Inhibition of cAMP signalling cascade-mediated Ca2+ influx by a prothoracicostatic peptide (Mas-MIP I) via dihydropyridine-sensitive Ca2+ channels in the prothoracic glands of the silkworm, Bombyx mori

Insect Biochemistry and Molecular Biology 33 (2003) 219–228 www.elsevier.com/locate/ibmb

Inhibition of cAMP signalling cascade-mediated Ca2+ influx by a prothoracicostatic peptide (Mas-MIP I) via dihydropyridinesensitive Ca2+ channels in the prothoracic glands of the silkworm, Bombyx mori S.G. Dedos ∗, 1, H. Birkenbeil Saxon Academy of Sciences at Leipzig, AG Prof. Dr. H. Penzlin, Erbertstr. 1, PF 100322, 07703 Jena, Germany Received 16 July 2002; received in revised form 3 October 2002; accepted 8 October 2002

Abstract Measurements of Ca2+ influx in Fura-2/AM loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, after application of forskolin or the cAMP analogue, 8-bromo-cAMP, showed a steady increase in [Ca2+]i, which was of extracellular origin and was inhibited, in both cases, by the dihydropyridine (DHP) derivative, nitrendipine. Nitrendipine also inhibited the abrupt S(-)·Bay K 8644-mediated increase in [Ca2+]i and its effects were mimicked by a myoinhibitory/prothoracicostatic peptide (Mas-MIP I/PTSP), which was isolated from Manduca sexta and was found to possess ecdysteroidostatic activity in Bombyx mori PGs. This peptide blocked both the forskolin and S(-)·Bay K 8644-mediated increase in [Ca2+]i of PG cells. It was ineffective, however, in blocking the recombinant prothoracicotropic hormone (rPTTH)-stimulated high increase in [Ca2+]i of PG cells suggesting that distinct and independently regulated Ca2+ influx mechanisms operate in the PG cells of Bombyx mori. The dependence of DHP-sensitive Ca2+ channels on the cAMP-signalling cascade was further corroborated by the inabilitity of nitrendipine to block the thapsigarginstimulated high increase in [Ca2+]i after depletion of Ca2+ from the intracellular stores. This, together with the inability of thapsigargin to stimulate the cAMP levels of PG cells suggest that there is a tightly regulated cross-talk mechanism between the two signalling cascades of Ca2+ and cAMP. The combined results suggest a cAMP-mediated regulation of the opening-state of DHP-sensitive Ca2+ channels and stimulation of [Ca2+]i increases and ecdysteroid secretion by a positive feedback mechanism. Mas-MIP I/PTSP interferes with this mechanism by blocking DHP-sensitive Ca2+ channels. This regulatory mechanism appears to be autonomously stimulating ecdysteroidogenesis by the PGs, it is regulated by Mas-MIP I/PTSPS, and it is not involved in other Ca2+ influx mechanisms that operate within the PG cells of Bombyx mori.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Bombyx mori; Prothoracic glands; Prothoracicostatic peptide; Mas-MIP I; Fura-2; Ca2+ channels; Prothoracicotropic hormone; Ecdysteroid

1. Introduction The process of moulting and metamorphosis in insects is regulated by ecdysteroids which are secreted by specialized glands (see reviews by Gilbert et al., 1996, 2002). The process of ecdysteroid synthesis and secretion by these glands is regulated by neuropeptide hormones that Corresponding author. Tel.: +44-1223-334062; fax: +44-1223334040. E-mail address: [email protected] (S.G. Dedos). 1 Present address: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK ∗

are released from the brain-corpora cardiaca-corpora allata (Br-CC-CA) complex (see reviews by Bollenbacher and Granger, 1985; Ga¨de et al., 1997). The net result is an orchestrated increase of ecdysteroid in the haemolymph which initiates the moulting or metamorphosis processes (see review by Riddiford, 1994). Due to the critical importance of the ecdysteroid secreting glands for the development of insects, much research has focused on the regulatory mechanisms of ecdysteroidogenesis and the neuropeptide hormones that regulate this process (see reviews by Smith, 1995; Gilbert et al., 1996, 2002). Such research has identified not only positively regulating but also negatively regulating ecdysteroido-

0965-1748/03/$ - see front matter  2002 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0965-1748(02)00206-0

