Signal Transduction in Eclosion Hormone-induced
Secretion of Ecdysis-triggering Hormone*
Timothy G.
Kingan
§¶,
Richard A.
Cardullo
, and
Michael E.
Adams
§
From the Departments of
Cell Biology/Neuroscience,
§ Entomology, and
Biology,
University of California, Riverside, California 92521
Received for publication, March 19, 2001
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ABSTRACT |
Inka cells of insect epitracheal glands (EGs)
secrete preecdysis and ecdysis-triggering hormones (PETH and ETH) at
the end of each developmental stage. Both peptides act in the central nervous system to evoke the ecdysis behavioral sequence, a stereotype behavior during which old cuticle is shed. Secretion of ETH is stimulated by a brain neuropeptide, eclosion hormone (EH). EH evokes
accumulation of cGMP followed by release of ETH from Inka cells, and
exogenous cGMP evokes secretion of ETH. The secretory responses to EH
and cGMP are inhibited by the broad-spectrum kinase inhibitor
staurosporine, and the response to EH is potentiated by the phosphatase
inhibitor calyculin A. Staurosporine did not inhibit EH-evoked
accumulation of cGMP. Changes in cytoplasmic Ca2+ in
Inka cells during EH signaling were monitored via fluorescence ratioing
with fura-2-loaded EGs. Cytoplasmic Ca2+ increases within
30-120 s after addition of EH to EGs, and it remains elevated for at
least 10 min, corresponding with the time course of secretion.
Secretion is increased in dose-dependent manner by the
Ca2+-ATPase inhibitor thapsigargin, a treatment that does
not elevate glandular cGMP above basal levels. The secretory response
to EH is partially inhibited in glands loaded with EGTA, while cGMP levels are unaffected. These findings suggest that EH activates second
messenger cascades leading to cGMP accumulation and Ca2+
mobilization and/or influx and that both pathways are required for a
full secretory response. cGMP activates a staurosporine-inhibitable protein kinase. We propose that Ca2+ acts via a parallel
cascade with a time course that is similar to that for cGMP activation
of a cGMP-dependent protein kinase.
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INTRODUCTION |
Growth and differentiation of tissues during development of
insects is orchestrated by polyhydroxylated steroids, the ecdysones, and their interplay with the sesquiterpene juvenile hormones, acting
through nuclear receptors to direct transcription. An important outcome
of this interplay is the production of cuticular exoskeleton. After new
cuticle is produced at the end of each stadium, old cuticle must be
shed. This shedding of old cuticle, termed ecdysis, occurs in a
stereotype sequence of behaviors (1). These behaviors, most thoroughly
studied in the lepidopterous insect Manduca sexta, are
initiated and completed in 60-70 minutes by actions of the peptide hormones preecdysis and ecdysis-triggering hormones (PETH and
ETH)1 (2, 3) and eclosion
hormone (EH) (4). The findings from a number of laboratories suggest
that the ecdysis sequence is set in motion following peripheral release
of PETH and ETH (2, 3), and the behaviors are driven by multiple loci
in the CNS (5-7).
ETH is secreted by Inka cells of the segmentally distributed
epitracheal glands (3) in response to EH (8). EH is secreted hormonally
by peripheral neurohemal endings and centrally from axons, both of the
brain-centered "ventromedial" neurosecretory cells (9, 10),
probably in response to ETH (7, 11). Together these findings suggest a
model for endocrine events in ecdysis in which ETH and EH are secreted
in mutually positive feedback, leading to near depletion in stores of
both peptides (8, 11). Support for this model comes from several lines of evidence, including the appearance of PETH and ETH in hemolymph at
the onset of the behavior (2). It has not yet been possible to
adequately determine the timing of EH secretion, because of its very
low concentrations in hemolymph. Nevertheless, cGMP, a second messenger
in the action of EH (12, 13), accumulates in epitracheal glands during
the ecdysis sequence (8, 11). Since ETH is secreted during EH
signaling, elucidating events in the transduction cascade is critical
for understanding the endocrinology of ecdysis.
Cyclic GMP increases in epitracheal glands in response to EH, and if
added to culture medium, cGMP will evoke a secretory response (8);
however, the mechanism by which it causes secretion or if it is even
necessary for secretion is not known. In addition, findings to date
have not addressed a possible role for Ca2+ in ETH
secretion; Ca2+ is known to participate in secretory events
by neurons and neuroendocrine cells (14, 15). Here we report the
results of pharmacological studies with epitracheal glands showing that
cGMP is likely to act in secretion via activation of a protein kinase.
In addition, we show that Ca2+ levels increase in Inka
cells during EH signaling and that this increase is required for a full
secretory response.
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EXPERIMENTAL PROCEDURES |
Materials--
Staurosporine and cGMP were obtained from
Sigma; calyculin A and thapsigargin were from Biomol Research
Laboratories (Plymouth Meeting, PA). EGTA-AM was obtained from
Calbiochem. Inhibitors were dissolved in dry dimethyl sulfoxide
(Me2SO) and diluted into Weever's saline (8)
containing 0.3% BSA (Weever's/BSA) for use; final
Me2SO was 0.1% and did not affect basal or evoked
secretion. EGTA-AM was diluted in Weever's/BSA to 0.1 mM, and glands were loaded for 1 h before activation
with EH.
