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Interactions between Ca2+ and cAMP in ecdysteroid secretion from the prothoracic glands of Bombyx mori
Molecular and Cellular Endocrinology 154 (1999) 63 – 70 www.elsevier.com/locate/mce
Interactions between Ca2 + and cAMP in ecdysteroid secretion from the prothoracic glands of Bombyx mori Skarlatos G. Dedos 1, Hajime Fugo * Department of Biological Production, Tokyo Uni6ersity of Agriculture and Technology, Fuchu-shi, Tokyo 183, Japan Received 12 December 1998; accepted 22 March 1999
Abstract The interaction between Ca2 + and cAMP in the mediation of ecdysteroid secretion from prothoracic glands (PGs) of Bombyx mori was investigated in vitro. Omission of Ca2 + from the PGs’ incubation medium decreased basal ecdysteroid secretion from day 3 until day 6. On day 6, the ability of forskolin or 3-isobutyl-1-methylxanthine (IBMX) to stimulate ecdysteroid secretion was affected by the omission of Ca2 + from the medium. The cAMP agonist Sp-adenosine 3%,5%-cyclic monophosphothioate (Sp-cAMPS) and the cAMP analogue dibutyryl cyclic AMP (dbcAMP) stimulated ecdysteroid secretion even in the absence of Ca2 + from the medium. The Sp-cAMPS-stimulated ecdysteroid secretion was inhibited by the cAMP antagonist Rp-adenosine 3%,5%-cyclic monophosphothioate (Rp-cAMPS) and the L-type Ca2 + channel blocker verapamil. Both the Ca2 + ionophore A23187 and the L-type Ca2 + channel agonist S(-) Bay K 8644 could stimulate ecdysteroid secretion. The A23187-induced ecdysteroid secretion was partially inhibited by Rp-cAMPS. The combined results indicate that Ca2 + and cAMP signaling pathways can cooperatively, as well as independently, stimulate ecdysteroid secretion from the PGs. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ecdysone; Ca2 + ; cAMP; Prothoracic gland; Bombyx mori
1. Introduction The prothoracic glands (PGs) of insects synthesize and secrete ecdysteroids which regulate their postembryonic development (Riddiford, 1985) Ecdysteroidogenesis in the PGs is regulated by a family of cerebral neuropeptide, the prothoracicotropic hormones (PTTHs) (Bollenbacher and Granger, 1985). Stimulation of PG cells by PTTHs and subsequent secretion of ecdysteroid involve signaling cascades that utilize two common second messengers, Ca2 + and cAMP (Smith and Gilbert, 1989; Gilbert et al., 1996). In the tobacco hornworm, Manduca sexta, the two forms of Manduca PTTHs were shown to mediate their signaling cascades via cAMP as a second messenger (Smith, 1993; Watson
* Corresponding author. Tel.: +81-423-675734; fax: + 81-423608830. E-mail address: [email protected] (H. Fugo) 1 Present address: Department of Biotechnology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan.
et al., 1993). It was also demonstrated that the Manduca big PTTH raises glandular cAMP levels by increasing glandular Ca2 + levels. Elevated Ca2 + levels stimulate a Ca2 + /calmodulin-dependent adenylate cyclase (Meller et al., 1988; Smith et al., 1989; Girgenrath and Smith, 1996). Gu et al. (1996) reported PTTH-mediated increases of cAMP levels in PGs of Bombyx mori. In this study, we investigated the ability of agents that increase intracellular Ca2 + and cAMP levels to stimulate ecdysteroid secretion from the PGs of B. mori. Before reaching any conclusions about the signaling cascades of PTTHs in the PGs of B. mori, it was necessary to reveal how Ca2 + and cAMP interact as second messengers in the ecdysteroidogenic process. Thus, we avoided the use of PTTHs in this study. We have also applied literature methods (Watson et al., 1993; Hayes et al., 1995) originally used in Manduca PGs, to compare ecdysteroidogenic processes in the PGs of the two insect species. Our results suggest that both Ca2 + - and cAMP-mediated signaling pathways
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regulate ecdysteroid secretion, and a considerable degree of crosstalk between these pathways exists in the PGs of B. mori.
2. Materials and methods
2.1. Animals All experiments used larvae from the hybrid J106× DAIZO. The larvae were reared on mulberry leaves under a 12:12-L:D photoperiod at 25 9 1°C and 60% relative humidity (Dedos and Fugo, 1996). Larvae were staged after every larval ecdysis, and the day of each ecdysis was designated as day 0. Since the larvae mainly moult to the final (fifth) instar during the scotophase, all the larvae that ecdysed during the scotophase were segregated immediately after the photophase. This time was designated as 0 h of the fifth instar. In this particular hybrid, the fifth instar period lasts about : 208 h. The onset of pupal commitment occurs after 60 h (day 3) and the onset of wandering behavior occurs 144 h (day 6) after the final larval ecdysis. Female larvae were exclusively used in this study.
2.2. Reagents Graces’s medium was purchased from GIBCO-BRL (Grand Island, NY, USA).Vermapil, Ca2 + ionophore A23187, dibutyryl cyclic AMP (dbcAMP), and 3isobutyl-1-methylxanthine (IBMX) were purchased from Sigma (St Louis, MO, USA). Forskolin, Spadenosine 3%,5%-cyclic monophosphothioate (SpcAMPS), Rp-adenosine 3%,5%-cyclic monophosphothioate (Rp-cAMPS) and S(-) Bay K 8644 were purchased from Research Biochemicals International (Natick, MA, USA). EGTA was purchased from Wako (Japan). Stock solutions of forskolin, IBMX, Ca2 + ionophore A23187 and S(-) Bay K 8644 were prepared in dimethylsulfoxide (DMSO).
experiments requiring a Ca2 + -free medium, Ringer saline (Shirai et al., 1994) was prepared using NaCl (4.5 mM) in place of the standard 4.5 mM CaCl2. Hepes buffer (0.01 M, pH 6.8) and either EGTA (0.1 mM) or the indicated amount of Ca2 + (in the form of CaCl2) were added prior to use (Hayes et al., 1995). Incubations were carried out at 259 1°C in high humidity in plastic 96-multiwell plates (Wako, Japan) for the time periods shown in the figure legends. After each designated incubated period, the incubation medium was removed, and an aliquot of the medium was subjected to radioimmunoassay (RIA) for quantification of ecdysteroid content.
2.4. Radioimmunoassay The content of ecdysteroid in the incubation medium was quantified by RIA as described by Takeda et al. (1986) and Dedos and Fugo (1996). The antiserum for 20-hydroxyecdysone (20E) was kindly donated by Dr Satoshi Takeda, National Institute of Sericultural and Entomological Science, Tsukuba, Japan. The internal standard ecdysteroid for RIA was 20E. Radiolabeled ecdysone, [23,24-3H]ecdysone (sp. act. 53 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA, USA).
2.5. Statistical analyses The statistical significance of differences among means was determined by analysis of variance (ANOVA) or Student’s t-test. For most experiments, ANOVA was followed by Tukey multiple comparisons test (Watson et al., 1993). Test results are shown in the figure legends. Statistical analyses were conducted with GraphPad Prism™ 2.0 computer software.