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tropic peptides from insects (Kataoka et al., 1991; Ga¨ de et al., 1997; Hua et al., 1999; Gilbert et al., 2000). Among these neuropeptide hormones, a neuropeptide (PTSP) has been identified in the brains of the silkworm, Bombyx mori, and has been shown to inhibit ecdysteroid biosynthesis in the prothoracic glands (PGs) of this insect in the 5th instar (Hua et al., 1999). This peptide is identical to a myoinhibitory peptide (Mas-MIP I) isolated from the ventral nerve cord of the tobacco hornworm, Manduca sexta (Blackburn et al., 1995, 2001). It is also different by only one amino acid residue from Cam-AST B6 isolated from brains of the stick insect, Carausius morosus (Lorenz et al., 2000) and shares sequence similarities with the vertebrate peptide galanin (Hua et al., 1999). Previous research on the mechanism of inhibition of ecdysteroid synthesis by this peptide has shown that Ca2+ is involved (Dedos et al., 2001). Incubation of PGs in the presence of PTSP abolished the S(-)·Bay K 8644mediated stimulation of ecdysteroid secretion while the ecdysteroidostatic activity of PTSP was abolished in the absence of extracellular Ca2+ (Dedos et al., 2001). In this study, based on the previous findings, we used intracellular [Ca2+]i measurements of Fura-2/AM loaded PG cells to identify the mechanism that transduces the prothoracicostatic peptide’s inhibitory action. Here, we report a regulatory mechanism that involves cAMP-mediated activation of Ca2+ influx through dihydropyridine (DHP)-sensitive Ca2+ channels. These DHP-sensitive Ca2+ currents are blocked by PTSP and are not involved in the prothoracicotropic hormone (Gilbert et al., 1996, 2002) (PTTH)-stimulated Ca2+ influx or the Ca2+-activated Ca2+ influx upon depletion of intracellular stores by thapsigargin in the PG cells of Bombyx mori.

2. Materials and methods 2.1. Insects All experiments were conducted using B. mori larvae from the hybrid J106xDAIZO. The larvae were reared on an artificial diet (Yakult Co., Japan) under a 12:12L:D photoperiod at 25 ± 1°C and 60% relative humidity. 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 (5th) instar during the scotophase, all the larvae that ecdysed during the scotophase were segregated immediately after the onset of photophase. This time was designated as 0 h of the 5th instar. In this particular hybrid, the 5th 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.

2.2. Reagents Synthetic Mas-MIP I (AWQDLNSAW-NH2, M.W. 1089.81; Blackburn et al., 1995, 2001), which is identical to a prothoracicostatic peptide (PTSP; Hua et al., 1999), was a gift from Dr. M. Blackburn (Insect Biocontrol Lab., ARS, USDA). Since these two peptides are identical and neither myoinhibitory activity of PTSP nor prothoracicostatic activity of Mas-MIP I has been reported in the insect species they have been isolated from, we, as a convention, will be using the term MasMIP I/PTSP to describe the same peptide, throughout the text. The peptide was dissolved in aliquots of Ringer’s saline (Shirai et al., 1994) or Ca2+-free Ringer’s saline and stored at ⫺20°C. Bombyx mori recombinant prothoracicotropic hormone (rPTTH; Ishibashi et al., 1994) was a gift from Dr. H. Kataoka (The University of Tokyo, Japan). It was dissolved in aliquots of Ringer’s saline and stored at ⫺20°C. Fura-2/AM, forskolin, thapsigargin, nitrendipine, 8-bromoadenosine 3’,5’-cyclic monophosphate (8-Br-cAMP), isobutyl-methylxanthine (IBMX) and S(-)·Bay K 8644 were purchased from Calbiochem (Bad Soden, Germany). All other reagents were from Sigma (Deisenhofen, Germany). Stock solutions of thapsigargin, forskolin, IBMX, nitrendipine and S(-)·Bay K 8644 were prepared in dimethylsulfoxide (DMSO). 2.3. Cell loading with Fura-2/AM and microfluorometry Due to the autofluorescence of Grace’s medium (GIBCO-BRL, Grand Island, NY, USA), dissected Bombyx mori PGs were loaded with 40 µM of Fura-2/AM in 1 ml of Ringer’s saline for 60 min at 25°C in the dark. Then glands were rinsed in Ringer’s saline for 5 minutes and placed in 50 µl of the same saline on a coverslip in a tight chamber. In experiments requiring a Ca2+-free medium, Ringer’s saline 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 EGTA (0.1 mM) were added prior to use. Dual wavelength measurements were made with a Zeiss (Oberkochen, Germany) microscope-photometer MPM 200 mounted on an inverted Zeiss microscope ‘Axiovert 135’ (with an objective ‘Fluar’ 10x/0.50). Emitted fluorescence was collected through an aperture adjusted to the size of prothoracic gland cells. Background fluorescence was estimated with unloaded cells. The measurements of fluorescence intensity (F) at the two excitation wavelengths were used to calculate [Ca2+]i, assuming a Kd of 224 nM (Grynkiewicz et al., 1985). Free intracellular Ca2+ was measured in nine identified cells of each prothoracic gland and was recorded as mean ± SEM. Addition of all reagents was made by replacement of the whole bathing medium or by addition of 10 µl saline with a pipette. All reagents added were tested for autofluorescence.

