We used insect epidermis and cultured cells to define the role of the GBP signaling pathway in the enhancement of TH and DDC gene expression.. The downstream signaling pathways diverge to
Trang 1cascades regulate two separate groups of target genes
Yosuke Ninomiya1, Maiko Kurakake2, Yasunori Oda2, Seiji Tsuzuki2and Yoichi Hayakawa2
1 Graduate School of Environmental Earth Science, Hokkaido University, Sapporo, Japan
2 Department of Applied Biological Sciences, Saga University, Japan
In animals, tyrosine hydroxylase (TH, EC 1.14.16.2)
and 3,4-dihydroxy-l-phenylalanine (Dopa)
decarboxyl-ase (DDC, EC 4.1.1.26) are required for the
produc-tion of Dopa and dopamine [1,2] Because Dopa is
required for tanning of newly formed cuticle and
dopa-mine has been shown to exert many neurohormonal
functions in insects, both enzymes are essential for the
maintenance of life [3–6] Furthermore, Dopa and
dopamine are also oxidized by a system of
phenoloxid-ases and cofactors to form melanins that produce the
various black and brown patterns of the cuticles in a
broad range of insect species [7,8]
We previously demonstrated that transcription lev-els as well as enzyme activities of TH and DDC in larval cuticles are elevated during molt periods in the armyworm, Pseudaletia separata [9,10] Morpho-logical analysis showed preferential distribution of both enzymes in the epidermal cells beneath the black stripes in the dorsal surface of armyworm lar-vae Because the black stripes become much darker and wider after each larval ecdysis, both enzymes are thought to contribute to production of melanins during molts [9] Furthermore, the periodic expression of both enzyme genes was found to be
Keywords
calcium ion; Dopa decarboxylase;
extracellular signal-regulated kinase;
growth-blocking peptide; tyrosine hydroxylase
Correspondence
Y Hayakawa, Department of Applied
Biological Science, Saga University, Honjo-1,
Saga 840-8502, Japan
Fax ⁄ Tel: 81 952 28 8747
E-mail: hayakayo@cc.saga-u.ac.jp
(Received 9 October 2007, revised 4
December 2007, accepted 18 December
2007)
doi:10.1111/j.1742-4658.2008.06252.x
Growth-blocking peptide (GBP) is a 25 amino acid insect cytokine found
in lepidopteran insects that has diverse biological activities, such as larval growth regulation, paralysis induction, cell proliferation, and stimulation
of immune cells GBP also enhances expression of the tyrosine hydroxylase (TH, EC 1.14.16.2) and 3,4-dihydroxy-l-phenylalanine (Dopa) decarboxyl-ase (DDC, EC 4.1.1.26) genes, which elevate dopamine levels in insect epi-dermal cells We used insect epidermis and cultured cells to define the role
of the GBP signaling pathway in the enhancement of TH and DDC gene expression It has been recently reported that robust expression of the DDC gene requires activation of extracellular signal-regulated kinase (ERK) in epidermal cells of wounded Drosophila embryos This study con-firmed that GBP activates ERK, but this activation is not directly linked to the enhancement of TH and DDC gene expression One of the GBP path-way components is phospholipase C, whose activation is essential for the activation of ERK and elevation of expression of both enzyme genes The downstream signaling pathways diverge to ERK activation through acti-vated protein kinase C and expression of the enzyme genes through inositol triphosphate receptor-mediated Ca2+ influx from extracellular fluid Our data indicate that the diverged GBP signaling pathways enable GBP to exert completely different biological functions, even in a single cell type
Abbreviations
DAG, diacylglycerol; DDC, Dopa decarboxylase (EC 4.1.1.26); Dopa, 3,4-dihydroxy- L -phenylalanine; ERK, extracellular signal-regulated kinase; GBP, growth-blocking peptide; IP 3 , inositol triphosphate; MAP, mitogen-activated protein; MEK, mitogen-activated protein kinase kinase; PKC, protein kinase C; PLC, phospholipase C; PTTH, prothoracicotropic hormone; TH, tyrosine hydroxylase (EC 1.14.16.2).