3. Results
2.3. In 6itro prothoracic gland assay
3.1. Effects of Ca 2 + on basal ecdysteroid secretion
Larvae were anaesthetized by submersion in water and PGs were dissected rapidly (:2 min/animal) from each larva in sterile saline (0.85% NaCl). The glands were pre-incubated in Grace’s medium for 15 – 30 min. A paired gland design was used in some experiments. One gland of the pair was incubated in 20 ml of medium containing an experimental agent(s); the other gland of the pair was incubated in 20 ml medium containing solvent. When DMSO was used as solvent, the incubation medium contained 1% DMSO. In experiments where the dose response to an experimental agent was investigated, glands from a pooled group were randomly selected for incubation at the indicated doses. In
Initially, we assessed the Ca2 + requirement for basal ecdysteroidogenesis in the PGs of B. mori. Glands from each day of the fifth instar and the first day of the pupal stage were incubated in vitro for 2 h in Ca2 + -free or normal Ringer saline (Fig. 1). The ecdysteroid secretion in the presence of Ca2 + was significantly higher than that in its absence only from day 3 until day 6 (P B0.05; Fig. 1). The difference in ecdysteroid secretion in the presence versus absence of Ca2 + was expressed as activation ratio (Ar, see inlet of Fig. 1). The greatest difference was observed on day 6 (Fig. 1). Thus, all further experiments were conducted on day 6.
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3.2. Effects of Ca 2 + on the abilities of intracellular cAMP modulating agents to induce ecdysteroid secretion Next, we compared efficiencies of chemicals that increase intracellular cAMP levels to stimulate ecdys-
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teroid secretion in the presence or absence of Ca2 + . The abilities of forskolin (an activator of adenylyl cyclase) and IBMX (a phosphodiesterase inhibitor) to stimulate ecdysteroid secretion were significantly attenuated in the absence of Ca2 + (PB0.05; Fig. 2). On the
Fig. 1. Effect of external Ca2 + on basal ecdysteroid secretion from Bombyx mori PGs in vitro. One gland of a pair was incubated in Ca2 + -free Ringer saline, while its contralateral was incubated in normal Ringer saline for 2 h. The inlet shows the activation ratio of basal ecdysteroid secretion in the presence of external Ca2 + over basal secretion in the absence of external Ca2 + . Each point represents the mean9SEM of eight glands. Tests of the difference between experimental and control glands (t-tests) revealed the means to be significantly different on days 3, 4, 5 and 6 of the fifth instar (PB 0.05).
Fig. 2. Comparison between the presence and absence of external Ca2 + on the ability of intracellular cAMP modulating agents to stimulate ecdysteroid secretion from day 6 PGs in normal or Ca2 + -free Ringer saline. One gland of a pair was incubated in normal Ringer saline (open bars) while its contralateral was incubated in Ca2 + -free Ringer saline (closed bars) for 2 h. Each point represents the mean9SEM of six glands. Tests of the difference between experimental and control glands (t-tests) revealed that only the stimulation of ecdysteroid secretion by forskolin and IBMX was significantly reduced in Ca2 + -free Ringer saline (PB 0.05).
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Fig. 3. Effect of Sp-cAMPS or dbcAMP on basal ecdysteroid secretion in Ca2 + -free Ringer saline. Individual glands of day 6 were incubated for 2 h with the indicated concentrations of Sp-cAMPS or dbcAMP. Each point is the mean 9 SEM of 5 – 6 glands. Results of Tukey multiple comparisons (pairs of means enclosed by the range of a bracket are not significantly different, P\ 0.05): [1.44 – 2.32], [1.98 – 3.04], [6.04–7.08], [7.07–8.51].
contrary, the ability of the cAMP analogue, dbcAMP, to stimulate ecdysteroid secretion was not affected by the absence of Ca2 + (P \ 0.05; Fig. 2).
3.3. Effect of Sp-cAMPS or dbcAMP on ecdysteroid secretion in the absence of Ca 2 + Glands of day 6 were incubated in Ca2 + -free Ringer saline in the presence of various concentrations of the cAMP agonist, Sp-cAMPS or the cAMP analogue, dbcAMP. Sp-cAMPS dose-dependently stimulated ecdysteroid secretion in the Ca2 + -free Ringer saline (EC50 : 0.47 mM; Fig. 3). The cAMP analogue, dbcAMP was less potent than Sp-cAMPS in stimulating ecdysteroid secretion in the Ca2 + -free Ringer saline (EC50 : 1.62 mM; Fig. 3). The highest concentration of Sp-cAMPS that was tested (5mM) stimulated ecdysteroid secretion to 8.30 ng/gland and this value was not significantly different (P \0.05) from that observed in the presence of 5 mM dbcAMP (7.07 ng/gland) in the Ca2 + -free Ringer saline (Fig. 3). Similar results were obtained when these experiments were conducted with Grace’s medium. For example, 5 mM Sp-cAMPS in Grace’s medium stimulated ecdysteroid secretion to 8.22 ng/gland (data not shown).
3.4. Effect of Rp-cAMPS, and 6erapamil Sp-cAMPS-stimulated ecdysteroid secretion
on
The ability of Sp-cAMPS to stimulate ecdysteroid secretion was competitively inhibited by the cAMP
antagonist Rp-cAMPS. Glands were incubated in Grace’s medium with Sp-cAMPS (1mM) in the absence or presence of Rp-cAMPS (0–10 mM; Fig. 4). In the absence of Rp-cAMPS, Sp-cAMPS stimulated an increase above basal levels of 8.05 ng/gland but this stimulation gradually decreased as the concentration of Rp-cAMPS in the medium was increased (Fig. 4). In an experimental design adopted by Hayes et al. (1995), the L-type Ca2 + channel blocker, verapamil, could also inhibit the Sp-cAMPS-stimulated ecdysteroid secretion (Fig. 5). One gland of a pair was incubated in Grace’s medium while its contralateral was incubated in either 5 mM Sp-cAMPS or 5 mM SpcAMPS+ 10 mM verapamil (Fig. 5). Basal ecdysteroid secretion by the PGs was relatively high so the stimulation by Sp-cAMPS did not reach the levels observed in Fig. 4. However, the Sp-cAMPS-stimulated ecdysteroid secretion was blocked by verapamil and the secretory activity of the treated glands was similar to basal levels (P\0.05; Fig. 5).
