- Email: [email protected]
Signal transduction in the inhibition of juvenile hormone biosynthesis by allatostatins: roles of diacylglycerol and calcium
ELSEVIER
Molecular and Cellular Endocrinology 105 (1994) 89-96
Signal transduction in the inhibition of juvenile hormone biosynthesis by allatostatins: roles of diacylglycerol and calcium A. Rachinskya, J. Zhangb, S.S. Tobebs* ati Entwicklungsphysiologie, Zoologisches hstitut, Universitiit Tiibingen, Auf der Morgenstelle 28, D-72076 TWngen, Germany bDepartment of Zoology, University of Toronto, Toronto, Ontario, MSS IAI, Canada Received 18 May 1994; accepted 25 July 1994
Abstract
The effects of pharmacological agents that interfere with the 1,4,5inositol trisphosphate (IPs)/diacylglycerol (DAG) pathway on juvenile hormone (JH) biosynthesis by corpora allata (CA) of the cockroach Diplopteru punctata have been investigated. These effects were assessed in the presence of the inhibitory neuropeptides, allatostatins, with a view to elucidating the pathway for signal transduction in the inhibition of JH biosynthesis. Treatment of CA with inhibitors of DAG kinase to elevate the concentration of DAG within the CA cells, resulted in a significant, dose-dependent decrease in JH biosynthesis. Simultaneous treatment of glands with both DAG kinase inhibitors and allatostatins further enhanced this effect, suggesting that DAG is an intermediate in the allatostatin-induced inhibition of JH production. The inhibitory actions of the phorbol ester activator of PKC, PDBu, or of allatostatin on JH biosynthesis were partially blocked by pre-incubating the CA with PKC inhibitors. Treatment of CA with the calcium-mobilizing drug thapsigargin resulted in a significant stimulation in JH biosynthesis in glands from mated females producing JH at high rates. Thapsigargin was also able to reverse the effect of allatostatins in high-activity mated CA. This suggests an involvement of the other product of phosphoinositide hydrolysis, IP3, in the modulation of JH biosynthesis at specific developmental times and in glands showing specific levels of activity. Keywords:
Diacylglycerol
kinase inhibitors;
Protein
kinase C inhibitors; Calcium; Juvenile hormone biosynthesis;
Allatostatins;
Cockroach; Insect
1. Introduction In the cockroach Diploptera punctata, the activity of the juvenile hormone (JH)-producing corpora allata (CA) is, in part, under control of inhibitory neuropeptides. A family of seven structurally related allatostatins which are biologically active in the in vitro assay for JH biosynthesis (Pratt and Tobe, 1974; Tobe and Pratt, 1974) has been isolated from D. punctata brains (Woodhead et al., 1989, 1994; Pratt et al, 1991). A gene encoding all seven known allatostatins plus six previously unknown allatostatins has recently been cloned and shown to be expressed in medial neurosecretory cells of the brain of D. punctata (Donly et al., 1993). The detection of allatostatin-immunoreactive pathways between brain and retrocerebral complex, and
*Corresponding 3522.
author. email [email protected]. Fax 416 978
0167-8140/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDl 0167-8140(94)03367-3
of specific allatostatin receptors in brain, fat body and CA membrane preparations (Cusson et al., 1991), emphasizes the importance of these peptides in regulation of CA but also suggests other possible regulatory functions (Lange et al., 1993; Stay and Woodhead, 1993). In addition, the callatostatins, a group of five neuropeptides isolated from Calliphora vomitoria with strong structural similarity to the D. punctata allatostatins, show pronounced allatostatic activity in D. punctara, callatostatin 5 being the most potent neuropeptide inhibitor of JH biosynthesis so far tested in this species (Duve et al., 1993). Allatostatins presumably stimulate signal transduction pathway(s) after binding to membrane receptors of CA cells. The generation of intracellular second messengers may then act to modulate enzymatic activity in the JH biosynthetic pathway (Tobe et al., 1994). Most neuropeptide receptors studied to date interact with G proteins to exert their effects via second messengers such as D-
A. Rachinsky
90
et al. /Molecular
and Cellular
inositol 1,4,5triphosphate (IPs) and diacylglycerol (DAG). In D. punctutu, correlations exist between allatostatic action of brain extracts and elevated CAMP levels within the CA, and pharmacologically elevated CAMP levels also inhibit JH release (Meller et al., 1985; Aucoin et al., 1987; Tobe, 1990). Despite these observations, synthetic D. puncruru allatostatins did not elicit any significant changes in the levels of cyclic nucleotides in isolated CA (Cusson et al., 1992), suggesting that these compounds may not be directly involved in the mediation of allatostatin action. Other possible candidates for signal transduction in the regulation of JH biosynthesis are IPs and DAG which are produced by receptor-mediated hydrolysis of phosphatidylinositol-4,5bisphosphate. IPs causes the release of Ca from intracellular storage sites, whereas DAG activates protein kinase C (PKC) (Berridge, 1993). The IPs second messenger pathway has been shown to be present in insects and is, for example, involved in mobilization of intracellular calcium in blowfly salivary glands (Berridge, 1993) and PKC activation in Manduca sextu brains (Qiu et al., 1990a,b). In CA of adult M. sextu, an allatotropin neuropeptide induces the formation of inositol phosphates (Reagan et al., 1992). Furthermore, several pharmacological agents that act at different sites in the IPs pathway also stimulate JH biosynthesis, suggesting an activation of this second messenger system by the allatotropin (Reagan et al., 1992). In D. punctutu, phorbol ester activators of PKC proved to be potent inhibitors of JH biosynthesis, thus implicating PKC in the regulation of CA activity (Feyereisen and Farnsworth, 1987). If activation of PKC is an essential event in allatostatin-induced inhibition of JH biosynthesis in D. punctutu, an experimentally evoked increase in diacylglycerol concentration should mimic the effect of allatostatins. We therefore studied the effects of DAG kinase inhibitors on JH release. On the other hand, compounds that inhibit PKC activity should antagonize allatostatin-evoked inhibition and accordingly, we investigated the effects of various PKC inhibitors. Furthermore, to obtain information on the role of IP, in regulation of JH biosynthesis, we have determined the effects of thapsigargin, a compound known to mimic the effects of IP, on intracellular Ca stores. 2. Materials
and methods
2.1. Animals The viviparous cockroach D. punctutu was reared as previously described (Stay and Coop, 1973). We used 2day-old virgins and 2, 4 and 5 day mated females for our experiments. Virgin females, whose CA show relatively constant low rates of JH biosynthesis (Stay et al., 1991), were obtained by isolating last instar females from the colony and maintaining them in separate jars. In mated females, oocyte growth is strictly correlated with age and
Endocrinology
105 (1994) 89-96
rates of JH biosynthesis (Tobe et al., 1985). Therefore, to ensure similar physiological status of experimental animals, length of basal oocytes was checked before dissection of the CA. 2.2. Materials H7, H8, R59022, R59949 and tamoxifen citrate were purchased from Research Biochemicals Inc. (Natick, MA); phorbol 12,13-dibutyrate (PDBu) and trifluoperazine from Sigma Chemical Corp. (St. Louis, MO). Bisindolylmaleimide, calphostin C, I-0-hexadecyl-2-0methyl-rut-glycerol, polymyxin B and D-erythrosphingosine were obtained from Calbiochem Corp. (La Jolla, CA); thapsigargin from LC Services Corp. (Woburn, MA). Synthetic Dipstatin7 (Dip7; previous designation AST 1; for allatostatin nomenclature, see Donly et al., 1993) and callatostatin 5 (CAST 5) were obtained from University of Iowa Protein Structure Facility or Core Facility, Insect Biotech Canada. All solvents used were of the highest grade available from commercial suppliers. Bisindolylmaleimide, calphostin C, I-0-hexadecyl-20-methyl-rut-glycerol, PDBu, R59949 and thapsigargin were prepared as stock solutions in DMSO. Tamoxifen citrate and R59022 were prepared as stock solutions in ethanol. R59949 and R59022 were diluted 1: 10 in 5 mN HCl before further dilution in incubation medium. Detythro-Sphingosine was dissolved in chloroform/methanol (1: 1). The amount required for assays was dried under nitrogen and dissolved in medium by sonication. All other reagents used were water soluble. Solvent controls were found to have no effect on JH release at final concentrations of 0.1% DMSO and 0.1% ethanol.
