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Different Ca2+ signalling cascades manifested by mastoparan in the prothoracic glands of the tobacco hornworm Manduca sexta and the silkworm Bombyx mori.

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52
Dedos et al.
Archives of Insect Biochemistry and Physiology 65:52–64 (2007)
Different Ca2+ Signalling Cascades Manifested
by Mastoparan in the Prothoracic Glands of the
Tobacco Hornworm, Manduca sexta, and the
Silkworm, Bombyx mori
Skarlatos G. Dedos,* Dieter Wicher, Sabine Kaltofen, and Heiner Birkenbeil
Application of the tetradecapeptide mastoparan to the prothoracic glands (PGs) of the tobacco hornworm, Manduca sexta, and
the silkworm, Bombyx mori, resulted in increases in intracellular Ca2+ ([Ca2+]i). In M. sexta, Gi proteins are involved in the
mastoparan-stimulated increase in [Ca2+]i. However, there is no involvement of Gi proteins in the mastoparan-stimulated
increase in [Ca2+]i in prothoracic gland cells from B. mori. Unlike in M. sexta prothoracic glands, in B. mori prothoracic
glands mastoparan increases [Ca2+]i even in the absence of extracellular Ca2+. Pharmacological manipulation of the Ca2+
signalling cascades in the prothoracic glands of both insect species suggests that in M. sexta prothoracic glands, mastoparan’s
first site of action is influx of Ca2+ through plasma membrane Ca2+ channels while in B. mori prothoracic glands, mastoparan’s
first site of action is mobilization of Ca2+ from intracellular stores. In M. sexta, the combined results indicate the presence of
mastoparan-sensitive plasma membrane Ca2+ channels, distinct from those activated by prothoracicotropic hormone or the IP3
signalling cascade, that coordinate spatial increases in [Ca2+]i in prothoracic gland cells. We propose that in B. mori, mastoparan
stimulates Ca2+ mobilization from ryanodine-sensitive intracellular Ca2+ stores in prothoracic gland cells. Arch. Insect Biochem.
Physiol. 65:52–64, 2007. © 2007 Wiley-Liss, Inc.
KEYWORDS: Manduca sexta; Bombyx mori; prothoracic glands; mastoparan; Ca2+ channels; ryanodine
INTRODUCTION
Changes in intracellular Ca2+ ([Ca2+]i) in prothoracic gland cells are regulated by the prothoracicotropic hormone, a brain neurosecretory protein that
stimulates the prothoracic glands to secrete ecdysteroid by stimulating Ca2+ influx (Fellner et al.,
2005; Priester and Smith, 2005). This protein acts
through a transduction cascade requiring extracellular Ca2+ (Smith, 1995) and results in activation of
a Ca 2+/calmodulin dependent adenylyl cyclase
(Meller et al., 1988; Chen et al., 2001) and increased
cAMP levels (Smith, 1995). Although the regulatory
mechanisms responsible for increases in [Ca2+]i in
prothoracic gland cells are not well understood,
pharmacological manipulation of [Ca2+]i can help
identify and distinguish between the various mechanisms underlying the regulation of [Ca2+]i in these
cells (Fellner et al., 2005; Priester and Smith, 2005;
Dedos et al., 2005). One pharmacological agent is
mastoparan, which produces increases in [Ca2+]i in
prothoracic gland cells of M. sexta (Birkenbeil, 2000).
Saxon Academy of Sciences at Leipzig, Department of Neurohormones, Jena, Germany
*Correspondence to: Skarlatos G. Dedos, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
E-mail: sd323@cam.ac.uk
© 2007 Wiley-Liss, Inc.
DOI: 10.1002/arch.20180
Published online in Wiley InterScience (www.interscience.wiley.com)
Archives of Insect Biochemistry and Physiology
June 2007
doi: 10.1002/arch.
Ca2+ Mobilization by Mastoparan in PGs
Mastoparan, a 14-residue peptide toxin present
in the venom of hymenopteroid wasps, directly activates G proteins (Higashijima et al., 1988).
Mastoparan directly triggers Gi and Go activation
and induces production of inositol 1,4,5-trisphosphate (IP3) by activating phospholipase C (PLC)
in a way similar to receptor activation (Legendre
et al., 1993; Berridge et al., 2000). Therefore, the
[Ca2+]i rise induced by mastoparan depends on
PLC-catalyzed IP3 production and Ca2+ mobilization, a pathway regulated by G proteins in many
cases. Most of the effects of mastoparan have been
linked to the activation of Go and Gi proteins (Higashijima et al., 1988; Weingarten et al., 1990; Tomita
et al., 1991), ATP- and GTP-degrading nucleotidases
(Denker et al., 1991) and small GTP-binding proteins (Koch et al., 1991). However, a small number of studies have revealed an association of
mastoparan with glycogen phosphorylase, ryanodine receptors and glycogenolysis (Longland et al.,
1999; Hirata et al., 2000, 2003).