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2.4. 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 solution (0.85% NaCl). For reasons of consistency with previously reported results, determinations of ecdysteroid secretion and glandular cAMP levels (see below) were done using Grace’s medium. The glands were pre-incubated in Grace’s medium for 10–15 min and then transferred and incubated in 20 µl of Grace’s medium containing the experimental agent(s). Since DMSO was used as solvent for some treatments, the incubation medium contained 1% DMSO in all experiments. Incubations were carried out at 25±1°C in high humidity (90%) in 96-well microplates (Greiner, Frickenhausen, Germany) for 30 min. Then the incubation medium was removed and an aliquot of the medium (5 µl) was subjected to radioimmunoassay (RIA) for quantification of ecdysteroid content. The results are expressed as total ecdysteroid production per incubation period. 2.5. Quantification of cAMP The content of cAMP in prothoracic glands was quantified by enzyme immunoassay (EIA) using a kit and protocol available from Cayman Chemical Company (Ann Arbor, MI, USA). Cyclic AMP was extracted from prothoracic glands as follows: Glands were removed from the incubation medium after 30 min and homogenized in 100 µl acidified ethanol (ethanol/1 N HCl, 100:1 v/v). The homogenate was centrifuged (2000xg, 15 min) and the supernatant was removed and saved. The pellet was resuspended in 200 µl ethanol/DW (2:1 v/v) and centrifuged as above. The supernatants were combined and dried in vacuo. The residue was dissolved in phosphate buffer. The resulting solution was assayed according to the instructions provided with the kit. Acetylation of standards or samples by acetic anhydride was performed according to the instructions in the kit.

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experiments, ANOVA was followed by Tukey multiple comparisons test. Statistical analyses are shown in appropriate figure legends.

3. Results 3.1. Effects of forskolin on [Ca2+]i of the PG cells on day 6 of the 5th instar of Bombyx mori Previous research with PGs of Bombyx mori on day 6 of the 5th instar has shown that the forskolin-stimulated ecdysteroid secretion by the PGs is dependent on extracellular Ca2+ (Dedos and Fugo, 1999). Namely, the ability of forskolin to stimulate ecdysteroid secretion in Ringer’s saline was significantly decreased when extracellular Ca2+ was omitted from the incubation medium (Dedos and Fugo, 1999). Therefore, we determined whether the previously reported decreased ability of forskolin to stimulate ecdysteroid secretion in the absence of extracellular Ca2+ is related to a cAMP-mediated Ca2+ influx in the PG cells. Addition of forskolin in Fura2/AM loaded PG cells resulted in increased [Ca2+]i within 10 min which steadily increased thereafter to a level of 181.2 nM [Ca2+]i in 20 min (Fig. 1). To test whether the forskolin-mediated influx of Ca2+ has extracellular origin or not, we repeated the same experiment using Ca2+-free Ringer’s saline. The results showed that the forskolin-mediated Ca2+ influx is of extracellular origin since no increase in [Ca2+]i was recorded and the

2.6. Radioimmunoassay The amount of ecdysteroid in the incubation medium was quantified by radioimmunoassay (RIA) as described (Takeda et al., 1986). Sensitivity of the RIA was approximately 0.01 ng/assay. Radiolabeled ecdysone [(23,24-3H) ecdysone (sp. act. 53 Ci/mmol)], was purchased from New England Nuclear (Boston, MA, USA). 2.7. Statistical analyses GraphPad Prism 2.0 computer software was used for all statistical analyses. Statistical significance of the results was determined by analysis of variance (ANOVA) or Student’s unpaired t-tests. For most

Fig. 1. Effects of forskolin on the [Ca2+]i of PG cells in the presence or absence of extracellular Ca2+. Day 6 prothoracic glands from 5th larval instar of Bombyx mori were incubated in Ca2+-free or plain Ringer’s saline in the presence of forskolin (50 µM). The arrow indicates the time of addition of forskolin. Each value is the mean ± SEM of at least nine identified PG cells. Results of Tukey multiple comparisons tests (pairs of means enclosed by the range of a bracket are not significantly different, P ⬎ 0.05): (1) Forskolin in plain Ringer’s saline [142.4–151.6],[150.9–162.2],[162.2–171.3],[171.3–181.1]; (2) Forskolin in Ca2+-free Ringer’s saline: [79.4–80.2],[83.8–96.0].

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[Ca2+]i of PG cells dropped to 79.4 nM after 20 min (Fig. 1). 3.2. Effects of nitrendipine on forskolin-stimulated [Ca2+]i in the PG cells Based on another previous finding that the DHP-sensitive Ca2+ channel inhibitor, verapamil, was able to block the Sp-cAMPS-mediated increase in ecdysteroid secretion (Dedos and Fugo, 1999), we determined the effects of nitrendipine on the forskolin-stimulated Ca2+ influx in PG cells. Although addition of forskolin in PG cells resulted in increased [Ca2+]i levels (Fig. 2), preincubation of PGs with nitrendipine (100 µM) for 5 min before the addition of forskolin resulted in a pattern of [Ca2+]i levels not different from that recorded when PGs were incubated in plain Ringer’s saline (Fig. 2). This showed that nitrendipine diminished the forskolinmediated increase in [Ca2+]i for the next 20 min (Fig. 2), an indication of a possible link between the cAMPsignalling cascade and DHP-sensitive Ca2+ channels in the cellular membrane of PG cells. 3.3. Effects of 8-bromo-cAMP on [Ca2+]i of PG cells To determine whether the previously described effects of forskolin on Ca2+ influx are the product of activation of the cAMP signalling cascade, we recorded the effects