2.3. Assay for CA activity Rates of JH biosynthesis were determined by the in vitro radiochemical method of Feyereisen and Tobe (1981) and Tobe and Clarke (1985). Single CA or one pair of CA were usually incubated for 3 h at 27°C in 100~1 of methionine-free medium 199 (Gibco; supplemented with 1.3 mM calcium chloride and 2% (w/v) Fi~011) containing L-[methyl-i4C]methionine (Amersham; 55-57 mCi/mmol) in the incubation medium to a final concentration of 50pM to monitor biosynthesis of radiolabeled JH. At the end of the incubation time, CA were removed from the medium before extraction and quantification of JH. Thus the values represent rates of JH released into the medium. Pharmacological agents were usually added to the labeled medium immediately before starting the incubations. For experiments with potential PKC inhibitors, CA were pre-incubated for 20 min in 45 ~1 labeled medium containing 10pM inhibitor (except for 2,uM for calphostin C, due to its solubility), before addition of 5 ~1 of either 10pM PDBu or 1 PM Dip7. Glands were then incubated for another 40 min before assaying the incubation
A. Rachinskyet al. I Molecularand CellularEndocrinology105 (1994) 89-96
01’
I
0
IO-Q
lo-8
Concentration
I
I
lo-'
of R 59022
IO-6
lo-5
(M)
Fig. 1. Dose-response characteristics of in vitro JH release by CA of 2day-old virgin (0)and S-day-old mated females (m) (inset) as influenced by the DAG kinase inhibitor R59022. In both stages, R59022 caused a dose-dependent inhibition of JH release. Asterisks denote significant differences from untreated controls (t-test; ***P < 0.001). Means f SEM of 5-10 replicate assays.
medium for radiolabeled JH. For tests on reversibility of DGK inhibitors, glands were first incubated for 3 h in control medium to determine basal rates of JH release, then for 3 h in medium containing the inhibitors, and finally for another 3 h in control medium without inhibitors. At the end of each incubation period, the medium was assayed for radiolabeled JH.
91
tostatin Dip7 were tested simultaneously. Relative to basal rates (measured in the first 3 h of incubation), rates of JH release significantly decreased following transfer of glands into medium containing either R59022 or Dip7 in the second incubation period (data not shown). In both cases, JH release recovered after transfer of the glands into medium without inhibitor. For the third incubation period, JH release was not significantly different from basal rates. To examine the combined effects of R59022 and inhibitory neuropeptides, CA of 2-day-old virgin females were incubated with different concentrations of the DAG kinase inhibitor and either Dip7 or CAST 5. R59022 and Dip7 clearly showed an additive effect. Treatment of the glands with 1O-7M Dip7 alone caused a significant inhibition of JH release (46%). Addition of R59022 to these Dip7-treated glands resulted in a further significant decrease in JH release (Fig. 2A). Even at a threshold concentration, R59022 (lo-8 M) markedly decreased JH release by Dip7-treated glands, suggesting a synergistic effect (Fig. 2A). Maximal inhibition achieved by a combination of R59022 and Dip7 was 81%. Inhibition of JH
T
0
R59022
l
R59022+10-'M
Dip7
2.4. Statistical analysis All values are reported as mean + standard error of the mean (SEM). Statistical significance of data was determined by Student’s t-test (paired or unpaired; P < 0.05).
3. Results 3. I. Effects of diacylglycerol
B kinase inhibitors
R59022 and R59949 inhibit DAG kinase in vertebrate systems, leading to accumulation of DAG and an increase in PKC activity (Chaffoy de Courcelles et al., 1985, 1989). In CA from %-day-old virgin D. punctara females, treatment with R59022 resulted in a significant and dosedependent inhibition of JH release (Fig. l), showing maximum inhibition of 60% (in comparison to untreated controls) at 1O-5M. In CA from 5-day-old mated females, R59022 inhibited JH release to about the same degree (63% inhibition at 1O-5M; see inset Fig. 1). However, in these glands, inhibition was not statistically significant because of higher variation in the data. In contrast to R59022, the structurally similar R59949 did not affect JH release from CA of 2-day virgins, even at concentrations as high as 1tY5M(data not shown). The reversibility of inhibition of JH release from CA of 2-day-old virgin females by R59022 was also determined. For comparative purposes, R59022 and the alla-
T
6
*
l
I
R59022
.
R59022+10"MCAST5
i““h.,
:
0
0
,,
”
I
lo'
**
**
**
l
*
5
I
IO-8
Concentration
1
IO-'
I
104
I
10-S
of R 59022 (M)
Fig. 2. (A) Individual and combined effects of 10m7M Dip7 and different concentrations of DAG kinase inhibitor R59022 on JH release by CA of 2day-old virgin females. R59022 significantly further decreased JH release by Dip7-treated glands (r-test; *P $0.05). Means f SEM of 5 replicates. (B) Individual and combined effects of 10T9M callatostatin 5 and different concentrations of DAG kinaae inhibitor R59022 on JH release by CA of 2-day-old virgin females. R59022 signifv’antly further decreased JH release by CAST 5-treated glands (ttest; *P S 0.05;**P S 0.01). Means i SEM of 9 replicates.
92
A. Rachrnsky et al. I Mr~leculur und Cellular
DIP7
CONT
DIP7 R59022
Endocrinology
10.5 (1994)
PDBu
DIP7 R59022
CONT
Fig. 3. Comparison of individual and combined effects on JH release inhibitor R59022 and either Dip7, CAST 5 or phorbol ester PDBu and 6 M PDBu and 10m8 to lo-’ M R59022. Suboptimal concentrations clearly showed additive effects. Figures within histograms represent treatment with both compounds (I-test; *P 5 0.05; **P 5 0.01). Means
PDBu R59022
CAST5
PDBu R59022
CONT
CAST5
by CA of 2-day-old virgin females controls (CONT). Concentrations of either inhibitory neuropeptides percent inhibition of JH release. of 5-9 replicates.
release as a result of treatment of CA with high concentrations of Dip7 (lo+ M; 77% inhibition) could not be increased by addition of lO+j M R59022 (data not shown). Following exposure of CAST 5-treated glands to R59022, a similar effect was observed (Fig. 2B). Treatment of CA with 1O-9 M CAST 5 alone resulted in significant inhibition of JH release (54%). A combination of R59022 and CAST 5 further decreased JH release significantly, also resulting in 81% maximal inhibition. As in the: case of Dip7, the addition of inhibitory concentrations of the DAG kinase inhibitor did not further reduce JH release, and a threshold concentration of R59022 also synergized the CAST 5 effect (Fig. 2B). It is clear from Fig. 3 that the additive effects of suboptimal concentrations of R59022 with either Dip7 or CAST 5 were identical to those observed following treatment of CA with a combination of R59022 and a suboptimal concentration of the phorbol ester PDBu (IO” M). PDBu is a direct activator of PKC and treatment of CA with this compound alone results in a dose-dependent inhibition of JH biosynthesis, showing maximal inhibition of about 60% at 1O-5 M (data not shown), and similar to that reported by Feyereisen and Farnsworth (1987). Treatment of glands with suboptimal concentrations of these compounds thus permits the assessment of the contribution of each component to the inhibition of JH biosynthesis. 3.2. Effects of protein kinase C inhibitors To determine whether allatostatins act on JH production by activating PKC, we employed PKC inhibitors in several experiments. We used the following compounds that have been shown to block PKC activity specifically in vertebrate and also some invertebrate systems: bisindolylmaleimide, calphostin C, H7, H8, I-0-hexadecyl-2-
X9-96
CAST5 R59022 R59022
of suboptimal concentrations of DAG kinase used were lo-’ M Dip7, 10W9M CAST 5, loor PKC activator PDBu and DGK inhibitor Asterisks denote significant differences from
0-methyl-rut-glycerol, polymyxin B, sphingosine, tamoxifen and trifluoperazine. Since information on the inhibitory action of these agents on PKC activity comes mainly from studies on vertebrate systems, we initially screened these inhibitors for potency in CA of D. punctata. For this purpose, we pre-incubated CA from 2-dayold virgin females with the PKC inhibitors prior to the addition of the PKC activator PDBu. PDBu effectively inhibits JH production in these glands (Fig. 3 and unpubControls 50 r
PKC inhibitors + PDBu
1
Fig. 4. Effects of PKC inhibitors on inhibition of JH release by the PKC activator PDBu. CA of 2-day-old virgin females were preincubated for 20 min with PKC inhibitors ( 10m5 M, except for 2 X IO’ M for calphostin C), before lOA M PDBu were added for an additional 40 min incubation. Bisindolylmaleimide and calphostin C significantly reduced the inhibitory effect of PDBu on JH release (t-test; *P 5 0.05). Means ? SEM of 5-l 2 replicates.
A. Rachinsky et al. /Molecular and Cellular Endocrinology IO5 (1994) 89-96
Controls
PKC inhibitors + Dip7
2z 8 B z ‘ij d p! %
93
*
3020 lol
*
0 -’
5 s 50
-10
.-’
”
-
-
z
-20 -
d 8
-30 -
5
-40 / CAST 5
Thapsigargin
Thapsigargin + CAST 5
Fig. 7. Individual and combined effects of callatostatin 5 and thapsigargin on JH release by CA of 4-day-old mated females. lo-’ M thapsigargin completely reversed the CAST 5-induced inhibition of JH release. Asterisks denote significant differences from untreated controls (r-test; *P 20.05; **P IO.01). Means of 10 replicate assays. Fig. 5. Effects of PKC inhibitors on inhibition of JH release by Dip7. CA of 2-day-old virgin females were pre-incubated for 20 min with PKC inhibitors (10e5 M, except for 2 x lo4 M for calphostin C), before lo-’ M Dip7 was added for an additional 40 min incubation. Bisindolylmaleimide and HS significantly reduced the inhibitory effect of Dip7 on JH release (t-test; *P 20.05). Means f SEM of 6 replicates.
lished data). Fig. 4 shows that bisindolylmaleimide, calphostin C, H7, H8 and l-O-hexadecyl-2-U-methyl-rucglycerol all partially blocked inhibition of JH production by PDBu. However, only in the case of bisindolylmaleimide and calphostin C was JH release significantly higher than in PDBu-treated glands. The same five compounds also partially blocked inhibition of JH production 140 0
130 - 0
Day 4 mated Day 2 virgin
*
T
120
*
*
by Dip7 (Fig. 5), with the effects of bisindolylmaleimide and H8 being statistically significant. 3.3. Effects of thapsigargin To explore the role of possible IP3-induced changes in intracellular Ca*+ levels on regulation of JH biosynthesis, we studied the effects of the sesquiterpene lactone thapsigargin on JH release by CA of D. punctutu. Thapsigargin mimics the effects of IP3 by releasing Ca*+ from intracellular stores (Thastrup et al., 1990). Whereas JH release from CA of 2-day-old virgin females was not affected by this compound, that from CA of 4-day-old mated females resulted in a significant increase in JH release following treatment with lO_’ M and higher concentrations of thapsigargin (Fig. 6). In these glands, thapsigargin and CAST 5 also clearly showed antagonistic effects. Thus, the addition of lO_’ M thapsigargin (29% stimulation of JH release) completely reversed the inhibitory effect of 1O-9 M CAST 5 (34% inhibition) to control levels (Fig. 7).