The overarching aim of our study was to identify and compare [Ca2+]i increase mechanisms in the
prothoracic gland cells of M. sexta and B. mori. Our
approach was to use mastoparan as a pharmacological probe to identify if the observed effects in
M. sexta prothoracic glands (Birkenbeil, 2000) can
also be extended to B. mori prothoracic glands. This
will give us a better understanding of the links between Ca2+ signalling, the activation of the different
signalling pathways in prothoracic glands (Smith,
1995), and the role that Ca2+ plays in mediating
the overall function of prothoracic glands.
MATERIALS AND METHODS
Insects
Tobacco hornworms, Manduca sexta, were reared
on an artificial diet (Hoffman et al., 1966) at 25°C
and >60% relative humidity under a 16:8 L:D photoperiod and staged by the last larval moult and
by the beginning of wandering behaviour on day
5 of the last larval instar. Experimental animals
were used in the last instar,1 day after wandering.
Silkworms, Bombyx mori (hybrid J106xDAIZO)
were reared on an artificial diet (Nihon Nosan
Archives of Insect Biochemistry and Physiology
June 2007
doi: 10.1002/arch.
53
Kogyo Co., Yokohama, Japan) kindly provided by
Dr. S. Cappellozza (Istituto Sperimentale per la
Zoologia Agraria, Sezione Specializzata per la
Bachicoltura, Padova, Italy), under a 12:12 L:D
photoperiod at 25 ± 1°C and 60% relative humidity. Larvae were staged after every larval ecdysis, and
the day of each ecdysis was designated as day 0.
Since the larvae mainly moulted to the final (5th)
instar during the scotophase, all the larvae that
ecdysed during the scotophase were segregated immediately after the onset of photophase. This time
was designated as 0 h of the 5th instar. In this particular hybrid, the 5th instar period lasts about 208
h. The onset of wandering behavior occurred 144
h (day 6) after the final larval ecdysis.
Reagents
2-Aminoethoxydiphenyl borate (2-APB), U
73122, 4-amino-5-(4-methylphenyl)-7-(t-butyl)
pyrazolo[3,4-D]-pyrimidine (PP1), Fura-2/AM,
mastoparan, nifedipine, nitrendipine, ryanodine,
and thapsigargin were purchased from Calbiochem
(Bad Soden, Germany). Mastoparan was dissolved
in aliquots in either Ringer’s saline (Shirai et al.,
1994) or Ca2+-free Ringer’s saline (see below) and
stored at –20°C until use. Influx™ pinocytotic cellloading reagent was from Molecular Probes (Eugene, OR). Grace’s medium was from GIBCO-BRL
(Grand Island, NY). Guanosine 5′-[β-thio]diphosphate trilithium salt (GDPβS), pertussis toxin
(PTX), heparin, gadolinium chloride, dimethyl sulfoxide (DMSO), Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) and all
other reagents were from Sigma (Deisenhofen, Germany). Stock solutions of nifedipine, nitrendipine,
and thapsigargin were prepared in DMSO.
Photomultiplier-Based Microfluorometry
Due to the autofluorescence of Grace’s medium,
dissected B. mori prothoracic glands were loaded
with 40 µM Fura-2/AM in 1 ml of Ringer’s saline
for 60 min at 25°C in the dark. Then glands were
rinsed in Ringer’s saline for 5 min and placed in 50
µl of the same saline on a cover slip in a tight cham-
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Dedos et al.
ber. In experiments requiring Ca2+-free medium,
Ringer’s saline was prepared using NaCl (4.5 mM)
in place of the standard 4.5 mM CaCl2. HEPES
buffer (0.01 M, pH 6.8) and EGTA (0.1 mM) were
added prior to use. DMSO, when present, did not
interfere with the fluorescence recordings and did
not affect intracellular Ca2+ levels in the concentrations used (1%). Dual wavelength measurements
were made with a Zeiss (Oberkochen, Germany)
microscope-photometer MPM 200 mounted on an
inverted Zeiss microscope “Axiovert 135” (with an
objective “Fluar” 10×/0.50). Emitted fluorescence
was collected through an aperture adjusted to the
size of prothoracic gland cells. Background fluorescence was estimated with unloaded cells. The measurements of fluorescence intensity (F) at the two
excitation wavelengths were used to calculate [Ca2+]i,
assuming a Kd of 224 nM (Grynkiewicz et al., 1985).
Free intracellular Ca2+ was measured in 9 identified
cells of each prothoracic gland and was recorded as
mean ± S.E.M. Addition of all reagents was made
by replacement of the whole bathing medium or
by addition of 10 µl saline with a pipette. All reagents added were tested for autofluorescence.
