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First published online May 1, 2009
Journal of Experimental Biology 212, 1535-1543 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.030197
Sympathetic outflow activates the venom gland of the snake Bothrops jararaca by regulating the activation of transcription factors and the synthesis of venom gland proteins
1 Laboratório de Farmacologia, Instituto Butantan, Av. Vital Brazil 1500,
05503-900, São Paulo, Brazil
2 Departamento de Fisiologia, Instituto de Biociências, Universidade de
São Paulo, Rua do Matão, travessa 14, 05508-900, São
Paulo, Brazil
* Author for correspondence (e-mail: norma{at}butantan.gov.br)
Accepted 12 March 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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1- and β-adrenoceptors in the venom gland. In this
study, we show that venom removal leads to the activation of transcription
factors NF
B and AP-1 in the venom gland. In dispersed secretory cells,
noradrenaline activated both NFB and AP-1. Activation of NFB and
AP-1 depended on phospholipase C and protein kinase A. Activation of
NFB also depended on protein kinase C. Isoprenaline activated both
NFB and AP-1, and phenylephrine activated NFB and later AP-1. We
also show that the protein composition of the venom gland changes during the
venom production cycle. Striking changes occurred 4 and 7 days after venom
removal in female and male snakes, respectively. Reserpine blocks this change,
and the administration of 1- and β-adrenoceptor
agonists to reserpine-treated snakes largely restores the protein composition
of the venom gland. However, the protein composition of the venom from
reserpinized snakes treated with 1- or β-adrenoceptor
agonists appears normal, judging from SDS-PAGE electrophoresis. A sexual
dimorphism in activating transcription factors and activating venom gland was
observed. Our data suggest that the release of noradrenaline after biting is
necessary to activate the venom gland by regulating the activation of
transcription factors and consequently regulating the synthesis of proteins in
the venom gland for venom production.
Key words: sympathetic innervation, transcription factors, protein synthesis, exocrine gland, snake, Bothrops jararaca
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). This
venom gland has two important characteristics: the presence of a central lumen
where most of the venom produced is stored, and a long venom production cycle
that lasts around 30–50 days, with distinct steps which include an
active stage after loss of venom from the lumen, and a quiescent stage when
the lumen is filled (Kochva,
1960; Kochva,
1987; Mackessy,
1991). Because the activation of the venom gland can be explored
step by step, it can be used to elucidate the mechanisms involved in the
activation of exocrine glands. Besides, snake venom is a rich source of active
molecules for bioprospecting studies and, therefore, understanding how the
venom is produced allows us to establish a functional secretory cell line in
order to obtain large-scale venom production in vitro.
After venom is lost from the lumen of the gland, a cycle of venom
production and secretion is initiated, which starts with morphological and
biochemical changes of the secretory epithelium. The epithelial cells change
their shape from cuboid to columnar, the cisternae of the rough endoplasmatic
reticulum expand, and venom is synthesized. The maximal synthetic activity of
the secretory cells and the highest mRNA concentrations are observed after
4–8 days. Afterward, the synthetic activity decreases, and venom
gradually accumulates in the gland lumen, while the epithelium returns to the
quiescent stage (Ben-Shaul et al.,
1971; Carneiro et al.,
1991; De Lucca et al.,
1974; Kochva,
1978; Oron and Bdolah,
1973; Rotenberg et al.,
1971). Thus, a complete venom production cycle is much longer than
the protein production cycle in mammalian salivary and pancreatic glands
(Amsterdam et al., 1969;
Jamieson and Palade, 1967a;
Jamieson and Palade,
1967b).
We have shown that the noradrenergic innervation present in the venom gland
has an essential role in triggering the venom production cycle. Both
1- and β-adrenoceptors are involved in this process
(Yamanouye et al., 1997;
Yamanouye et al., 2000;
Kerchove et al., 2004). The
1-adrenoceptor is desensitized immediately after venom
removal, suggesting that noradrenaline is released in the venom gland at this
time, and is resensitized 30 days later
(Kerchove et al., 2004). The
sensitivity of this receptor for noradrenaline and phenylephrine is low
(Kerchove et al., 2004). It is
coupled to a Gq protein, and triggers the venom production cycle by activating
the phosphatidylinositol 4,5-bisphosphate and extracellular signal-regulated
kinase (ERK) signalling pathways (Kerchove
et al., 2008). Like mammalian adrenoceptors, the
β-adrenoceptor is coupled to Gs protein, as its stimulation increases
cyclic AMP production, but its sensitivity to drugs differs from that of
mammalian adrenoceptors. This receptor seems to become uncoupled from its
second messenger system in activated cells
(Yamanouye et al., 2000).