3.5. Effects of S( -) Bay K 8644 and the Ca 2 + ionophore A 23187 on ecdysteroid secretion from day 6 PGs The existence of an L-type Ca2 + channel in the PGs was further investigated using a specific agonist, S(-) Bay K 8644 (Bergamaschi et al., 1988). S(-) Bay K 8644 (10 mM) could stimulate ecdysteroid secretion (Table 1). The same concentration of the Ca2 +
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ionophore A23187, however, gave results dependent on the incubation medium that was used. Glands were incubated with 10 mM A23187 in Ringer saline in the
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presence or absence of external Ca2 + (Table 1). In the presence of external Ca2 + (normal saline) the ecdysteroid secretory activity by PGs was 2.29 ng/gland
Fig. 4. Effects of Rp-cAMPS on Sp-cAMPS-stimulated ecdysteroid secretion by day 6 larval PGs. One gland of a pair was incubated in Grace’s medium containing Sp-cAMPS (1 mM) 9 Rp-cAMPS (0–10 mM) while its contralateral was incubated in Grace’s medium alone. Incubations were carried out for 2 h. The amount of ecdysteroid secreted from the experimental gland (Sp-cAMPS (1 mM) 9 Rp-cAMPS (0 – 10 mM)) minus the amount of ecdysteroid secreted from its contralateral is shown. Each point represents the mean 9 SEM of four gland pairs/dose. Tests of the difference between experimental and control glands (t-tests) revealed all the means were significantly different except when 10 mM Rp-cAMPS were present (P \0.05). Results of Tukey multiple comparisons for the effects of Rp-cAMPS on Sp-cAMPS-stimulated ecdysteroid secretion (pairs of means enclosed by the range of a bracket are not significantly different, P \0.05): [4.55 – 5.10], [5.10 – 7.15], [7.15 – 8.05].
Fig. 5. Effect of verapamil on Sp-cAMPS-stimulated ecdysteroid secretion by day 6 PGs. Open bars: Glands incubated in Grace’s medium alone. Closed bars: their contralaterals incubated in Grace’s medium containing either Sp-cAMPS (5 mM) or Sp-cAMPS (5 mM) and verapamil (10 mM). Incubations were carried out for 2 h. Each point represents the mean 9 SEM of six glands. One-way analysis of variance revealed that verapamil inhibited the Sp-cAMPS-stimulated ecdysteroid secretion (PB0.05). Results of Tukey multiple comparisons (pairs of mean enclosed by the range of a bracket are not significantly different, P\ 0.05): [4.80 – 5.38].
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Table 1 Effects of S(-) Bay K 8644 and A23187 on ecdysteroid secretion by day 6 prothoracic glandsa Agents
Ecdysteroid (ng /gland)
S(-) Bay K 8644 (10 mM)
A23187 (10 mM)
Basal
Experimental
Ca2+-free ringer
Normal ringer
5.149 0.71
13.40 90.45
0.93 90.08
2.29 9 0.14
a S(-) Bay K 8644 One gland of a pair was incubated without S(-) Bay K 8644 (Basal) and its contralateral was incubated with (Experimental) the indicated concentration of this agent for 2 h in Grace’s medium. A23187 One gland of a pair was incubated in Ca2+-free Ringer saline, while its contralateral was incubated in normal Ringer saline for 2 h. The A23187-stimulated ecdysteroid secretion in Grace’s medium is shown in Fig. 5. Each value is the mean9SEM of six glands. Tests of the difference between experimental and control glands (t-tests) revealed the means were significantly different (PB0.05).
(Table 1). This value was statistically similar (P \0.05) to the basal ecdysteroid secretion by day 6 PGs in normal Ringer saline (see Fig. 1). In the absence of external Ca2 + , the amount of ecdysteroid secreted by the A23187-stimulated PGs reached 0.93 ng/gland (Table 1). This value was statistically similar (P \ 0.05) to the basal ecdysteroid secretion on day 6 in Ca2 + -free Ringer saline (see Fig. 1). However, day 6 PGs incubated with 10 mM A23187 in Grace’s medium had a secretory activity of 9.96 ng/gland (Fig. 6). This ability of A23187 to stimulate ecdysteroid secretion was significantly decreased but not completely inhibited by 5 mM Rp-cAMPS (PB0.05; Fig. 6). 4. Discussion The results in this study suggest that Ca2 + and
cAMP play complex roles in the regulation of ecdysteroid secretion from the PGs of the silkworm, B. mori. Both Ca2 + -mediated and cAMP-mediated signaling pathways independently stimulate ecdysteroidogenesis in the PGs, but there is also a high degree of crosstalk and dependence of each signaling pathway on the other. Ca2 + by itself is a stimulator of ecdysteroid secretion because its omission resulted in a significant decrease in ecdysteroid secretion from day 3 until day 6 of the fifth larval instar (Fig. 1). Ecdysteroid secretion, however, was not dependent on external Ca2 + from day 7 until day 0 of the pupal stage (Fig. 1). This observation is in agreement with data presented by Smith et al. (1985) for Manduca day 0 pupal PGs. These authors suggested that the absence of extracellular Ca2 + is not toxic per se to the glands, nor does its omission inhibit ecdys-
Fig. 6. Effect of Rp-cAMPS on A23187-stimulated ecdysteroid secretion by day 6 PGs. Open bars: Glands incubated in Grace’s medium alone. Closed bars: their contralaterals incubated for the first 30 min in 10 ml of Grace’s medium containing or lacking Rp-cAMPS (5 mM). Then, to those glands preincubated in the presence of Rp-cAMPS was added 10 ml Grace’s medium containing Rp-cAMPS (5mM) and A23187 (20 mM), after which the glands were incubated for 1.5 h. To those glands preincubated in the absence of Rp-cAMPS was added 10 ml A23187 (20 mM), after which the glands were incubated for 1.5 h. Each point represents the mean 9SEM of six glands. One-way analysis of variance revealed that Rp-cAMPS inhibited the A23187-stimulated ecdysteroid secretion (PB 0.05). Results of Tukey multiple comparisons (pairs of means enclosed by the range of a bracket are not significantly different, P\ 0.05): [2.63 – 2.80].