In order to assess the effects of the non-hydrolysable GDP analog, guanosine 5′-[β-thio]diphosphate, dissected B. mori prothoracic glands were
loaded with 40 µM of Fura-2/AM in 1 ml of Ringer’s
saline for 60 min at 25°C in the dark. Then, the
influx™ pinocytic cell-loading reagent was used to
load prothoracic gland cells with Guanosine 5′-[βthio]diphosphate. Essentially following the manufacturer’s protocol, the hypertonic solution was
made with 5 ml Ringer’s solution. Control glands
were incubated for 10 min at room temperature in
this hypertonic solution and then transferred to the
hypotonic solution for 2 min. For experimental
glands, guanosine 5′-[β-thio]diphosphate was dissolved in the hypertonic solution (5 mg/ml).
Fluorescence Optic Measurement of
Intracellular Calcium
Prothoracic gland cells of M. sexta were loaded
with 40 µM Fura-2/AM as described in the previous section for 20 min. Light exciting Fura-2 at 340
and 380 nm, provided by Polychrome II (T.I.L.L.
Photonics, Gräfelfing, Germany), was coupled via
an epifluorescence condenser into an Axioskop FS
(Carl Zeiss, Jena, Germany) equipped with a water immersion objective (LUMPFL 40×W/IR/0.8;
Olympus, Hamburg, Germany). Emitted light was
separated by a 400-nm dichroic mirror and filtered
with a 420-nm long-pass filter. The free intracellular Ca2+ concentration [Ca2+]i was calculated according to the equation [Ca2+]i = Keff(R-Rmin)/(Rmax-R).
The Keff, Rmin, and Rmax were determined using prothoracic gland cells permeabilized with 10 µM
ionomycin and three solutions with different concentrations of free Ca2+ (Ca2+ free; 5 mM Ca2+; 500
nM Ca2+; the composition of the 500 nM solution
was calculated with WEBMAXC v.2.20; Patton et
al., 2004). The values of Keff, Rmin, and Rmax were
3.7 µM, 0.38, and 4.9, respectively. Fluorescence
images were acquired using a cooled CCD camera
controlled by the software TILLVision 4.0 (T.I.L.L.
Photonics). The resolution was 640 × 480 pixels
in a frame size of 175 × 130 µm (40×/IR/0.8 objective). Image pairs were obtained by excitation
for 50 ms at 340 and 380 nm; for recording of
image series intervening intervals of 10 sec were
chosen. Background fluorescence was subtracted.
For calculation of calcium kinetics, regions of interest were defined in the cells.
Statistical Analyses
GraphPad Prism™ 4.0 computer software was
used for all statistical analyses. Statistical significance of the results was determined by analysis of
variance (ANOVA) followed by Tukey multiple
comparisons test.
RESULTS
Effects of Mastoparan on [Ca2+]i of the Prothoracic
Gland Cells on Day 6 5th Instar B. mori and M. sexta
Research with prothoracic glands of M. sexta
(Birkenbeil, 2000) showed that mastoparan increases [Ca2+]i of prothoracic gland cells. Therefore,
initial experiments were designed to determine
whether mastoparan has similar activity in the proArchives of Insect Biochemistry and Physiology
June 2007
doi: 10.1002/arch.
Ca2+ Mobilization by Mastoparan in PGs
thoracic gland cells of B. mori. Using 100 µM, we
observed rapid and large increases in [Ca2+]i that
peaked at ~400 nM after ~1 min (Fig. 1A). The
increase in [Ca2+]i was transient and decreased
thereafter. At 10 µM of mastoparan, we recorded
statistically significant increases in [Ca2+]i after 4-
55
min incubation with mastoparan with peak [Ca2+]i
of ~260 nM, 14 min after its addition (Fig. 1A).
This concentration was used in all subsequent experiments since the 100-µM concentration with its
rapid onset and its rapid decline was judged to be
inappropriate to determine the pharmacological
bases of mastoparan’s action (Fig. 1A). Using the
same concentrations as in B. mori prothoracic
glands, a dose-dependent relationship between
mastoparan concentration and [Ca2+]i was not observed in M. sexta prothoracic gland cells (Fig. 1B).
However, for consistency, the concentration of 10
µM was used for all subsequent experiments with
M. sexta prothoracic glands, similar to that used in
a previous study (Birkenbeil, 2000).
β S or Pertussis Toxin on MastoparanEffects of GDPβ
Stimulated [Ca2+]i in the Prothoracic Gland Cells
Fig. 1. Time- and dose-dependent stimulation of [Ca2+]i
by mastoparan in the prothoracic gland cells of B. mori
(A) and M. sexta (B). Day 6 prothoracic glands from 5th
larval instar of B. mori and M. sexta were loaded with Fura2/AM and then incubated in Ringer’s saline with three
different concentrations of mastoparan. The arrows indicate the time of addition of mastoparan. Results of Tukey
multiple comparisons test revealed that whereas 100 µM
of mastoparan stimulated a statistically significant increase
in [Ca2+]i within 1 min, at a concentration of 10 µM it
took 9 min for mastoparan to produce statistically significant increases in [Ca2+]i in B. mori prothoracic gland
cells. Each data point is the mean ± SEM of 9–18 independent measurements of prothoracic gland cells.