Animals and Dissections--
Tobacco hornworms (Manduca
sexta) were reared on artificial diet as described previously (8).
Epitracheal glands were removed, attached to a short segment of
trachea, from pharate pupae under Weever's saline, and cultured
individually in 40 µl of medium. For most experiments we used glands
from insects at "anterior shrink" stage (AS), ~3.5-4 h before
pupation (16). Enzyme inhibitors were loaded by placing glands in
medium with inhibitor for periods of 30-90 min, after which aliquots
for ETH assay were removed and EH for activation of glands was added.
Glands were cultured for an additional 20-30 min, and the medium was
removed for assay. In some experiments glands were first removed from
the medium and placed in ethanol:1 N HCl, 100:1, for
homogenization and quantification of cGMP; the remaining medium was set
aside for ETH determination.
Assays and Data Analysis--
ETH and cGMP were quantified by
enzyme immunoassay as described previously (8). Values for ETH
in the medium after stimulation were corrected for the generally low
level of ETH secreted during loading of inhibitor. Statistical
comparisons of data sets were done by the Mann-Whitney test of medians.
Ca2+ Imaging--
Calcium levels in individual Inka
cells were quantified using the calcium indicator dye fura-2 and a
video-enhanced microscope system. EGs were dissected from AS pharate
pupae. Some glands were selected in which the Inka cell was separated
on the trachea from the other gland cells (3, 17). Glands were loaded
for 30 min with 2-10 µM fura-2-AM (Molecular Probes,
Eugene, OR) in Weever's/BSA medium and then rinsed twice for 5 min in Weever's/BSA. Glands were mounted on
poly-L-lysine-coated "no. 0" coverslips in 90 µl of
Weever's/BSA, the trachea bearing the gland adhered to the
coverslip to provide an unobstructed view of the Inka cell.
A Nikon TE-300 inverted microscope equipped with Neofluar optics and a
xenon light source was used for fluorescence imaging. The microscope
was outfitted with a 10-position filter wheel (Sutter Instruments),
which contained both 340- and 380-nm bandpass filters for fura-2
excitation. The filter cube contained a dichroic mirror and a 510-nm
longpass filter. Ten microliters of medium containing EH was added and
mixed. An intensified charge couple device camera (Princeton
Instruments) was used to capture images every 3 or 6 s during
excitation at 340 and 380 nm. An image processor (MetaFluor; Universal
Imaging) averaged four frames at each wavelength to increase
signal-to-noise prior to storage of 340 and 380 images and the
340/380 ratio. Backgrounds were subtracted at each wavelength from cell-free fields, and the relative calcium concentration was
calculated from a calibration curve generated from known calcium standards (Molecular Probes).
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RESULTS |
We previously showed that cGMP accumulates in epitracheal glands
during ETH secretion in response to EH exposure and that exogenous cGMP
and its 8-bromo analogue activate secretion of ETH by glands in
vitro (8). To address the possibility that cGMP acts by regulating
the activity of a protein kinase and the phosphorylation state of a
protein in the transduction cascade, we tested the action of protein
kinase and phosphatase inhibitors in basal and stimulated secretion. We
also considered the possibility that protein kinases act upstream of
cGMP accumulation. In these experiments, individually cultured glands
were removed from medium and extracted for cGMP determination, and the
medium was collected for ETH determination.
Staurosporine, an alkaloid from Streptomyces sp., is a
potent inhibitor of protein kinase C (18), as well as most other kinases (19). We tested the ability of staurosporine to inhibit EH-evoked secretion of ETH by EGs in vitro. While 0.1 µM inhibitor was without effect, inhibition developed at
higher concentrations; the EC50 was 0.2-0.4
µM. (Fig. 1). Staurosporine
did not have a measurable effect on the low basal rate of secretion
(data not shown).

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Fig. 1.
Effect of staurosporine on EH-evoked release
of ETH from EGs of pharate pupae. Glands from "AS" pharate
pupae (see "Experimental Procedures") were incubated for 30 min in
the indicated concentrations of staurosporine and then activated for an
additional 30 min with 100 pM EH. Secretion by unstimulated
glands was 0.200 pmol per gland. Values are average ± S.D.;
n = 8 for each dose.
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Because EH evokes an increase in glandular cGMP, and exogenous cGMP
evokes a small secretory response (8), we wanted to know if the
staurosporine finding reflects inhibition of a
cGMP-dependent protein kinase (PKG) required for secretion.
As an indirect measure, we tested the action of staurosporine in
secretion evoked by exogenous cGMP. While the response was variable
(Table I) it appears that staurosporine
reduces secretion evoked by cGMP and 8-Br-cGMP, suggesting that
elevation of endogenous cGMP during EH stimulation would lead to
secretion in a staurosporine-inhibitable manner. Together, these
considerations suggest that EH stimulation leads to activation of a PKG
in the transduction cascade, and that this activation plays an
important role in secretion.