Sympathetic outflow stimulates the synthesis of salivary proteins in
mammals (Barka et al., 1986;
Woon et al., 1993;
Ann and Lin, 1997;
Ann and Lin, 1998), and
sympathetic stimulation with isoprenaline changes the gene expression pattern
in the salivary gland (Ten Hagen et al.,
2002). These data led us to the hypothesis that noradrenergic
innervation could be important for the synthesis of venom proteins and/or
proteins involved in venom production. Thus, the aim of the present study was
to determine how noradrenergic innervation triggers the venom production
cycle. Here, we show that venom removal and noradrenaline activate the
transcription factors NFB and AP-1. We also show for the first time
that noradrenergic innervation is a key activator of this exocrine gland.
Noradrenergic stimulation is required for the production of venom gland
proteins, unlike in the mammalian salivary gland, where noradrenaline is
directly involved in the synthesis of salivary proteins.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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B and AP-1 consensus
oligonucleotides were purchased from Promega (Madison, WI, USA). T4
polynucleotide kinase was purchased from Invitrogen (Carlsbad, CA, USA).
Poly(dI-dC), [
32P]-ATP nucleotide and MicroSpin G-25 columns
were purchased from GE Healthcare Life Sciences (Piscataway, NJ, USA). NP40,
U-73122, myristoylated cell permeable protein kinase A inhibitor 14-22 amide
(PKI), H89 dihydrochloride and staurosporine from Streptomyces sp.
were purchased from Calbiochem (La Jolla, CA, USA). The protein assay reagent
was purchased from Bio-Rad Laboratories (Hercules, CA, USA). Sodium
pentobarbital was purchased from Cristália (São Paulo, SP,
Brazil). Other chemicals were of analytical or reagent grade, and were
purchased from commercial suppliers.
Animals and venom gland
Bothrops jararaca (Wied 1824) adults of both sexes
(N=88), weighing 150–400 g, were classified by the Laboratory
of Herpetology of the Instituto Butantan, and kept in a room under controlled
conditions instead of maintained as described by Breno and colleagues
(Breno et al., 1990). Snakes
were kept without access to food for 40 days before the start of the
experiment to make sure that most of the cells in the venom gland were in the
quiescent stage. Fasting periods of 1–2 months are common in snakes
living in the wild, but fasts can exceed 1 year
(Secor and Nagy, 1994). Animal
care and procedures used were in accordance with guidelines of the Animal
Ethics Committee of the Instituto Butantan and the Biomedical Science
Institute of the University of São Paulo.
Snakes were anaesthetized with sodium pentobarbital (30 mg
kg–1, s.c.) and decapitated, and the venom gland was removed
and freed from connective tissue
(Yamanouye et al., 2007). To
remove the venom, snakes were anaesthetized with sodium pentobarbital (20 mg
kg–1, s.c.) and the venom was removed manually
(Belluomini, 1968).
Preparation of dispersed cells
Venom glands were dissected from female snakes from which no venom had been
removed for at least 40 days in order to obtain cells in a quiescent stage.
Quiescent secretory cells were dispersed as previously described
(Yamanouye et al., 2000;
Kerchove et al., 2004;
Yamanouye et al., 2007) and
resuspended in Krebs-Hepes solution (composition in mmol l–1:
NaCl 120; KCl 4; MgSO4 1.2; KH2PO4 1.2; Hepes
15; CaCl2 2.5; with ascorbic acid 0.01% and glucose 10; pH 7.4) and
used immediately.