Fig. 2. Nitrendipine-mediated inhibition of forskolin-stimulated Ca2+ influx. Each value is the mean ± SEM of at least nine identified PG cells. The arrow indicates the time of addition of nitrendipine (100 µM) and forskolin (50 µM) or forskolin alone (50 µM) in plain Ringer’s saline. The fluctuation of [Ca2+]i levels of PG cells in plain Ringer’s saline for the length of a typical experiment is shown for comparison. Results of Tukey multiple comparisons tests (pairs of means enclosed by the range of a bracket are not significantly different, P ⬎ 0.05): (1) Nitrendipine and forskolin in Ringer’s saline [132.0– 141.7], [141.7–153.3], [152.5–155.3]; (2) Plain Ringer’s saline [139.9– 158.3]; (3) Forskolin in plain Ringer’s saline [139.8–164.0], [164.0– 192.6]. Two-way analysis of variance revealed that the fluctuations of [Ca2+]i levels of PG cells in the presence of both nitrendipine and forskolin were not different from those in plain Ringer’s saline (P ⬎ 0.05).

of a cAMP analogue, 8-bromo-cAMP, on Ca2+ influx. The results (Fig. 3), showed that 8-bromo-cAMP stimulates a steady increase in intracellular [Ca2+]i also within 10 min of its addition in the incubation medium. This increase in [Ca2+]i was not abrupt and reached the same levels as those observed with forskolin within 20 min (181.1 for forskolin (Fig. 1) vs. 181.2 for 8-bromocAMP (Fig. 3)). In a similar way as observed with forskolin, this escalating increase of [Ca2+]i of PG cells was diminished when PG cells were pre-incubated for 5 min in the presence of nitrendipine (Fig. 3; P ⫽ 0.14). 3.4. Effects of S(-)·Bay K 8644 on Ca2+ influx in PG cells The previous results showed an ability of cAMP-modulating agents to stimulate Ca2+ influx in PG cells. In both cases, however, a small and gradual increase in [Ca2+]i was observed (Figs. 1 and 3). To determined whether DHP-sensitive Ca2+ channels are, upon activation, producing such small and gradually elevating increases in [Ca2+]i, we used the DHP-sensitive Ca2+ channel agonist S(-)·Bay K 8644. This Ca2+ influx stimulating agent produced very abrupt and high increases in [Ca2+]i of the PG cells within 1 min of its addition. As a result of its addition, the S(-)·Bay K 8644-stimulated increase in [Ca2+]i reached a level of 268 nM within 15 min (Fig. 4), unlike the forskolin or 8-bromo-cAMPmediated effects. On the other hand, nitrendipine was able to partially block the high increase in [Ca2+]i in the presence of S(-)·Bay K 8644 (10 µM) and only an increase of 23 nM in [Ca2+]i was observed after 20 min (Fig. 4). It is worth mentioning that the S(-)·Bay K 8644-

Fig. 3. Nitrendipine-mediated inhibition of 8-bromo-cAMP-stimulated Ca2+ influx. Each value is the mean ± SEM of at least nine identified PG cells. The arrow indicates the time of addition of nitrendipine (100 µM) or 8-bromo-cAMP (5 mM). Results of Tukey multiple comparisons tests (pairs of means enclosed by the range of a bracket are not significantly different, P ⬎ 0.05): (1) 8-bromo-cAMP and nitrendipine [119.6–136.7],[133.1–144.8]; (2) 8-bromo-cAMP: [113.3– 125.3],[122.4–129.3],[147.1–156.1],[172.4–181.2].

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Fig. 4. Nitrendipine-mediated inhibition of S(-)·Bay K 8644-stimulated Ca2+ influx. Each value is the mean ± SEM of at least nine identified PG cells. The arrow indicates the time of addition of nitrendipine (100 µM) or S(-)·Bay K 8644 (10 µM). Results of Tukey multiple comparisons tests (pairs of means enclosed by the range of a bracket are not significantly different, P ⬎ 0.05): (1) S(-)·Bay K 8644 [126.3– 138.2],[199.2⫺213.4],[213.4–254.6],[254.6–267.9]; (2) S(-)·Bay K 8644 and nitrendipine [141.3–148.4],[168.9–178.3].

stimulated increase in [Ca2+]i of PG cells was dosedependent since at a concentration of 20 µM, this agent produced a massive increase in [Ca2+]i which reached 284 nM within 1 min and levelled off at 317 nM after 15 min (data not shown).

