The objective of this study was to determine which intracellular second messenger systems are activated by prothoracicotropic hormone in the prothoracic glands (PGs) of Bombyx mori. Recombinant prothoracicotropic hormone (rPTTH) could stimulate ecdysteroid synthesis and secretion from day 6 PGs of the 5th instar of Bombyx mori within 30 min of in vitro incubation. However, rPTTH did not stimulate any increases in the glandular content of inositol 1,4,5-trisphosphate and cAMP during this short incubation period. Extracellular Ca2 influenced the basal and rPTTH-stimulated ecdysteroid synthesis and release in a dose-dependent manner. The L-type Ca2 channel antagonist, nitrendipine, inhibited the rPTTH-stimulated ecdysteroid synthesis and secretion (IC50 ∼28 μM). The phospholipase C inhibitor, 2-nitro-4-carboxyphenylN, N-diphenylcarbamate, inhibited the rPTTH-stimulated ecdysteroid synthesis (IC50 ∼19 μM). The protein kinase C inhibitor, chelerythrine chloride, inhibited the rPTTH-stimulated ecdysteroid synthesis (IC50∼14 μM). The protein kinase C activator, phorbol-12-myristate 13-acetate (PMA), could stimulate basal ecdysteroid synthesis and secretion (EC50∼1 μM) and its inactive α-isomer (4 α-PMA) was ineffective. The combined results suggest that the PTTH-stimulated ecdysteroid synthesis and release in the PGs of Bombyx is dependent on extracellular Ca2 and the bifurcating second messenger signalling cascade of inositol 1,4,5-trisphosphate and diacylglycerol.
INTRODUCTION
The synthesis and secretion of ecdysteroid hormone from prothoracic glands (PGs) of insects is regulated by a family of cerebral neuropeptides, the prothoracicotropic hormones (PTTHs) (Gilbert et al., 1996). In the tobacco hornworm, Manduca sexta, two size variants of PTTH have been identified: big PTTH and small PTTH (Gilbert et al., 1996). Both forms of Manduca PTTH were shown to mediate their signalling cascade via cAMP as a second messenger (Smith, 1993; Watson et al., 1993). The signalling cascade of Manduca big PTTH does not involve any increases in glandular inositol phosphate production (Girgenrath and Smith, 1996). Furthermore, phorbol esters (protein kinase C activators) were not found to stimulate basal and big PTTH-stimulated ecdysone synthesis from Manduca PGs, although protein kinase C activity was found in the PGs of this insect (Smith, 1993).
In the silkworm, Bombyx mori, only the 30,000-dalton PTTH was found to stimulate ecdysteroid synthesis and secretion from the PGs of this insect (Kiriishi et al., 1992). In the present study, we used the recombinant form of Bombyx PTTH (rPTTH; Ishibashi et al., 1994), which was shown to stimulate ecdysteroid synthesis and release from 5th instar PGs (Dedos et al., 1999a). Our objective in this study was to determine which intracellular second messenger systems are activated by Bombyx PTTH in the PGs and how such activation is regulated. We present evidences to suggest that activation of ecdysone synthesis and secretion by PTTH is dependent on the presence of extracellular Ca2+ and on the generation and action of the second messengers, inositol-1,4,5 trisphosphate (IP3) and diacylglycerol which are generated from the bifurcating signalling system of receptor activated phospholipase C.
MATERIALS AND METHODS
Animals
All experiments used larvae from the hybrid J106xDAIZO. The larvae were reared on mulberry leaves under a 12:12-L:D photoperiod at 25±1°C and 60% relative humidity. Larvae were staged as we previously described (Dedos et al., 1999a). In this particular hybrid, the 5th instar period lasts about ∼208 hr. The onset of wandering behaviour occurs ∼144 hr (day 6) after the final larval ecdysis. Female larvae of day 6 were exclusively used in this study.