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teroid secretion from PGs. These conclusions may also apply to the pupal day 0 PGs of B. mori. Therefore, we believe that Ca2 + -dependent mechanisms regulate ecdysteroid secretion from the PGs between day 3 and day 6. In a similar way, Meller et al. (1990) have shown that from day 1 to day 5 of the fifth instar of M. sexta the presence of extracellular Ca2 + alone can result in a several fold increase in ecdysteroid secretion in vitro. This dependency of ecdysteroid secretion on Ca2 + may be an indication for a site of action of extracellular Ca2 + at a level prior to cAMP formation. Indeed, the ability of forskolin and IBMX to stimulate ecdysteroid secretion was substantially attenuated in the absence of Ca2 + (Fig. 2). These results are consistent with the notion put forward by Hayes et al. (1995) for Manduca PGs, that Ca2 + acts in the cAMP-signaling pathway at the site prior to the formation of cAMP. The ability of the cAMP analogue, dbcAMP, to stimulate ecdysteroid secretion, however, was not attenuated in the absence of external Ca2 + (Fig. 2). Furthermore, both dbcAMP and the cAMP agonist, Sp-cAMPS, dose-dependently stimulated ecdysteroid secretion and this stimulation was not dependent on the presence or absence of Ca2 + (Fig. 3). The cAMP antagonist, Rp-cAMPS competitively inhibited the SpcAMPS stimulated ecdysteroid secretion (Fig. 4), indicating that activation of a cAMP-dependent protein kinase A (PKA) is directly associated with an increase in ecdysteroid secretion. It is worth mentioning that quantitatively similar decreases in Sp-cAMPS- and A23187-stimulated ecdysteroid secretions were observed in the presence of 5 mM Rp-cAMPS (Fig. 4 and Fig. 6). These decreases were also quantitatively similar to the differences in forskolin-stimulated and IBMX-stimulated ecdysteroid secretion in Ca2 + -free and normal Ringer saline (Fig. 2). The Sp-cAMPS-stimulated ecdysteroid secretion was inhibited by the L-type Ca2 + channel blocker, verapamil (Fig. 5). The same experiment performed in Manduca PGs showed that verapamil did not affect the Sp-cAMPS-stimulated ecdysteroid secretion (Hayes et al., 1995). This discrepancy between the two experimental data suggests that there are different PKA-mediated regulatory mechanisms in the PGs of the two insect species. Although Ca2 + influx activates adenylyl cyclase in both insect species (Fig. 2; Smith et al., 1985), the PKA-mediated ecdysteroid secretion is dependent on the activation of a plasma membrane-located L-type Ca2 + channel in B. mori (Fig. 5) but not in M. sexta (Hayes et al., 1995). A similar inhibition of angiotensin II and potassium-stimulated aldosterone production from adrenal glomerulosa cells by verapamil has been originally reported by Fakunding and Catt (1980) but their findings have been re-examined because verapamil has a number of actions unrelated to voltage-dependent Ca2 + channels (Aguilera and Catt 1986). Furthermore,
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verapamil has been reported to inhibit voltage-dependent K + channels and Na + channels (cited by Koch et al., 1992). Therefore, although verapamil assayed in Grace’s medium did not inhibit basal ecdysteroid secretion (data not shown), we believe that more research is required to determine any relation between PKA and a plasma membrane-located L-type Ca2 + channel. The presence of an L-type Ca2 + channel in the plasma membrane of the Bombyx PGs cells is indicated by the ability of the Ca2 + channel agonist S(-) Bay K 8644 to stimulate ecdysteroid secretion on day 6 (Table 1). Similar activation of ecdysteroid secretion was observed when PGs were incubated with the Ca2 + ionophore A23187 in Grace’s medium (Fig. 6). In normal Ringer saline the A23187-stimulated ecdysteroid secretion was substantially lower (Table 1) than in Grace’s medium, although basal ecdysteroid secretion was similar in the two incubation mediums (see Fig. 1 and Fig. 6). Ca2 + influx into the cytosol can be triggered when intracellular Ca2 + stores are artificially emptied by Ca2 + ionophores (Smith et al., 1989; Hoth and Penner, 1992; Berridge, 1993). Ionophore induced increases of intracellular free Ca2 + have been found to be relatively inefficient in regulating adenylyl cyclase activity (Cooper et al., 1995). Current evidence suggests a strong linkage between Ca2 + influx through plasma membrane ion channels and the control of adenylyl cyclase activity (Antoni, 1997). It is possible that cAMP formation in the PG cells is stimulated by a high concentration of free cytosolic Ca2 + resulting from the cumulative effects of Ca2 + influx through plasma membrane ion channels and the release of sequestered Ca2 + from inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular stores. Thus, the different A23187-mediated ecdysteroid secretions, observed between Grace’s medium and Ringer saline, might be an indication that protein phosphorylation or other second messengers are responsible for mobilizing Ca2 + from the intracellular stores leading to increased ecdysteroid secretion. A candidate might be PKA because the cAMP antagonist, Rp-cAMPS, inhibited the A23187-stimulated ecdysteroid secretion (Fig. 6). Phosphorylation of IP3 receptors by PKA has been reported to lead to IP3-induced Ca2 + mobilization (Wojcikiewicz and Luo, 1998) although cAMP-mediated inhibition of Ca2 + oscillation has also been reported (Tsunoda, 1993). The inability of Rp-cAMPS to completely block the A23187-stimulated ecdysteroid secretion (Fig. 6) supports the hypothesis that in Bombyx PGs, Ca2 + -mediated stimulation of ecdysteroid secretion can be independent of the cAMPsignaling pathway. A similar hypothesis has been proposed for Manduca PGs (Hayes et al., 1995). Further investigation is required to identify the role PKA plays in the regulation of L-type Ca2 + channels and/or intracellular Ca2 + mobilization in the PGs of B. mori.
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Acknowledgements We would like to thank Dr Satoshi Takeda, National Institute of Sericultural and Entomological Science, Tsukuba, Japan, for donating the antiserum for 20-hydroxyecdysone. This study was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (No. 08276206, 09265204 and 10161205).
References Aguilera, G., Catt, J.K., 1986. Participation of voltage-dependent calcium channels in the regulation of adrenal glomerulosa function by angiotensin II and potassium. Endocrinology 118, 112 – 117. Antoni, F.A., 1997. Calcium regulation of adenylyl cyclase, Trends Endocrinol. Metab. 8, 7–14 Bergamaschi, S., Govoni, S., Cominetti, P., Parenti, M., Trabucchi, M., 1988. Direct coupling of a G-protein to dihydropyridine binding sites. Biochem. Biophys. Res. Commun. 156, 1279 – 1286. Berridge, M.J., 1993. Inositol trisphosphate and calcium signalling. Nature 361, 315 – 325. Bollenbacher, W.E., Granger, N.A., 1985. Endocrinology of the prothoracicotropic hormone. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon Press, New York, pp. 109–151. Koch, B.D., Faurot, G.F., Kopanitsa, M.V., Swinney, D. C., 1992. Pharmacology of a Ca2 + -influx pathway activated by emptying the intracellular Ca2 + stores in HL–60 cells: evidence that a cytochrome P-450 is not involved. Biochem. J. 302, 187–190. Cooper, M.F.D., Mons, N., Karpen, J.W., 1995. Adenylyl cyclases and the interaction between calcium and cAMP signaling. Nature 374, 421 – 424. Dedos, S.G., Fugo, H., 1996. Effects of fenoxycarb on the secretory activity of the prothoracic glands in the fifth instar of the silkworm, Bombyx mori. Gen. Comp. Endocrinol. 104, 213–224. Fakunding, J.L., Catt, K.J., 1980. Dependence of aldosterone stimulation in adrenal glomerulosa cells on calcium uptake: effects of lanthanum and verapamil. Endocrinology 107, 1345–1353. Gilbert, L.I., Rybczynski, R., Tobe, S.S., 1996. Endocrine cascade in insect metamorphosis. In: Gilbert, L.I., Tata, J.R., Atkinson, G.B. (Eds.), Metamorphosis: Postembryonic Reprogramming of Gene Expression in Amphibian and Insect Cells. Academic Press, San Diego, pp. 59 – 107. Girgenrath, S., Smith, W.A., 1996. Investigation of presumptive mobilization pathways for calcium in the steroidogenic action of big prothoracicotropic hormone. Insect Biochem. Molec. Biol. 26, 455 – 463.
