (also W. Ludewig, unpublished). Moreover, our results show that InsPs (10 FM) has no effect in saponin-permeabilized larvae. Permeabilization did not impair subsequent induction with Cs+, but we do not know whether normal bio.logical response is possible in the presence of the permeabilizing agent. However, considering all the findings we agree with Freeman and Ridgway (1987) in stating that Ca2+mobilization is necessary but not sufficient to bring about later events in metamorphosis. It is tempting to suggest a role for Ca2+ in epithelial muscular cell contraction or exocytosis, both of which are important events preceding the metamorphosis proper (Weis and I~USS,1987). A more detailed interpretation of the measured changes in the amount of metabolites of the PI-cycle is impeded because larvae consist of few, but more than one, cell types. In this pilot study we were able to measure only overall values. Only changes occurring in numerically dominant and in metabolically active cells are expressed in the figures. At present we are not able to assign biochemical events to defined cell types within these animals. Possibly InsP3 production takes place in epithelial muscular and/or gland cells. A prominent overall change was also measured in the amount of labeled InsP2 after long-term (l- and 3-hr) exposure of the larvae to Cs+. This points to another interesting correlation ‘between the metabolism of phosphatidylinositol (pha’sphates) and metamorphosis. InsP2 was the only studied compound which was significantly elevated even after 1 and 3 hr of stimulation. According to the experiments of several groups (reviewed in Berridge and Irvine, 1984) InsP2 does not elevate cytosolic Ca2+ in the hitherto studied biological systems. InsP2 is either a product of a phosphomonoesterase reaction with InsPi (Michell et aZ.,1981; Berridge et ab, 1983) or it is form.ed from phosphatidylinositol monophosphate (PtdInsP) by the action of phospholipase C. (Phosphorylation of InsP2 seems to play no role in InsPz formation since InsPI is mainly dephosphorylated to yield inositol for resynthesis of phosphatidylinositol (Berridge et aZ., 1983; Berridge and Irvine, 1984; Parthasarathy and Eisenberg, 1986) (see Fig. 4)). If cleavage of PtdInsP by phospholipase C is realized then diacylglycerol production would result. According to the theory of Majerus et ah, (1985) the Cs+ stimulus would lead to the generation of InsP, and some diacylglycerol from PtdlnsP2. However, the majority of diacylglycerol would result from phosphodiesteratic cleavage of PtdInsP (see also Hokin-Neaverson a:nd Sadeghian, 1984). We are currently investigating these possibilities by examining the lipid metabolism of the larvae during initiation of metamorphosis and by introducing Li+ as an inhibitor of InsPI phosphomonoesterase (review by Parthasarathy and Eisenberg, 1986). The latter study is complicated by

87

in Hydractinia t

lnositol+

P,

FIG. 4. PI-cycle and possible pathways for the formation of diacylglycerol during the initiation of metamorphosis. (a) Second to minute response; (b) hour response.

the fact that Li+ itself induces metamorphosis (Mtiller and Buchal, 1973; Spindler and Mtiller, 1972). Whereas InsPs seems to trigger merely preparatory steps of metamorphosis, the diacylglycerol/protein kinase C pathway appears to be more directly involved in metamorphosis since phorbol esters induce metamorphosis in H. echinata (Mtiller, 1985). Although Mtiller (1985) did not find significant activity of protein kinase C this was probably due to the low amount of material available or to the cells involved. The synthetic diacylglycerol 1-oleoyl-2-acetyl-sn-glycerol (OAG) had no effect on planula larvae (Miiller, 1985). As shown in the present investigation another synthetic diacylglycerol, 1,2-dioctanoyl-rat-glycerol (diCs) is active in inducing metamorphosis (Fig. 3). This is in line with the observations of Shen and Burgart (1986) on the activation of the sea urchin egg. Whereas PMA and diCS caused a rise in pHi, OAG did not. Moreover, diCs proved most effective in activating protein kinase C (Ganong et ak, 1986). In planula larvae of IS. echinata 98% metamorphosis was achieved by the application of 100 @f di&. This is a relatively high concentration but it is in the range required for the activation of the sea urchin egg (see Shen and Burgart, 1986). The moderate effectiveness may be accounted for partly by the use of a racemic compound since protein kinase C is stereospecifically activated by an-1,2-diacylglycerols (for a review, see Bell, 1986). Another point to keep in mind is that diacylglycerols are rapidly metabolized by diacylglycerol kinase(s) (Nishizuka, 1984; Ashendel, 1985; Schwantke et aZ.,1985; Takai et al, 1985; Bell, 1986). Therefore in the present study a recently developed inhibitor of diacylglycerol kinase, R 59022 (de Chaffoy de Courcelles et al., 1985), was used to inhibit the metabolism of diCs and to possibly potentiate its effect on the induction of metamorphosis. As shown in Table 1, R 59022 was ineffective at concentrations of