Reagents
Recombinant Bombyx PTTH (rPTTH) (Ishibashi et al., 1994) was dissolved in either Grace's medium (GIBCO-BRL, Grand Island, NY, USA) or Ca2+-free Ringer saline and stored at –20°C until use. Nitrendipine (Calbiochem, La Jolla, CA, USA), chelerythrine chloride (Research Biochemicals International, Natick, MA, USA), 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) (Sigma, St. Louis, MO, USA), phorbol 12-myristate 13-acetate (PMA) and 4α-phorbol 12-myristate 13-acetate (4α-PMA) (GIBCO-BRL, Grand Island, NY, USA) were prepared as stock solutions in dimethylsulfoxide (DMSO). Ethylene glycol bis(β-aminoethyl ether)- N,N,N′,N′-tetraacetic acid (EGTA) was purchased from Wako (Osaka, Japan).
in vitro prothoracic gland assay
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 μl of medium containing one or more experimental agent(s); the other gland of the pair was incubated in 20 μl medium containing solvent. When DMSO was used as solvent, all PGs were incubated in medium containing 1% DMSO. The presence of 1% DMSO does not affect basal or rPTTH-stimulated ecdysteroid synthesis and release. 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 experiments requiring a Ca2+-free medium, Ringer's 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 25±1°C in high humidity in 96-multiwell plates (Wako, Osaka, Japan). After each designated incubation period, the medium was removed, and an aliquot of the medium was subjected to radio-immunoassay for quantification of ecdysteroid content.
Quantification of inositol 1,4,5-trisphosphate and cAMP
The content of inositol 1,4,5-trisphosphate (IP3) in PGs was quantified by a [3H]radioreceptor assay using the kit and protocol available from New England Nuclear Corp (Boston, MA, USA) as we previously described (Dedos et al., 1998). Recovery of IP3 from the glands was determined to be 90%. Sensitivity of the IP3 assay system was approximately 0.1 pmol.
The content of cAMP in PGs was quantified by enzyme immunoassay using the kit and protocol available from Cayman Chemical Co. (Ann Arbor, MI, USA), as we previously described (Dedos et al., 1999a).
Radioimmunoassay
The amount of ecdysteroid in the incubation medium was quantified by radioimmunoassay, as we previously described (Dedos et al., 1999a). Radiolabeled ecdysone, [23,24-3H]ecdysone (sp. act. 53 Ci/mmol) was purchased from New England Nuclear Corp. (Boston, MA, USA).
Statistical analyses
Statistical significance of the results was determined by analysis of variance or Student's t-test. For most experiments, analysis of variance was followed by Tukey multiple comparisons tests. Test results are shown in figure legends. The statistical analyses were done with computer software (GraphPad Prism™ 2.0).
RESULTS
Stimulation of ecdysteroid synthesis and secretion by rPTTH and effects of rPTTH on glandular inositol 1,4,5-trisphosphate (IP3) and cAMP levels
To determine rPTTH potentiation of ecdysteroid synthesis and release from day 6 PGs, groups of glands were exposed to 1 ng of rPTTH for 30 min. The glands were then transferred to plain Grace's medium and incubated for an additional 90 min (Table 1). The results showed that rPTTH could stimulate ecdysteroid synthesis and secretion from day 6 PGs within the 30 min and even in the subsequent 90 min incubation period (Table 1).
Table 1
Stimulation of ecdysteroid synthesis and release from PGs of Bombyx mori by rPTTH
Next we determined whether rPTTH transduces its signalling cascade through activation of a phospholipase C (PLC), thus generating the second messenger inositol 1,4,5-trisphosphate (IP3). Groups of day 6 PGs were incubated in the presence of 1 ng rPTTH/gland and their IP3 contents were determined at various time intervals. As shown in Fig. 1, rPTTH could not stimulate any increase in glandular IP3 content in time intervals ranging from 30 sec to 2 hr (P=0.95).
Fig. 1
Effects of rPTTH on prothoracic gland inositol trisphosphate levels. Each point is the mean±S.E. of 4–5 glands. Data were analysed by one-way analysis of variance (P=0.95).
In a previous study, we showed that a cerebral prothoracicotropic factor, which is different from Bombyx PTTH, stimulates cAMP accumulation in the PGs of this insect (Dedos et al., 1999a). However, both a cerebral prothoracicotropic factor and rPTTH did not stimulate cAMP accumulation in day 6 PGs within a 30 min incubation (Dedos et al., 1999a). Since rPTTH stimulated ecdysteroid synthesis and secretion within a 30 min incubation (Table 1), we determined whether rPTTH transduces its signalling cascade through activation of an adenylate cyclase at time periods much shorter than 30 min. The results (Fig. 2) showed that 1 ng rPTTH did not change glandular cAMP content during incubation times ranging from 2 min to 1 hr (P=0.51; Fig. 2). The glandular content of another cyclic nucleotide, cGMP, was either not affected by rPTTH in day 6 PGs (data not shown).