Trang 2regulated by the insect cytokine, growth-blocking
peptide (GBP) [10]
GBP was initially identified as the factor responsible
for the reduced growth exhibited by P separata larvae
after parasitization by the parasitic wasp Cotesia
kar-iyai[11–13] Analysis of the mechanism by which GBP
retards larval growth revealed that GBP activates
DDC activities in epidermal cells and elevates
dopa-mine concentrations in the integuments [14–16]
Subse-quent studies provided evidence that part of the
increased dopamine in the integuments is released into
the hemolymph, and consequently retards the normal
development of armyworm larvae [15] Furthermore,
recent studies showed that the activation of DDC is
induced by its transcriptional enhancement through
GBP-induced elevation of cytoplasmic Ca2+
concen-trations in the epidermal cells [10] Therefore, we
spec-ulated that the increased level of cytoplasmic Ca2+
affects a certain transcription enhancer that stimulates
expression of the DDC gene A previous study also
suggested the involvement of mitogen-activated protein
(MAP) kinases in epidermal DDC expression: in
wounded Drosophila embryos, activation of
extracellu-lar signal-regulated kinase (ERK) is required for the
activation of DDC gene expression near wounded sites
in the epidermal integument [17]
In this study, we characterized the signal
transduc-tion pathway of GBP to clarify whether GBP can
acti-vate ERK and whether its activation is required for
the expression of TH and DDC genes Using insect
integuments and cultured cells, we demonstrated that
GBP activates ERK but that its activation is not
nec-essary for the induction of expression of either enzyme
gene
Results
GBP-induced activation of ERK in integuments
Prior studies showed that expression of TH and DDC
genes was enhanced in integuments isolated from
armyworm larvae when they were incubated with GBP
[10] We examined whether GBP induces dual
phos-phorylation (activation) of ERK in the larval
integu-ments under the same conditions As shown in
Fig 1A, dual phosphorylation of ERK was clearly
enhanced when the integuments were incubated with
10 nm GBP for 5 min, but this phosphorylation was
completely blocked by U0126, a MAP kinase kinase
(MEK) inhibitor that reduces the phosphorylation and
activation of the ERK–MAP kinase In contrast,
addi-tion of U0126 to the incubaaddi-tion medium did not
pre-vent GBP-induced expression of TH and DDC genes
(Fig 1B), thus indicating that activated ERK is not required for GBP-induced gene expression of either enzyme in epidermal integuments isolated from army-worm larvae
GBP-induced ERK activation in MaBr4 cells For the detailed characterization of transcriptional reg-ulatory inputs involved in GBP-dependent activation
of expression of TH and DDC genes, we used cultured
dpERK
ERK
TH
DDC
Actin
4 3 2
1 0
*
0 n M GBP 10 n M GBP 10 n M GBP/U0126
0 n M GBP 10 n M GBP 10 n M GBP/U0126
A
B
Fig 1 Effect of GBP on ERK activation and expression of the TH and DDC genes in the dorsal integument of day 1 last instar larvae
of the armyworm (A) Western blots of ERK The antibody employed specifically recognizes dually phosphorylated (activated) ERKs Phosphorylation of ERK was observed in the integument after incubation with 10 n M GBP at 25 C for 5 min A MEK inhibi-tor (10 l M , U0126) inhibited GBP-induced phosphorylation of ERK Each bar indicates the mean ± SD of three independent determina-tions *Significantly different from control (0 n M GBP; P < 0.05, Student’s t-test) (B) RT-PCR analysis of TH and DDC gene expres-sion Clear bands of TH and DDC were expressed in the integu-ment after incubation with 10 n M GBP, and expression of both enzyme genes was not inhibited by U0126 dpERK, dually phos-phorylated ERK *Significantly different from control (0 n M GBP;
P < 0.05, Student’s t-test).