Archives of Insect Biochemistry and Physiology
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We initially tested guanosine 5′-[β-thio]diphosphate (see also Dedos et al., 2005) and introduced
it into the cells with the aid of the influx™ pinocytotic cell-loading reagent (Fig. 2). After inducing
an osmotic shock to either control cells (loaded
with just influx™ pinocytotic cell-loading reagent)
or to guanosine 5′-[β-thio]diphosphate-loaded cells
to release it immediately before the start of the experiment, mastoparan (10 µM) was added 6 min
later (Fig. 2). No statistically significant difference
was observed in the mastoparan-stimulated increase
in [Ca2+]i whether guanosine 5′-[β-thio]diphosphate
was present or not (Fig. 2). To unequivocally demonstrate that guanosine 5′-[β-thio]diphosphate
once in the cells does not prevent the mastoparanstimulated increase in [Ca2+]i in B. mori prothoracic gland cells, we injected guanosine 5′-[β-thio]
diphosphate in prothoracic gland cells in vitro as
described (Birkenbeil, 2000). The results were similar to those shown in Figure 2 (data not shown).
These results suggested that there might be a different mechanism of action for mastoparan in B.
mori prothoracic gland cells from that shown for
M. sexta prothoracic glands (Birkenbeil, 2000). To
test this assumption, we used pertussis toxin in B.
mori prothoracic glands in combination with
mastoparan. Despite using an excessively high con-
56
Dedos et al.
Fig. 2. Effect of GDPβS on mastoparan-stimulated increases in [Ca2+]i of B. mori prothoracic gland cells. Prothoracic gland cells were loaded with either plain or
guanosine 5′-[β-thio]diphosphate-containing (5 mg/ml)
influx™ pinocytotic cell-loading reagent before addition
of mastoparan (see Materials and Methods section for details). The arrows indicate the time of addition of each
reagent. Results of unpaired t-tests revealed that guanosine
5′-[β-thio]diphosphate did not affect the mastoparanstimulated increase in [Ca2+]i (P > 0.05). Each data point
is the mean ± SEM of 9–18 independent measurements
of prothoracic gland cells. Inset: Effect of pertussis toxin
(PTX) on mastoparan-stimulated increases in [Ca2+]i of
B. mori prothoracic gland cells. The arrows indicate the
time of addition of each reagent. Results of Tukey multiple comparisons test revealed that there was a statistically significant increase in [Ca2+]i only after a 9-min
incubation with mastoparan (10 µM) in the presence of
PTX (P < 0.05). Each data point is the mean ± SEM of 9–
18 independent measurements of prothoracic gland cells.
centration of pertussis toxin to insure its quick entrance to the prothoracic gland cells, we did not
record an inhibition of mastoparan-stimulated Ca2+
influx (Fig. 2, Inset).
We used heparin (100 µM), a specific blocker
of IP3 receptors, in combination with mastoparan
but did not abolish a mastoparan-stimulated increase in [Ca2+]i (data not shown). Since heparin
was effective in abolishing the prothoracicotropic
hormone-mediated increase in [Ca2+]i (Dedos et al.,
2005), it seems reasonable to infer that in B. mori
prothoracic gland cells the mastoparan-stimulated
increase in [Ca2+]i does not result from mobilization of Ca2+ from IP3-sensitive intracellular stores
via the activation of a G protein–coupled PLC.
Using U 73122 (Fig. 3A) or heparin (Fig. 3B)
in combination with mastoparan in M. sexta prothoracic gland cells, we did not record an inhibi-
tion of the mastoparan-stimulated increase in
[Ca2+]i (Fig. 3A and 3B). Moreover, PP1 did not
inhibit the mastoparan-stimulated increase in
[Ca2+]i (Fig. 3C).
Effects of Intracellular Ca2+ Modulating Agents on
Mastoparan-Stimulated [Ca2+]i in the Prothoracic
Gland Cells
We used thapsigargin in combination with
mastoparan in M. sexta (Fig. 4A) and B. mori (Fig.
4B). In both insect species, thapsigargin increased
[Ca2+]i in prothoracic gland cells within 4 min (Fig.