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Table I
Inhibition of cyclic nucleotide-evoked release by staurosporine
Glands were preincubated in staurosporine for 60 min; an aliquot of
medium was then removed for ETH assay and nucleotide was added for an
additional 30 min. Values shown, corrected for release prior to
addition of nucleotide, are in fmol/EG ± S.D.
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If phosphorylation of substrate proteins is required in EH signaling,
the activity of a protein phosphatase may limit the secretory response.
Accordingly, we tested the action of calyculin A, a sponge toxin that
inhibits phosphatases (20) with selectivity for types 1 and 2A (21).
Calyculin A increases both basal secretion and potentiates, by
~5-fold, secretion resulting from threshold doses of EH (Fig.
2). The type II pyrethroid cypermethrin,
a protein phosphatase type 2B (calcineurin) inhibitor (22), was without effect in evoked secretion (data not shown). Thus, it is likely that
basal and EH-stimulated secretion is quantitatively determined, at
least in part, by a balance between the activities of kinases and a
protein phosphatase similar to the mammalian type 1 or 2A enzyme.

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Fig. 2.
Effect of calyculin A on basal and EH-evoked
secretion of ETH. Glands were incubated with or without calyculin
A for 30 min and then for an additional 30 min in the presence of 30 pM EH at which time medium from control and stimulated
glands was collected. Values shown are average ± S.D.; the number
of determinations is shown in parentheses. *,
p < 0.001; **, p < 0.001.
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The suggestion above that staurosporine acts on a PKG does not rule out
an action on other kinases. To test the possibility that such a kinase
could regulate cGMP production, we measured EH-evoked cGMP accumulation
in the presence of staurosporine. As already shown (Fig. 1),
staurosporine inhibits evoked secretion (Fig.
3A); however, cGMP
accumulation was not reduced (Fig. 3B). This suggests that
staurosporine does not inhibit kinases that stimulate synthesis of
cGMP.

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Fig. 3.
Action of staurosporine in EH-evoked
secretion of ETH and accumulation of cGMP in epitracheal glands.
Glands from pharate pupae were preincubated for 1 h with 2 µM staurosporine and then stimulated with 0.01 or 0.03 nM EH. At the end of the second period glands were removed
for extraction and cGMP determination, and medium was then removed for
ETH determination. Values shown are average ± S.D. 10-12
determinations. A, ETH secretion; *, p = 0.0014; **, p = 0.015. B, cGMP accumulation; *,
p = 0.03.
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Exocytotic secretion in neuroendocrine cells and neurons is triggered
by elevation of cytoplasmic Ca2+ (14, 23). To begin testing
the possibility that Ca2+ also participates in secretion
from Inka cells, we investigated the effect of EH on cytoplasmic
Ca2+. Fura-2-loaded glands were monitored for fluorescence
with alternate 340 nm and 380 nm excitation. When 340/380 ratios
were monitored for 10 min without application of EH, no change was
observed (data not shown). In 10 preparations containing a total of 17 Inka cells, 12 cells responded to EH (Fig.
4). Upon application of EH ratios began
to increase within 15-195 s, with higher concentrations leading to
shorter onsets to measurable increases (Fig. 4, C and F). A plateau in the 340/380 ratio was reached within
~6 min, and it remained elevated for the duration of 10-min
experiments. With × 20 magnification the ratio appeared to
increase uniformly across the surface of the cell (Fig. 4, A
and B). At × 40 magnification, however, it was
apparent that the highest ratios were attained at the cell margin,
while lower ratios were found in underlying cortical regions (Fig. 4,
D and E). In this preparation, a slight reduction
of the ratio occurred over the trachea adjacent to the Inka cell (Fig.
4, E and F). Over the Inka cell the ratio
increased 60-175% above that found prior to application of EH.
Increases of this magnitude, when compared with the calibration set,
are found to correspond to ~90-350% increases in Ca2+.
Ratios obtained for Ca2+ solutions from a calibration set
are shown in Table II.
Determinations of absolute values of Ca2+ in Inka cells
from this set were not made because of an uncertain basis for comparing
the environment for fluorescence in the large (200 µm,
diameter) Inka cell with that in the saline of the calibration set. Nevertheless, it appeared that Ca2+ rose maximally to
~1 µM in the response to EH.

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Fig. 4.
Mobilization of Ca2+ in Inka
cells from pharate pupae during stimulation by EH. False
color images were produced in MetaFluor from 340/380 ratios
(see "Experimental Procedures"); purple represents the
lowest ratios, increasing to blue, green,
yellow, and red. A and B
represent the beginning and ending images from a 10-min exposure to 1 nM EH in which 340/380 rises from 0.4 to ~0.7.
C, the time course of 340/380, collected with a × 20 objective and recorded every 3 s, is shown for the margin of
the Inka cell, circled 1, as well as a control region,
circled 2, over the trachea. D and E
are the beginning and ending images from a 10-min exposure to 2 nM EH in which images were collected with a × 40 objective every 6 s. F, the time course of
340/380 is shown for a region at the margin of the Inka cell,
circled 1, and over the trachea, circled 2.