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Gu, S.H., Chow, Y.S., Lin, F.J., Wu, J.L., Ho, R.J., 1996. A deficiency in prothoracicotropic hormone transduction pathway during the early last larval instar of Bombyx mori. Mol. Cell. Endocrinol. 120, 99 – 105. Hayes, G.C., Muehleisen, D.P., Bollenbacher, W.E., Watson, R.D., 1995. Stimulation of ecdysteroidogenesis by small prothoracicotropic hormone: role of calcium. Mol. Cell Endocrinol. 115, 105 – 112. Hoth, M., Penner, R., 1992. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 355, 353–356. Meller, V.H., Combest, W.L., Smith, W.A., Gilbert, L.I., 1988. A calmodulin sensitive adenylate cyclase in the prothoracic glands of the tobacco hornworm, Manduca sexta. Mol. Cell. Endocrinol. 59, 67 – 76. Meller, V., Sakurai, S., Gilbert, L.I., 1990. Developmental regulation of calmodulin-dependent adenylate cyclase activity in an insect endocrine gland. Cell Regul. 1, 771 – 780. Riddiford, L.M., 1985. Hormone action at the cellular level. In: Kerkut, G.A., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology. Pergamon Press, New York, pp. 37 – 84. Shirai, Y., Iwasaki, T., Matsubara, F., Aizono, Y., 1994. The carbachol-induced release of prothoracicotropic hormone from braincorpus cardiacum-corpus allatum complex of the silkworm, Bombyx mori. J. Insect Physiol. 40, 469 – 473. Smith, J.B., Zheng, T., Lyu, R.-M., 1989. Ionomycin releases calcium from the sarcoplasmic reticulum and activates Na + /Ca2 + exchange in vascular smooth muscle cells. Cell Calcium 10, 125– 134. Smith, W.A., Gilbert, L.I., Bollenbacher, W.E., 1985. Calcium-cyclic AMP interactions in prothoracicotropic hormone stimulation of ecdysone synthesis. Mol. Cell. Endocrinol. 39, 71 – 78. Smith, W.A., Gilbert, L.I., 1989. Early events in peptide-stimulated ecdysteroid secretion by the prothoracic glands of Manduca sexta. J. Exper. Zool. 252, 264 – 270. Smith, W.A., 1993. Second messengers and the action of prothoracicotropic hormone in Manduca sexta. Am. Zool. 33, 330– 339. Takeda, S., Kiuchi, M., Ueda, S., 1986. Preparation of anti-20-hydroxyecdysone serum and its application for radioimmunoassay of ecdysteroids in silkworm haemolymph. Bull. Seric. Sci. Stn. 30, 361 – 374. Tsunoda, Y., 1993. Receptor-operated Ca2 + signaling and crosstalk in stimulus secretion coupling. Biochim. Biophys. Acta 1154, 105 – 156. Watson, R.D., Yeh, W.E., Muehleisen, D.P., Watson, C.J., Bollenbacher, W.E., 1993. Stimulation of ecdysteroidogenesis by small prothoracicotropic hormone: role of cAMP. Mol. Cell. Endocrinol. 92, 221 – 228. Wojcikiewicz, R.J., Luo, G.S., 1998. Phosphorylation of inositol 1,4,5-trisphosphate receptors by cAMP-dependent protein kinase. J. Biol. Chem. 273, 5670 – 5677.
Interactions between Ca2 + and cAMP in ecdysteroid secretion from the prothoracic glands of Bombyx mori Skarlatos G. Dedos 1, Hajime Fugo * Department of Biological Production, Tokyo Uni6ersity of Agriculture and Technology, Fuchu-shi, Tokyo 183, Japan Received 12 December 1998; accepted 22 March 1999
Abstract The interaction between Ca2 + and cAMP in the mediation of ecdysteroid secretion from prothoracic glands (PGs) of Bombyx mori was investigated in vitro. Omission of Ca2 + from the PGs’ incubation medium decreased basal ecdysteroid secretion from day 3 until day 6. On day 6, the ability of forskolin or 3-isobutyl-1-methylxanthine (IBMX) to stimulate ecdysteroid secretion was affected by the omission of Ca2 + from the medium. The cAMP agonist Sp-adenosine 3%,5%-cyclic monophosphothioate (Sp-cAMPS) and the cAMP analogue dibutyryl cyclic AMP (dbcAMP) stimulated ecdysteroid secretion even in the absence of Ca2 + from the medium. The Sp-cAMPS-stimulated ecdysteroid secretion was inhibited by the cAMP antagonist Rp-adenosine 3%,5%-cyclic monophosphothioate (Rp-cAMPS) and the L-type Ca2 + channel blocker verapamil. Both the Ca2 + ionophore A23187 and the L-type Ca2 + channel agonist S(-) Bay K 8644 could stimulate ecdysteroid secretion. The A23187-induced ecdysteroid secretion was partially inhibited by Rp-cAMPS. The combined results indicate that Ca2 + and cAMP signaling pathways can cooperatively, as well as independently, stimulate ecdysteroid secretion from the PGs. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ecdysone; Ca2 + ; cAMP; Prothoracic gland; Bombyx mori
1. Introduction The prothoracic glands (PGs) of insects synthesize and secrete ecdysteroids which regulate their postembryonic development (Riddiford, 1985) Ecdysteroidogenesis in the PGs is regulated by a family of cerebral neuropeptide, the prothoracicotropic hormones (PTTHs) (Bollenbacher and Granger, 1985). Stimulation of PG cells by PTTHs and subsequent secretion of ecdysteroid involve signaling cascades that utilize two common second messengers, Ca2 + and cAMP (Smith and Gilbert, 1989; Gilbert et al., 1996). In the tobacco hornworm, Manduca sexta, the two forms of Manduca PTTHs were shown to mediate their signaling cascades via cAMP as a second messenger (Smith, 1993; Watson
* Corresponding author. Tel.: +81-423-675734; fax: + 81-423608830. E-mail address: [email protected] (H. Fugo) 1 Present address: Department of Biotechnology, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan.
et al., 1993). It was also demonstrated that the Manduca big PTTH raises glandular cAMP levels by increasing glandular Ca2 + levels. Elevated Ca2 + levels stimulate a Ca2 + /calmodulin-dependent adenylate cyclase (Meller et al., 1988; Smith et al., 1989; Girgenrath and Smith, 1996). Gu et al. (1996) reported PTTH-mediated increases of cAMP levels in PGs of Bombyx mori. In this study, we investigated the ability of agents that increase intracellular Ca2 + and cAMP levels to stimulate ecdysteroid secretion from the PGs of B. mori. Before reaching any conclusions about the signaling cascades of PTTHs in the PGs of B. mori, it was necessary to reveal how Ca2 + and cAMP interact as second messengers in the ecdysteroidogenic process. Thus, we avoided the use of PTTHs in this study. We have also applied literature methods (Watson et al., 1993; Hayes et al., 1995) originally used in Manduca PGs, to compare ecdysteroidogenic processes in the PGs of the two insect species. Our results suggest that both Ca2 + - and cAMP-mediated signaling pathways
0303-7207/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 3 - 7 2 0 7 ( 9 9 ) 0 0 0 8 2 - 9
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regulate ecdysteroid secretion, and a considerable degree of crosstalk between these pathways exists in the PGs of B. mori.