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Fig. 5. Mas-MIP I/PTSP-mediated inhibition of forskolin or S(-)·Bay K 8644-stimulated Ca2+ influx. Each value is the mean ± SEM of at least nine identified PG cells. The arrow indicates the time of addition of Mas-MIP I/PTSP (5 ng), forskolin (50 µM) or S(-)·Bay K 8644 (10 µM). Results of Tukey multiple comparisons tests (pairs of means enclosed by the range of a bracket are not significantly different, P ⬎ 0.05): (1) Mas-MIP I/PTSP and forskolin [139.3–145.7],[142.2– 152.8]; (2) Mas-MIP I/PTSP and S(-)·Bay K 8644 [132.8– 146.4],[146.4–151.4],[166.0–175.3].

teroid secretion by the PGs (Dedos et al., 1999) and its ecdysteroidogenic activity is dependent on extracellular Ca2+(Gu et al., 1998). Addition of 1 ng rPTTH to Fura2/AM loaded PG cells resulted in an increase of [Ca2+]i within 4 min (Fig. 6). Incubation of PG cells for 10 min

3.5. Effects of Mas-MIP I/PTSP on forskolin and S(-)·Bay K 8644-mediated Ca2+ influx in PG cells Since a previous study (Dedos et al., 2001) has reported an inhibition of the S(-)·Bay K 8644-stimulated ecdysteroid secretion by PTSP, we investigated the effects of this peptide on the S(-)·Bay K 8644 or forskolin-mediated Ca2+ influx in PG cells. Pre-incubation of PG cells with Mas-MIP I/PTSP eliminated the forskolinstimulated increase of [Ca2+]i (Fig. 5) and produced, upon addition of S(-)·Bay K 8644, a [Ca2+]i pattern (Fig. 5) that resembled that of the nitrendipine-mediated inhibition of S(-)·Bay K 8644-stimulated Ca2+ influx in PG cells (see Fig. 4). 3.6. Involvement of Ca2+ influx mechanisms in the Mas MIP I/PTSP-mediated inhibition of DHP-sensitive Ca2+ channels The previous results have shown that there is a regulatory mechanism in PG cells which connects the activation of the cAMP signalling pathway with increased [Ca2+]i from DHP-sensitive Ca2+ channels that are, in turn, blocked by Mas-MIP I/PTSP. Is Ca2+ influx from DHP-sensitive Ca2+ channels the only mechanism of Ca2+ influx in PG cells? To answer this question we have used rPTTH that has been shown to stimulate ecdys-

Fig. 6. Mas-MIP I/PTSP has no effect on basal [Ca2+]i or rPTTHstimulated Ca2+ influx in PG cells. Each value is the mean ± SEM of at least nine identified PG cells. The arrow indicates the time of addition of Mas-MIP I/PTSP (5 ng) and rPTTH (1 ng) or rPTTH (1 ng) alone. Results of Tukey multiple comparisons tests (pairs of means enclosed by the range of a bracket are not significantly different, P ⬎ 0.05): (1) Mas-MIP I/PTSP [132.5–142.7],[164.3–170.9]; (2) Mas-MIP I/PTSP and rPTTH [140.8–143.9],[300.6–311.6]. One-way analysis of variance revealed that rPTTH did not stimulate a statistically significant increase of [Ca2+]i levels within 1 min, but only after 4 min of its addition (P ⬍ 0.05) both in the presence or absence of Mas-MIP I/PTSP.

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with Mas-MIP I/PTSP before the addition of rPTTH had no effect on the rPTTH-mediated increase in [Ca2+]i which reached a level of 307 nM within 4 min (Fig. 6). Moreover, the presence of Mas-MIP I/PTSP alone for 25 min did not produce any inhibition in [Ca2+]i (Fig. 6), an indication that this peptide does not affect the steady state of intracellular Ca2+ regulation but blocks the activation of DHP-sensitive Ca2+ channels of PG cells. 3.7. Effects of nitrendipine on thapsigargin-stimulated increase of [Ca2+]i of PG cells To determine whether DHP-sensitive Ca2+ channels in PG cells are activated and participate in the replenishing of intracellular Ca2+ stores, we have used a combination of nitrendipine and thapsigargin in the following experimental set up. Different PG cells were incubated in the presence or absence of nitrendipine before the addition of thapsigargin 5 min later. The results (shown in Fig. 7) showed that DHP-sensitive Ca2+ channels in PG cells are neither involved in Ca2+-release activated Ca2+ influx nor participate in replenishment of intracellular Ca2+ stores, after they are emptied by thapsigargin, because nitrendipine had no effect in the thapsigargin-stimulated high increase in [Ca2+]i. Similar levels of [Ca2+]i were recorded in the presence of thapsigargin whether nitrendipine was present or absent from the incubation medium (Fig. 7). 3.8. Effects of cAMP and Ca2+ modulating agents on short-term ecdysteroid secretion by the PG cells Since forskolin was shown to stimulate a small increase in [Ca2+]i (Fig. 1) it was interesting to determine