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Table II
340/380 values for Ca2+ standards
Standards containing the indicated amounts of Ca2+ were
prepared on microscope slides and coverslipped. Images were collected
at 340 and 380 nm excitation (see "Experimental Procedures") and
ratios calculated.
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We showed earlier that removal of extracellular Ca2+ does
not diminish EH-evoked secretion (8). To address the possibility that
Ca2+ mobilized from intracellular stores drives secretory
events in the Inka cell, we tested the effect of thapsigargin, an
inhibitor of ATP-dependent Ca2+ pumps found in
cardiac sarcoplasmic and endoplasmic reticulum (24). While 0.01 µM thapsigargin was without effect, ETH release occurred
at 0.1 and 1.0 µM inhibitor (Fig.
5A). At 1.0 µM
thapsigargin release was already evident at 30 min, and further release
apparently did not occur; 2.1 pmol/gland released represents ~15% of
the total ETH. In separate experiments we also tested the ability of
thapsigargin to affect accumulation of cGMP during evoked secretion. If
Ca2+ is required as a direct or indirect activator upstream
of guanylyl cyclase, an accumulation of cGMP might parallel
thapsigargin-evoked secretion of ETH. However, thapsigargin did not
significantly alter the amount of cGMP at the end of a 30-min
incubation (Fig. 5B). This finding does not rule out a role
for a PKC in regulating guanylyl cyclase, however, since production of
neutral lipid (diacylglycerol, a second messenger in PKC activation)
would not be expected in thapsigargin treatment. These considerations
aside, our findings suggest that Ca2+ mobilization can
evoke ETH release without participation of cGMP.

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Fig. 5.
Effect of thapsigargin on ETH release and
cGMP accumulation. A, glands were incubated for 90 min
in the presence of 0.01-1.0 µM thapsigargin, and the
medium was sampled at 30, 60, and 90 min. Values are the
average ± S.D. of six or seven determinations. Values at 0.1 µM are offset 2 min in the x axis for clarity.
*, p = 0.09; **, p = 0.005. B, in an experiment separate from that shown in
A, glands were extracted for cGMP determination following
incubation in thapsigargin. Values shown are average ± S.D.;
control, n = 11; thapsigargin, n = 13;
*, p < 0.001; **, p = 0.08.
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To test the possibility that mobilization of Ca2+ is
required in secretion, we preloaded glands with the membrane-permeable ester of EGTA (see "Experimental Procedures"). In the presence of 3 mM Ca2+ in the extracellular medium, a small
inhibition of release was observed in EGTA-loaded glands (Fig.
6). When glands were incubated in 0 Ca2+ medium, EGTA again reduced evoked secretion; the small
additional effectiveness of EGTA in the absence of Ca2+ may
not be significant. Therefore, while Ca2+ influx may occur
and participate in secretion, it seems not to be required for a full
response (Fig. 6; see also Ref. 8). To further address the possibility
of cross-talk between Ca2+ and cGMP pathways, we tested the
effect of EGTA loading on EH-evoked cGMP accumulation. EH (100 pM) evoked accumulation of 87 ± 74 fmol cGMP (S.D.,
n = 11); when glands were preincubated for 50 min in
0.01 or 0.1 mM EGTA-AM and then rinsed before EH
activation, they accumulated 105 ± 70 (n = 11)
and 75.1 ± 64 (n = 12) fmol cGMP, respectively;
these values are not significantly different from those determined in
the absence of EGTA. Therefore, the data suggest that cGMP accumulation
is unaffected by EGTA, while ETH secretion is reduced ~36% (Fig.
6).

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Fig. 6.
Effect of EGTA on EH-evoked release.
Glands were loaded with 0.1 mM EGTA-AM for 60-90 min in
Weever's saline with or without 3 mM
Ca2+ and then activated with 10 pM EH for an
additional 21-25 min. Values shown are average ± S.D.; the
number of determinations is shown in parentheses. *,
p = 0.11; **, p = 0.04.
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A summary of our findings and a model for signal transduction in Inka
cells is shown in Fig. 7. A cell surface
receptor is coupled to a guanylyl cyclase such as that described from
M. sexta CNS (25). The cyclase produces cGMP in
response to EH. A phosphodiesterase limits secretion in unstimulated
cells (8), presumably by its action on cGMP that would otherwise
accumulate. A PKG, inhibitable with staurosporine, promotes
phosphorylation of a substrate protein, while a phosphatase, sensitive
to calyculin A, limits basal and evoked secretion, presumably by
reversing the action of the PKG. Either in parallel or in sequence with
cGMP production, cytoplasmic Ca2+ increases with a time
course that is similar to that of cGMP accumulation (8). The
Ca2+ increase during secretion plateaus at low micromolar
concentrations, suggesting high affinity effectors. This increase may
occur either 1) in parallel with activation of guanylyl cyclase, for
instance, via separate activation of receptors in intracellular stores
leading to mobilization of Ca2+, or 2) in sequence with
guanylyl cyclase, by a PKG-mediated activation of a substrate protein,
which then participates in Ca2+ mobilization. While we do
not yet know if cGMP can affect Ca2+ mobilization, findings
from our EGTA and thapsigargin experiments suggest that
Ca2+ does not act in a pathway leading to cGMP
accumulation. Proteins that function as effectors of second messenger
cGMP and Ca2+ have not been identified, but could include
effectors of translocation of granules to a readily releasable pool as
well as mediators of docking and/or fusion immediately prior to
exocytosis.