110 4. Discussion
100 90
4.1. Effects of DAG kinase inhibitors 1
30 20 10 r
O-10-5 0 1O-8
10-7
10-6
Concentration of thapsigargin (M) Fig. 6. Effects of thapsigargin on JH release by CA of 2-day-old virgin (0) and 4-day-old mated females (Cl). Only in mated females was JH release significantly stimulated by thapsigargin (r-test; *P IO.05). Means f SEM of 6-14 replicates.
The present data implicate the phosphoinositide second messenger system in the regulation of JH biosynthesis in D. punctatu. Several pharmacological agents known to act at different sites in this second messenger cascade affect JH release from CA in vitro. Addition of the DAG kinase inhibitor R59022 resulted in reduced JH release from glands of both low (virgin females) and high activity (mated females) in a dose-dependent manner (Fig. 1). Since JH release was fully restored after removal of the DAG kinase inhibitor from the incubation medium, a specific, reversible action of the agent on a DAG kinase in CA cells, rather than a non-specific, cytotoxic effect, is probably responsible for the observed inhibition of JH
94
A. Rachinsky et al. I Molecular and Cellular Endocrinology 105 (1994) 89-96
release. The reversibiIity of the effect of R59022 is similar to that obtained with allatostatins (compare Woodhead et al., 1989). This result by itself does not necessarily imply that both compounds act on JH release via the same signal transduction mechanisms. However, evidence for a synergistic or additive effect of R59022 on the action of neuropeptide inhibitors of JH release comes from our additivity studies. The combination of submaximal concentrations of R59022 and either inhibitory neuropeptides, such as Dip7 and CAST 5, or an agent known to act at a different site in the same second messenger system, such as the phorbol ester activator of PKC, PDBu, caused additive inhibition of JH release, up to a maximum of about 80% (Fig. 3). This maximum could not be exceeded by addition of higher concentrations of R59022. In CA of D. punctutu which are producing JH at low rates (virgin females and mated females early and late in the reproductive cycle), JH release is never completely blocked. Inhibition of JH release by about 80% appears to be the maximum that can be realized by incubation with either brain extracts (Paulson and Stay, 1987), allatostatins (Woodhead et al., 1989; Cusson et al., 1992), or PKC activators (Feyereisen and Farnsworth, 1987). This upper limit for inhibition renders it difficult to test ph~macological agents for synergistic effects on JH release. However, threshold concentrations of DAG kinase inhibitor that by themselves failed to elicit an effect on JH release were able to amplify markedly the inhibitory effects of either Dip7 or CAST 5. The question remains as to whether these synergistic effects arise from action of the two compounds on two different second messenger system or one and the same second messenger system. For example, synergistic effects on chicken growth hormone release were caused by two secretagogues that are known to act via generation of CAMP and phosphoinositide breakdown respectively (Perez et al., 1990). R59022 was unable to elicit the ‘full’ response of about 80% inhibition of JH release, showing only about 60% inhibition of JH release at the highest concentrations. A possible explanation is that since allatostatins, DAG kinase inhibitors and PKC activators all have different sites of action within the phosphoinositide cascade, signals of different intensity are generated. In D. punctatu CA, allatostatin is thought to bind to specific membrane receptors (Cusson et al., 1991; Stay et al., 1994), and these signals are amplified through several steps of a second messenger cascade, finally resulting in the production of multiple second messenger molecules (for example, DAG). On the other hand, phorbol esters mimic DAG effects by directly activating PKC in a dose-dependent manner (Nishizuka, 1984) whereas DAG kinase inhibitors cause an increase in intracellular DAG by preventing its degradation (Chaffoy de Courcelles et al., 1985). Thus, the sites of action and the intensity of responses to DAG in CA cells are correlated with the amount of DAG generated in the target cell.
4.2. effects ofPKC i~hi~ito~~ Addition of PKC inhibitors to CA from virgin female D. punctata reduces the ability of PKC activators and allatostatins to inhibit JH release. Of all PKC inhibitors tested, five partially blocked the effects of the phorbol ester PDBu: H7, H8, bisindolylmaIeimide, calphostin C, and 1-U-hexadecyl-2-~-methyl-r~c-glycerol. The latter three compounds have been described as highly selective and potent inhibitors of PKC in vertebrate cells (Kramer et al., 1989; Toullec et al., 1991; Bergstrand et al., 1992). Calphostin C and 1-0-hexadecyl-2-O-methyl-rac-glycerol block reversibly the activation caused by phorbot ester binding to PKC, whereas bisindolylmaleimide inhibits PKC exclusively via the ATP-binding site (Kobayashi et al., 1989; Kramer et al., 1989; Toullec et al., 1991). In CA of D. punctatu, bisindolylmaleimide, calphostin C and I-0-hexadecyl-2-U-methyl-rat-glycerol also p~ially blocked the effects of Dip7 or PDBu on JH release (Figs. 4 and 5), and thus implicating PKC in the regulation of JH biosynthesis. Blocking of inhibition of JH release by PDBu as a result of treatment with either H7 or H8 was less pronounced. However, H7 and especially H8 act not only on PKC but also inhibit cyclic nucleotide-dependent protein kinases (Hidaka et al., 1984). H8 is more potent in inhibiting cyclic nucleotide-dependent protein kinases than PKC and our findings that this compound also significantly blocked the effect of Dip7 on D. punctata CA is surprising. Recent investigations have shown that synthetic D. punctata allatostatins, including Dip7, do not appear to elicit an increase in CAMP or cGMP within CA, suggesting that these compounds may not act directly as second messengers of these peptides (Cusson et al., 1992; see however Stay et al., 1994; Tobe et al., 1994). However, our resufts on the ability of H8 to block the effect of Dip7 suggest a role for cyclic nucleotide~dependent protein kinases in allatostatin-induced inhibition of JH release. In fact, brain extracts and adenylate cyclase activators such as forskolin act to elevate CAMP levels in CA and concurrently to inhibit JH biosynthesis, and analogues of the cyclic nucleotides are also potent inhibitors of JH biosynthesis (Meller et al., 1985; Aucoin et al., 1987; Tobe, 1990). In addition, phenothiazines, which block calcium binding to calmodulin and thus affect activity of cyclic nucleotide-dependent protein kinases, can modulate JH biosynthesis (Tobe, 1990). In addition, simultaneous treatment of CA with subthreshold concentrations of forskolin and allatostatin does not result in additive inhibition of JH biosynthesis (Cusson et al., 1992), suggesting two distinct sites of actions of these effecters. Thus, cyclic nucleotides can play a role in the regulation of JH production although the allatostatins themselves appear unable to effect detectable changes in their levels within CA. Although polymyxin B, sphingosine, tamoxifen and trifluoperazine are potent PKC inhibitors in both verte-
A. Rachinsky et al. I Molecular and Cellular Endocrinology 105 (1994) 89-96