2. Materials and methods
2.1. Animals All experiments used larvae from the hybrid J106× DAIZO. The larvae were reared on mulberry leaves under a 12:12-L:D photoperiod at 25 9 1°C and 60% relative humidity (Dedos and Fugo, 1996). Larvae were staged after every larval ecdysis, and the day of each ecdysis was designated as day 0. Since the larvae mainly moult to the final (fifth) instar during the scotophase, all the larvae that ecdysed during the scotophase were segregated immediately after the photophase. This time was designated as 0 h of the fifth instar. In this particular hybrid, the fifth instar period lasts about : 208 h. The onset of pupal commitment occurs after 60 h (day 3) and the onset of wandering behavior occurs 144 h (day 6) after the final larval ecdysis. Female larvae were exclusively used in this study.
2.2. Reagents Graces’s medium was purchased from GIBCO-BRL (Grand Island, NY, USA).Vermapil, Ca2 + ionophore A23187, dibutyryl cyclic AMP (dbcAMP), and 3isobutyl-1-methylxanthine (IBMX) were purchased from Sigma (St Louis, MO, USA). Forskolin, Spadenosine 3%,5%-cyclic monophosphothioate (SpcAMPS), Rp-adenosine 3%,5%-cyclic monophosphothioate (Rp-cAMPS) and S(-) Bay K 8644 were purchased from Research Biochemicals International (Natick, MA, USA). EGTA was purchased from Wako (Japan). Stock solutions of forskolin, IBMX, Ca2 + ionophore A23187 and S(-) Bay K 8644 were prepared in dimethylsulfoxide (DMSO).
experiments requiring a Ca2 + -free medium, Ringer saline (Shirai et al., 1994) was prepared using NaCl (4.5 mM) in place of the standard 4.5 mM CaCl2. Hepes buffer (0.01 M, pH 6.8) and either EGTA (0.1 mM) or the indicated amount of Ca2 + (in the form of CaCl2) were added prior to use (Hayes et al., 1995). Incubations were carried out at 259 1°C in high humidity in plastic 96-multiwell plates (Wako, Japan) for the time periods shown in the figure legends. After each designated incubated period, the incubation medium was removed, and an aliquot of the medium was subjected to radioimmunoassay (RIA) for quantification of ecdysteroid content.
2.4. Radioimmunoassay The content of ecdysteroid in the incubation medium was quantified by RIA as described by Takeda et al. (1986) and Dedos and Fugo (1996). The antiserum for 20-hydroxyecdysone (20E) was kindly donated by Dr Satoshi Takeda, National Institute of Sericultural and Entomological Science, Tsukuba, Japan. The internal standard ecdysteroid for RIA was 20E. Radiolabeled ecdysone, [23,24-3H]ecdysone (sp. act. 53 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA, USA).
2.5. Statistical analyses The statistical significance of differences among means was determined by analysis of variance (ANOVA) or Student’s t-test. For most experiments, ANOVA was followed by Tukey multiple comparisons test (Watson et al., 1993). Test results are shown in the figure legends. Statistical analyses were conducted with GraphPad Prism™ 2.0 computer software.
3. Results
2.3. In 6itro prothoracic gland assay
3.1. Effects of Ca 2 + on basal ecdysteroid secretion
Larvae were anaesthetized by submersion in water and PGs were dissected rapidly (:2 min/animal) from each larva in sterile saline (0.85% NaCl). The glands were pre-incubated in Grace’s medium for 15 – 30 min. A paired gland design was used in some experiments. One gland of the pair was incubated in 20 ml of medium containing an experimental agent(s); the other gland of the pair was incubated in 20 ml medium containing solvent. When DMSO was used as solvent, the incubation medium contained 1% DMSO. In experiments where the dose response to an experimental agent was investigated, glands from a pooled group were randomly selected for incubation at the indicated doses. In
Initially, we assessed the Ca2 + requirement for basal ecdysteroidogenesis in the PGs of B. mori. Glands from each day of the fifth instar and the first day of the pupal stage were incubated in vitro for 2 h in Ca2 + -free or normal Ringer saline (Fig. 1). The ecdysteroid secretion in the presence of Ca2 + was significantly higher than that in its absence only from day 3 until day 6 (P B0.05; Fig. 1). The difference in ecdysteroid secretion in the presence versus absence of Ca2 + was expressed as activation ratio (Ar, see inlet of Fig. 1). The greatest difference was observed on day 6 (Fig. 1). Thus, all further experiments were conducted on day 6.
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3.2. Effects of Ca 2 + on the abilities of intracellular cAMP modulating agents to induce ecdysteroid secretion Next, we compared efficiencies of chemicals that increase intracellular cAMP levels to stimulate ecdys-
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teroid secretion in the presence or absence of Ca2 + . The abilities of forskolin (an activator of adenylyl cyclase) and IBMX (a phosphodiesterase inhibitor) to stimulate ecdysteroid secretion were significantly attenuated in the absence of Ca2 + (PB0.05; Fig. 2). On the
Fig. 1. Effect of external Ca2 + on basal ecdysteroid secretion from Bombyx mori PGs in vitro. One gland of a pair was incubated in Ca2 + -free Ringer saline, while its contralateral was incubated in normal Ringer saline for 2 h. The inlet shows the activation ratio of basal ecdysteroid secretion in the presence of external Ca2 + over basal secretion in the absence of external Ca2 + . Each point represents the mean9SEM of eight glands. Tests of the difference between experimental and control glands (t-tests) revealed the means to be significantly different on days 3, 4, 5 and 6 of the fifth instar (PB 0.05).
Fig. 2. Comparison between the presence and absence of external Ca2 + on the ability of intracellular cAMP modulating agents to stimulate ecdysteroid secretion from day 6 PGs in normal or Ca2 + -free Ringer saline. One gland of a pair was incubated in normal Ringer saline (open bars) while its contralateral was incubated in Ca2 + -free Ringer saline (closed bars) for 2 h. Each point represents the mean9SEM of six glands. Tests of the difference between experimental and control glands (t-tests) revealed that only the stimulation of ecdysteroid secretion by forskolin and IBMX was significantly reduced in Ca2 + -free Ringer saline (PB 0.05).
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Fig. 3. Effect of Sp-cAMPS or dbcAMP on basal ecdysteroid secretion in Ca2 + -free Ringer saline. Individual glands of day 6 were incubated for 2 h with the indicated concentrations of Sp-cAMPS or dbcAMP. Each point is the mean 9 SEM of 5 – 6 glands. Results of Tukey multiple comparisons (pairs of means enclosed by the range of a bracket are not significantly different, P\ 0.05): [1.44 – 2.32], [1.98 – 3.04], [6.04–7.08], [7.07–8.51].
contrary, the ability of the cAMP analogue, dbcAMP, to stimulate ecdysteroid secretion was not affected by the absence of Ca2 + (P \ 0.05; Fig. 2).