the relative contribution it has on ecdysteroid secretion during a short-term incubation of PGs. Moreover, since baculovirus-expressed PTTH of Bombyx mori was shown to be unable to stimulate a glandular adenylate cyclase (Chen et al., 2001), it was important to determine the relative contribution of the cAMP signalling cascade activation on the PTTH-mediated Ca2+ influx and subsequent ecdysteroid secretion. In the same line of thought we determined the effects of two agents used in this study, thapsigargin and S(-)·Bay K 8644 on ecdysteroid secretion and cAMP levels of PG cells. The results (presented in Table 1) show that forskolin was relatively inefficient in stimulating ecdysteroid secretion within 30 min. This effect is probably caused by the presumed high activity of a glandular cAMP-degrading phosphodiesterase since the phosphodiesterase inhibitor, IBMX, was able to stimulate a higher increase in ecdysteroid secretion (Table 1). Recombinant PTTH stimulated ecdysteroid secretion but not cAMP levels within 30 min (Table 1). Its effects, both on ecdysteroid secretion and glandular cAMP levels, were greatly augmented in the presence of IBMX (Table 1). This is an indication that the cAMP signalling pathway is involved in the PTTHstimulated ecdysteroid secretion, probably through a Ca2+-mediated activation of a cellular Ca2+/calmodulinsensitive adenylate cyclase, as has been shown in both Bombyx mori and Manduca sexta PGs (Chen et al., 2001; Meller et al., 1988; Meller et al., 1990). In the same set of experiments, we determined the effects of thapsigargin and S(-)·Bay K 8644 on both the glandular cAMP content and the ecdysteroid secretion during a 30 min incubation period in an attempt to determine the relative contribution of [Ca2+]i-modulating agents (other Table 1 Effects of cAMP or Ca2+ modulating agents on glandular cAMP levels and ecdysteroid secretion by the prothoracic glands of Bombyx mori Agents

Basal Forskolin (50 µM) IBMX (50 µM) rPTTH (1 ng) IBMX (50 µM) + rPTTH (1 ng) Thapsigargin (10 µM) S(-)·Bay K 8644 (10 µM) Fig. 7. Nitrendipine has no effect on thapsigargin-stimulated Ca2+ influx in PG cells. Each value is the mean ± SEM of at least nine identified PG cells. The arrow indicates the time of addition of of nitrendipine (100 µM) or thapsigargin (10 µM). Results of Tukey multiple comparisons tests (pairs of means enclosed by the range of a bracket are not significantly different, P ⬎ 0.05): (1) Thapsigargin [145.5–154.6],[266.9–284.8]; (2) Nitrendipine and thapsigargin [133.3–144.6],[248.1–259.1].

cAMP (fmol/gland)

Ecdysteroid (ng/gland/30 min)

201.3±15.0 3978.0±272.1 1672.5±76.7 240.6±12.9 3170.3±224.2

0.85±0.14 1.21±0.12 2.55±0.11 3.02±0.16 4.37±0.35

163.6±16.7

3.76±0.23

238.8±12.0

2.68±0.09

Glands were incubated in Grace’s medium alone (Basal) or in the presence of the indicated agents for 30 min. Each value is the mean ± SEM of six glands. Student’s unpaired t-tests revealed that all agents significantly stimulated ecdysteroid secretion above basal levels (P ⬍ 0.05). Student’s unpaired t-tests revealed that rPTTH, thapsigargin and S(-)·Bay K 8644 did not significantly stimulate glandular cAMP levels above basal level (P ⬎ 0.05).

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than PTTH) on the cAMP-signalling cascade and the relative feedback that Ca2+ influx has on the intracellular levels of another second messenger (cAMP). Neither thapsigargin nor S(-)·Bay K 8644 could greatly influence the glandular cAMP levels although they both produced significant increases of ecdysteroid secretion by the PGs (Table 1).