Dependence of basal and rPTTH-stimulated ecdysteroid synthesis and secretion on extracellular Ca2+
Prothoracic glands from day 6 were incubated for 2 hr in Ringer's saline, in which the indicated concentration of CaCl2 was added with or without 1 ng rPTTH/gland (Fig. 3). Both, basal and rPTTH-stimulated ecdysteroid secretions were influenced by extracellular Ca2+ in a dose-dependent manner. When glands were incubated in Ca2+-free Ringer's saline, the rPTTH-stimulated ecdysteroid synthesis and secretion reached levels similar to the basal ecdysteroid secretion in the absence of extracellular Ca2+. The higher the concentration of CaCl2 in saline, the more rPTTH could stimulate ecdysteroid synthesis and release above basal levels (Fig. 3).
Fig. 3
Effect of external Ca2+ on basal and rPTTH-stimulated ecdysteroid synthesis and release by day 6 PGs. Each point is the mean±S.E. of 5 to 6 glands. Results of Tukey multiple comparisons (pairs of means enclosed by the range of a bracket are not significantly different, P>0.05): Basal; [0.56–0.97], [3.01–3.14] [3.14–3.99]; rPTTH; [0.77–1.73], [8.55–8.63], [10.49–11.47].
Effects of nitrendipine on basal and rPTTH-stimulated ecdysteroid synthesis and secretion
Prothoracic glands of day 6 were incubated for 2 hr in the presence of various concentrations of the L-type Ca2+ channel antagonist, nitrendipine, and 1 ng rPTTH/gland. The results in Figure 4 indicate that nitredipine competetively inhibited the rPTTH-stimulated ecdysteroid synthesis and release in vitro (IC50∼28 μM). Nitrendipine did not inhibit the basal secretory activity of the glands (P=0.11; Fig. 4). The rPTTH-stimulated ecdysteroid synthesis and release was inhibited in a similar way by the phenylalkylamine derivative, verapamil but not by the benzothiazepine, diltiazem (data not shown).
Fig. 4
Effect of nitrendipine on basal and rPTTH-stimulated ecdysteroid synthesis and release by day 6 PGs. Individual glands were incubated for 2 hr with the indicated concentrations of nitrendipine alone (basal) or in the simultaneous presence of 1 ng rPTTH/gland (rPTTH). Each point is the mean±S.E. of 5 to 6 glands. Data for the effects of nitrendipine on basal ecdysteroid synthesis and release were analyzed by one-way analysis of variance (P=0.11). Results of Tukey multiple comparisons for the effects of nitrendipine on rPTTH-stimulated ecdysteroid synthesis and release (pairs of means enclosed by the range of a bracket are not significantly different, P>0.05): [1.28–2.33], [9.74–11.43].
Dependence of rPTTH-stimulated ecdysteroid synthesis and release on the generation of inositol 1,4,5-trisphosphate (IP3) in the PGs
Two-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC) is known as a potent inhibitor of PLC (Takei et al., 1991). Glands of day 6 were incubated for 2 hr in the presence of 1 ng rPTTH/gland or 1 ng rPTTH+50 μM NCDC (Table 2). Recombinant PTTH stimulated ecdysteroid synthesis and release but NCDC inhibited the rPTTH-stimulated ecdysteroid synthesis and release (P>0.05; Table 2). The IP3 content of the PGs remained unchanged for 2 hr (Table 2; P=0.48).
Table 2
Effect of rPTTH on ecdysteroid synthesis and release and IP3 levels of day 6 larval PGs of Bombyx mori
In a different approach, PGs of day 6 were incubated for 2 hr in the presence of various concentrations of NCDC and 1 ng rPTTH/gland. The results in Figure 5 indicate that NCDC competetively inhibited the rPTTH-stimulated ecdysteroid synthesis and release (IC50∼19 μM), although NCDC did not inhibit the basal secretory activity (P=0.11; Fig. 5).
Fig. 5
Effect of NCDC on basal and rPTTH-stimulated ecdysteroid synthesis and release by day 6 PGs. Individual glands were incubated for 2 hr with the indicated concentrations of NCDC alone (basal) or in the simultaneous presence of 1 ng rPTTH/gland (rPTTH). Each point is the mean±S.E. of 5 to 6 glands. Data for the effects of NCDC on basal ecdysteroid level were analyzed by one-way analysis of variance (P=0.11). Results of Tukey multiplecomparisons for the effects of NCDC on rPTTH-stimulated ecdysteroid synthesis and release (pairs of means enclosed by the range of a bracket are not significantly different, P>0.05): [0.74–1.95], [6.62–8.77], [8.84–9.15].