Trang 3insect cells instead of the epidermal integuments,
because we had observed that addition of chemicals
and long incubation caused unexpected damage to the
isolated integument To select the best cultured cells
for following studies, we tested both enzyme gene
expression and ERK activation of some cultured cells,
including Sf9, MaBr3, and MaBr4 cells, and found
that only MaBr4 cells show clear expression of both
enzyme genes and the ERK activation, as shown in
Fig 2 Activation of ERK was observed 8 min after
the cells were simply transferred from 4 to 25C
(Fig 2A), and at that time, the TH and DDC genes were clearly expressed in the cells (Fig 2B) Further-more, the GBP-induced gene expression of both enzymes was highly enhanced only in the Ca2+ -con-taining medium (Fig 2C)
To check whether GBP elevates cytoplasmic Ca2+ concentrations in MaBr4 cells, Ca2+ concentrations in those cells were monitored using a laser confocal microscope Addition of GBP to the incubation med-ium elevated the cytoplasmic Ca2+ concentrations in MaBr4 cells, but BSA did not change the Ca2+ con-centrations at all (Fig 3), indicating that, in analogy
to the epidermal cells, MaBr4 cells have the property
of increasing cytoplasmic Ca2+ concentrations by GBP [10]
GBP showed a dose-dependent capacity to activate ERK, as shown in Fig 4A The activation of ERK was roughly proportional to the concentration of GBP
up to 100 nm Furthermore, GBP-induced activation
of ERK was detected with the presence of EGTA (Fig 4B) Whereas a slight enhancement of ERK phosphorylation was also observed when the concen-tration of Ca2+added to the medium exceeded that of EGTA (Fig 4B), the calcium ionophore A23187 did not activate ERK at all (Fig 4C) These results imply that GBP stimulates more than one signaling pathway leading to activation of ERK, and that among them,
at least one pathway is Ca2+-independent
GBP-induced ERK activation requires MEK The GBP-induced activation of ERK was observed transiently 5 min after addition of GBP to the culture medium of MaBr4 cells (Fig 5A) This GBP-depen-dent ERK activation was completely blocked by 10 lm U0126 (MEK inhibitor), indicating that the GBP sig-naling pathway requires MEK activity (Fig 5B) Furthermore, to test whether the GBP-induced expression of TH and DDC genes requires ERK acti-vation, expression of both enzyme genes was measured
in MaBr4 cells stimulated by GBP in the presence of U0126 GBP clearly enhanced expression of both enzyme genes even in the presence of 10 lm U0126, indicating that the GBP signaling pathway for the stimulation of TH and DDC gene expression does not require the activated ERK (Fig 5C)
Analysis of GBP signaling pathways
It is now apparent that the GBP signaling pathways diverge from a certain component into at least two pathways: one towards ERK activation, and the other towards TH and DDC gene expression To
On ice for 1h/25o C incubation time
dpERK
ERK
Marker TH DDC Actin
TH
DDC
Actin
6 min 8 min
* *
10 n M GBP
10 n M GBP 10 n M BSA
3 m M CaCl23 m M CaCl2 3 m M CaCl2
2 m M EGTA 2 m M EGTA 2 m M EGTA 2 m M EGTA 2 m M EGTA
5
4
3
2
1
0
A
B
C
Fig 2 ERK activation and expression of TH and DDC genes in
MaBr4 cells (A) Western blots of ERK Phosphorylation of ERK in
Mabr4 cells was observed 8 min after transfer from 4 to 25 C (B)
RT-PCR analysis of expression of TH and DDC genes RNAs were
prepared from MaBr4 cells 8 min after transfer from 4 to 25 C (C)
RT-PCR analysis of GBP-induced expression of TH and DDC genes.
Total RNA was prepared from MaBr4 cells after incubation with
the indicated chemicals at 25 C for 6 h Each bar indicates the
mean ± SD of three independent determinations *Significantly
different from control (0 n M GBP; P < 0.05, Student’s t-test).
**Significantly different from control (0 n M GBP; P < 0.01,
Student’s t-test).