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Fig. 7.
Summary of pharmacological findings and model
for activation of Inka cells in ETH secretion. EH stimulates a
receptor, which is followed by activation of a guanylyl cyclase
(GC) and mobilization of Ca2+ from intracellular
stores. cGMP accumulates and activates a staurosporine
(staur)-inhibitable protein kinase (PKG), which
phosphorylates an as yet unidentified substrate protein
(SP); the phosphorylation state of substrate protein is
partially regulated by the action of a calyculin A-inhibitable protein
phosphatase (PP). Ca2+ is mobilized, probably
from thapsigargin (thaps)-sensitive stores, concurrent with
cGMP accumulation. Elevation of intracellular Ca2+ levels
may also receive a contribution from influx. Receptor proteins for
Ca2+ have not been identified. cGMP and Ca2+
pathways may independently promote secretion.
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DISCUSSION |
We have shown that release of ETH from Inka cells of insect
epitracheal glands requires participation of a protein kinase that
likely is activated by cGMP during EH signaling. The extent of
secretion during stimulation is limited by a calyculin A-inhibitable protein phosphatase. In addition, we find that cytoplasmic
Ca2+ increases during signaling and that its time course is
similar to that of secretion. Thapsigargin, an effective inhibitor of Ca2+-dependent ATPase in endoplasmic reticulum,
increases basal secretion. Intracellular EGTA, a Ca2+
chelator, blunts stimulated secretion, showing that Ca2+
mobilization participates in secretion.
A Role for Protein Kinases in EH-evoked
Secretion--
Staurosporine is effective in inhibiting EH-evoked
secretion (IC50 = 0.2-0.4 µM), indicating a
requirement for a protein kinase in ETH release. Moreover, secretion
evoked by exogenous cGMP and 8-Br-cGMP is also inhibited by
staurosporine. While the percent inhibition is lower than for EH-evoked
secretion (compare Fig. 1, Table I), some of the difference may be
accounted for by basal and staurosporine-insensitive secretion in both
sets of glands. The current findings with staurosporine, together with
our earlier demonstration that cGMP accumulates during EH signaling
(8), suggests that a PKG would be activated in EH signaling and
participate in the transduction cascade leading to ETH secretion. In an
additional test of a role for PKG, we assayed the PKG inhibitors
(Rp)-8-(para-chlorophenylthio)-guanosine-3',5'-cyclic monophosphorothioate and
(Rp)-8-Br-guanosine-3',5'-cyclic
monophosphorothioate (26, 27) for activity in inhibiting
EH-evoked secretion. However, neither was effective in inhibiting
evoked secretion (data not shown). This apparently negative result must
be viewed in the context of our observation that 8-Br-cGMP is only
weakly active in evoking secretion (Table I), suggesting that insect
and mammalian kinases differ in their pharmacological sensitivities or
that these exogenous analogues are excluded from access to the enzyme compartment. Additional evidence indicating a role for protein kinases
in secretion is the finding that the protein phosphatase inhibitor
calyculin A potentiates basal and EH-evoked secretion. Inhibition of
phosphatases presumably unmasks or synergizes with a basal level of
protein kinase activity, allowing accumulation of a phosphoprotein that
is a component in the transduction cascade. We found earlier that the
phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine potentiates basal secretion (8), presumably by unmasking activity of a
guanylyl cyclase. Together these findings indicate that a guanylyl
cyclase and one or more protein kinases are active in unstimulated
cells, but that secretion is held in check by hydrolytic enzymes. In
EH-stimulated cells the kinase is then able to outpace the phosphatase.
This conclusion does not rule out, however, the possibility that the
phosphodiesterase or phosphatase is negatively regulated to affect
secretion in stimulated cells. That phosphatases may be important
regulators of secretion was shown in studies with adrenal chromaffin
cells, in which calyculin A treatment lead to increased
phosphorylation of vimentin, redistribution of granules toward the cell
periphery, and enhanced basal secretion (28). However, the kinase in
these events is likely to be a PKC, rather than a PKG (29).
Characterized PKGs have low Ka values, 0.05-1.0
µM (30). cGMP is low in unstimulated Inka cells, 1-3
fmol in 200-µm diameter cells from pharate pupae (8). If contained in
the outer 20% of cell volume as suggested by immunocytochemical
findings (11), the basal concentration of cGMP would be ~0.5
µM, well within the range for activation of identified
PKGs. Activated cells contain 10-50 times greater cGMP.
A role for PKGs in EH signaling in the CNS of M. sexta was
suggested earlier by the observation that two 54-kDa proteins are phosphorylated in response to EH and cGMP (31). The identity of these
proteins and their role in signaling, or whether they might also be
present in epitracheal glands, has not been determined. Identified PKGs
in insects include DG2, which has been implicated in regulating
foraging behavior in Drosophila melanogaster (32), although
substrate proteins for DG2 or the tissue expressing enzyme required for
the phenotype have not been identified. In addition, insect PKGs have
not been characterized pharmacologically.