Nuclear protein extraction
Nuclear extracts were prepared as described by Rong and Baudry
(Rong and Baudry, 1996) with
some modifications. Briefly, dissected venom glands were homogenized (tissue
homogenizer with Teflon pestle, Thomas Scientific, Swedesboro, NJ, USA) in
cold lysis buffer (2.6 µl buffer mg–1 of wet tissue,
buffer composition: 10 mmol l–1 Hepes pH 7.5, 10 mmol
l–1 KCl, 0.1 mmol l–1 EDTA pH 8.0, 10%
glycerol, 1.0 mmol l–1 DTT, protease inhibitor cocktail
dilution 1:100) and incubated on ice for 15 min. A volume of 25 µl of 10%
nonidet P-40 was added to a final volume of 400 µl and the mixture was
vortexed for 10 s, and centrifuged at 12,000 g for 2 min at
4°C. The pellet was washed with lysis buffer and recentrifuged at 12,000
g for 2 min at 4°C. The nuclear pellet was resuspended in
nuclear extraction buffer (10 mmol l–1 Hepes pH 7.5, 0.5 mmol
l–1 KCl, 1 mmol l–1 EDTA pH 8.0, 10%
glycerol, 1.0 mmol l–1 DTT, protease inhibitor cocktail
dilution 1:100) at 0.7 µlmg–1 wet tissue, incubated on ice
for 15 min, and then centrifuged at 13,000 g for 20 min at
4°C. The supernatant containing the nuclear proteins was stored at
–70°C until use. The protein concentration of the nuclear extract of
venom gland was determined by the method described by Bradford
(Bradford, 1976).
To prepare nuclear extracts, 1.5x106–3x106 dispersed quiescent secretory cells were incubated in 400 µl cold lysis buffer and broken by repeated pipetting. The nuclear pellet was resuspended in 100 µl of nuclear extraction buffer. Other procedures were the same as for the nuclear extract of venom gland except the washing step, which was omitted.
Electrophoretic mobility shift assay
Double-stranded NFB (5'-AGTTGAGGGGACTTTCCCAGGC-3') or
AP-1 (5'-CGCTTGATGAGTCAGCCGGAA-3') consensus oligonucleotides were
end-labelled with [-32P]-ATP (specific activity of 3000 Ci
mmol–1) in the presence of 10 units T4 polynucleotide kinase
(10 min, 37°C). Unincorporated nucleotides were removed by passing the
reaction mixture through a Microspin G-25 column. A gel shift assay was
performed as described by Staal and colleagues
(Staal et al., 1990) with some
modifications. A total of 7–15 µg of nuclear extract protein or 15
µl of nuclear extract of secretory cells was incubated for 20 min at room
temperature with 2 µl gel shift binding buffer (50 mmol
l–1 NaCl, 0.2 mmol l–1 EDTA pH 8.0, 0.5 mmol
l–1 DTT, 10% glycerol, 10 mmol l–1 Tris-HCl
pH7.5) and 1 µg poly(dI-dC) in a final reaction mix volume of 22 µl.
Each sample was then incubated for 30 min at room temperature with
30,000–50,000 c.p.m. of purified 32P-labelled probe.
Protein–DNA complexes were separated by electrophoresis through a 6%
non-denaturing acrylamide:bis-acrylamide (37.5:1) gel in Tris-borate/EDTA (TBE
buffer 0.5x) at 150 V for 1.5 h at room temperature. The gels were
vacuum dried, and exposed to Kodak MS film for 4–7 days at
–70°C. Autoradiography films were scanned and analysed
densitometrically with MCDI M4 image analysis 3.0 software (Imaging Research,
St Catharine, Ontario, Canada). For competition studies, unlabelled NFB
or AP-1 double-strand oligonucleotide was added in molar excess over
radiolabelled probe.
Preparation of extract of the venom gland
Venom gland extracts were prepared based on the protocol described by
Gonçalves and colleagues
(Gonçalves et al.,
1997). Briefly, venom glands from which all venom had been removed
from the lumen were cut into 250 µm slices (McIlwain Tissue Chopper,
Vibratome Company, O'Fallon, MO, USA) and homogenized (tissue homogenizer,
Teflon pestle, Thomas Scientific); 15 g of homogenate was mixed with 100 ml
ice-cold solution containing 0.32 mol l–1 sucrose, 1 mmol
l–1 EDTA, 3 mmol l–1 MgCl2 and 1
mmol l–1 PMSF. The homogenate was centrifuged at 14,000
g for 40 min at 4°C. The supernatant containing cytosolic
proteins was recovered and stored at –20°C until use.