Trang 4characterize these pathways, the possible participation
of some components of a classic signal transduction
cascade was tested Neither elevation of the gene
expression of both enzymes nor ERK activation was
observed after the addition of 2 lm U73122, a
phos-pholipase C (PLC) inhibitor, to the MaBr4 cell culture
medium (Fig 6) However, whereas the addition of
10 lm chelerythrine chloride, a protein kinase C
(PKC) inhibitor, did not abolish the GBP-dependent
expression of both enzyme genes, it did block the
ERK activation (Fig 6) In contrast, the addition of
10 lm TMB-8, an inositol triphosphate (IP3) receptor
antagonist, did not inhibit the GBP-induced ERK
acti-vation, but abolished the elevation of expression of
both enzyme genes (Fig 6)
These results clearly indicate that the GBP signaling
events initially activated PLC, which activates ERK
through activation of PKC Furthermore, IP3produced
by activated PLC initiates the signaling cascade that
ultimately triggers Ca2+ entry from the extracellular
fluid The elevation of the cytoplasmic Ca2+
concen-tration enhances the expression of TH and DDC genes (Fig 7)
Discussion
In the present study, we demonstrated that GBP induces dual phosphorylation (activation) of ERK in the integuments of armyworm larvae This observa-tion, together with a previous report indicating that robust induction of DDC gene expression requires activated ERK–MAP kinase in wounded Drosophila embryos [17], suggested the possibility that the GBP-induced enhancement of expression of TH and DDC genes also requires activated ERK To assess this pos-sibility, the GBP–ERK pathway was examined in epi-dermal integuments Addition of the MEK inhibitor U0126 to the isolated integument culture medium com-pletely blocked GBP-induced ERK phosphorylation in the tissues, but expression of the TH and DDC genes was enhanced These results were interpreted to mean that the GBP signaling pathway towards the
A
B
Fig 3 Monitoring cytoplasmic Ca2+concentrations in MaBr4 cells incubated with BSA (A) and GBP (B) Ca2+concentrations in MaBr4 cells containing Fluo-3AM were monitored at 5 min after addition of 10 n M BSA or GBP (a) Laser transmission images (b) Ca 2+ indicator Fluo-3AM fluorescence images (green) (c) Fluorescence images overlaid with the laser transmission images.
Trang 5expression of both enzyme genes is mediated
indepen-dently of ERK activation To assess this interpretation,
follow-up analysis was conducted using cultured
MaBr4 insect cells
Although MaBr4 cells were originally derived from
the larval fat body of Mamestra brassicae, a species
closely related to P separata, their morphology is
rather hemocyte-like [18] GBP-induced activation of
dpERK ERK
dpERK ERK
4
2
0
4
2
0
4
2
0
*
*
*
*
dpERK ERK
0 n M GBP 1 n M GBP 10 n M GBP 100 n M GBP
2 m M EGTA 2 m M EGTA
10 n M GBP
2 m M EGTA/3 m M CaCl2
10 n M GBP
10 n M GBP 0 0.5 5
DMSO
A23187 concentration (µ M )
A
B
C
Fig 4 Effect of Ca 2+ on GBP-induced activation of ERK in MaBr4
cells (A) Effects of various concentrations of GBP on ERK
phos-phorylation MaBr4 cells were incubated with GBP at 25 C for
5 min *Significantly different from control (0 n M GBP; P < 0.05,
Student’s t-test) (B) Requirement of Ca 2+ for GBP-induced
phos-phorylation of ERK MaBr4 cells were incubated with the indicated
chemicals at 25 C for 5 min **Significantly different from control
(0 n M GBP; P < 0.01, Student’s t-test) (C) Effect of various
concen-trations of the calcium ionophore A23187 on ERK phosphorylation.