Effects of chelerythrine chloride on basal and rPTTH-stimulated ecdysteroid synthesis and release
Prothoracic glands of day 6 were incubated for 2 hr in the presence of various concentrations of the protein kinase C (PKC) inhibitor chelerythrine chloride (Herbert et al., 1990) and 1 ng rPTTH/gland. The results in Fig. 6 indicate that chelerythrine chloride competetively inhibited the steroidogenic effect of rPTTH (IC50∼14 μM). Chelerythrine chloride did not inhibit the basal secretory activity of the glands (P=0.41; Fig. 6). Similar results were obtained with another PKC inhibitor, calphostin C (data not shown).
Fig. 6
Effect of chelerythrine chloride on basal and rPTTH-stimulated ecdysteroid synthesis and release by day 6 PGs. Individual glands were incubated for 2 hr with the indicated concentrations of chelerythrine chloride alone (basal) or in the simultaneous presence of 1 ng rPTTH/gland (rPTTH). Each point is the mean±S.E. of 5 to 6 glands. Data for the effects of chelerythrine chloride on basal ecdysteroid level were analyzed by one-way analysis of variance (P=0.41). Results of Tukey multiple comparisons for the effects of chelerythrine chloride on rPTTH-stimulated ecdysteroid synthesis and release (pairs of means enclosed by the range of a bracket are not significantly different, P>0.05): [4.81–7.76], [10.49–11.33].
Effect of phorbol 12-myristate 13-acetate (PMA) and 4α-phorbol 12-myristate 13-acetate (4α-PMA) on basal ecdysteroid synthesis and release
In initial experiments, day 6 PGs were incubated in the presence of varying concentrations (0.1 nM ∼ 0.1 mM) of PMA and 4α-PMA for 2 hr. During this incubation period, however, no statistically significant change in ecdysteroid synthesis and release was observed with PMA or 4α-PMA (data not shown). Statistical analysis showed that PMA did not alter basal ecdysteroid synthesis and release (P=0.14) and similar results were also observed with 4α-PMA (P=0.11; data not shown). Since the possibility remained that the 2 hr incubation period was too short to observe any PMA-mediated effects on basal ecdysteroid synthesis and release, the incubation period was extended to 5 hr. At 5 hr incubation period, PMA stimulated basal ecdysteroid synthesis and release (EC50∼1 μM; Fig. 7). Under similar conditions, 4α-PMA did not alter basal ecdysteroid synthesis and release (P=0.21; Fig. 7). These results were verified using a paired gland design at 5 hr incubation. Glands incubated in 1 μM PMA in Grace's medium secreted 12.3 ng ecdysone/gland while their contralaterals secreted 7.6 ng/gland (n=4), but such an effect was not observed during a 2 hr incubation (data not shown).
Fig. 7
Effect of phorbol 12-myristate 13-acetate (PMA) and 4α-PMA on basal ecdysteroid synthesis and release by day 6 PGs. Individual glands were incubated for 5 hr with the indicated concentrations of PMA or 4α-PMA. Each point is the mean±S.E. of 5 to 6 glands. Data for the effects of 4α-PMA on basal ecdysteroid level were analyzed by one-way analysis of variance (P=0.21). Results of Tukey multiple comarisons for the effects of PMA on basal ecdysteroid synthesis and release (pairs of means enclosed by the range of a bracket are not significantly different, P>0.05): [7.31–9.35], [9.05–11.03],[14.50–15.70].
DISCUSSION
The results in the present study demonstrate that the steroidogenic effect of Bombyx PTTH in PGs is dependent on extracellular Ca2+ and the bifurcating second messenger system that generates the two intracellular messengers, inositol 1,4,5-trisphosphate and diacylglycerol.