4A and B). Subsequent addition of mastoparan in
prothoracic gland cells treated with thapsigargin
did not produce any statistically significant increases in [Ca2+]i (Fig. 4A and B). Reversing the order in which the two compounds were added to
Archives of Insect Biochemistry and Physiology
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Ca2+ Mobilization by Mastoparan in PGs
57
Fig. 3. Effect of the PLC inhibitor, U 73122 (A), the IP3R inhibitor, heparin (B), or the Src kinase
inhibitor, PP1 (C) on mastoparanstimulated increases in [Ca2+]i of
M. sexta prothoracic gland cells.
The arrows indicate the time of addition of each reagent. Results of
the Tukey multiple comparisons
test revealed that none of these
reagents had any effect on the
mastoparan-stimulated increase in
[Ca2+]i (P > 0.05). Each data point
is the mean ± SEM of 9–18 independent measurements of prothoracic gland cells.
the prothoracic glands incubation medium, i.e.,
adding mastoparan first followed by addition of
thapsigargin did not produce any further statistically significant increases in [Ca 2+]i (data not
shown). Additional experiments revealed that: (1)
thapsigargin’s action was dependent on extracellular Ca2+ because when prothoracic glands were preincubated for 5 min in Ca2+-free Ringer’s saline,
the addition of thapsigargin did not result in any
increase in [Ca2+]i (data not shown); and (2) in
Archives of Insect Biochemistry and Physiology
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experiments where prothoracic gland cells were
incubated in Ca2+-free Ringer’s saline in which
thapsigargin and mastoparan were sequentially
added, we observed no further increase in [Ca2+]i
in the presence of mastoparan (data not shown).
We used intracellular Ca2+ modulating agents
in combination with mastoparan to block any increases in [Ca2+]i. Neither nifedipine (Fig. 4C) nor
nitrendipine (Fig. 4D), which does not inhibit the
mastoparan-stimulated increase in [Ca2+]i in M.
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Dedos et al.
Figure 4.
Archives of Insect Biochemistry and Physiology
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Ca2+ Mobilization by Mastoparan in PGs
sexta prothoracic gland cells (Birkenbeil, 2000), inhibited the mastoparan-stimulated increase in
[Ca2+]i, an indication that L-type voltage gated Ca2+
channels, although present in the prothoracic gland
cells of both insect species (Girgenrath and Smith,
1996; Dedos and Birkenbeil, 2003), do not mediate the mastoparan-stimulated increase in [Ca2+]i.
We tested ryanodine in combination with
mastoparan in prothoracic glands of both M. sexta
(Fig. 4E) and B. mori (Fig.4F). Ryanodine (100 µM)
had no effect on the mastoparan-induced increase
in [Ca2+]i in M. sexta prothoracic glands (Fig. 4E)
and did not effect [Ca2+]i of B. mori prothoracic
gland cells (Fig. 4F) but in its presence, mastoparan
was rendered completely incapable of stimulating
any increases in [Ca2+]i (Fig. 4F). This result identifies the source of the mastoparan’s initial site of
action as ryanodine-sensitive Ca2+ stores in prothoracic gland cells of B. mori.
2-Aminoethoxydiphenyl borate (2-APB), an inhibitor of store-operated Ca2+ entry and also an
inhibitor of Ca2+ release from IP3-gated intracellular stores (Bootman et al., 2002), had no effect on
the mastoparan-induced increase in [Ca2+]i in both
M. sexta prothoracic glands (Fig. 4G) and B. mori
prothoracic glands (Fig. 4H), although it has been
shown to inhibit any increase in [Ca2+]i by either
thapsigargin or prothoracicotropic hormone at a
concentration of 100 µM in B. mori prothoracic
gland cells (Dedos et al., 2005).
Finally, we used the trivalent cation gadolinium
Fig. 4. Effects of intracellular Ca2+ modulating agents on
mastoparan-stimulated increases in [Ca2+]i of B. mori and
M. sexta prothoracic gland cells. The arrows indicate the
time of addition of each reagent. Results of Tukey multiple comparisons test revealed that: (1) In M. sexta prothoracic gland cells, only Gd3+ significantly inhibited the
mastoparan-stimulated increase in [Ca2+]i (P < 0.05), (2)
In B. mori prothoracic gland cells, ryanodine inhibited the
mastoparan-stimulated increase in [Ca2+]i (P < 0.05) and
had no effect on the basal [Ca2+]i of prothoracic gland
cells (P > 0.05), 3) None of the other tested reagents had
any effect on the mastoparan-stimulated increase in [Ca2+]i
(P > 0.05). Each data point is the mean ± SEM of 9–18
independent measurements of prothoracic gland cells.
Archives of Insect Biochemistry and Physiology
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(Gd3+), a blocker of capacitative Ca2+ entry, by preventing Ca2+ influx through the plasma membrane
(Broad et al., 1999). When added before mastoparan
either in M. sexta prothoracic glands (Fig. 4I) or B.
mori prothoracic glands (Fig. 4J), this cation could
completely abolish the mastoparan-stimulated increase in [Ca2+]i in M. sexta prothoracic glands (Fig.