Do Kinases Act Up- or Downstream of Guanylyl Cyclase?--
In
addition to determining the effects of kinase inhibitors on ETH
secretion, we also quantified their effects on cGMP accumulation. We
found that staurosporine does not affect EH-evoked cGMP accumulation, indicating that its action in ETH release is downstream of guanylyl cyclase. Tamoxifen has also been shown to inhibit EH-evoked cGMP accumulation in M. sexta transverse nerves, a finding that
suggested the action of a PKC in regulating EH-activated guanylyl
cyclase (33). However, the specificity of these inhibitors in insect preparations has not been addressed.
A Role for Ca2+ in EH Signaling--
EH evoked an
increase in Ca2+ in the Inka cell; the time to onset was
concentration-dependent. With 5 nM EH the time
to onset was ~20 s, while with 0.1 nM onset occurred at
~200 s. The Ca2+ response occurred as a sustained
plateau, rising to at most 1-2 µM in concentration. The
relatively slow onsets in Inka cells are similar to those observed in a
variety of nonexcitable cells in which two receptor classes, either
seven-transmembrane domain-containing receptors or receptor tyrosine
kinases, are coupled through G-proteins to phospholipase C (34). In
Inka cells, the elevation in Ca2+ appeared to sweep
uniformly across the surface of the cell during the rising phase.
However, use of a × 40 objective clearly revealed higher
concentrations of Ca2+ in the cortex of the cell,
suggesting either influx or local "hot" areas of mobilization from
subplasmalemmal intracellular stores (Fig. 5). We interpret this result
with caution, however, because of uncertainty in focal plane thickness
and contribution from curvature of the large diameter (200 µm) cell.
It will be important to record the response in smaller cells from
earlier developmental stages and to use optical sectioning of confocal microscopy to reveal regional details of mobilization. Nevertheless, in
other preparations mobilization from intracellular stores activates Ca2+ influx by "capacitative calcium entry" (35), and
our observation of higher 340/380 in cortical regions of Inka
cells is consistent with the prediction of influx.
We addressed the significance of Ca2+ elevation in two
ways: first, we found that thapsigargin treatment leads to secretion of
ETH. Thapsigargin treatment leads to elevation of cytoplasmic levels of
Ca2+ (36, 37) and activation of effector proteins via its
inhibition of ATP-dependent Ca2+ pumps with net
release from intracellular stores (38). Second, we showed that EGTA
blunts, but does not abolish, EH-evoked secretion. This finding
indicates that a portion of secretion is Ca2+-independent
(and presumably cGMP-dependent). However, a conclusion on
the role of Ca2+ will first require quantifying the
effectiveness of EGTA in blunting Ca2+ mobilization during
secretion as well as quantifying secretion by and
Ca2+ mobilization in individual glands. Nevertheless, our
findings indicate that Ca2+ mobilization is both sufficient
for partial secretion of available stores and necessary for full
secretion in response to EH.
The range of times to onset of Ca2+ mobilization was
similar to onset in cGMP increase determined earlier (8), although the temporal resolution in cGMP measurements was relatively crude in
comparison with the Ca2+ measurements reported here. These
observations lead us to consider the possibility that one second
messenger could affect the accumulation of the other. While we have not
yet addressed this directly, the lack of effect of thapsigargin and
EGTA on cGMP levels suggests that Ca2+ does not directly
regulate cGMP accumulation. In this regard, Inka cells differ from
other preparations, e.g. rat pancreatic acini, in which
thapsigargin does evoke an increase in cGMP (37). In pancreatic acini,
however, Ca2+ activates nitric-oxide synthase, which then
leads to cGMP production (37), a pathway that does not function in Inka
cells (8). These considerations also raise the converse question that
has not yet been addressed: does cGMP play a role in Ca2+
mobilization or influx in Inka cells?
The importance of elevated Ca2+ in regulating exocytotic
secretion by aminergic and peptidergic endocrine cells has long been known (14, 39). A high degree of cooperativity for the action of
Ca2+ in secretion indicates activity at multiple steps,
from early events associated with translocation of vesicles to a
readily releasable pool (40) to late events following docking and
associated with fusion (41). In early events the slower kinetics of
rearrangements in cytoskeleton and/or translocation of vesicles to
release sites allow for the involvement of both Ca2+ and
other second messengers such as cyclic nucleotides (39, 42). Late
events are mediated by "sensor" proteins found in vesicle and
plasma membranes, the activation of which by relatively high
[Ca2+] in neurons or lower [Ca2+] in
endocrine or neuroendocrine cells leads to fast fusion of an
immediately releasable pool of vesicles.