MaBr4 cells were incubated with various concentrations of A23187
at 25 C for 5 min Because A23187 was dissolved in
dimethylsulf-oxide, dimethylsulfoxide (final concentration 0.1%) was added to
the incubation medium Only the control incubation contained GBP
(10 n M ) **Significantly different from control (10 n M GBP;
P < 0.01, Student’s t-test) Each bar indicates the mean ± SD of
three independent determinations.
dpERK
ERK
dpERK
ERK
5 4 3 2 1 0
4
0 2
4
2
0
4
2
0 4
0 2
TH DDC Actin
*
A
B
C
Fig 5 Effect of MEK inhibitor on GBP-induced ERK activation and enhancement of TH and DDC gene expression inMaBr4 cells (A) Effect of incubation time on GBP-induced phosphorylation of ERK MaBr4 cells were incubated with or without 10 n M GBP in the medium containing 0.1% dimethylsulfoxide at 25 C Phosphoryla-tion of ERK was observed 5 min after addiPhosphoryla-tion of GBP to the MaBr4 incubation medium Closed and open bars: with and without GBP, respectively **Significantly different from control (0 min;
P < 0.01, Student’s t-test) (B) Effect of MEK inhibitor on GBP-induced ERK phosphorylation U0126 (10 l M ), a MEK inhibitor, completely inhibited GBP-induced ERK phosphorylation Other explanations as in (A) (C) Effect of MEK inhibitor on enhancement
of TH and DDC gene expression MaBr4 cells were incubated at
25 C for 6 h after addition of the indicated chemicals, and total RNA was prepared The GBP-induced expression of TH and DDC mRNAs was not blocked by the MEK inhibitor *Significantly differ-ent from control (0 n M GBP; P < 0.05, Student’s t-test) Each bar indicates the mean ± SD of three independent determinations.
Trang 6ERK was transiently observed at 5 min after addition
of GBP to the incubation medium of MaBr4 cells The
involvement of a MAP kinase kinase (MEK) in the
ERK activation pathway [19] was tested by evaluating
the effect of U0126 U0126 completely abolished the
temperature-dependent activation of ERK, indicating
that ERK activation in MaBr4 cells proceeds through
the common pathway characterized in mammalian
cells
Prior studies indicated that GBP induces Ca2+
influx into brain synaptosomes and epidermal cells
[10,20] Especially in the latter, the enhancement of
TH and DDC gene expression was demonstrated to be
accompanied by GBP-induced elevation of the cyto-plasmic Ca2+concentration We examined whether the GBP-induced activation of ERK requires Ca2+ influx into MaBr4 cells Excess amounts of EGTA did not block the GBP-induced ERK activation Furthermore, the calcium ionophore A23187 did not activate ERK
at all, even at a concentration of 5 lm, suggesting that the GBP-dependent activation of ERK does not require Ca2+ influx into MaBr4 cells However, the GBP-induced enhancement of TH and DDC gene expression was completely abolished by EGTA Fur-thermore, the GBP-dependent expression of both enzyme genes was observed in the presence of the MEK inhibitor, thereby demonstrating that the GBP-dependent enhancement of expression of the enzyme genes in MaBr4 cells is independent of the ERK–MAP kinase pathway
As U73122 (PLC inhibitor) has been shown to block the formation of IP3 in several insect tissues [21,22],
we applied it to MaBr4 cells The inhibitory effect of U72122 on ERK and expression of the enzyme genes clearly demonstrated that GBP signaling events ini-tially activate PLC The PLC-triggered metabolism of membrane phospholipids produces diacylglycerol (DAG) and IP3 Because chelerythrine chloride, a PKC inhibitor, abolished GBP-dependent activation of ERK, the PLC-produced DAG would contribute to activation of PKC [23–25] In contrast, the observation that TMB-8, an IP3receptor antagonist, blocked
GBP-TH
DDC
Actin
Control PLC inhibitor IP 3 R antagonist PKC inhibitor
*
*
*
*
5
4
3
2
1
0
Control PLC inhibitor IP 3 R antagonist PKC inhibitor
**
**
dpERK
ERK
5
4
3
2
1
0
Fig 6 ERK activation and TH and DDC gene expression in the
presence of various inhibitors MaBr4 cells were incubated with or
without 10 n M GBP in the medium containing 0.1%
dimethylsulfox-ide with 2 l M U73122 (PLC inhibitor), 10 l M TMB-8 (IP3receptor
antagonist), or 10 l M chelerythrine chloride (PKC inhibitor) at 25 C
for 6 h, and total RNA was prepared from cells after incubation.