4I), but in B. mori prothoracic gland cells, a small,
transient increase in [Ca2+]i was still observed after
addition of mastoparan.
To determine that mastoparan’s action is directed towards release of Ca2+ from the intracellular stores in B. mori prothoracic gland cells, we
investigated its effect on [Ca2+]i in the absence of
extracellular Ca2+ (Fig. 5). Prothoracic glands were
pre-incubated for 5 min in Ca2+-free Ringer’s saline, then transferred into fresh Ca2+-free Ringer’s
saline before the start of the experiment (Fig. 5)
Fig. 5. Effect of extracellular Ca2+ on mastoparan-stimulated increases in [Ca2+]i of B. mori and M. sexta prothoracic gland cells. Glands were pre-incubated in Ca2+-free
Ringer’s saline for 5 min before transferral to fresh Ca2+free Ringer’s saline immediately before the start of the
experiment. Results of Tukey multiple comparisons test
revealed that: (1) In M. sexta prothoracic gland cells, the
absence of extracellular Ca2+ inhibited the mastoparanstimulated increase in [Ca2+]i (P > 0.05), (2) In B. mori
prothoracic gland cells, there was a statistically significant
increase in [Ca2+]i after 9-min incubation with mastoparan
in the absence of extracellular Ca2+ (P < 0.05). The arrows indicate the time of addition of each reagent. Each
data point is the mean ± SEM of 9–18 independent measurements of prothoracic gland cells.
60
Dedos et al.
and then upon addition of mastoparan a small but
stable increase in [Ca2+]i was recorded (Fig. 5). Intracellular Ca2+ reached 130 nM within 9 min after the addition of mastoparan, considerably less
than that recorded in the presence of extracellular
Ca2+ (compare Figs. 1A and 5). Most notably, the
kinetics of mastoparan’s stimulated increase in
[Ca2+]i, in the absence of extracellular Ca2+, were
completely different from those of thapsigargin,
which did not produce any further increase in
[Ca2+]i in the absence of extracellular Ca2+ (data
not shown). The same experiment (Fig. 5) conducted in M. sexta prothoracic glands showed that
upon addition of mastoparan, there was no increase in [Ca2+]i (Fig. 5).
Spatial Aspects in [Ca2+]i in Mastoparan-Stimulated
Prothoracic Gland Cells of M. sexta
Ca2+ imaging experiments (Fig. 6) revealed that
after addition of mastoparan (10 µM; Fig. 6A and
B), an increase in [Ca2+]i in M. sexta prothoracic
gland cells was observed in a spatially restricted
region near the plasma membrane. The region of
this spatially restricted increase in [Ca2+]i was consistently near the pole of the cell throughout the
course of the observation (5 min). By isolating regions of interest and determining [Ca2+]i in them
throughout the course of the observation, we recorded distinct increases in [Ca2+]i in one region
and no increase in [Ca2+]i in another (Fig. 6C), but
we never recorded globally homogeneous [Ca2+]i
throughout the cell (Fig. 6C).
Pre-incubation of M. sexta prothoracic gland
cells with 100 µM Gd3+ (Fig. 6D) abolished the
effect of mastoparan (10 µM; Fig. 6E), throughout
the course of the observation (5 min), and this
suggests that the source of mastoparan’s initial site
of action is a capacitative Ca2+ entry channel in
prothoracic gland cells of M. sexta. The absence of
response to mastoparan in the presence of 100 µM
Gd3+ was evident also when regions of interest were
selected and analysed throughout the course of the
observation (Fig. 6F).
Addition of thapsigargin in M. sexta prothoracic
gland cells gave different results (Fig. 6G and H)
from those observed by mastoparan. Within 10 min
after its application, thapsigargin (10 µM) induced
large increases in [Ca2+]i that were global and homogeneous in appearance and extended throughout
the cell with the notable exception of the regions
immediately near the plasma membrane. Figure 6I
shows the overall time course of increase in [Ca2+]i
at 2 different regions of a prothoracic gland cell
after addition of thapsigargin.
DISCUSSION
The ability of mastoparan to increase [Ca2+]i was
originally reported in M. sexta prothoracic gland
cells (Birkenbeil, 2000). In that study, the mastoparan-stimulated increase in [Ca2+]i was abolished
in the presence of pertussis toxin (Birkenbeil,
2000). This is an indication that the mastoparanstimulated increase in [Ca2+]i is directed towards
activation of a pertussis toxin-sensitive G protein
in prothoracic gland cells of M. sexta. This interpretation fits with the consensus on mastoparan
as activator of G proteins (Holler et al., 1999). But
in contrast with results presented for M. sexta prothoracic glands (Birkenbeil, 2000), the mode of
action of mastoparan in the prothoracic gland cells
of B. mori appears to be different although it produces increases in [Ca2+]i.
