Chem. Senses 25: 173-180,
2000
© Oxford University Press 2000
Suppression of S-methylglutathione-induced Tentacle Ball Formation by Peptides and Nullification of the Suppression by TGF-ß in Hydra
Laboratory of Nutrition Chemistry, Division of Applied Life Science, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 and 1 Laboratory of Physics, Kyoto Prefectural University of Medicine, Kyoto 603-8334, Japan
Correspondence to be sent to: Kazumitsu Hanai, Biosensing Laboratory, Department of Physics, Kyoto Prefectural University of Medicine, Nishitakatsukasa-cho 13, Taishogun, Kita-ku, Kyoto 603-8334, Japan. e-mail: hanai{at}koto.kpu-m.ac.jp
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Abstract |
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Tentacle ball formation (TBF) in Hydra elicited by S-methylglutathione (GSM) was modulated by a number of biologically active peptides. Hydra fed on Artemia, which had been hatched in a common salt solution supplemented with LiCl and ZnCl2, easily induced TBF in response to GSM after pretreatment with trypsin. After Hydra were treated with 100 pg/ml trypsin for 10 min, the response to GSM (TBF) was sensitively suppressed by acidic fibroblast growth factor and other biologically active peptides for >10 h. Various peptides, but not transforming growth factor beta (TGF-ß), suppressed GSM-induced TBF in a specific pattern for each peptide. However, TGF-ß was unique in that it did not suppress the response to GSM, but nullified the suppressive effect of other peptides. Only active TGF-ß nullified the suppressive effect of the peptides, and the latent form of TGF-ß neither suppressed GSM-induced TBF nor nullified the suppressive effect of other peptides. Members of the TGF-ß family suppressed GSM-induced TBF. These results indicate that all peptides examined, except for TGF-ß suppressed the response to GSM in a manner specific to each peptide. This assay system would be useful in identification of biologically active peptides.
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Introduction |
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S-Methylglutathione (GSM), a stimulant as potent as reduced glutathione, elicited the tentacle ball formation (TBF) response to GSM, is a component of the feeding behavior of Hydra and the response is modified by the presence of various peptides. Of all the components of Hydra feeding behaviors, only TBF was modified by the presence of various peptides. Interestingly, TBF is sensitively modulated by various biologically active peptides including platelet-derived growth factor (PDGF) (Hanai et al., 1987
) and acidic fibroblast growth factor (aFGF) (Hanai et al., 1989). Modulation by the peptides (mainly suppression) was observed in the presence of GSM at concentrations specific to each peptide. Recently we found that trypsin treatment of live Hydra is required for GSM-induced TBF (Hanai and Matsuoka, 1995). It is likely that a subtle change in culture conditions of Hydra affects the behavior elicited by GSM, since GSM induces so many different but mutually related behavioral components in Hydra. In this study, we report the culture conditions under which TBF is induced efficiently in Hydra by GSM, and also the modulation of the response to GSM by aFGF and other synthetic peptides. Further, we describe a novel effect of TGF-ß, which specifically nullifies the suppressive effect of active peptides on GSM-induced TBF. We also examined in detail the effects of TGF-ß and closely related peptides on GSM-induced TBF.
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Materials and methods |
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Hydra culture
Hydra japonica were cultured as previously described (Hanai, 1998). Every 2 days, they were fed Artemia naupli (Argentemia Gold, Argent, Redmond, WA), which had been hatched in a solution of 30 g/l common salt (>99% NaCl, Japan Tobacco, Tokyo, Japan) containing 1 ng/l ZnCl2 and 0.3 g/l LiCl (except for the experiment to examine the effect of salt composition), with aeration for 1 day at 28–30°C. A portion of Hydra from the mass culture was transferred to a new glass tray, and cultured for another 4 days before the behavioral test. The Hydra were fed on the first and the third day with A. naupli which had been hatched in a solution of 30 g/l common salt supplemented with 4.0 g/l MgCl2·6H2O with aeration for 1 day at 28–30°C. TBF of Hydra that were fed on Artemia hatched in the medium supplemented with MgCl2 just before experimental use was easily modulated by peptides.
Effect of salt composition of Artemia hatching solution
Artemia were hatched in a solution containing 30 g/l common salt (Japan Tobacco), 0.3 g/l LiCl and 10 ng/l of one of the following metal salts: NaVO3, MnCl2·4H2O, FeCl3·6H2O, CoCl2·6H2O, NiCl2·6H2O, CuSO4·5H2O, ZnCl2, NaSeO3 and Na2MoO4. The effects of various concentrations of ZnCl2 were examined in the presence or absence of 0.3 g/l LiCl. TBF of Hydra fed on Artemia, which had been grown in solutions containing each salt, was examined in the presence of 0.1 or 10 µM GSM for 10 h, after a 10 min pretreatment with trypsin (crystallized, porcine pancreas, 100 pg/ml; Wako Pure Chemical Co. Ltd, Osaka, Japan). In this experiment, Hydra in mass culture were fed on Artemia that had been hatched in the common salt solution without any supplements.