brate (Oishi et al., 1988; Spacey et al., 1990) and invertebrate systems (Qiu et al., 1990a,b), these compounds were unable to block the action of PDBu on JH release from D. punctutu CA. These findings are not surprising since PKCs are a large family of proteins, with different isoforms showing individual enzymological characteristics, patterns of tissue expression and intracellular localization. Such isoforms show differences in sensitivity to the various types of PKC inhibitors and therefore, probably have particular/unique functions in transducing external signals (Nishizuka, 1988). 4.3. Effects of thapsigargin Thapsigargin causes the release of calcium from intracellular stores without generation of inositol phosphates (Thastrup et al., 1990). In most vertebrate systems studies to date, thapsigargin evokes a sustained Ca*+ rise to a new elevated steady state level. However, in a vertebrate neuronal cell line, it induced a transient rise, resembling hormonally induced increases in Ca*+ (Thastrup et al., 1990). In M. sextu, thapsigargin mimicked the stimulatory effect of the species-specific allatotropin on JH biosynthesis without altering the basal level of inositol phosphates within the CA (Reagan et al., 1992), suggesting that the drug was effecting the direct mobilization of intracellular Ca*+. Whereas the DAG kinase inhibitor R59022 affected JH release from CA of both virgin and mated females of D. punctutu, thapsigargin clearly showed stage-specific differences in its action. Thapsigargin stimulated JH release only in glands from 4-day-old mated females, a physiological stage characterized by high and increasing rates of JH release (maximum rates for JH release are reached on day 5; Tobe et al., 1985) and which is relatively insensitive to inhibitory influences (Stay et al., 1991). Changes in electrical properties of CA of mated females have been documented, including apparent changes in potassium and calcium membrane conductance (McQuiston and Tobe, 1991) supporting a changing role for calcium, via calcium flux, in the regulation of JH biosynthesis. Although extracellular, and most likely intracellular, calcium levels are important for JH biosynthesis and for the mediation of inhibition of JH biosynthesis provoked by brain extracts in CA from 2-day-old virgin females (Kikukawa et al., 1987; Aucoin et al., 1987) these glands proved to be insensitive to stimulation by thapsigargin. Our results indicate that in CA from virgin females, it is difficult to effect thapsigargin-mediated calcium release from intracellular stores. Thapsigargin discharges intracellular calcium stores by specific inhibition of the endoplasmic Ca *+-ATPase, resulting in calcium influx as a consequence of depletion of intracellular stores (Thastrup et al., 1990). As suggested for IPs-receptor-mediated release of intracellular calcium (Berridge, 1993), variations in sensitivity of release mechanisms to the respective ef-
95
fector might explain the observed stage-specific differences in thapsigargin action in CA. Receptor-mediated hydrolysis of phosphatidylinositol leads to the simultaneous generation of two intracellular messengers, DAG and IPs. Our experiments with DAG kinase inhibitors, PKC activators and PKC inhibitors indicate that DAG and PKC are involved in the decline of JH biosynthesis and likely function as signal transducers for allatostatin and callatostatin in both high and low activity CA of D. punctatu. The observation that CA from mated females at the end of the first reproductive cycle also show high sensitivity to the phorbol ester 12myristate 13-acetate (Feyereisen and Farnsworth, 1987), lends further support to this conclusion. The stage-specific differences in thapsigargin action raise questions about the possible role of IP,. In CA from 4-day mated females, thapsigargin stimulated JH release and also antagonized the effects of low concentrations of inhibitory neuropeptide, but it was ineffective in CA from virgin females. Since treatment with either thapsigargin or IPs elevates intracellular Ca*+ levels, we assume that IPs is probably only effective at certain physiological stages. This phenomenon might offer an explanation for earlier observed stage-specific differences in sensitivity to allatostatins; whereas in O-8 day virgin females, the CA show constant high rates of inhibition by allatostatins, in mated females, sensitivity to inhibition by allatostatins declines dramatically on day 4, i.e. shortly before the glands reach maximal rates of JH biosynthesis (Stay et al., 1991). From our results we conclude that in D. punctuta CA, DAG and IPs are two of the intracellular messengers for Dip7 and CAST 5. We further hypothesize that the intracellular action of IPs might be specifically regulated, modulating the inhibitory effects of DAG by acting in an antagonistic fashion. Such interaction between the two second messengers could result in either reduced sensitivity to allato&tins at physiological stages in which high CA activity is required, as for example during vitellogenesis, or in enhanced sensitivity to allatostatins, at times of reduced gland activity, such as in postvitellogenic or virgin females. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Ra 549/1-l) and by an operating grant from the Natural Sciences and Engineering Research Council of Canada (SST). We thank Dr. W.G. Bendena and Dr. R. Rybczynski for critical comments on the manuscript. References Aucoin, R.R., Rankin, S.M.. Stay, B. and Tobe, S.S. (1987) Insect Biochem. 17,965969.
96
A. Rachinsky
et ul. I Moleculur
und Cellular
Bergstrand, H., Lundquist, B., Karabclas, K. and Michelsen, P. (1992) J. Pharmacol. Exp. Ther. 260, 1028-1037. Berridge, M. J. (1993) Nature 361,315-325. Chaffoy de Courcelles, D. de, Roevens, P. and Van Belle, H. (1985) J. Biol. Chem. 260, 15762-15770. Chaffoy de Courcelles, D. de, Roevens, P., Van Belle, H., Kennis, L., Somers Y. and DeClerck F. (1989) J. Biol. Chem. 264,3274-3285. Cusson, M., Prestwich, G.D., Stay, B. and Tobe, S.S. (1991) Biochem. Biophys. Res. Commun. 181.736-742. Cusson, M., Yagi, K.J., Guan, Xue-Chen and Tobe, S.S. (1992) Mol. Cell. Endocrinol. 89, 121-125. Donly, B.C., Ding, Q., Tobe, S.S. and Bendena, W.G. (1993) Proc. Natl. Acad. Sci. USA 90, 8807-8811. Duve, H., Johnsen, A.H., Scott, A.G., Yu, C.G., Yagi, K.J., Tobe, S.S. and Thorpe, A. (1993) Proc. Natl. Acad. Sci. USA 90.2456-2460. Feyereisen, R. and Farnsworth, D.E. (1987) FEBS Lett. 222.345-348. Feyereisen, R. and Tobe, S.S. (1981) Anal. Biochem. Ill, 372-375. Hidaka, H., lnagaki, M., Kawamoto, S. and Sasaki, Y. (1984) Biochemistry 23, 5036-5041. Kobayashi, E., Nakano, H., Morimoto, M. and Tamaoki, T. (1989) Biochem. Biophys. Res. Commun. 159.548-553. Kramer, I.M., van der Bend, R.L., Tool, A.T.J., van Blitterswijk, W.J., Roos, D. and Verhoeven, A.J. (1989) J. Biol. Chem. 264, 58765884. Kikukawa, S., Tobe, S.S., Solowiej, S. Rankin, S.M. and Stay, B. (1987) Insect Biochem. 17, 179-187. Lange, A.B., Ghan, K.K. and Stay, B. (1993). Arch. Insect Biochem. Physiol. 24.79-92. McQuiston, A.R. and Tobe, S.S. (1991) J. Insect Physiol. 37, 223-229. Meller, V.H., Aucoin, R.R., Tobe, S.S. and Feyereisen, R. (1985) Mol. Cell. Endocrinol. 43, 155-163. Nishizuka, Y. (1984). Nature 308, 693-697. Nishizuka, Y. (1988) Nature 334,661465. Oishi, K., Raynor, R.L., Charp, P.A. and Kuo, J.F. (1988) J. Biol. Chem. 263,68654871. Paulson, C.R. and Stay, B. (1987) Mol. Cell, Endocrinol. 51, 243-252. Perez, F.M., Malamed, S. and Scanes, C.G. (1990) Gen. Comp. Endocrinol. 80, 181-188.
Endocrinology
105 (1994) 89-96
Pratt, G.E. and Tobe, S.S. (1974) Life Sci. 14, 575-586. Pratt, G.E., Farnsworth, D.E., Fok, K.F., Siegel, N.R., McCormack, A.L., Shabanowitz, J., Hunt, D.F. and Feyereisen, R. (1991) Proc. Nat]. Acad. Sci. USA 88, 2412-2416. Qiu. T.H., Combest. W.L. and Gilbert, L.I. (1990a) Insect Biochem. 20. 405-411. Qiu. T.H., Combest. W.L. and Gilbert. L.O. (1990b) Insect Biochem. 20.413420. Reagan, J.D., Miller, W.H. and Kramer, S.J. (1992) Arch. Insect Biothem. Physiol. 20, 145-155. Spacey, G.D., Bonser, R.W., Randall, R.W. and Garland, L.G. (1990) Cell. Signal. 2, 329-338. Stay, B. and Coop, A. (1973) J. Insect Physiol. 19, 147-171. Stay, B. and Woodhead, A.P. (1993) Am. Zool. 33.357-364. Stay, B., Joshi. S. and Woodhead, A.P. (1991) J. Insect Physiol. 37,6370. Stay, B., Tobe, S.S. and Bendena, W.G. (1994) Adv. Insect Physiol. 25, 267-338. Thastrup, 0.. Cullen, P. I., Drobak. B. K., Hanley, M. R. and Dawson, A.P. (1990) Proc. Natl. Acad. Sci. USA 87, 2466-2470. Tobe, S.S. (1990) In Progress in Comparative Endocrinology (Epple A., Scanes C.G. and Stetson M.H., eds.), pp. 174-179, Wiley Liss, New York. Tobe. S.S. and Pratt. G.E. (1974) Biochem. J. 144, 107-l 13. Tobe, S.S. and Clarke, N. (1985) Insect Biochem. 15, 175-179. Tobe, S.S., Ruegg, R.P., Stay, B.A., Baker, F.C., Miller, CA. and Schooley, D.A. (1985) Experientia 41, 1028-1034. Tobe, S.S., Yu, C.G. and Bendena, W.G. (1994) In Perspectives in Endocrinology (Davey K.G., Peter R.E., Tobe S.S., eds.), pp. 1219, National Research Council of Canada, Ottawa. Toullec, D., Pianetti, P., Coste, H., Belleverge. P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D. and Kirilovsky. J. (1991) J. Biol. Chem. 266, 15771-15781. Woodhead, A.P., Stay, B., Seidel, S.L., Khan, M.A. and Tobe, S.S. (1989) Proc. Nat]. Acad. Sci. USA 86, 5997-6001. Woodhead, A.P., Khan, M.A., Stay, B. and Tobe, S.S. (1994) Insect Biochem. Mol. Biol. 24, 257-263.