ETH secretion begins at the onset of preecdysis behavior and increases
by positive feedback from EH to maximal activation, driving the
behavioral sequence to its conclusion (3, 8, 11). Receptor activation
leads to accumulation of cGMP (8, 11) and elevation of Ca2+
(this report). The signaling events leading to Ca2+
elevation have not been identified, but could follow production of
inositol 1,4,5-trisphosphate, shown to accumulate in CNS tissue in
response to EH (43, 44). Moreover, since cGMP production in CNS is
decreased by phospholipase C inhibitors (44), EH receptor activation
may lead to cGMP and Ca2+ mobilization through a common
PLC, which, once activated, leads to accumulation of each second
messenger independent of the other. Earlier work showed that EH is
likely to activate a PKG in the CNS (31). Our findings with
staurosporine suggest that EH acts similarly in Inka cells. If so,
identifying the kinases and their substrates, the Ca2+
effector proteins, and the functions of these proteins in the transduction cascade represents an exciting challenge for the immediate future.
 |
FOOTNOTES |
*
This work was supported by United States
Department of Agriculture Grant CSREES 9802582, National Institutes of
Health Grant AI 40555, and National Science Foundation Grant
IBN-9514678.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Entomology, University of California, 5419 Boyce Hall, Riverside, CA 92521. Tel.: 909-787-4369; Fax: 909-787-3087; E-mail:
tkingan@citrus.ucr.edu.
Published, JBC Papers in Press, April 19, 2001, DOI 10.1074/jbc.M102421200
 |
ABBREVIATIONS |
The abbreviations used are:
PETH, preecdysis-triggering hormone;
ETH, ecdysis-triggering hormone;
EH, eclosion hormone;
CNS, central nervous system;
BSA, bovine serum
albumin;
AS, anterior shrink stage;
PKG, cGMP-dependent
protein kinase;
PKC, protein kinase C;
EG, epitracheal gland.
 |
REFERENCES |
1.
|
Weeks, J. C.,
and Truman, J. W.
(1984)
J. Comp. Physiol. A
155,
407-422
|
2.
|
Zitnan, D.,
Ross, L. S.,
Zitnanova, I.,
Hermesman, J. L.,
Gill, S. S.,
and Adams, M. E.
(1999)
Neuron
23,
523-535[Medline]
[Order article via Infotrieve]
|
3.
|
Zitnan, D.,
Kingan, T. G.,
Hermesman, J. L.,
and Adams, M. E.
(1996)
Science
271,
88-91[Abstract]
|
4.
|
Truman, J. W.,
Taghert, P. H.,
Copenhaver, P. F.,
Tublitz, N. J.,
and Schwartz, L. M.
(1981)
Nature
291,
70-71
|
5.
|
Zitnan, D.,
and Adams, M. E.
(2000)
J. Exp. Biol.
203,
1329-1340[Abstract/Free Full Text]
|
6.
|
Novicki, A.,
and Weeks, J. C.
(1996)
J. Exp. Biol.
199,
1757-1769[Abstract/Free Full Text]
|
7.
|
Gammie, S. C.,
and Truman, J. W.
(1999)
J. Exp. Biol.
202,
343-352[Abstract/Free Full Text]
|
8.
|
Kingan, T. G.,
Gray, W.,
Zitnan, D.,
and Adams, M. E.
(1997)
J. Exp. Biol.
200,
3245-3256[Abstract/Free Full Text]
|
9.
|
Hewes, R. S.,
and Truman, J. W.
(1991)
J. Comp. Physiol.
168,
697-707[Medline]
[Order article via Infotrieve]
|
10.
|
Truman, J. W.,
and Copenhaver, P. F.
(1989)
J. Exp. Biol.
147,
457-470
|
11.
|
Ewer, J.,
Gammie, S. C.,
and Truman, J. W.
(1997)
J. Exp. Biol.
200,
869-881[Abstract/Free Full Text]
|
12.
|
Morton, D. B.,
and Truman, J. W.
(1985)
J. Comp. Physiol. A
157,
423-432[Medline]
[Order article via Infotrieve]
|
13.
|
Truman, J. W.,
Mumby, S. M.,
and Welch, S. K.
(1979)
J. Exp. Biol.
84,
201-212[Abstract]
|
14.
|
Douglas, W. W.
(1968)
Br. J. Pharmacol.
34,
451-474[Medline]
[Order article via Infotrieve]
|
15.
|
Knight, D. E.,
von Grafenstein, H.,
and Athayde, C. M.
(1989)
Trends Neurosci.
12,
451-458[CrossRef][Medline]
[Order article via Infotrieve]
|
16.
|
Truman, J. W.,
Taghert, P. H.,
and Reynolds, S. E.
(1980)
J. Exp. Biol.
88,
327-337
|
17.
|
Klein, C.,
Kallenborn, H. G.,
and Radlicki, C.
(1999)
J. Insect Physiol.
45,
65-73[CrossRef][Medline]
[Order article via Infotrieve]
|
18.
|
Tamaoki, T.,
Nomoto, H.,
Takahashi, I.,
Kato, Y.,
Morimoto, M.,
and Tomita, F.
(1986)
Biochem. Biophys. Res. Commun.
135,
397-402[Medline]
[Order article via Infotrieve]
|
19.
|
Rüegg, U. T.,
and Burgess, G. M.