Trypsin treatment
Hydra were treated with 10, 100 or 1000 pg/ml trypsin for 10 min in 1 mM HEPES buffer containing 1 mM NaHCO3, and 1 mM CaCl2 (pH 7.7)—HEPES-buffered BC (Hanai and Matsuoka, 1995). After the designated period, ten Hydra individuals were transferred to a dish (35 mm in diameter) containing 2 ml PIPES solution (1 mM PIPES, 1 mM CaCl2, pH 6.2), and the response to 10 µM GSM was examined in the presence or absence of 1 pg aFGF (Toyobo, Osaka, Japan).
Induction of TBF by GSM
After a brief rinse, ten Hydra individuals were transferred into a dish containing 2 ml of PIPES (1 mM PIPES, 1 mM CaCl2, pH 6.2) and 1 µl of the test peptide in 0.2% Prionex (Merck, Darmstadt, Germany). The dish was placed on the stage of a binocular microscope, which was kept at 20°C by circulating temperature-regulated water. After 5 min, a small amount (<10 µl) of concentrated GSM solution was applied to make final concentrations of 0.1, 0.3, 3, 10 and 50 µM. At these concentrations of GSM, we observed the largest suppression of GSM-induced TBF by various biologically active peptides (Hanai et al., 1987). After a gentle swirl, the number of Hydra that showed TBF was counted every minute for 10 min under a binocular microscope (x8). The response to GSM (TBF) was shown by the sum of the numbers of Hydra with TBF counted at each minute from 6 to 10 min divided by the total number of Hydra (Hanai and Matsuoka, 1995).
Sampling of rat cerebrospinal fluid (CSF)
Eight-week-old male Sprague–Dawley rats (Japan Charles River, Yokohama, Japan) were kept at 22 ± 2°C and 60% relative humidity under a 12 h:12 h light:dark cycle. The rats had free access to food and water. The rats were starved overnight before the day of the experiment and were anesthetized with pentobarbital. CSF was then collected from the cistema magna.All animals received humane care as outlined in the Guide for the Care and Use of Laboratory Animals (Kyoto University Animal Care Committee, according to NIH No. 86-23, revised 1985).
Biologically active peptides used as modulators of GSM-induced TBF
The peptides tested were: aFGF and basic fibroblast growth factor (bFGF); PDGF (Becton Dickinson Laboware, Bedford, MA); calcitonin gene-related peptide (CGRP, human); cholecystokinin tetrapeptide (CCK4, 30–33); cholecystokinin octapeptide (CCK8, 26–33, non-sulfated form); corticotropin releasing factor (CRF, human); growth hormone releasing factor (GRF, human); neuropeptide Y (NPY, human); and substance P (human). All synthetic peptides were the products of Peptide Institute Inc. (Osaka, Japan).
Effect of TGF-ß on GSM-induced TBF
CRF was used to suppress GSM-induced TBF because it is easily available and shows a stable suppressive effect at the highest GSM concentration (50 µM). The mixtures of CRF (1 ng) and TGF-ß1, -ß2 and -ß3 (1 ng) were added to Hydra assay medium (2 ml) containing 50 µM GSM and TBF was observed.
Effect of the members of TGF-ß superfamily on GSM-induced TBF
Latent TGF-ß1 (1 ng, recombinant human latent TGF-ß1, R&D Systems MN, USA) or active TGF-ß1 (1 ng) was added to the Hydra assay medium (2 ml) containing GSM at various concentrations and TBF was observed. The effect of TGF-ß1 on TBF in the presence of both GSM and 1 µl of rat CSF (diluted to 104 with 0.2% Prionex solution) was also examined. Rat CSF, which contains various biologically active peptides, strongly suppressed TBF induced by GSM at each concentration, as observed previously (Hanai et al., 1989; Inoue et al., 1999). The effect of the mixture of latent or active TGF-ß1 and diluted rat CSF was also examined.