Molecular and Cellular Endocrinology 105 (1994) 89-96
Signal transduction in the inhibition of juvenile hormone biosynthesis by allatostatins: roles of diacylglycerol and calcium A. Rachinskya, J. Zhangb, S.S. Tobebs* ati Entwicklungsphysiologie, Zoologisches hstitut, Universitiit Tiibingen, Auf der Morgenstelle 28, D-72076 TWngen, Germany bDepartment of Zoology, University of Toronto, Toronto, Ontario, MSS IAI, Canada Received 18 May 1994; accepted 25 July 1994
Abstract
The effects of pharmacological agents that interfere with the 1,4,5inositol trisphosphate (IPs)/diacylglycerol (DAG) pathway on juvenile hormone (JH) biosynthesis by corpora allata (CA) of the cockroach Diplopteru punctata have been investigated. These effects were assessed in the presence of the inhibitory neuropeptides, allatostatins, with a view to elucidating the pathway for signal transduction in the inhibition of JH biosynthesis. Treatment of CA with inhibitors of DAG kinase to elevate the concentration of DAG within the CA cells, resulted in a significant, dose-dependent decrease in JH biosynthesis. Simultaneous treatment of glands with both DAG kinase inhibitors and allatostatins further enhanced this effect, suggesting that DAG is an intermediate in the allatostatin-induced inhibition of JH production. The inhibitory actions of the phorbol ester activator of PKC, PDBu, or of allatostatin on JH biosynthesis were partially blocked by pre-incubating the CA with PKC inhibitors. Treatment of CA with the calcium-mobilizing drug thapsigargin resulted in a significant stimulation in JH biosynthesis in glands from mated females producing JH at high rates. Thapsigargin was also able to reverse the effect of allatostatins in high-activity mated CA. This suggests an involvement of the other product of phosphoinositide hydrolysis, IP3, in the modulation of JH biosynthesis at specific developmental times and in glands showing specific levels of activity. Keywords:
Diacylglycerol
kinase inhibitors;
Protein
kinase C inhibitors; Calcium; Juvenile hormone biosynthesis;
Allatostatins;
Cockroach; Insect
1. Introduction In the cockroach Diploptera punctata, the activity of the juvenile hormone (JH)-producing corpora allata (CA) is, in part, under control of inhibitory neuropeptides. A family of seven structurally related allatostatins which are biologically active in the in vitro assay for JH biosynthesis (Pratt and Tobe, 1974; Tobe and Pratt, 1974) has been isolated from D. punctata brains (Woodhead et al., 1989, 1994; Pratt et al, 1991). A gene encoding all seven known allatostatins plus six previously unknown allatostatins has recently been cloned and shown to be expressed in medial neurosecretory cells of the brain of D. punctata (Donly et al., 1993). The detection of allatostatin-immunoreactive pathways between brain and retrocerebral complex, and
*Corresponding 3522.
author. email [email protected]. Fax 416 978
0167-8140/94/$07.00 0 1994 Elsevier Science Ireland Ltd. All rights reserved SSDl 0167-8140(94)03367-3
of specific allatostatin receptors in brain, fat body and CA membrane preparations (Cusson et al., 1991), emphasizes the importance of these peptides in regulation of CA but also suggests other possible regulatory functions (Lange et al., 1993; Stay and Woodhead, 1993). In addition, the callatostatins, a group of five neuropeptides isolated from Calliphora vomitoria with strong structural similarity to the D. punctata allatostatins, show pronounced allatostatic activity in D. punctara, callatostatin 5 being the most potent neuropeptide inhibitor of JH biosynthesis so far tested in this species (Duve et al., 1993). Allatostatins presumably stimulate signal transduction pathway(s) after binding to membrane receptors of CA cells. The generation of intracellular second messengers may then act to modulate enzymatic activity in the JH biosynthetic pathway (Tobe et al., 1994). Most neuropeptide receptors studied to date interact with G proteins to exert their effects via second messengers such as D-
A. Rachinsky
90
et al. /Molecular
and Cellular
inositol 1,4,5triphosphate (IPs) and diacylglycerol (DAG). In D. punctutu, correlations exist between allatostatic action of brain extracts and elevated CAMP levels within the CA, and pharmacologically elevated CAMP levels also inhibit JH release (Meller et al., 1985; Aucoin et al., 1987; Tobe, 1990). Despite these observations, synthetic D. puncruru allatostatins did not elicit any significant changes in the levels of cyclic nucleotides in isolated CA (Cusson et al., 1992), suggesting that these compounds may not be directly involved in the mediation of allatostatin action. Other possible candidates for signal transduction in the regulation of JH biosynthesis are IPs and DAG which are produced by receptor-mediated hydrolysis of phosphatidylinositol-4,5bisphosphate. IPs causes the release of Ca from intracellular storage sites, whereas DAG activates protein kinase C (PKC) (Berridge, 1993). The IPs second messenger pathway has been shown to be present in insects and is, for example, involved in mobilization of intracellular calcium in blowfly salivary glands (Berridge, 1993) and PKC activation in Manduca sextu brains (Qiu et al., 1990a,b). In CA of adult M. sextu, an allatotropin neuropeptide induces the formation of inositol phosphates (Reagan et al., 1992). Furthermore, several pharmacological agents that act at different sites in the IPs pathway also stimulate JH biosynthesis, suggesting an activation of this second messenger system by the allatotropin (Reagan et al., 1992). In D. punctutu, phorbol ester activators of PKC proved to be potent inhibitors of JH biosynthesis, thus implicating PKC in the regulation of CA activity (Feyereisen and Farnsworth, 1987). If activation of PKC is an essential event in allatostatin-induced inhibition of JH biosynthesis in D. punctutu, an experimentally evoked increase in diacylglycerol concentration should mimic the effect of allatostatins. We therefore studied the effects of DAG kinase inhibitors on JH release. On the other hand, compounds that inhibit PKC activity should antagonize allatostatin-evoked inhibition and accordingly, we investigated the effects of various PKC inhibitors. Furthermore, to obtain information on the role of IP, in regulation of JH biosynthesis, we have determined the effects of thapsigargin, a compound known to mimic the effects of IP, on intracellular Ca stores. 2. Materials
and methods
2.1. Animals The viviparous cockroach D. punctutu was reared as previously described (Stay and Coop, 1973). We used 2day-old virgins and 2, 4 and 5 day mated females for our experiments. Virgin females, whose CA show relatively constant low rates of JH biosynthesis (Stay et al., 1991), were obtained by isolating last instar females from the colony and maintaining them in separate jars. In mated females, oocyte growth is strictly correlated with age and
Endocrinology
105 (1994) 89-96
rates of JH biosynthesis (Tobe et al., 1985). Therefore, to ensure similar physiological status of experimental animals, length of basal oocytes was checked before dissection of the CA. 2.2. Materials H7, H8, R59022, R59949 and tamoxifen citrate were purchased from Research Biochemicals Inc. (Natick, MA); phorbol 12,13-dibutyrate (PDBu) and trifluoperazine from Sigma Chemical Corp. (St. Louis, MO). Bisindolylmaleimide, calphostin C, I-0-hexadecyl-2-0methyl-rut-glycerol, polymyxin B and D-erythrosphingosine were obtained from Calbiochem Corp. (La Jolla, CA); thapsigargin from LC Services Corp. (Woburn, MA). Synthetic Dipstatin7 (Dip7; previous designation AST 1; for allatostatin nomenclature, see Donly et al., 1993) and callatostatin 5 (CAST 5) were obtained from University of Iowa Protein Structure Facility or Core Facility, Insect Biotech Canada. All solvents used were of the highest grade available from commercial suppliers. Bisindolylmaleimide, calphostin C, I-0-hexadecyl-20-methyl-rut-glycerol, PDBu, R59949 and thapsigargin were prepared as stock solutions in DMSO. Tamoxifen citrate and R59022 were prepared as stock solutions in ethanol. R59949 and R59022 were diluted 1: 10 in 5 mN HCl before further dilution in incubation medium. Detythro-Sphingosine was dissolved in chloroform/methanol (1: 1). The amount required for assays was dried under nitrogen and dissolved in medium by sonication. All other reagents used were water soluble. Solvent controls were found to have no effect on JH release at final concentrations of 0.1% DMSO and 0.1% ethanol.
2.3. Assay for CA activity Rates of JH biosynthesis were determined by the in vitro radiochemical method of Feyereisen and Tobe (1981) and Tobe and Clarke (1985). Single CA or one pair of CA were usually incubated for 3 h at 27°C in 100~1 of methionine-free medium 199 (Gibco; supplemented with 1.3 mM calcium chloride and 2% (w/v) Fi~011) containing L-[methyl-i4C]methionine (Amersham; 55-57 mCi/mmol) in the incubation medium to a final concentration of 50pM to monitor biosynthesis of radiolabeled JH. At the end of the incubation time, CA were removed from the medium before extraction and quantification of JH. Thus the values represent rates of JH released into the medium. Pharmacological agents were usually added to the labeled medium immediately before starting the incubations. For experiments with potential PKC inhibitors, CA were pre-incubated for 20 min in 45 ~1 labeled medium containing 10pM inhibitor (except for 2,uM for calphostin C, due to its solubility), before addition of 5 ~1 of either 10pM PDBu or 1 PM Dip7. Glands were then incubated for another 40 min before assaying the incubation
A. Rachinskyet al. I Molecularand CellularEndocrinology105 (1994) 89-96
01’
I
0
IO-Q
lo-8
Concentration
I
I
lo-'
of R 59022
IO-6
lo-5
(M)
Fig. 1. Dose-response characteristics of in vitro JH release by CA of 2day-old virgin (0)and S-day-old mated females (m) (inset) as influenced by the DAG kinase inhibitor R59022. In both stages, R59022 caused a dose-dependent inhibition of JH release. Asterisks denote significant differences from untreated controls (t-test; ***P < 0.001). Means f SEM of 5-10 replicate assays.
medium for radiolabeled JH. For tests on reversibility of DGK inhibitors, glands were first incubated for 3 h in control medium to determine basal rates of JH release, then for 3 h in medium containing the inhibitors, and finally for another 3 h in control medium without inhibitors. At the end of each incubation period, the medium was assayed for radiolabeled JH.