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10 and 100 nM, but 1 pM R 59022 increased the percentage of metamorphosis induced by 5 pM diCs. Similarly, 100 nM R 59022 increased the rate of metamorphoses induced by 30 mM Cs+. Therefore, this result can be interpreted as evidence for the formation of diacylglycerols after induction of metamorphosis with Cs+. Taken together the results of our experiments point to a role for PI-cycling and protein kinase C in the initiation of metamorphosis in Hydractinia echinata. Possibly protein kinase C is more directly involved in signal transduction from the stimulus to the events leading to complete metamorphosis whereas the InsP3/Ca2+ pathway is associated with exocytosis or epitheliomuscular contraction occurring during the initial stages of metamorphosis. But how does activation of protein kinase C lead to further events of metamorphosis? Protein kinase C is thought to be involved in the regulation of ion channels, in particular of the Na+/H+ exchanger both in vertebrate and invertebrate systems (Berridge and Irvine. 1984). With respect to the metamorphosis of H. echinata it was hypothesized that membrane-associated ionic events are involved (Mi.iller and Buchal, 1973; May and Mi.iller, 1975; Miiller, 1985). To elucidate the possible importance of the Na+/H+ exchanger we are currently investigating the influence of ionophores and other drugs on the initiation of metamorphosis in H. echinata. This study received financial support by Grant Mu 222118-7accorded to W.A.M. by the DFG. REFERENCES ASHENDEL,C. L. (1985). The phorbol ester receptor: A phospholipidregulated protein kinase. Biochim. Biophys. Acta 822,219-242. BA’ITY, I. A., NAHORSKI,S. R., and IRVINE,R. F. (1985).Rapid formation of inositol1,3,4,5-tetrakisphosphate following stimulation of rat cerebral cortical slices. B&hem J. 232,211-215. BELL, R. M. (1986).Protein kinase C activation by diacylglycerol second messengers. Cell 45,631-632. BERKING,S. (1984). Metamorphosis of Hydractinia echinata, Insights into pattern formation in hydroids. Wil&Zm Roux’s Arch 193,370378. BERRIDGE,M. J. (1984a). Inositol trisphosphate and diacylglycerols as second messengers. Biochem. J. 220,345-360. BERRIDGE,M. J. (1984b). Oncogenes, inositol lipids and cellular proliferation. Biotechnology 2, 541-546. BERRIDGE,M. J. (1985). Calcium-mobilizing receptors. In “Calcium in Biological Systems” (R. P. Rubin, G. B. Weiss, and J. W. Putney, Eds.), pp. 37-44. Plenum, New York/London. BERRIDGE,M. J. (1986a). Cell signalling through phospholipid metabolism. J. Cell Sci 4, Suppl., 137-153. BERRIDGE,M. J. (1986b). Intracellular signalling through inositol trisphosphate and diacylglycerol. BioL Chem. Hoppe Seyler 367, 447456. BERRIDGE,M. J., BROWN,K. D., IRVINE, R. F., and HESLOP,J. P. (1985). Phosphoinositides and cell proliferation. J. Cell Sci. 3, Suppl., 187198. BERRIDGE,M. J., DAWSON,R. M. C., DOWNES,C. P., HESLOP,J. P., and

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IRVINE, R. F. (1983). Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. B&hem. J. 212,473-482. BERRIDGE,M. J., and IRVINE, R. F. (1984). Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature (London) 312,315-321. BROCKERHOFF, H. (1986). Membrane protein-lipid hydrogen-bonding: Evidence from protein kinase C, diglyceride and tumor promoters. FEBS L&t. 201, l-4. DE CHAFFOYDECOURCELLES, D., ROEVENS,P., and VANBELLE,H. (1985). R 59022,a diacylglycerol kinase inhibitor. J. BioL Chem. 260,15’76215770. EXTON,J. H. (1985).Role of calcium and phosphoinositides in the actions of certain hormones and neurotransmitters. J. Clin. Invest. 75,1753-

1101. FAIN, J. N. (1984).Activation of plasma membrane phosphatidylinositol turnover by hormones. Vi&m. Harm 41,117-160. FISHER, S. i., VAN ROOIJEN,L. A. A., and AGRANOFF,B. W. (1984). Renewed interest in the polyphosphoinositides. Trends Bio&em. Sci 8, 53-56. FREEMAN,G., and RIDGWAY,E. B. (1987). Endogenous photoproteins, calcium channels, and calcium transients during metamorphosis in hydrozoans. Roux’s Arch. Dev. BioL 196,30-50. GANONG,B. R., LOOMIS,C. R., HANNUN, Y. A., and BELL, R. M. (1986). Specificity and mechanism of protein kinase C activation by sn-1,2diacylglycerols. Proc. NatL Acad Sci USA 83,1184-1188. HIRASAWA,K., and NISHIZUKA,Y. (1985).Phosphatidylinositol turnover in receptor mechanism and signal transduction. Annu. Rev. Pharmacol ToxicoL 25,147-170.

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