Phosphorylation of ERK was observed 5 min after addition of
10 n M GBP to the MaBr4 incubation medium Each bar indicates
the mean ± SD of three independent determinations *Significantly
different from control (0 n M GBP; P < 0.05, Student’s t-test)
**Sig-nificantly different from control (0 n M GBP; P < 0.01, Student’s
t-test).
Fig 7 Schematic representation of hypothesized GBP signaling pathways in MaBr4 cells Stimulation of the putative GBP receptor activates PLC DAG, newly formed by activated PLC, stimulates PKC, and activated PKC induces ERK activation Independently of this signaling pathway, the IP 3 produced by activated PLC stimu-lates the IP3receptor to release Ca 2+ from the endoplasmic retic-ulum, which leads to the extracellular Ca 2+ influx that induces robust enhancement of TH and DDC gene expression ER, endo-plasmic reticulum; GBPR, growth-blocking peptide receptor; IP3R,
IP3receptor.
Trang 7dependent expression of both enzyme genes suggested
that the IP3 receptor-mediated Ca2+ influx from the
extracellular fluid plays an essential role in the
tran-scriptional enhancement of TH and DDC genes
(Fig 7) Although the GBP signaling pathways were
characterized by analyzing MaBr4 cells, it is
reason-able to assume that these pathways are employed in
armyworm epidermal cells It has been reported that
prothoracicotropic hormone (PTTH) stimulates Ca2+
influx into the prothoracic gland and also activates
ERK in the tobacco hornworm Manduca sexta
[26–28] PTTH-induced ERK activation is strongly
dependent on external Ca2+, and ERK activation is
induced by substitution of the calcium ionophore
A23187 for PTTH Therefore, the PTTH-induced
events in the prothoracic gland seem to be completely
different from those observed in the GBP signaling
pathway in the epidermis and MaBr4 cells
Once GBP was identified as a growth inhibitory
pep-tide in insects, a series of follow-up studies revealed its
diverse biological functions, including paralysis
induc-tion, cell proliferainduc-tion, and stimulation of immune cells
[29–31] This study clearly characterized two different
signaling pathways of GBP in epidermal cells and
cul-tured MaBr4 cells Although the multiple activities
reported for GBP may reflect differences in the types
of GBP receptors expressed by specific tissues or
devel-opmental stages of insects, divergence of the GBP
signaling pathway must also enable GBP to exert
different activities in the same cell
In summary, this study provides unequivocal
evi-dence that GBP stimulates Ca2+ influx into cultured
MaBr4 cells and subsequent enhancement of
expres-sion of the TH and DDC genes Furthermore, GBP
stimulates ERK phosphorylation, which is not
essen-tial for the enhancement of expression of either
enzyme gene One of the GBP pathway components
is PLC, from which the downstream signaling
path-ways diverge to ERK activation through PKC and
enzyme gene expression through IP3
receptor-medi-ated Ca2+ influx Therefore, it is reasonable to
con-clude that the GBP signaling pathway to expression
of the TH and DDC genes is unique in its ability to
function independently of the ERK–MAP kinase
kinase pathway
Experimental procedures
Animals
Pseudaletia separata larvae were reared on an artificial diet
at 25 ± 1C with a 16 h light ⁄ 8 h dark photoperiod [10]
Penultimate instar larvae undergoing ecdysis between 4 and
4.5 h after starting the light period were designated as day 0 last instar larvae
Chemicals Polyclonal antibody to MAP kinase (ERK-1, ERK-2), U0126, U73122, TMB-8 and chelerythrine chloride were purchased from Sigma-Aldrich Co (St Louis, MO, USA) Monoclonal antibody to active MAP kinase was obtained from Promega Co (Madison, WI, USA) A23187 (calcium ionophore) and Grace’s medium (Cat No 11595-030) were purchased from Nacalai Tesque Co (Kyoto, Japan) and Invitrogen Co (Carlsbad, CA, USA), respectively All other chemicals were of reagent grade