3.3. Effect of Sp-cAMPS or dbcAMP on ecdysteroid secretion in the absence of Ca 2 + Glands of day 6 were incubated in Ca2 + -free Ringer saline in the presence of various concentrations of the cAMP agonist, Sp-cAMPS or the cAMP analogue, dbcAMP. Sp-cAMPS dose-dependently stimulated ecdysteroid secretion in the Ca2 + -free Ringer saline (EC50 : 0.47 mM; Fig. 3). The cAMP analogue, dbcAMP was less potent than Sp-cAMPS in stimulating ecdysteroid secretion in the Ca2 + -free Ringer saline (EC50 : 1.62 mM; Fig. 3). The highest concentration of Sp-cAMPS that was tested (5mM) stimulated ecdysteroid secretion to 8.30 ng/gland and this value was not significantly different (P \0.05) from that observed in the presence of 5 mM dbcAMP (7.07 ng/gland) in the Ca2 + -free Ringer saline (Fig. 3). Similar results were obtained when these experiments were conducted with Grace’s medium. For example, 5 mM Sp-cAMPS in Grace’s medium stimulated ecdysteroid secretion to 8.22 ng/gland (data not shown).
3.4. Effect of Rp-cAMPS, and 6erapamil Sp-cAMPS-stimulated ecdysteroid secretion
on
The ability of Sp-cAMPS to stimulate ecdysteroid secretion was competitively inhibited by the cAMP
antagonist Rp-cAMPS. Glands were incubated in Grace’s medium with Sp-cAMPS (1mM) in the absence or presence of Rp-cAMPS (0–10 mM; Fig. 4). In the absence of Rp-cAMPS, Sp-cAMPS stimulated an increase above basal levels of 8.05 ng/gland but this stimulation gradually decreased as the concentration of Rp-cAMPS in the medium was increased (Fig. 4). In an experimental design adopted by Hayes et al. (1995), the L-type Ca2 + channel blocker, verapamil, could also inhibit the Sp-cAMPS-stimulated ecdysteroid secretion (Fig. 5). One gland of a pair was incubated in Grace’s medium while its contralateral was incubated in either 5 mM Sp-cAMPS or 5 mM SpcAMPS+ 10 mM verapamil (Fig. 5). Basal ecdysteroid secretion by the PGs was relatively high so the stimulation by Sp-cAMPS did not reach the levels observed in Fig. 4. However, the Sp-cAMPS-stimulated ecdysteroid secretion was blocked by verapamil and the secretory activity of the treated glands was similar to basal levels (P\0.05; Fig. 5).
3.5. Effects of S( -) Bay K 8644 and the Ca 2 + ionophore A 23187 on ecdysteroid secretion from day 6 PGs The existence of an L-type Ca2 + channel in the PGs was further investigated using a specific agonist, S(-) Bay K 8644 (Bergamaschi et al., 1988). S(-) Bay K 8644 (10 mM) could stimulate ecdysteroid secretion (Table 1). The same concentration of the Ca2 +
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ionophore A23187, however, gave results dependent on the incubation medium that was used. Glands were incubated with 10 mM A23187 in Ringer saline in the
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presence or absence of external Ca2 + (Table 1). In the presence of external Ca2 + (normal saline) the ecdysteroid secretory activity by PGs was 2.29 ng/gland
Fig. 4. Effects of Rp-cAMPS on Sp-cAMPS-stimulated ecdysteroid secretion by day 6 larval PGs. One gland of a pair was incubated in Grace’s medium containing Sp-cAMPS (1 mM) 9 Rp-cAMPS (0–10 mM) while its contralateral was incubated in Grace’s medium alone. Incubations were carried out for 2 h. The amount of ecdysteroid secreted from the experimental gland (Sp-cAMPS (1 mM) 9 Rp-cAMPS (0 – 10 mM)) minus the amount of ecdysteroid secreted from its contralateral is shown. Each point represents the mean 9 SEM of four gland pairs/dose. Tests of the difference between experimental and control glands (t-tests) revealed all the means were significantly different except when 10 mM Rp-cAMPS were present (P \0.05). Results of Tukey multiple comparisons for the effects of Rp-cAMPS on Sp-cAMPS-stimulated ecdysteroid secretion (pairs of means enclosed by the range of a bracket are not significantly different, P \0.05): [4.55 – 5.10], [5.10 – 7.15], [7.15 – 8.05].
Fig. 5. Effect of verapamil on Sp-cAMPS-stimulated ecdysteroid secretion by day 6 PGs. Open bars: Glands incubated in Grace’s medium alone. Closed bars: their contralaterals incubated in Grace’s medium containing either Sp-cAMPS (5 mM) or Sp-cAMPS (5 mM) and verapamil (10 mM). Incubations were carried out for 2 h. Each point represents the mean 9 SEM of six glands. One-way analysis of variance revealed that verapamil inhibited the Sp-cAMPS-stimulated ecdysteroid secretion (PB0.05). Results of Tukey multiple comparisons (pairs of mean enclosed by the range of a bracket are not significantly different, P\ 0.05): [4.80 – 5.38].
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Table 1 Effects of S(-) Bay K 8644 and A23187 on ecdysteroid secretion by day 6 prothoracic glandsa Agents
Ecdysteroid (ng /gland)
S(-) Bay K 8644 (10 mM)
A23187 (10 mM)
Basal
Experimental
Ca2+-free ringer
Normal ringer
5.149 0.71
13.40 90.45
0.93 90.08
2.29 9 0.14
a S(-) Bay K 8644 One gland of a pair was incubated without S(-) Bay K 8644 (Basal) and its contralateral was incubated with (Experimental) the indicated concentration of this agent for 2 h in Grace’s medium. A23187 One gland of a pair was incubated in Ca2+-free Ringer saline, while its contralateral was incubated in normal Ringer saline for 2 h. The A23187-stimulated ecdysteroid secretion in Grace’s medium is shown in Fig. 5. Each value is the mean9SEM of six glands. Tests of the difference between experimental and control glands (t-tests) revealed the means were significantly different (PB0.05).
(Table 1). This value was statistically similar (P \0.05) to the basal ecdysteroid secretion by day 6 PGs in normal Ringer saline (see Fig. 1). In the absence of external Ca2 + , the amount of ecdysteroid secreted by the A23187-stimulated PGs reached 0.93 ng/gland (Table 1). This value was statistically similar (P \ 0.05) to the basal ecdysteroid secretion on day 6 in Ca2 + -free Ringer saline (see Fig. 1). However, day 6 PGs incubated with 10 mM A23187 in Grace’s medium had a secretory activity of 9.96 ng/gland (Fig. 6). This ability of A23187 to stimulate ecdysteroid secretion was significantly decreased but not completely inhibited by 5 mM Rp-cAMPS (PB0.05; Fig. 6). 4. Discussion The results in this study suggest that Ca2 + and
cAMP play complex roles in the regulation of ecdysteroid secretion from the PGs of the silkworm, B. mori. Both Ca2 + -mediated and cAMP-mediated signaling pathways independently stimulate ecdysteroidogenesis in the PGs, but there is also a high degree of crosstalk and dependence of each signaling pathway on the other. Ca2 + by itself is a stimulator of ecdysteroid secretion because its omission resulted in a significant decrease in ecdysteroid secretion from day 3 until day 6 of the fifth larval instar (Fig. 1). Ecdysteroid secretion, however, was not dependent on external Ca2 + from day 7 until day 0 of the pupal stage (Fig. 1). This observation is in agreement with data presented by Smith et al. (1985) for Manduca day 0 pupal PGs. These authors suggested that the absence of extracellular Ca2 + is not toxic per se to the glands, nor does its omission inhibit ecdys-
Fig. 6. Effect of Rp-cAMPS on A23187-stimulated ecdysteroid secretion by day 6 PGs. Open bars: Glands incubated in Grace’s medium alone. Closed bars: their contralaterals incubated for the first 30 min in 10 ml of Grace’s medium containing or lacking Rp-cAMPS (5 mM). Then, to those glands preincubated in the presence of Rp-cAMPS was added 10 ml Grace’s medium containing Rp-cAMPS (5mM) and A23187 (20 mM), after which the glands were incubated for 1.5 h. To those glands preincubated in the absence of Rp-cAMPS was added 10 ml A23187 (20 mM), after which the glands were incubated for 1.5 h. Each point represents the mean 9SEM of six glands. One-way analysis of variance revealed that Rp-cAMPS inhibited the A23187-stimulated ecdysteroid secretion (PB 0.05). Results of Tukey multiple comparisons (pairs of means enclosed by the range of a bracket are not significantly different, P\ 0.05): [2.63 – 2.80].