91
tostatin Dip7 were tested simultaneously. Relative to basal rates (measured in the first 3 h of incubation), rates of JH release significantly decreased following transfer of glands into medium containing either R59022 or Dip7 in the second incubation period (data not shown). In both cases, JH release recovered after transfer of the glands into medium without inhibitor. For the third incubation period, JH release was not significantly different from basal rates. To examine the combined effects of R59022 and inhibitory neuropeptides, CA of 2-day-old virgin females were incubated with different concentrations of the DAG kinase inhibitor and either Dip7 or CAST 5. R59022 and Dip7 clearly showed an additive effect. Treatment of the glands with 1O-7M Dip7 alone caused a significant inhibition of JH release (46%). Addition of R59022 to these Dip7-treated glands resulted in a further significant decrease in JH release (Fig. 2A). Even at a threshold concentration, R59022 (lo-8 M) markedly decreased JH release by Dip7-treated glands, suggesting a synergistic effect (Fig. 2A). Maximal inhibition achieved by a combination of R59022 and Dip7 was 81%. Inhibition of JH
T
0
R59022
l
R59022+10-'M
Dip7
2.4. Statistical analysis All values are reported as mean + standard error of the mean (SEM). Statistical significance of data was determined by Student’s t-test (paired or unpaired; P < 0.05).
3. Results 3. I. Effects of diacylglycerol
B kinase inhibitors
R59022 and R59949 inhibit DAG kinase in vertebrate systems, leading to accumulation of DAG and an increase in PKC activity (Chaffoy de Courcelles et al., 1985, 1989). In CA from %-day-old virgin D. punctara females, treatment with R59022 resulted in a significant and dosedependent inhibition of JH release (Fig. l), showing maximum inhibition of 60% (in comparison to untreated controls) at 1O-5M. In CA from 5-day-old mated females, R59022 inhibited JH release to about the same degree (63% inhibition at 1O-5M; see inset Fig. 1). However, in these glands, inhibition was not statistically significant because of higher variation in the data. In contrast to R59022, the structurally similar R59949 did not affect JH release from CA of 2-day virgins, even at concentrations as high as 1tY5M(data not shown). The reversibility of inhibition of JH release from CA of 2-day-old virgin females by R59022 was also determined. For comparative purposes, R59022 and the alla-
T
6
*
l
I
R59022
.
R59022+10"MCAST5
i““h.,
:
0
0
,,
”
I
lo'
**
**
**
l
*
5
I
IO-8
Concentration
1
IO-'
I
104
I
10-S
of R 59022 (M)
Fig. 2. (A) Individual and combined effects of 10m7M Dip7 and different concentrations of DAG kinase inhibitor R59022 on JH release by CA of 2day-old virgin females. R59022 significantly further decreased JH release by Dip7-treated glands (r-test; *P $0.05). Means f SEM of 5 replicates. (B) Individual and combined effects of 10T9M callatostatin 5 and different concentrations of DAG kinaae inhibitor R59022 on JH release by CA of 2-day-old virgin females. R59022 signifv’antly further decreased JH release by CAST 5-treated glands (ttest; *P S 0.05;**P S 0.01). Means i SEM of 9 replicates.
92
A. Rachrnsky et al. I Mr~leculur und Cellular
DIP7
CONT
DIP7 R59022
Endocrinology
10.5 (1994)
PDBu
DIP7 R59022
CONT
Fig. 3. Comparison of individual and combined effects on JH release inhibitor R59022 and either Dip7, CAST 5 or phorbol ester PDBu and 6 M PDBu and 10m8 to lo-’ M R59022. Suboptimal concentrations clearly showed additive effects. Figures within histograms represent treatment with both compounds (I-test; *P 5 0.05; **P 5 0.01). Means
PDBu R59022
CAST5
PDBu R59022
CONT
CAST5
by CA of 2-day-old virgin females controls (CONT). Concentrations of either inhibitory neuropeptides percent inhibition of JH release. of 5-9 replicates.
release as a result of treatment of CA with high concentrations of Dip7 (lo+ M; 77% inhibition) could not be increased by addition of lO+j M R59022 (data not shown). Following exposure of CAST 5-treated glands to R59022, a similar effect was observed (Fig. 2B). Treatment of CA with 1O-9 M CAST 5 alone resulted in significant inhibition of JH release (54%). A combination of R59022 and CAST 5 further decreased JH release significantly, also resulting in 81% maximal inhibition. As in the: case of Dip7, the addition of inhibitory concentrations of the DAG kinase inhibitor did not further reduce JH release, and a threshold concentration of R59022 also synergized the CAST 5 effect (Fig. 2B). It is clear from Fig. 3 that the additive effects of suboptimal concentrations of R59022 with either Dip7 or CAST 5 were identical to those observed following treatment of CA with a combination of R59022 and a suboptimal concentration of the phorbol ester PDBu (IO” M). PDBu is a direct activator of PKC and treatment of CA with this compound alone results in a dose-dependent inhibition of JH biosynthesis, showing maximal inhibition of about 60% at 1O-5 M (data not shown), and similar to that reported by Feyereisen and Farnsworth (1987). Treatment of glands with suboptimal concentrations of these compounds thus permits the assessment of the contribution of each component to the inhibition of JH biosynthesis. 3.2. Effects of protein kinase C inhibitors To determine whether allatostatins act on JH production by activating PKC, we employed PKC inhibitors in several experiments. We used the following compounds that have been shown to block PKC activity specifically in vertebrate and also some invertebrate systems: bisindolylmaleimide, calphostin C, H7, H8, I-0-hexadecyl-2-
X9-96
CAST5 R59022 R59022
of suboptimal concentrations of DAG kinase used were lo-’ M Dip7, 10W9M CAST 5, loor PKC activator PDBu and DGK inhibitor Asterisks denote significant differences from
0-methyl-rut-glycerol, polymyxin B, sphingosine, tamoxifen and trifluoperazine. Since information on the inhibitory action of these agents on PKC activity comes mainly from studies on vertebrate systems, we initially screened these inhibitors for potency in CA of D. punctata. For this purpose, we pre-incubated CA from 2-dayold virgin females with the PKC inhibitors prior to the addition of the PKC activator PDBu. PDBu effectively inhibits JH production in these glands (Fig. 3 and unpubControls 50 r
PKC inhibitors + PDBu
1
Fig. 4. Effects of PKC inhibitors on inhibition of JH release by the PKC activator PDBu. CA of 2-day-old virgin females were preincubated for 20 min with PKC inhibitors ( 10m5 M, except for 2 X IO’ M for calphostin C), before lOA M PDBu were added for an additional 40 min incubation. Bisindolylmaleimide and calphostin C significantly reduced the inhibitory effect of PDBu on JH release (t-test; *P 5 0.05). Means ? SEM of 5-l 2 replicates.
A. Rachinsky et al. /Molecular and Cellular Endocrinology IO5 (1994) 89-96
Controls
PKC inhibitors + Dip7
2z 8 B z ‘ij d p! %
93
*
3020 lol
*
0 -’
5 s 50
-10
.-’
”
-
-
z
-20 -
d 8
-30 -
5
-40 / CAST 5
Thapsigargin
Thapsigargin + CAST 5
Fig. 7. Individual and combined effects of callatostatin 5 and thapsigargin on JH release by CA of 4-day-old mated females. lo-’ M thapsigargin completely reversed the CAST 5-induced inhibition of JH release. Asterisks denote significant differences from untreated controls (r-test; *P 20.05; **P IO.01). Means of 10 replicate assays. Fig. 5. Effects of PKC inhibitors on inhibition of JH release by Dip7. CA of 2-day-old virgin females were pre-incubated for 20 min with PKC inhibitors (10e5 M, except for 2 x lo4 M for calphostin C), before lo-’ M Dip7 was added for an additional 40 min incubation. Bisindolylmaleimide and HS significantly reduced the inhibitory effect of Dip7 on JH release (t-test; *P 20.05). Means f SEM of 6 replicates.
lished data). Fig. 4 shows that bisindolylmaleimide, calphostin C, H7, H8 and l-O-hexadecyl-2-U-methyl-rucglycerol all partially blocked inhibition of JH production by PDBu. However, only in the case of bisindolylmaleimide and calphostin C was JH release significantly higher than in PDBu-treated glands. The same five compounds also partially blocked inhibition of JH production 140 0
130 - 0
Day 4 mated Day 2 virgin
*
T
120
*
*
by Dip7 (Fig. 5), with the effects of bisindolylmaleimide and H8 being statistically significant. 3.3. Effects of thapsigargin To explore the role of possible IP3-induced changes in intracellular Ca*+ levels on regulation of JH biosynthesis, we studied the effects of the sesquiterpene lactone thapsigargin on JH release by CA of D. punctutu. Thapsigargin mimics the effects of IP3 by releasing Ca*+ from intracellular stores (Thastrup et al., 1990). Whereas JH release from CA of 2-day-old virgin females was not affected by this compound, that from CA of 4-day-old mated females resulted in a significant increase in JH release following treatment with lO_’ M and higher concentrations of thapsigargin (Fig. 6). In these glands, thapsigargin and CAST 5 also clearly showed antagonistic effects. Thus, the addition of lO_’ M thapsigargin (29% stimulation of JH release) completely reversed the inhibitory effect of 1O-9 M CAST 5 (34% inhibition) to control levels (Fig. 7).