Fig. 6. Mastoparan and thapsigargin produce spatially
different increases in [Ca2+]i in M. sexta prothoracic gland
cells. Subcellular Ca2+ signals were acquired and analysed
with the imaging technique. Cells were loaded with Fura2/
AM and excited at 340 and 380 nm (100 ms). A–C: Distribution of [Ca2+]i in a prothoracic gland cell before (A)
and 10 min after application of 10 µM mastoparan (B).
The scaling bar in A = 10 µm. Representative kinetics in
[Ca2+]i in the frames defined in A are shown in C) (n = 6
cells). D–F: Distribution of [Ca2+]i in a prothoracic gland
cell pre-incubated with 100 µM Gd3+ before (D) and 10
min after application of 10 µM mastoparan (E). Representative kinetics in [Ca2+]i in the frames defined in D
are shown in F (n = 6 cells). E–H: Distribution of [Ca2+]i
in a prothoracic gland cell before (G) and 10 min after
application of 10 µM thapsigargin (H). Representative kinetics in [Ca2+]i in the frames defined in G are shown in I
(n= 6 cells).
Archives of Insect Biochemistry and Physiology
June 2007
doi: 10.1002/arch.
61
Figure 6.
Ca2+ Mobilization by Mastoparan in PGs
Archives of Insect Biochemistry and Physiology
June 2007
doi: 10.1002/arch.
62
Dedos et al.
In this study, we further explored the effects of
mastoparan on M. sexta prothoracic gland cells and
show that mastoparan acts on Ca2+ channels in the
plasma membrane of M. sexta prothoracic gland
cells to generate localized high increases in [Ca2+]i
by a mechanism that has been shown (Birkenbeil,
2000) to involve pertussis toxin-sensitive G proteins. Such pertussis toxin-sensitive G proteins have
been documented to exist in M. sexta prothoracic
gland cells and their activation has been shown to
be irrelevant to the prothoracicotropic hormone
signalling cascade (Girgenrath and Smith, 1996).
Previous research has identified two compounds
that have an effect on increases in [Ca2+]i in M.
sexta prothoracic gland cells (Fellner et al., 2005;
Priester and Smith, 2005). Namely, U 73122, a specific PLC inhibitor, was shown by Fellner et al.
(2005) to block the prothoracicotropic hormonemediated increase in [Ca2+]i. Also, 4-amino-5-(4methylphenyl)-7-(t-butyl)pyrazolo[3,4-D]-pyrimidine
(PP1), a specific Src kinase inhibitor, was shown
by Priester and Smith, (2005) to inhibit the
prothoracicotropic hormone-mediated increases in
[Ca2+]i. Therefore, we reasoned that if mastoparan
is pharmacologically activating components of the
signalling cascade of prothoracicotropic hormone,
the physiological activator of prothoracic gland
cells, these compounds will block mastoparan’s action. The combined results suggest that in M. sexta
prothoracic gland cells, mastoparan does not
stimulate an increase in [Ca2+]i via the PLC signalling cascade and generation of IP3 and 1,2-diacylglycerol (Fellner et al., 2005) or the Src kinase
signalling cascade, which is involved in the prothoracicotropic hormone-mediated increase in
[Ca2+]i (Priester and Smith, 2005). Yet, the present
and previous results (Birkenbeil, 2000) suggest that
in M. sexta, the mastoparan-stimulated increase in
[Ca2+]i is Gi dependent.
To identify the nature of the plasma membrane
2+
Ca channels that participate in the mastoparanstimulated increase in [Ca2+]i, we have used several voltage-gated Ca2+ channel inhibitors and
capacitative Ca2+ entry channel blockers. Our results show that Gd3+ and La3+ (data not shown)
could block the mastoparan-stimulated increase in
[Ca2+]i, while blockers of T-type Ca2+ channels such
as flunarizine (100 µM), bepridil (100 µM), or
mibefradil (100 µM) had no effect on the mastoparan-stimulated increase in [Ca2+]i in M. sexta prothoracic gland cells (data not shown). These results
suggest that mastoparan acts on pertussis toxin-sensitive G proteins, activates them, and this directly
leads to opening of plasma membrane Ca2+ channels that have a pharmacological profile similar to
capacitative Ca2+ entry channels. Therefore, the
mastoparan-stimulated increase in [Ca2+]i in M.
sexta prothoracic gland cells does not exhibit a bimodal profile and it is the result of strictly localized Ca2+ influx in the cells in sharp contrast to
the globally homogeneous increase in [Ca2+]i observed in the presence of thapsigargin.
In B. mori prothoracic gland cells, there appears
to be a bimodal profile in the mechanism that
mastoparan stimulates increases in [Ca2+]i and in
a previous study we have shown that this is also
the way prothoracicotropic hormone stimulates
increases in [Ca2+]i (Birkenbeil and Dedos, 2002).