Analysis of protein composition
The protein concentration of venom gland extracts and venom was determined
by the method of Bradford (Bradford,
1976) using Bio-Rad reagents and bovine serum albumin as a
standard. Venom (10 µg protein) or venom gland extract (30 µg protein)
was denatured in sample buffer (Laemmli,
1970) for 5 min at 100°C. The proteins were separated by
SDS-PAGE (12% or 15%) with the buffer system described by Laemmli
(Laemmli, 1970). The proteins
were stained with Coomassie Brilliant Blue. The gel was scanned and the
density of the bands was quantified with Quantity One software (Bio-Rad).
Statistical analysis
The results are expressed as means ± s.e.m. of the indicated number
of experiments. Statistical comparisons were made by one-way analysis of
variance (ANOVA) followed by Newman–Keuls test for multiple comparisons
with Graph-Pad Prism 3.0 software (San Diego, CA, USA). A probability of less
than 0.05 was considered statistically significant.
Design of experiments
Time course of transcription factor activation after venom removal Venom
glands were obtained from female and male snakes (N=5 for each group)
from which no venom was removed (quiescent stage) and from snakes that had
their venom removed 30, 60 or 120 min before they were killed by decapitation.
Nuclear extract of the venom gland was prepared, and the activation of the
NFB and AP-1 transcription factors was analysed by the electrophoretic
mobility shift assay as described above.
Activation of transcription factors by stimulation of adrenoceptors in venom gland
Nuclear extracts were prepared from dispersed secretory cells obtained from
venom glands in the quiescent stage from female snakes (N=18). A
total of 1.5x106–3.0x106 cells was
incubated for 30 or 60 min at 30°C in Krebs-Hepes solution containing
noradrenaline (0.1 mmol l–1), phenylephrine (0.3 mmol
l–1) or isoprenaline (0.3 mmol l–1) in a
final volume of 500 µl. The concentrations used were based on the
EC50 of these agonists determined by functional studies using
microphysiometry (Kerchove et al.,
2004; M. B. Zablith and N.Y., unpublished data from our
laboratory).
To verify the upstream pathway, cells were incubated at 30°C with inhibitors of phospholipase C (PLC; U73122, 10 µmol l–1 for 60 min), protein kinase C (PKC; staurosporine, 100 nmol l–1, 30 min), PKA/PKC (H89, 90 µmol l–1, 30 min) or protein kinase A (PKA; PKI, 10 µmol l–1, 30 min). Noradrenaline (0.1 mmol l–1) was then added and the cells were incubated for another 30 min. The activation of transcription factors was analysed by the electrophoretic mobility shift assay described above and normalized to 1x106 cells. The venom gland of male snakes was not used because their smaller gland yielded fewer secretory cells.
Effect of adrenoceptor stimulation on venom composition
The venom of snakes was manually removed to start the venom production
cycle. Six snakes were treated with reserpine (20 mg kg–1,
s.c., 24 h before venom removal, followed by daily injections of 5 mg
kg–1, s.c. for 15 days). This dose of reserpine completely
eliminates noradrenaline from the venom gland
(Yamanouye et al., 1997). Some
reserpine-treated snakes received phenylephrine (100 mg kg–1,
s.c.) or isoprenaline (100 mg kg–1, s.c.) (Nunez-Burgos et
al., 1993) just after the removal of venom. A second sample of venom was
collected 15 days after the first collection. The protein composition of the
samples was analysed by SDS-PAGE. The first sample was used as a control to
reduce effects of inter-individual variation on venom composition
(Meier, 1986;
Chippaux et al., 1991;
Monteiro et al., 1998a;
Monteiro et al., 1998b).
Time course of changes in venom gland proteins
Venom glands were obtained from female and male snakes in which venom was
not removed previously (quiescent stage, N=2) and female and male
snakes that had their venom removed manually 4, 7 or 15 days (activated gland,
N=2 for each group) before they were killed by decapitation. The
venom gland proteins were separated by SDS-PAGE.
Effect of adrenoceptor stimulation on the composition of the venom gland
Four female and four male snakes were treated with reserpine (20 mg
kg–1, s.c., 24 h before the first venom removal, followed by
daily s.c. injections of 5 mg kg–1). Some reserpine-treated
snakes received phenylephrine (100 mg kg–1, s.c.) and
isoprenaline (100 mg kg–1, s.c.), just after the removal of
venom. Female snakes were killed 4 days later, male snakes were killed 7 days
later. The venom glands were dissected and the venom gland proteins were
separated by SDS-PAGE. Control snakes had their venom removed but were not
treated with reserpine, and they were killed 4 days (females) or 7 days
(males) later.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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B and AP-1 in the venom gland of Bothrops jararaca). Noradrenaline starts the venom production cycle by
stimulating both 1- and β-adrenoceptors
(Yamanouye et al., 1997;
Yamanouye et al., 2000;
Kerchove et al., 2004). We
examined whether venom removal could activate transcription factors such as
NFB and AP-1.