110 4. Discussion
100 90
4.1. Effects of DAG kinase inhibitors 1
30 20 10 r
O-10-5 0 1O-8
10-7
10-6
Concentration of thapsigargin (M) Fig. 6. Effects of thapsigargin on JH release by CA of 2-day-old virgin (0) and 4-day-old mated females (Cl). Only in mated females was JH release significantly stimulated by thapsigargin (r-test; *P IO.05). Means f SEM of 6-14 replicates.
The present data implicate the phosphoinositide second messenger system in the regulation of JH biosynthesis in D. punctatu. Several pharmacological agents known to act at different sites in this second messenger cascade affect JH release from CA in vitro. Addition of the DAG kinase inhibitor R59022 resulted in reduced JH release from glands of both low (virgin females) and high activity (mated females) in a dose-dependent manner (Fig. 1). Since JH release was fully restored after removal of the DAG kinase inhibitor from the incubation medium, a specific, reversible action of the agent on a DAG kinase in CA cells, rather than a non-specific, cytotoxic effect, is probably responsible for the observed inhibition of JH
94
A. Rachinsky et al. I Molecular and Cellular Endocrinology 105 (1994) 89-96
release. The reversibiIity of the effect of R59022 is similar to that obtained with allatostatins (compare Woodhead et al., 1989). This result by itself does not necessarily imply that both compounds act on JH release via the same signal transduction mechanisms. However, evidence for a synergistic or additive effect of R59022 on the action of neuropeptide inhibitors of JH release comes from our additivity studies. The combination of submaximal concentrations of R59022 and either inhibitory neuropeptides, such as Dip7 and CAST 5, or an agent known to act at a different site in the same second messenger system, such as the phorbol ester activator of PKC, PDBu, caused additive inhibition of JH release, up to a maximum of about 80% (Fig. 3). This maximum could not be exceeded by addition of higher concentrations of R59022. In CA of D. punctutu which are producing JH at low rates (virgin females and mated females early and late in the reproductive cycle), JH release is never completely blocked. Inhibition of JH release by about 80% appears to be the maximum that can be realized by incubation with either brain extracts (Paulson and Stay, 1987), allatostatins (Woodhead et al., 1989; Cusson et al., 1992), or PKC activators (Feyereisen and Farnsworth, 1987). This upper limit for inhibition renders it difficult to test ph~macological agents for synergistic effects on JH release. However, threshold concentrations of DAG kinase inhibitor that by themselves failed to elicit an effect on JH release were able to amplify markedly the inhibitory effects of either Dip7 or CAST 5. The question remains as to whether these synergistic effects arise from action of the two compounds on two different second messenger system or one and the same second messenger system. For example, synergistic effects on chicken growth hormone release were caused by two secretagogues that are known to act via generation of CAMP and phosphoinositide breakdown respectively (Perez et al., 1990). R59022 was unable to elicit the ‘full’ response of about 80% inhibition of JH release, showing only about 60% inhibition of JH release at the highest concentrations. A possible explanation is that since allatostatins, DAG kinase inhibitors and PKC activators all have different sites of action within the phosphoinositide cascade, signals of different intensity are generated. In D. punctatu CA, allatostatin is thought to bind to specific membrane receptors (Cusson et al., 1991; Stay et al., 1994), and these signals are amplified through several steps of a second messenger cascade, finally resulting in the production of multiple second messenger molecules (for example, DAG). On the other hand, phorbol esters mimic DAG effects by directly activating PKC in a dose-dependent manner (Nishizuka, 1984) whereas DAG kinase inhibitors cause an increase in intracellular DAG by preventing its degradation (Chaffoy de Courcelles et al., 1985). Thus, the sites of action and the intensity of responses to DAG in CA cells are correlated with the amount of DAG generated in the target cell.
4.2. effects ofPKC i~hi~ito~~ Addition of PKC inhibitors to CA from virgin female D. punctata reduces the ability of PKC activators and allatostatins to inhibit JH release. Of all PKC inhibitors tested, five partially blocked the effects of the phorbol ester PDBu: H7, H8, bisindolylmaIeimide, calphostin C, and 1-U-hexadecyl-2-~-methyl-r~c-glycerol. The latter three compounds have been described as highly selective and potent inhibitors of PKC in vertebrate cells (Kramer et al., 1989; Toullec et al., 1991; Bergstrand et al., 1992). Calphostin C and 1-0-hexadecyl-2-O-methyl-rac-glycerol block reversibly the activation caused by phorbot ester binding to PKC, whereas bisindolylmaleimide inhibits PKC exclusively via the ATP-binding site (Kobayashi et al., 1989; Kramer et al., 1989; Toullec et al., 1991). In CA of D. punctatu, bisindolylmaleimide, calphostin C and I-0-hexadecyl-2-U-methyl-rat-glycerol also p~ially blocked the effects of Dip7 or PDBu on JH release (Figs. 4 and 5), and thus implicating PKC in the regulation of JH biosynthesis. Blocking of inhibition of JH release by PDBu as a result of treatment with either H7 or H8 was less pronounced. However, H7 and especially H8 act not only on PKC but also inhibit cyclic nucleotide-dependent protein kinases (Hidaka et al., 1984). H8 is more potent in inhibiting cyclic nucleotide-dependent protein kinases than PKC and our findings that this compound also significantly blocked the effect of Dip7 on D. punctata CA is surprising. Recent investigations have shown that synthetic D. punctata allatostatins, including Dip7, do not appear to elicit an increase in CAMP or cGMP within CA, suggesting that these compounds may not act directly as second messengers of these peptides (Cusson et al., 1992; see however Stay et al., 1994; Tobe et al., 1994). However, our resufts on the ability of H8 to block the effect of Dip7 suggest a role for cyclic nucleotide~dependent protein kinases in allatostatin-induced inhibition of JH release. In fact, brain extracts and adenylate cyclase activators such as forskolin act to elevate CAMP levels in CA and concurrently to inhibit JH biosynthesis, and analogues of the cyclic nucleotides are also potent inhibitors of JH biosynthesis (Meller et al., 1985; Aucoin et al., 1987; Tobe, 1990). In addition, phenothiazines, which block calcium binding to calmodulin and thus affect activity of cyclic nucleotide-dependent protein kinases, can modulate JH biosynthesis (Tobe, 1990). In addition, simultaneous treatment of CA with subthreshold concentrations of forskolin and allatostatin does not result in additive inhibition of JH biosynthesis (Cusson et al., 1992), suggesting two distinct sites of actions of these effecters. Thus, cyclic nucleotides can play a role in the regulation of JH production although the allatostatins themselves appear unable to effect detectable changes in their levels within CA. Although polymyxin B, sphingosine, tamoxifen and trifluoperazine are potent PKC inhibitors in both verte-
A. Rachinsky et al. I Molecular and Cellular Endocrinology 105 (1994) 89-96