Effect of the members of TGF-ß superfamily on GSM-induced TBF
We examined the effects of TGF-ß1, BMP4 (recombinant, Xenopus laevis, a kind gift from Dr Naoto Ueno) (Nishimatsu et al., 1992), human glial-derived neurotrophic factor (GDNF, Alomone Labs Ltd, Jerusalem, Israel), inhibin (human follicular fluid, Biogenesis Ltd, Poole, UK) and activin A (recombinant bovine activin A, Innogenetics NV, Zwijndrecht, Belgium) on GSM-induced TBF. The stock solutions of these peptides were prepared according to the manufacturers’ directions and added after dilution with 0.2% Prionex. Peptides (1 ng) were added to the Hydra assay medium (2 ml) containing GSM at various concentrations, and TBF was observed.
Statistics
All statistical tests were carried out using StatView (SAS Institute Inc., Cary, NC).
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Results |
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Effect of metal ions in Artemia hatching solution on GSM-induced TBF
In preliminary experiments we found that the salt composition of the solution in which Artemia had been hatched affected GSM-induced TBF in Hydra. Since the common salt from Japan Tobacco was better for induction than the reagent grade NaCl, we assumed that minor heat-stable components contained in the former salt had a positive effect on GSM-induced TBF. We examined metal salts that are contained in the biological tissues as cofactors of transport proteins and enzymes.
Various salts were added to the Artemia hatching solution. Multivalent cations such as Fe3+ and Zn2+ effectively promoted TBF in response to 0.1 µM GSM (Figure 1). A similar effect of these ions was observed in the response to higher concentrations of GSM, although the effect was less apparent (data not shown). The effect of ZnCl2 was stronger in the presence of LiCl, especially at lower concentrations of ZnCl2 (Figure 2). After extensive examinations, 1 ng/l ZnCl2 and 0.3 g/l LiCl were added to the common salt. In Hydra fed on Artemia hatched in the solution containing FeCl3, GSM-induced TBF tended to be less modulated by peptides (unpublished observation).
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Modulation of GSM-induced TBF by peptides
When Hydra were treated with trypsin at concentrations <1 µg/ml for 10 min, they developed a new repertory of GSM-induced behavior, TBF, as reported earlier (Hanai and Matsuoka, 1995; Hanai, 1998). When the concentration of trypsin was 100 pg/ml, TBF was suppressed by aFGF for >8 h (Table 1). When the trypsin concentration was 10 pg/ml, the suppression was observed a few hours later. On the other hand, when the trypsin concentration was 1 ng/ml, the suppression was observed during the first few hours, but not thereafter.
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We examined TBF induced by GSM at various concentrations in the presence of PDGF, aFGF or bFGF (Figure 3). In this experiment, Hydra were pretreated with 100 pg/ml trypsin for 10 min. Only TBF induced by GSM at the concentration specific for each factor was suppressed by that factor, as reported earlier, when Hydra were not pretreated with trypsin. In our previous experiments, trypsin was not required to produce GSM-induced TBF for reasons which are unclear (Hanai et al., 1987
, 1989). The above factors seem to suppress GSM-induced TBF similarly, either with or without pretreatment with trypsin.
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We also examined the effect of some synthetic peptides on GSM-induced TBF. Figure 4 shows the effects of cholecystokinin peptides (CCK), CGRP, CRF, GRF, NPY and substance P. The effects of these peptides were examined at a concentration of 0.5 ng/ml. The minimum concentration of CRF required to suppress TBF was 0.5 fg/ml. GSM-induced TBF was suppressed by the synthetic peptides only when the GSM concentration was at the range specific to the peptide (Figure 4).
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Effect of TGF-ß on GSM-induced TBF
CRF suppressed TBF induced by 50 µM GSM (Figure 5). However, when TGF-ß2 was added to the medium together with CRF, the suppressive effect of CRF was nullified. Three distinct forms of TGF-ß have been identified in mammals (TGF-ß1, -ß2 and -ß3) (Massague, 1990), and any one of them nullified the suppressive effect of CRF similarly. No difference in the suppression-nullifying effects was observed among the three TGF-ß isoforms (Figure 5).
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We examined the effect of TGF-ß1 on GSM-induced TBF that was suppressed by CSF. CSF is known to suppress TBF induced by GSM (Hanai et al., 1989
; Inoue et al., 1999). Different concentrations of TGF-ß1 were required to nullify the suppressive effect of CSF depending on the concentration of GSM used to induce TBF (Figure 6). The concentration of TGF-ß1 required to nullify the suppressive effect of CSF was >200, >20, >0.2, >20 and >0.06 fM in the presence of 0.1, 0.3, 3, 10 and 50 µM GSM, respectively. The suppressive effect of CSF was the most sensitive to TGF-ß1 when TBF was induced by 50 µM GSM.