The transcription factors NFB and AP-1 were present in the nuclear
extract of the quiescent venom gland. Competition studies using unlabelled
NFB and AP-1 double-strand oligonucleotides showed that binding was
specific (Fig. 1A;
Fig. 2A).
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B in the venom gland
(Fig. 1A). As shown in
Fig. 1B, the activation of
NFB was increased in nuclear extracts of venom gland 30 min (% above
baseline level: 33.21±7.16, N=5 and 16.43±6.8,
N=4), 60 min (% above baseline level: 16.21±3.83, N=5
and 38.86±9.61, N=4) and 120 min (% above baseline level:
12.67±4.8, N=5 and 11.54±2.09, N=4) after
venom removal in female and male snakes, respectively. NFB activation
peaked 30 min after venom removal in female snakes and 60 min after venom
removal in male snakes (Fig.
1B). Venom removal also caused AP-1 activation (Fig. 2A). As shown in Fig. 2B, the activation of AP-1 was increased in nuclear extracts of venom gland 30 min (% above baseline level: 12.51±7.85, N=4 and 15.04±7.05, N=5), 60 min (% above baseline level: 91.96±25.65, N=4 and 56.07±14.96, N=5) and 120 min (% above baseline level: 42.67±18.89, N=4 and 31.08±9.67, N=5) after venom removal in female and male snakes, respectively. AP-1 activation peaked 60 min after venom removal (Fig. 2B, P<0.05) and was more pronounced in female than in male snakes (P<0.05).
Stimulation of 1- and β-adrenoceptors activates NFB and AP-1 in secretory cells
To examine the role of sympathetic outflow in the activation of
transcription factors, we used dispersed secretory cells from quiescent venom
glands of female snakes and stimulated them for 30 min with noradrenaline,
phenylephrine or isoprenaline. The venom glands of male snakes were not used
because they are smaller than female glands and yield few secretory cells.
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B (24.60±8.83% above baseline level, N=6;
Fig. 3B,C). Both the
-agonist phenylephrine (0.3 mmol l–1) and the
β-agonist isoprenaline (0.3 mmol l–1) also stimulated
NFB activation (% above baseline level: 19.31±5.29, N=6
and 30.35±4.58, N=8, respectively;
Fig. 3A,C) in quiescent
secretory cells stimulated for 30 min. Noradrenaline (0.1 mmol l–1 for 30 or 60 min) also caused the activation of AP-1 (% above baseline level: 12.01±4.21, N=3 and 19.19±5.92, N=4, respectively; Fig. 4A–D). Isoprenaline increased the activation of AP-1 in quiescent secretory cells stimulated for 30 and 60 min (% above baseline level: 25.49±7.73, N=3 and 25.52±12.30, N=5, respectively; Fig. 4A–D). However, phenylephrine only increased the activation of AP-1 after 60 min of stimulation (13.93±6.81% above baseline level, N=6; Fig. 4C,D). After 30 min of stimulation, phenylephrine decreased the activation of AP-1 (12.67±2.94% below baseline level, N=9; Fig. 4A,B).
|
In order to verify the upstream signalling pathway, we measured the effect
of inhibitors of PLC (U73122, 10 µmol l–1), PKC
(staurosporine, 100 nmol l–1), PKC/PKA (H89, 90 µmol
l–1) and PKA (PKI, 10 µmol l–1) on the
response to noradrenaline. As shown in
Table 1, U73122, staurosporine,
H89 and PKI significantly reduced the activation of NFB by
noradrenaline (0.1 mmol l–1, 30 min). All inhibitors tested
except staurosporine significantly reduced the activation of AP-1. We did not
examine upstream signalling pathways activated by 60 min of incubation with
noradrenaline because the effect was different from the sum of the adrenergic
agonists, suggesting the activation of additional mechanisms.