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teroid secretion from PGs. These conclusions may also apply to the pupal day 0 PGs of B. mori. Therefore, we believe that Ca2 + -dependent mechanisms regulate ecdysteroid secretion from the PGs between day 3 and day 6. In a similar way, Meller et al. (1990) have shown that from day 1 to day 5 of the fifth instar of M. sexta the presence of extracellular Ca2 + alone can result in a several fold increase in ecdysteroid secretion in vitro. This dependency of ecdysteroid secretion on Ca2 + may be an indication for a site of action of extracellular Ca2 + at a level prior to cAMP formation. Indeed, the ability of forskolin and IBMX to stimulate ecdysteroid secretion was substantially attenuated in the absence of Ca2 + (Fig. 2). These results are consistent with the notion put forward by Hayes et al. (1995) for Manduca PGs, that Ca2 + acts in the cAMP-signaling pathway at the site prior to the formation of cAMP. The ability of the cAMP analogue, dbcAMP, to stimulate ecdysteroid secretion, however, was not attenuated in the absence of external Ca2 + (Fig. 2). Furthermore, both dbcAMP and the cAMP agonist, Sp-cAMPS, dose-dependently stimulated ecdysteroid secretion and this stimulation was not dependent on the presence or absence of Ca2 + (Fig. 3). The cAMP antagonist, Rp-cAMPS competitively inhibited the SpcAMPS stimulated ecdysteroid secretion (Fig. 4), indicating that activation of a cAMP-dependent protein kinase A (PKA) is directly associated with an increase in ecdysteroid secretion. It is worth mentioning that quantitatively similar decreases in Sp-cAMPS- and A23187-stimulated ecdysteroid secretions were observed in the presence of 5 mM Rp-cAMPS (Fig. 4 and Fig. 6). These decreases were also quantitatively similar to the differences in forskolin-stimulated and IBMX-stimulated ecdysteroid secretion in Ca2 + -free and normal Ringer saline (Fig. 2). The Sp-cAMPS-stimulated ecdysteroid secretion was inhibited by the L-type Ca2 + channel blocker, verapamil (Fig. 5). The same experiment performed in Manduca PGs showed that verapamil did not affect the Sp-cAMPS-stimulated ecdysteroid secretion (Hayes et al., 1995). This discrepancy between the two experimental data suggests that there are different PKA-mediated regulatory mechanisms in the PGs of the two insect species. Although Ca2 + influx activates adenylyl cyclase in both insect species (Fig. 2; Smith et al., 1985), the PKA-mediated ecdysteroid secretion is dependent on the activation of a plasma membrane-located L-type Ca2 + channel in B. mori (Fig. 5) but not in M. sexta (Hayes et al., 1995). A similar inhibition of angiotensin II and potassium-stimulated aldosterone production from adrenal glomerulosa cells by verapamil has been originally reported by Fakunding and Catt (1980) but their findings have been re-examined because verapamil has a number of actions unrelated to voltage-dependent Ca2 + channels (Aguilera and Catt 1986). Furthermore,
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verapamil has been reported to inhibit voltage-dependent K + channels and Na + channels (cited by Koch et al., 1992). Therefore, although verapamil assayed in Grace’s medium did not inhibit basal ecdysteroid secretion (data not shown), we believe that more research is required to determine any relation between PKA and a plasma membrane-located L-type Ca2 + channel. The presence of an L-type Ca2 + channel in the plasma membrane of the Bombyx PGs cells is indicated by the ability of the Ca2 + channel agonist S(-) Bay K 8644 to stimulate ecdysteroid secretion on day 6 (Table 1). Similar activation of ecdysteroid secretion was observed when PGs were incubated with the Ca2 + ionophore A23187 in Grace’s medium (Fig. 6). In normal Ringer saline the A23187-stimulated ecdysteroid secretion was substantially lower (Table 1) than in Grace’s medium, although basal ecdysteroid secretion was similar in the two incubation mediums (see Fig. 1 and Fig. 6). Ca2 + influx into the cytosol can be triggered when intracellular Ca2 + stores are artificially emptied by Ca2 + ionophores (Smith et al., 1989; Hoth and Penner, 1992; Berridge, 1993). Ionophore induced increases of intracellular free Ca2 + have been found to be relatively inefficient in regulating adenylyl cyclase activity (Cooper et al., 1995). Current evidence suggests a strong linkage between Ca2 + influx through plasma membrane ion channels and the control of adenylyl cyclase activity (Antoni, 1997). It is possible that cAMP formation in the PG cells is stimulated by a high concentration of free cytosolic Ca2 + resulting from the cumulative effects of Ca2 + influx through plasma membrane ion channels and the release of sequestered Ca2 + from inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular stores. Thus, the different A23187-mediated ecdysteroid secretions, observed between Grace’s medium and Ringer saline, might be an indication that protein phosphorylation or other second messengers are responsible for mobilizing Ca2 + from the intracellular stores leading to increased ecdysteroid secretion. A candidate might be PKA because the cAMP antagonist, Rp-cAMPS, inhibited the A23187-stimulated ecdysteroid secretion (Fig. 6). Phosphorylation of IP3 receptors by PKA has been reported to lead to IP3-induced Ca2 + mobilization (Wojcikiewicz and Luo, 1998) although cAMP-mediated inhibition of Ca2 + oscillation has also been reported (Tsunoda, 1993). The inability of Rp-cAMPS to completely block the A23187-stimulated ecdysteroid secretion (Fig. 6) supports the hypothesis that in Bombyx PGs, Ca2 + -mediated stimulation of ecdysteroid secretion can be independent of the cAMPsignaling pathway. A similar hypothesis has been proposed for Manduca PGs (Hayes et al., 1995). Further investigation is required to identify the role PKA plays in the regulation of L-type Ca2 + channels and/or intracellular Ca2 + mobilization in the PGs of B. mori.
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Acknowledgements We would like to thank Dr Satoshi Takeda, National Institute of Sericultural and Entomological Science, Tsukuba, Japan, for donating the antiserum for 20-hydroxyecdysone. This study was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (No. 08276206, 09265204 and 10161205).
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