Dissection and culture of integument
A whole abdominal integument between the first and sec-ond segments was dissected from the test day 1 last instar larva of the armyworm Care was taken to remove all the adhering fat body tissue from the integument The dissected integument was separated into dorsal and ventral parts After being washed with NaCl⁄ Pi, the tissues were lightly blotted with filter paper, weighed, and immediately used for experiments
Pieces of dorsal larval integument were cultured in Grace’s medium with or without 10 nm GBP at 25C As
a control, 10 nm BSA was added to the medium To remove extracellular free Ca2+, Grace’s medium containing
1 mm EGTA was used A23187 was dissolved in dimethyl-sulfoxide and added to the medium
Cell culture The MaBr4 cell line established from M brassicae fat bodies was kindly provided by T Hiraoka (Tokyo Univer-sity of Agriculture and Technology), and maintained in Mitsuhashi⁄ Maramorosh insect medium (MM medium) at
25C [18]
RT-PCR and quantitative PCR Total RNA was isolated from integuments of test tissues or cultured cells using TRIzol reagent (Gibco-BRL, Rockville,
MD, USA), according to the manufacturer’s instructions Two micrograms of total RNA was reverse transcribed with oligo(dT) primer using ReverTra Ace (Toyobo, Osaka, Japan) The cDNA was amplified with a TH-specific primer pair (5¢-CAGCTGCCCAGAAGAACCGCGAGATG-3¢, +11 to +36 bp and 5¢-GAACTCCACGGTGAACCAGT-3¢, +1286 to +1305 bp), a DDC-specific primer pair (5¢-ATGGAGGCCGGAGATTTCAAAG-3¢, +1 to +22
bp and 5¢-ACGGGCTTTAAGTATTTCATCAGGC-3¢, +1405 to +1428 bp), and an actin primer pair (5¢-TTCG
Trang 8AGCAGGAGATGGCCACC-3¢ and 5¢-GAGATCCACAT
CTGYTGGAAGGT-3¢) PCR was conducted under the
following conditions: 25 cycles at 94C for 1 min, 50 C
for 1 min, and 72C for 2 min
Real-time quantitative PCR was used to determine the
relative expression levels of TH and DDC Quantitative
PCR was carried out with 2.5% of the reverse transcription
product in a 20 lL reaction volume of LightCycler Fast
DNA Master SYBR Green I (Roche Applied Science,
Indi-anapolis, IN, USA), using the Light-Cycler 1.2 instrument
and software (Roche Applied Science)
Immunoblotting analysis
Integuments dissected from larvae or cultured cells were
homogenized in 80 mm Tris⁄ HCl buffer (pH 8.8)
contain-ing 1% SDS and 2.5% 2-mercaptoethanol, and centrifuged
at 20 000 g for 10 min at 4C The supernatant was boiled
for 5 min and applied to SDS⁄ PAGE gel Proteins
sepa-rated by SDS⁄ PAGE were electrically transferred to a
poly(vinylidene difluoride) membrane filter, blocked, and
probed with the indicated primary antibody After being
washed thoroughly with 0.05% Tween-20 in Tris-buffered
saline (10 mm, 150 mm NaCl, pH 7.5), antigens were
detected using peroxidase-conjugated secondary antibody
and a 4-chloro-1-naphtol (4CN) Immun-Blot Colorimetric
Assay kit (Bio-Rad Laboratories, Hercules, CA, USA) [32]
All positive bands were quantified using imagej (NIH)
Confocal calcium imaging
MaBr4 cells were washed with Ca2+-free Carlson solution
(120 mm NaCl, 2.7 mm KCl, 0.5 mm MgCl2, 1.7 mm
NaH2PO4, 1.4 mm NaHCO3, 2.2 mm glucose), and loaded
with 10 lm Fluo-3AM (Dojindo Laboratories, Kumamoto,
Japan) at 25C for 30 min After loading, the cells were
washed twice in Ca2+-free Carlson solution by
sedimenta-tion and resuspension, and placed on the slide glass The
cells were stimulated with Grace’s medium with or without
1 nm GBP and immediately excited with light of 488 nm
wavelength by a confocal imaging system CellMap (Carl
Zeiss, Oberkochen, Germany)
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