During a 30 min incubation of day 6 PGs with recombinant PTTH there was a substantial increase of ecdysteroid synthesis and release (P<0.05;Table 1). This stimulation by rPTTH was not mediated through the generation of the second messenger IP3 since rPTTH did not increase glandular IP3 content even after 2 hr incubation (Fig. 1, Table 2). Moreover, the increase in ecdysteroid during 30 min incubation (Table 1) was not preceded or accompanied by any rPTTH-mediated increase in the glandular content of the second messenger cAMP, since rPTTH did not increase cAMP content even after 1 hr incubation (Fig. 2). Gu et al. (1996), using as source of PTTH medium in which brain complexes of Bombyx were incubated, found that PTTH could stimulate cAMP accumulation in the PGs of this insect. However, we observed that this cAMP stimulating activity in Bombyx PGs was mediated by another cerebral prothoracicotropic factor different from PTTH (Dedos et al., 1999a). Recombinant PTTH stimulated cAMP accumulation only in day 4 and day 5 Bombyx PGs, and did not increase the cAMP content after a 30 min incubation of day 6 PGs (Dedos et al., 1999a). In this study, it was further shown that rPTTH did not stimulate cAMP accumulation in PGs even at incubation periods much shorter than the 30 min used in previous experiments (Fig. 2; Dedos et al., 1999a). Previous research using day 6 Bombyx PGs showed that Ca2+ and cAMP signaling pathways can cooperatively, as well as independently, stimulate ecdysteroid synthesis and release from the PGs (Dedos and Fugo, 1999b). All these results suggested that there exists a high degree of complexity and stage-specific variability in the signaling cascades that mediate ecdysteroid synthesis and release by the PGs of Bombyx. Therefore, in order to explain the signaling cascade of rPTTH-mediated ecdysteroid synthesis and release, it was important to choose, for the present experiments, a developmental stage (day 6, 5th instar) at which the rPTTH-stimulated ecdysteroid synthesis and release does not involve multiple second messenger cascades.
Both basal and rPTTH-stimulated ecdysteroid secretions were found to be affected by the presence of extracellular Ca2+ in a dose-dependent manner (Fig. 3). A similar stage-specific dependence of basal ecdysteroid synthesis and release on the presence of extracellular Ca2+ was reported for the PGs of Manduca (Meller et al., 1990). The rPTTH-stimulated ecdysteroid synthesis was reduced to levels similar to basal release in the absence of Ca2+ from the incubation medium as shown in Fig. 3 and also by Gu et al. (1998).
These results suggest that a Ca2+-influx pathway into the cytosol of the PG cells may be the primary stimuli in the Bombyx PTTH signal transduction cascade. There are several pathways of Ca2+ influx activation by receptor agonists through the plasma membrane (Fasolato et al., 1994). Among them, the receptor-operated channels together with the voltage-operated channels provide brief and high intensity bursts of Ca2+ influx into the cytosol, while the store-operated channels provide a much smaller but sustained influx of Ca2+ (Berridge, 1997). The existence of receptor-operated Ca2+ channels was suggested for the PGs of Manduca (Girgenrath and Smith, 1996). These authors suggested that the Manduca big PTTH receptor mediates Ca2+-influx through an L-type Ca2+ channel based on the ability of the L-type Ca2+ channel antagonist, nitrendipine, to inhibit Manduca big PTTH-stimulated ecdysteroid synthesis and release (Girgenrath and Smith, 1996). This Manduca big PTTH-mediated Ca2+ channel was described to be nitrendipine-sensitive and verapamil-insensitive (Girgenrath and Smith, 1996), while Manduca small PTTH-stimulated ecdysteroid synthesis was inhibited by verapamil (Hayes et al., 1995). In this study, we showed that nitrendipine competitively inhibited only the rPTTH-stimulated ecdysteroid secretion, without affecting basal ecdysteroid synthesis and release in Bombyx (Fig. 4). We also suggest that binding of Bombyx PTTH to its cellular membrane receptor results in the opening of an L-type Ca2+ channel which promotes Ca2+ entry into the cytosol of PG cells.
Research on the intracellular Ca2+ modulation in the PGs of Galleria mellonella and Manduca (Birkenbeil, 1996; 1998) revealed that there are differences between insect species in the PTTH-mediated mobilization of intracellular Ca2+ in the PGs. For example, it was shown that the PTTH-mediated increase in intracellular Ca2+ in the PG cells of Galleria was abolished by the removal of extracellular Ca2+ or in the presence of the Ca2+ channel antagonists nicardipine and verapamil (Birkenbeil, 1996). Similar experiments with the PGs of Manduca showed that the PTTH-mediated increase in intra-cellular Ca2+ was not abolished by removal of extracellular Ca2+ or in the presence of nicardipine and verapamil (Birkenbeil, 1998). Our results suggest that the PTTH-mediated mobilization of intracellular Ca2+ in the PGs of Bombyx is different from those of Galleria and Manduca.