However, prothoracicotropic hormone-mediated
increases in [Ca2+]i are sensitive to heparin, insensitive to ryanodine, and never occur in the absence
of extracellular Ca2+ (Birkenbeil and Dedos, 2002).
Mastoparan exhibited the unique (for our experimental setup) ability to stimulate increases in
[Ca2+]i even when prothoracic glands were pre-incubated in Ca2+-free Ringer’s saline for 5 min. This
observation discredited our initial notion that in
B. mori prothoracic gland cells, the mastoparan and
prothoracicotropic hormone signalling pathways
are similar. Even thapsigargin, which was expected
to slightly and transiently increase [Ca2+]i in the
absence of extracellular Ca2+, did not do so when
prothoracic glands were pre-incubated in Ca2+-free
Ringer’s saline for 5 min (data not shown). Given
the fact that mastoparan has a different effect than
thapsigargin on [Ca2+]i in the absence of extracellular Ca 2+ in B. mori prothoracic gland cells,
mastoparan cannot be acting by the same mechanism as thapsigargin, i.e., inhibition of sarcoplasmic reticulum Ca2+ ATPases, as has been shown by
Longland et al. (1999). Mastoparan’s action in proArchives of Insect Biochemistry and Physiology
June 2007
doi: 10.1002/arch.
Ca2+ Mobilization by Mastoparan in PGs
thoracic gland cells is, therefore, directed towards
emptying intracellular Ca2+ stores that are thapsigargin-sensitive but IP3-insensitive. These Ca2+
stores are ryanodine-sensitive and this indicates
that the mastoparan-modulated increases in [Ca2+]i
in prothoracic gland cells occur from intracellular
Ca2+ stores that are not related and appear to be
distinct from any IP3-regulated Ca2+ stores that have
been identified previously (Dedos et al., 2005).
Moreover, the results clearly show that a large
increment of the mastoparan-stimulated increase in
[Ca2+]i in prothoracic gland cells is the result of Ca2+
influx from the extracellular domain. In other words,
there is a bimodal profile in the mastoparan-stimulated increases in [Ca2+]i in B. mori prothoracic gland
cells. The first phase consists of a transient mobilization of Ca2+ from ryanodine-sensitive intracellular Ca2+ stores and this phase is rapidly followed
by the second phase, which consists of Ca2+ influx
from the extracellular domain through the plasma
membrane.
To identify how the second phase of the mastoparan-stimulated increase in [Ca2+]i takes place, we
have used several voltage gated Ca2+ channel inhibitors and capacitative Ca2+ entry channel blockers.
Our results show that Gd3+ and La3+ (data not
shown) could block the mastoparan-stimulated increase in [Ca2+]i, while nitrendipine, a dihydropyridine derivative that inhibits L-type Ca2+ channels,
or a blocker of T-type Ca2+ channels (flunarizine,
100 µM; data not shown), could not prevent the
mastoparan-stimulated increase in [Ca2+]i. Based on
the ability of Gd3+ to inhibit the mastoparan-stimulated increases in [Ca2+]i in both M. sexta and B.
mori prothoracic gland cells, we believe that the
second phase of the mastoparan-stimulated increase in [Ca2+]i, i.e., the large and sustained increase in [Ca2+]i in B. mori prothoracic gland cells,
is identical to that observed in M. sexta prothoracic gland cells and it is mediated by pharmacologically identical regulatory elements, namely,
capacitative Ca2+ entry channels.
It has been shown that mastoparan binds to
glycogen phosphorylase in the heavy fraction of
sarcoplasmic reticulum (HSR), isolated from rabbit skeletal muscle, and removes the inhibition of
Archives of Insect Biochemistry and Physiology
June 2007
doi: 10.1002/arch.
63
glycogen phosphorylase on ryanodine receptors
(RyR) (Hirata et al., 2000, 2003). The subsequent
induction of Ca2+ release from RyRs present in HSR
can be inhibited by ryanodine (Hirata et al., 2000,
2003). As the activity of glycogen phosphorylase
on glycogenolysis is regulated by Ca2+ released from
SR, the ability of glycogen phosphorylase to inhibit the gating of RyR provides a hypothesis that
there may be a functional cross-talk between Ca2+
release from SR and glycogenolysis for energy supply mediated through glycogen phosphorylase
(Hirata et al., 2003).
Whether such a mechanism underlies the ability of ryanodine to inhibit the mastoparan-stimulated increases in [Ca2+]i that we observed in B. mori
prothoracic gland cells remains to be determined.
If this is true, then mastoparan can be a valuable
pharmacological tool in understanding how Ca2+
signalling mechanisms in prothoracic gland cells
control the overall function of these cells. This will
provide us with insights on how Ca2+ can regulate
mechanisms responsible for the development of
prothoracic glands and widen our understanding
of the function of these glands in insects.
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Archives of Insect Biochemistry and Physiology
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