4. Discussion The results of this study identify a regulatory mechanism in the prothoracic glands of the silkworm, Bombyx mori which involves a cAMP-mediated increase of [Ca2+]i in PG cells. This mechanism is regulated by a myoinhibitory/prothoracicostatic peptide (Mas-MIP I/PTSPS) and it is not related directly to other Ca2+ influx operating mechanisms in the PG cells. In a previous study, we have identified Ca2+ as the probable second messenger involved in the prothoracicostatic action of Mas-MIP I/PTSP (Dedos et al., 2001). Namely, Mas-MIP I/PTSP was able to block the S(-)·Bay K 8644-stimulated ecdysteroid secretion on day 6 of the 5th instar and its inhibitory effect was abolished in the absence of extracellular Ca2+ (Dedos et al., 2001). These two observations were investigated further in this study in an attempt to identify the signalling cascade(s) that are involved or influenced by the inhibitory action of Mas-MIP I/PTSP. Our results show that intracellular cAMP-modulating agents i.e. forskolin (Fig. 1) and 8bromo-cAMP (Fig. 3) can stimulate a small but steadily elevating increase in [Ca2+]i even within 10 min after their addition in the PGs incubation medium (Figs. 1and 3). This increase in [Ca2+]i has extracellular origin (Fig. 1) and it is presumably mediated by activation of a protein kinase A by the cAMP analogue, 8-bromo-cAMP (Fig. 3). The ability of nitrendipine to block both the forskolin (Fig. 2) and 8-bromo-cAMP-mediated (Fig. 3) increases in [Ca2+]i suggests that this increase in [Ca2+]i originates from Ca2+ influx through dihydropyridine (DHP)-sensitive Ca2+ channels on the cellular membrane of PG cells. This assumption is corroborated by the ability of S(-)·Bay K 8644 to stimulate a high increase in [Ca2+]i which was also quenched by nitrendipine (Fig. 4). The same concentration of nitrendipine, however, was unable to inhibit the rPTTH-stimulated increase of [Ca2+]i in PG cells (data not shown), an indication of the specificity of nitrendipine in regulating Ca2+ influx in PG cells. Mas-MIP I/PTSP was able to block both the forskolin and S(-)·Bay K 8644-stimulated increase in [Ca2+]i (Fig. 5). These results suggest that the Mas-MIP I/PTSP inhibitory cascade is directed towards blocking of DHP-sensitive Ca2+ channel(s). This Mas-MIP I/PTSP-mediated inhibition of DHP-sensitive Ca2+ channel(s) appears to be specific: neither Mas-MIP I/PTSP could inhibit the rPTTH-stimulated increase in

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[Ca2+]i (Fig. 6), nor nitrendipine had any influence on the very high thapsigargin-mediated increase in [Ca2+]i suggesting that in the PGs of Bombyx mori, the DHPsensitive Ca2+ channel(s) are not involved in replenishing [Ca2+]i upon depletion of the intracellular Ca2+ stores (Fig. 7). Furthermore, the inability of both S(-)·Bay K 8644 and thapsigargin to substantially increase intracellular cAMP levels in PG cells (Table 1) might be an indication that in PG cells, the plasma membrane DHP-sensitive Ca2+ channel(s) and the thapsigargin-sensitive intracellular Ca2+ stores are localised at cellular domains distal from those of Ca2+/calmodulinsensitive adenylate cyclase. Our results add another regulatory mechanism in the multiplicity of ecdysteroidogenic regulatory mechanisms that have been identified in Lepidopteran PGs (see review by Smith, 1995; Gilbert et al., 1996, 2002). Among the second messengers involved in the ecdysteroidogenic pathways of the PG cells, Ca2+ seems to be of paramount importance: it is required for the prothoracicotropic action of PTTH (Gilbert et al., 2000) and it is the second messenger that mediates the PTTH ecdysteroidogenic activity (see review by Smith, 1995; Gu et al., 1998; Gilbert et al., 2000, 2002). Ca2+ is required for activation of the cAMP signalling pathway via a Ca2+/calmodulin-sensitive adenylate cyclase in PG cells (Chen et al., 2001; Meller et al., 1988; Meller et al., 1990) giving rise to overtly interpreted activations of glandular cAMP levels by PTTH (Table 1). Our finding that activation of the cAMP signalling cascade leads to a further and steady increase in [Ca2+]i (Fig. 3) suggests that the above-described mechanism is even further augmented by Ca2+ influx through DHP-sensitive Ca2+ channels and presumably further activation of the Ca2+/calmodulin-sensitive adenylate cyclase. The negative control in this circular and bifurcating cascade of cAMP and Ca2+ is provided by Mas-MIP I/PTSP through its inhibition of further increases in [Ca2+]i levels of PG cells. This cascade of events, however, does not seem to influence two other Ca2+ influx mechanisms, namely the receptor-mediated Ca2+ influx by PTTH (Fig. 6) and the intracellular Ca2+ release-stimulated Ca2+ influx by thapsigargin (Fig. 7). Both of these Ca2+ influxes seem to operate via distinct pathways that nevertheless elicit ecdysteroidogenic activity (Table 1). The net result, however, if judged by measurements of ecdysteroid secretion can show that several ecdysteroidogenic pathways can converge and contribute in the secretory activity of the PGs. For example, Mas-MIP I/PTSP (Dedos et al., 2001) was capable of decreasing the thapsigargin-stimulated ecdysteroid secretion (Dedos et al., 2001) and this peptide was demonstrated to have an inhibitory effect on the rPTTH-stimulated ecdysteroid secretion that was strongly decreased in its presence (Hua et al., 1999). These findings are explained by the different assay durations and the different entities that