(1989)
Trends Pharmacol. Sci.
10,
218-220[CrossRef][Medline]
[Order article via Infotrieve]
|
20.
|
Kato, Y.,
Fusetani, N.,
Watabe, S.,
Hashimoto, K.,
Uemura, D.,
and Hartshorne, D. J.
(1986)
J. Am. Chem. Soc.
108,
2780-2781
|
21.
|
Ishihara, H.,
Martin, B. L.,
Brautigan, D. L.,
Karaki, H.,
Ozaki, H.,
Kato, Y.,
Fusetani, N.,
Watabe, S.,
Hashimoto, K.,
Uemura, D.,
and Hartshorne, D. J.
(1989)
Biochem. Biophys. Res. Commun.
159,
871-877[Medline]
[Order article via Infotrieve]
|
22.
|
Enan, E.,
and Matsumura, F.
(1992)
Biochem. Pharmacol.
43,
1777-1784[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Morgan, A.,
and Burgoyne, R. D.
(1997)
Cell Dev. Biol.
8,
141-149[CrossRef]
|
24.
|
Thastrup, O.,
Cullen, P. J.,
Drøbak, B.,
Hanley, M. R.,
and Dawson, A. P.
(1990)
Proc. Natl. Acad. Sci. U. S. A.
87,
2466-2470[Abstract]
|
25.
|
Nighorn, A.,
Byrnes, K. A.,
and Morton, D. B.
(1999)
J. Biol. Chem.
274,
2525-2531[Abstract/Free Full Text]
|
26.
|
Butt, E.,
Eigenthaler, M.,
and Genieser, H.-G.
(1994)
Eur. J. Pharmacol.
269,
265-268[CrossRef][Medline]
[Order article via Infotrieve]
|
27.
|
Nakazawa, M.,
and Imai, S.
(1994)
Eur. J. Pharmacol.
253,
179-181[CrossRef][Medline]
[Order article via Infotrieve]
|
28.
|
Gutierrez, L. M.,
Quitanar, J. L.,
Rueda, J.,
Viniergra, S.,
and Reig, J. A.
(1995)
Eur. J. Cell Biol.
68,
88-95[Medline]
[Order article via Infotrieve]
|
29.
|
Vitale, M. L.,
Rodríguez Del Castillo, A.,
and Trifaró, J.-M.
(1992)
Neuroscience
51,
463-474[CrossRef][Medline]
[Order article via Infotrieve]
|
30.
|
Lincoln, T. M.,
and Cornwell, T. L.
(1993)
FASEB J.
7,
328-338[Abstract/Free Full Text]
|
31.
|
Morton, D. B.,
and Truman, J. W.
(1986)
Nature
323,
264-267[Medline]
[Order article via Infotrieve]
|
32.
|
Osborne, K. A.,
Robichon, A.,
Burgess, E.,
Butland, S.,
Shaw, R. A.,
Coulthard, A.,
Pereira, H. S.,
Greenspan, R. J.,
and Sokolowski, M. B.
(1997)
Science
277,
834-836[Abstract/Free Full Text]
|
33.
|
Morton, D. B.
(1997)
Ann. N. Y. Acad. Sci.
814,
40-52[Medline]
[Order article via Infotrieve]
|
34.
|
Clapham, D. E.
(1995)
Cell
80,
259-268[Medline]
[Order article via Infotrieve]
|
35.
|
Putney, J. W., Jr.,
Bird, G.,
and St, J.
(1993)
Cell
75,
199-201[Medline]
[Order article via Infotrieve]
|
36.
|
Heemskerk, J. W. M.,
Feijge, M. A. H.,
Sage, S. O.,
and Walter, U.
(1994)
Eur. J. Biochem.
223,
543-551[Abstract]
|
37.
|
Xu, X.,
Star, R. A.,
Tortorici, G.,
and Muallem, S.
(1994)
J. Biol. Chem.
269,
12645-12653[Abstract/Free Full Text]
|
38.
|
Rutter, G. A.,
Theler, J.-M.,
and Wollheim, C. B.
(1994)
Cell Calcium
16,
71-80[Medline]
[Order article via Infotrieve]
|
39.
|
Zucker, R. S.
(1996)
Neuron
17,
1049-1955[Medline]
[Order article via Infotrieve]
|
40.
|
von Rüden, L.,
and Neher, E.
(1993)
Science
262,
1061-1065[Medline]
[Order article via Infotrieve]
|
41.
|
Peters, C.,
and Mayer, A.
(1998)
Nature
396,
575-579[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
Trifaró, J.-M.,
Vitale, M. L.,
and Rodríguez Del Castillo, A.
(1992)
Eur. J. Pharmacol.
225,
83-104[Medline]
[Order article via Infotrieve]
|
43.
|
Shibanaka, Y.,
Hayashi, H.,
Takai, M.,
and Fujita, N.
(1993)
Eur. J. Biochem.
211,
427-430[Abstract]
|
44.
|
Morton, D. B.,
and Simpson, P. J.
(1995)
J. Comp. Physiol. B
165,
417-427[Medline]
[Order article via Infotrieve]
|
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