brate (Oishi et al., 1988; Spacey et al., 1990) and invertebrate systems (Qiu et al., 1990a,b), these compounds were unable to block the action of PDBu on JH release from D. punctutu CA. These findings are not surprising since PKCs are a large family of proteins, with different isoforms showing individual enzymological characteristics, patterns of tissue expression and intracellular localization. Such isoforms show differences in sensitivity to the various types of PKC inhibitors and therefore, probably have particular/unique functions in transducing external signals (Nishizuka, 1988). 4.3. Effects of thapsigargin Thapsigargin causes the release of calcium from intracellular stores without generation of inositol phosphates (Thastrup et al., 1990). In most vertebrate systems studies to date, thapsigargin evokes a sustained Ca*+ rise to a new elevated steady state level. However, in a vertebrate neuronal cell line, it induced a transient rise, resembling hormonally induced increases in Ca*+ (Thastrup et al., 1990). In M. sextu, thapsigargin mimicked the stimulatory effect of the species-specific allatotropin on JH biosynthesis without altering the basal level of inositol phosphates within the CA (Reagan et al., 1992), suggesting that the drug was effecting the direct mobilization of intracellular Ca*+. Whereas the DAG kinase inhibitor R59022 affected JH release from CA of both virgin and mated females of D. punctutu, thapsigargin clearly showed stage-specific differences in its action. Thapsigargin stimulated JH release only in glands from 4-day-old mated females, a physiological stage characterized by high and increasing rates of JH release (maximum rates for JH release are reached on day 5; Tobe et al., 1985) and which is relatively insensitive to inhibitory influences (Stay et al., 1991). Changes in electrical properties of CA of mated females have been documented, including apparent changes in potassium and calcium membrane conductance (McQuiston and Tobe, 1991) supporting a changing role for calcium, via calcium flux, in the regulation of JH biosynthesis. Although extracellular, and most likely intracellular, calcium levels are important for JH biosynthesis and for the mediation of inhibition of JH biosynthesis provoked by brain extracts in CA from 2-day-old virgin females (Kikukawa et al., 1987; Aucoin et al., 1987) these glands proved to be insensitive to stimulation by thapsigargin. Our results indicate that in CA from virgin females, it is difficult to effect thapsigargin-mediated calcium release from intracellular stores. Thapsigargin discharges intracellular calcium stores by specific inhibition of the endoplasmic Ca *+-ATPase, resulting in calcium influx as a consequence of depletion of intracellular stores (Thastrup et al., 1990). As suggested for IPs-receptor-mediated release of intracellular calcium (Berridge, 1993), variations in sensitivity of release mechanisms to the respective ef-
95
fector might explain the observed stage-specific differences in thapsigargin action in CA. Receptor-mediated hydrolysis of phosphatidylinositol leads to the simultaneous generation of two intracellular messengers, DAG and IPs. Our experiments with DAG kinase inhibitors, PKC activators and PKC inhibitors indicate that DAG and PKC are involved in the decline of JH biosynthesis and likely function as signal transducers for allatostatin and callatostatin in both high and low activity CA of D. punctatu. The observation that CA from mated females at the end of the first reproductive cycle also show high sensitivity to the phorbol ester 12myristate 13-acetate (Feyereisen and Farnsworth, 1987), lends further support to this conclusion. The stage-specific differences in thapsigargin action raise questions about the possible role of IP,. In CA from 4-day mated females, thapsigargin stimulated JH release and also antagonized the effects of low concentrations of inhibitory neuropeptide, but it was ineffective in CA from virgin females. Since treatment with either thapsigargin or IPs elevates intracellular Ca*+ levels, we assume that IPs is probably only effective at certain physiological stages. This phenomenon might offer an explanation for earlier observed stage-specific differences in sensitivity to allatostatins; whereas in O-8 day virgin females, the CA show constant high rates of inhibition by allatostatins, in mated females, sensitivity to inhibition by allatostatins declines dramatically on day 4, i.e. shortly before the glands reach maximal rates of JH biosynthesis (Stay et al., 1991). From our results we conclude that in D. punctuta CA, DAG and IPs are two of the intracellular messengers for Dip7 and CAST 5. We further hypothesize that the intracellular action of IPs might be specifically regulated, modulating the inhibitory effects of DAG by acting in an antagonistic fashion. Such interaction between the two second messengers could result in either reduced sensitivity to allato&tins at physiological stages in which high CA activity is required, as for example during vitellogenesis, or in enhanced sensitivity to allatostatins, at times of reduced gland activity, such as in postvitellogenic or virgin females. Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Ra 549/1-l) and by an operating grant from the Natural Sciences and Engineering Research Council of Canada (SST). We thank Dr. W.G. Bendena and Dr. R. Rybczynski for critical comments on the manuscript. References Aucoin, R.R., Rankin, S.M.. Stay, B. and Tobe, S.S. (1987) Insect Biochem. 17,965969.
96
A. Rachinsky
et ul. I Moleculur
und Cellular
Bergstrand, H., Lundquist, B., Karabclas, K. and Michelsen, P. (1992) J. Pharmacol. Exp. Ther. 260, 1028-1037. Berridge, M. J. (1993) Nature 361,315-325. Chaffoy de Courcelles, D. de, Roevens, P. and Van Belle, H. (1985) J. Biol. Chem. 260, 15762-15770. Chaffoy de Courcelles, D. de, Roevens, P., Van Belle, H., Kennis, L., Somers Y. and DeClerck F. (1989) J. Biol. Chem. 264,3274-3285. Cusson, M., Prestwich, G.D., Stay, B. and Tobe, S.S. (1991) Biochem. Biophys. Res. Commun. 181.736-742. Cusson, M., Yagi, K.J., Guan, Xue-Chen and Tobe, S.S. (1992) Mol. Cell. Endocrinol. 89, 121-125. Donly, B.C., Ding, Q., Tobe, S.S. and Bendena, W.G. (1993) Proc. Natl. Acad. Sci. USA 90, 8807-8811. Duve, H., Johnsen, A.H., Scott, A.G., Yu, C.G., Yagi, K.J., Tobe, S.S. and Thorpe, A. (1993) Proc. Natl. Acad. Sci. USA 90.2456-2460. Feyereisen, R. and Farnsworth, D.E. (1987) FEBS Lett. 222.345-348. Feyereisen, R. and Tobe, S.S. (1981) Anal. Biochem. Ill, 372-375. Hidaka, H., lnagaki, M., Kawamoto, S. and Sasaki, Y. (1984) Biochemistry 23, 5036-5041. Kobayashi, E., Nakano, H., Morimoto, M. and Tamaoki, T. (1989) Biochem. Biophys. Res. Commun. 159.548-553. Kramer, I.M., van der Bend, R.L., Tool, A.T.J., van Blitterswijk, W.J., Roos, D. and Verhoeven, A.J. (1989) J. Biol. Chem. 264, 58765884. Kikukawa, S., Tobe, S.S., Solowiej, S. Rankin, S.M. and Stay, B. (1987) Insect Biochem. 17, 179-187. Lange, A.B., Ghan, K.K. and Stay, B. (1993). Arch. Insect Biochem. Physiol. 24.79-92. McQuiston, A.R. and Tobe, S.S. (1991) J. Insect Physiol. 37, 223-229. Meller, V.H., Aucoin, R.R., Tobe, S.S. and Feyereisen, R. (1985) Mol. Cell. Endocrinol. 43, 155-163. Nishizuka, Y. (1984). Nature 308, 693-697. Nishizuka, Y. (1988) Nature 334,661465. Oishi, K., Raynor, R.L., Charp, P.A. and Kuo, J.F. (1988) J. Biol. Chem. 263,68654871. Paulson, C.R. and Stay, B. (1987) Mol. Cell, Endocrinol. 51, 243-252. Perez, F.M., Malamed, S. and Scanes, C.G. (1990) Gen. Comp. Endocrinol. 80, 181-188.
Endocrinology
105 (1994) 89-96
Pratt, G.E. and Tobe, S.S. (1974) Life Sci. 14, 575-586. Pratt, G.E., Farnsworth, D.E., Fok, K.F., Siegel, N.R., McCormack, A.L., Shabanowitz, J., Hunt, D.F. and Feyereisen, R. (1991) Proc. Nat]. Acad. Sci. USA 88, 2412-2416. Qiu. T.H., Combest. W.L. and Gilbert, L.I. (1990a) Insect Biochem. 20. 405-411. Qiu. T.H., Combest. W.L. and Gilbert. L.O. (1990b) Insect Biochem. 20.413420. Reagan, J.D., Miller, W.H. and Kramer, S.J. (1992) Arch. Insect Biothem. Physiol. 20, 145-155. Spacey, G.D., Bonser, R.W., Randall, R.W. and Garland, L.G. (1990) Cell. Signal. 2, 329-338. Stay, B. and Coop, A. (1973) J. Insect Physiol. 19, 147-171. Stay, B. and Woodhead, A.P. (1993) Am. Zool. 33.357-364. Stay, B., Joshi. S. and Woodhead, A.P. (1991) J. Insect Physiol. 37,6370. Stay, B., Tobe, S.S. and Bendena, W.G. (1994) Adv. Insect Physiol. 25, 267-338. Thastrup, 0.. Cullen, P. I., Drobak. B. K., Hanley, M. R. and Dawson, A.P. (1990) Proc. Natl. Acad. Sci. USA 87, 2466-2470. Tobe, S.S. (1990) In Progress in Comparative Endocrinology (Epple A., Scanes C.G. and Stetson M.H., eds.), pp. 174-179, Wiley Liss, New York. Tobe. S.S. and Pratt. G.E. (1974) Biochem. J. 144, 107-l 13. Tobe, S.S. and Clarke, N. (1985) Insect Biochem. 15, 175-179. Tobe, S.S., Ruegg, R.P., Stay, B.A., Baker, F.C., Miller, CA. and Schooley, D.A. (1985) Experientia 41, 1028-1034. Tobe, S.S., Yu, C.G. and Bendena, W.G. (1994) In Perspectives in Endocrinology (Davey K.G., Peter R.E., Tobe S.S., eds.), pp. 1219, National Research Council of Canada, Ottawa. Toullec, D., Pianetti, P., Coste, H., Belleverge. P., Grand-Perret, T., Ajakane, M., Baudet, V., Boissin, P., Boursier, E., Loriolle, F., Duhamel, L., Charon, D. and Kirilovsky. J. (1991) J. Biol. Chem. 266, 15771-15781. Woodhead, A.P., Stay, B., Seidel, S.L., Khan, M.A. and Tobe, S.S. (1989) Proc. Nat]. Acad. Sci. USA 86, 5997-6001. Woodhead, A.P., Khan, M.A., Stay, B. and Tobe, S.S. (1994) Insect Biochem. Mol. Biol. 24, 257-263.