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were assayed in the previous studies vs. this study. Therefore, there can be no doubt that inhibition of DHPsensitive Ca2+ current activity by Mas-MIP I/PTSP can, in the long run, influence the general cascade of events that lead to ecdysteroid synthesis and secretion no matter if this secretion is a stimulated or non-stimulated one. Previous research in intracellular Ca2+ levels of the PGs from two other Lepidopteran species, Manduca sexta (Birkenbeil, 1998, 2000) and Galleria mellonella (Birkenbeil, 1996), has identified similar Ca2+ influx pathways. In both of these species a S(-)·Bay K 8644mediated increase in [Ca2+]i was recorded (Birkenbeil, 1996, 1998) and in Manduca sexta, the dibutyryl-cAMPstimulated increase in ecdysteroid secretion was inhibited by nitrendipine (Birkenbeil, 1998). Nitrendipine, however, had no effect on the Manduca brain extract (PTTH)-stimulated increase of [Ca2+]i (Birkenbeil, 1998) although previous research has shown that it could inhibit Manduca brain extract (PTTH)-stimulated increase in ecdysteroid secretion (Girgenrath and Smith, 1996). One possibility that is currently investigated and can not be ruled out by our results is that a cyclic nucleotide-activated Ca2+ channel, instead of a DHP-sensitive Ca2+ channel, is responsible for the recorded Ca2+ influx in the presence of 8-bromo-cAMP (Fig. 3), since 8bromo-cAMP has been found to directly activate such channels (Hagen et al., 1998). In a previous study (Dedos et al., 2001) it was shown that Mas-MIP I/PTSP inhibits ecdysteroid secretion by the PGs of 5th instar larvae specifically on 2 days of the 5th instar. One of these 2 days was day 3 of the 5th instar which is the first day after the onset of larval– pupal commitment and is characterized by potentiation of the cAMP signalling pathway in the PG cells and subsequently increased ecdysteroid secretion on that and the following days (Dedos et al., 1999). Since the well-coordinated completion of larval–pupal differentiation (Riddiford, 1994) is dependent on a steadily and timely increasing haemolymph ecdysteroid titer (Riddiford, 1994), we suggest that Mas-MIP I/PTSP action on day 3 PGs is directed towards dictating a steady pace of ecdysteroid secretion in the haemolymph. The other day of the 5th instar was day 6 when Mas-MIP I/PTSP could strongly inhibit ecdysteroid secretion by the PGs (Dedos et al., 2001). During this day, the haemolymph ecdysteroid titer is on a steep increase that reaches its peak the following day (Dedos et al., 1999) and initiates the metamorphosis to pupa. Therefore, if there has to be a regulatory mechanism for the timely and pre-metamorphic increase of the haemolymph ecdysteroid titer, we suggest that such a mechanism is exercised by Mas-MIP I/PTSP through its inhibition of the cAMP-mediated positive feedback in [Ca2+]i of the PG cells and the subsequent ecdysteroid secretion. Regulation of DHP-sensitive L-type Ca2+ channel activity by PKA-mediated phosphorylation is a ubiqui-

tously occurring mechanism (Brammar, 1999; Catterall, 2000). It has been found to occur in a variety of tissues and cell types of both vertebrates (Hell et al., 1995; Leiser and Fleischer, 1996; Davare et al., 2000) and invertebrates (MacPherson et al., 2001; Bhattacharya et al., 1999; Kits and Mansvelder, 1996; Wicher et al., 2001). This PKA-mediated phosphorylation is involved in the modulation of Ca2+ current amplitude by DHPsensitive L-type Ca2+ channels through promotion of the DHP-sensitive L-type Ca2+ channels’ upregulation and enhancement of the open probability of such Ca2+ channels (Carbone et al., 2001). Despite the fact that in Manduca PGs, cAMP-dependent protein kinase A (PKA)mediated phosphorylation substrates have been extensively studied (Song and Gilbert, 1994, 1995; Smith, 1995; Gilbert et al., 1996, 2002), none of these studies identified DHP-sensitive Ca2+ channels as PKA substrates. This together with the absence of relevant research on prothoracicostatic peptides, like Mas-MIP I/PTSP, in PGs of other Lepidopteran species renders premature the generalization of the regulatory cascade which is presented in this study and involves DHP-sensitive Ca2+ currents and their up-regulation by the cAMP signalling pathway. In conclusion, we believe that future research should be directed towards identifying the structural and electrophysiological characteristics of the DHP-sensitive Ca2+ channel(s) whose activity is inhibited by Mas-MIP I/PTSP. This will facilitate the identification of this type of Ca2+ channels as substrates of the glandular PKAmediated phosphorylation and modulation of Ca2+ current amplitude.

Acknowledgements We thank Dr. M. Blackburn (Insect Biocontrol Lab., ARS, USDA) for synthetic Mas-MIP I and Drs. S. Nagata and H. Kataoka, (The University of Tokyo, Japan) for donating recombinant PTTH. We thank S. Kaltofen for excellent technical assistance. The authors gratefully acknowledge that a part of the experimental work was conducted at the laboratory of Prof. Hajime Fugo (Laboratory of Insect Biochemistry), Department of Biological Production, Tokyo University of Agriculture and Technology, Tokyo, Japan.

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