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Effect of active and latent TGF-ß1 on GSM-induced TBF
Neither latent nor active TGF-ß1 suppressed GSM-induced TBF (Figure 7). Active TGF-ß1 greatly reduced the suppressive effect of rat CSF on GSM-induced TBF (Figure 8). However, latent TGF-ß1 did not reduce the suppressive effect, indicating that latent TGF-ß1 was ineffective. The strong suppression of 0.3 µM GSM-induced TBF by the simultaneous application of CSF and latent TGF-ß1 (Figure 8) may be an artifact derived from different batches of CSF samples in the control.
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Effect of members of TGF-ß superfamily on GSM-induced TBF
TGF-ß did not suppress GSM-induced TBF, while other TGF-ß superfamily peptides showed a suppressive effect (Figure 9). TBF induced by 10 µM GSM was suppressed by BMP4 at >5.8 fM and that induced by 3 and 10 µM GSM by 1.7 pM and 0.17 fM, respectively, that induced by 50 µM GSM by 0.2 fM activin and that induced by 50 µM GSM by 1.6 x 10–3 aM inhibin (Figure 10). None of the TGF-ß superfamily peptides reduced the suppressive effect of rat CSF on GSM-induced TBF (data not shown).
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Discussion |
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In this study, the conditions necessary for GSM-induced TBF were examined in detail. Hydra, fed on Artemia which had been hatched in a salt solution supplemented with ZnCl2, clearly showed TBF in response to GSM after trypsin treatment. The response to GSM after trypsin treatment was modulated by a number of biologically active peptides.
Direct addition of ZnCl2 to the culture medium was poisonous to Hydra at higher concentrations and ineffective at lower concentrations (unpublished observations). It was effective only when ZnCl2 was given through food, though we did not examine the ZnCl2 level in Artemia. It is noteworthy that Zn deficiency leads to hypogeusia in human (Atkin-Thor et al., 1978), although the mechanism of Zn requirement common to both humans and Hydra is unknown at present. A Zn-metalloprotein, gustin, from the human parotid gland has been reported to be carbonic anhydrase IV and to be correlated with the loss of the sense of taste (Thatcher et al., 1998). It is also interesting to note that incubation of Hydra with a bicarbonate solution after trypsin treatment is important in eliciting TBF (unpublished observation).
Many biologically active substances, such as nitric oxide (Colasanti et al., 1995, 1997), arachidonic acid and eicosanoids (Pierobon et al., 1997) have been reported to modulate the feeding response of Hydra. The effect of peptides on the response to GSM is outstanding: a large number of biologically active peptides seem to suppress the response to GSM in a specific way depending on individual peptides. Other peptides that were not examined in this study would also suppress the response to GSM. On the other hand, the low mol. wt classical transmitter substances, such as acetylcholine (Erzen and Brzin, 1978), biogenic amines (Hanai and Kitajima, 1984; Ventulini and Carolei, 1992) and 
-aminobutyric acid (Concas et al., 1998) have little to no effect on the response to GSM. It appears that Hydra, an animal with one of the most primitive nervous systems, extensively uses peptides as information-transmitting substances. The Hydra nervous system has been visualized by a number of antibodies to biologically active peptides (Grimmelikhuijzen and Westafall, 1995). Takahashi et al. (Takahashi et al., 1997) reported abundant peptides from Hydra tissues that were, potentially, biologically active.
The suppression-nullifying effect of TGF-ß was unique among the peptides examined. None of the peptides examined, including members of the TGF-ß superfamily, showed a similar effect. TGF-ß isoforms have diverse biological activities, including the regulation of cell proliferation and differentiation, stimulation of matrix formation, regulation of cell migration and stimulation of adhesion molecule expression (Massague et al., 1992). Furthermore, TGF-ß plays an important role as a modulator of the immune reaction, a mediator of tissue repair in bone formation and remodeling and processing of wound healing (Barnard et al., 1990; Grande, 1997; Clark and Coker, 1998; O’Kane and Ferguson, 1997). At present we do not understand why mammalian TGF-ß modified the response to GSM in Hydra. It is likely that Hydra, one of the most primitive organisms, has a system influenced by primordial TGF-ß. This unique effect of TGF-ß may be related to a mechanism involved in the suppression of the response to GSM as well as the possibly unique structural features of TGF-ß itself (Schlunegger and Grutter, 1992).
The sensitive, specific modulation of the response to GSM by biologically active peptides should be useful in the study of these peptides.
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Acknowledgments |
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The authors thank Dr Naoto Ueno for his kind gift of BMP4. This study was supported in part by a grant-in-aid from the Ministry of Education, Culture and Science (10640671), and by the ‘Research for the Future’ Program (JSPS-RFTF97L00906).
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Accepted November 18, 1999
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