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Effect of reserpine and stimulation of 1- and β-adrenoceptors on venom composition
To examine whether the stimulation of 1- and
β-adrenoceptors affects the composition of the venom, we used reserpine
to deplete endogenous catecholamine stores
(Yamanouye et al., 1997) and
injected 1- or β-adrenoceptor agonist (100 mg
kg–1 s.c. each) (Nunez-Burgos et al., 1993). Due to
inter-individual variation in venom composition
(Meier, 1986;
Chippaux et al., 1991;
Monteiro et al., 1998a;
Monteiro et al., 1998b), we
used the first sample of venom of the same snake as the control.
As shown in Fig. 5, the
venom protein profile of samples collected with a 15 day interval was similar
in untreated control snakes (lanes 1 and 2). Reserpine completely abolished
venom production, as shown previously
(Yamanouye et al., 1997). The
administration of isoprenaline to reserpine-treated snakes restored venom
production, and the composition of the venom was similar to that of the
control sample (lanes 3 and 4). Similarly, phenylephrine restored venom
production, and the composition of the venom was like that of the control
sample (lanes 5 and 6).
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Stimulation of 1- and β-adrenoceptors in reserpine-treated snakes is accompanied by regulation of levels of specific venom gland proteins
We investigated the role of 1- and β-adrenoceptors
in the changes that occur in the composition of the venom gland during the
venom production cycle by treating the snakes with reserpine and giving
1-plus β-adrenoceptor agonists. Venom-free venom glands
of female snakes were examined 4 days after venom removal, and of male snakes
7 days after venom removal, as the maximal changes in venom gland composition
occurred at these times. Fig. 7
shows that reserpine caused large changes in the cytosolic protein profile of
the venom gland. The protein profile of reserpine-treated glands appeared
similar to that of quiescent glands (compare
Fig. 6A with
Fig. 7A, and
Fig. 6B with
Fig. 7B), with a reduced
density of bands of approximately 81, 69, 47, 44, 41 and 38 kDa in female
snakes, and a reduced density of bands of 81, 69, 57, 54, 47 and 44 kDa in
male snakes. In female snakes, the administration of isoprenaline with
phenylephrine (100 mg kg–1 of each, s.c.) at the time of
venom removal resulted in a pattern that appeared similar to that observed in
controls not treated with reserpine (Fig.
7A). However, in male snakes, adrenoceptor stimulation only partly
reversed the effect of reserpine (bands of 57 and 54 kDa;
Fig. 7B).
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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B and AP-1
are present in the snake venom gland, and that they are activated by the
removal of venom. Similar transcription factor activation is induced by
noradrenaline, which is normally released after the loss of venom from the
venom gland. In addition, we showed that the removal of venom induces changes
in the protein composition of the venom gland tissue. This effect depends on
the release of noradrenaline, because it is blocked by reserpine, and
noradrenaline is necessary for the production of new venom. However,
noradrenaline does not change venom protein synthesis. Therefore, it seems
that the production of the complex mixture of venom proteins is determined by
a fixed programme of biochemical changes in the venom-producing cells. The
start of this programme is triggered by noradrenergic stimulation, and both
1- and β-adrenoceptors participate in this process.
Activation of transcription factors is a complex process and involves
multiple intracellular signalling factors, including kinases such as PKC and
PKA (Sheng et al., 1991;
Karin, 1995;
McBride and Nemer, 1998). In
Bothrops jararaca venom gland, stimulation of
1-adrenoceptors in quiescent secretory cells increases total
inositol phosphate, mobilizes calcium from intracellular stores, and activates
PKC and ERK (Kerchove et al.,
2008), and stimulation of β-adrenoceptors in quiescent
secretory cells selectively increases cAMP production and cytosolic calcium
concentration by activating voltage-operated and receptor-operated
Ca2+ channels (Yamanouye et
al., 2000; M. B. Zablith and N.Y., unpublished data from our
laboratory). These messengers may interact with additional proteins and
subsequently activate transcription factors, contributing to regulation of
gene expression.
Venom removal or noradrenaline can activate NFB and AP-1 in the
venom gland. NFB activation was stimulated by both 1-
and β-adrenoceptor agonists. AP-1 activation seemed to depend mostly on
β-adrenoceptors; the delayed AP-1 activation (after an initial
inhibition) induced by 1-adrenoceptor stimulation may be a
subsequent event. Inhibitors of PLC, PKC and PKA reduced the activation of
NFB by noradrenaline, whereas AP-1 activation was reduced by PLC and
PKA inhibitors, but not by inhibition of PKC.