Although rPTTH did not directly stimulate the generation of IP3 (Fig. 1), its ecdysteroidogenic action was dependent on the generation of this second messenger since the rPTTH-stimulated ecdysteroid secretion was competitively inhibited by NCDC (Table 2 and Fig. 5). These results suggest that the Ca2+ that is mobilized by PTTH into the PG cells is sequestered in IP3-sensitive intracellular Ca2+ stores and released in the ecdysteroidogenic process by the action of IP3. Thus, we believe that the PTTH signalling cascade in the PG cells involves keeping intracellular Ca2+ levels elevated during maintained stimulation and speeding the replenishment of intra-cellular Ca2+ stores. By doing so, filled internal Ca2+ stores are maintained, and their Ca2+ content is ready to be mobilized by IP3. Because the rPTTH-stimulated ecdysteroid synthesis and release was dependent on external Ca2+ (Fig. 3), it appears that an initial PTTH-mediated influx of Ca2+ requires the generation of IP3 (Table 2), and this explains why the NCDC-mediated inhibition of PLC eliminated the rPTTH-stimulated ecdysteroid synthesis and release (Fig. 5). The NCDC-mediated inhibition of PLC may eliminate increases in glandular IP3 content. This, in turn, would eliminate the IP3-mediated Ca2+ release from intracellular stores which would be the first event in the PTTH-mediated cascade of events that lead to replenishing of intracellular Ca2+ stores. Since a similar function in the replenishing of intracellular Ca2+ stores has been proposed for store-operated Ca2+ channels (Friel, 1996), we believe that if a receptor-operated Ca2+ channel is regulated by the Bombyx PTTH receptor, then this Ca2+ channel is probably in close proximity to IP3 receptors and it is also regulated by the state of filling of the IP3-sensitive intracellular Ca2+ stores.
Models for conformational coupling mechanisms between Ca2+ channels in the plasma membrane and cytosolic Ca2+ stores have been suggested to explain receptor-mediated (Rink, 1990; Tsunoda, 1993) and capacitative Ca2+ entries (Berridge, 1995; 1997, Parekh, 1997). Capacitative Ca2+ entry is the process in which Ca2+ enters the cytosol and replenishes intracellular Ca2+ stores which are emptied through the action of IP3 (Friel, 1996). The action of IP3 is essential for capacitative Ca2+ entry through store-operated Ca2+ channels (Friel, 1996) in the same way as the action of IP3 is essential for the PTTH-stimulated ecdysteroid synthesis and release. Moreover, since basal ecdysteroid synthesis and release decreased in the absence of external Ca2+ (Fig. 3), it is quite possible that store-operated Ca2+ channels exist in the PG cells to facilitate agonist-insensitive Ca2+ entry and replenishment of Ca2+ stores.
Chelerythrine chloride competitively inhibited the rPTTH-stimulated ecdysteroid synthesis and release (Fig. 6) whereas PMA stimulated basal ecdysteroid synthesis, but only after a 5 hr incubation (Fig. 7). One can assume that the PKC limb of the phosphoinositide signalling pathway is directly involved in the PTTH-evoked Ca2+ influx and increase of ecdysteroid synthesis and release. Protein kinase C has disparate and cell type specific effects on store-operated Ca2+ influx and voltage-dependent Ca2+ channel activity (Shearman et al., 1989). Activation of store-operated Ca2+ influx by PKC was documented (Tsunoda, 1993; Parekh and Penner, 1997) and this enzyme was found to positively regulate voltage-dependent Ca2+ channel activity (Shearman et al., 1989). Thus, if a PKC-mediated positive feedback mechanism operates in the PGs, a large rPTTH-stimulated ecdysteroid synthesis and release would not have been expected in the PGs in the presence of PKC inhibitors (Fig. 6). Alternatively, the PKC limb of the phosphoinositide signalling pathway may regulate ecdysteroid synthesis and secretion through an independent pathway. It may also converge with the PTTH-mediated signalling cascade at later stages of the ecdysteroidogenic process for maximal activation of ecdysteroid synthesis and release. Another possibility is that activation of PKC is required for a prolonged and sustained ecdysteroid synthesis and release as suggested for the Manduca PGs (Smith, 1993). Such a possibility may explain why a 5 hr incubation was required for PMA to stimulate ecdysteroid synthesis and release by the PGs of Bombyx (Fig. 7).
Acknowledgments
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).