Our finding that the stimulation of 1- and
β-adrenoceptors causes activation of NFB and AP-1 in the venom
gland is in line with several studies that show that the stimulation of
- and β-adrenoceptors induces c-fos expression in a
variety of cells, including cardiac myocytes, vascular smooth muscle cells,
neuroblastoma cells, brown adipocytes and hepatocytes
(Iwaki et al., 1990;
Shilling et al., 1991; Okazaki
et al., 1994; Thonberg et al.,
1994; Garcia-Sáinz and
Alcántara-Hernández, 1996;
Im et al., 1998;
Taimor et al., 2004). All
-adrenoceptor subtypes were able to induce the expression of
c-fos and c-jun, and this effect seems to be mediated by
PKC, but 1-adrenoceptor subtypes vary in the efficacy of
this induction (García-Saínz
et al., 1998). Activation of NFB can be induced by both
- and β-adrenoceptors (Meldrun et al., 1997;
Aksoy et al., 2001;
Chandrasekar et al., 2004;
Lymperopoulos et al.,
2006).
The administration of reserpine blocked the production of new venom
(Yamanouye et al., 1997). The
protein profile of the reserpine-treated gland suggests that reserpine caused
the gland to remain in the quiescent stage. Reserpine has been used to examine
the role of sympathetic innervation of the salivary gland in rats, but it
causes irreversible morphological and functional changes in the salivary
glands, similar to those observed in patients with cystic fibrosis
(Martinez et al., 1975a;
Martinez et al., 1975b;
Watson et al., 1984;
Johnson, 1988). In the snake,
the effect of reserpine does not seem to be due to damage to the gland, as
adrenoceptor stimulation reverses the effect of reserpine
(Yamanouye et al., 1997). The
reversible effect of reserpine may be due to the capacity of the venom gland
to maintain a quiescent stage. In contrast, the salivary gland is constantly
in the activated stage.
The similarity of the effects of 1- and
β-adrenoceptor stimulation on the composition of the venom is remarkable,
as these receptors are coupled to different G proteins, release different
second messengers, and activate different transcription factors. Nevertheless,
both induced the production of the full gamut of venom proteins.
The administration of 1- and β-adrenoceptor agonists
to reserpine-treated snakes restores venom gland activation, suggesting that
noradrenaline is important in triggering the cells to start the venom
production programme; in other words, to activate the secretory cells to
produce venom. It is known that sympathetic innervation plays an important
role in stimulating the synthesis and secretion of salivary proteins
(Mehansho and Carlson, 1983;
Barka et al., 1986;
Woon et al., 1993;
Ann and Lin, 1997;
Ann and Lin, 1998). In
contrast, in the venom gland of the snake, noradrenergic stimulation seems to
regulate the production of components of the machinery for venom production,
rather than directly stimulating the synthesis of venom toxins. In accordance
with our results, Nunez-Burgos and colleagues (Nunez-Burgos et al., 1993) have
also shown that chronic administration of isoproterenol to snakes alters the
composition of proteins of the quiescent venom gland.
The importance of β-adrenoceptors in the synthesis of salivary gland
proteins is well known. Chronic administration of isoprenaline in mammals
leads to hyperplasia and hypertrophy of acinar cells and changes in the
content of many proteins in the salivary gland by increasing DNA and RNA
synthesis (Barka, 1965;
Brown-Grant, 1961;
Schneyer, 1962;
Selye et al., 1961;
Vugman and Hand, 1995;
Woon et al., 1993). Using
microarray technology, Ten Hagen and colleagues
(Ten Hagen et al., 2002)
showed early changes in gene expression in the murine parotid gland exposed to
isoprenaline. Proteins in several functional classes were affected and could
be related to the hyperplasia or hypertrophy found before. However, there are
no studies that have examined the role of salivary gland -adrenoceptors
in protein synthesis.
Our data also showed a sexual dimorphism in the time of maximal activation
of NFB, and the intensity of activation of AP-1 in the venom gland. The
difference in AP-1 activity may be partially caused by differences in
NFB activation, which is known to affect AP-1 activity
(Fujioka et al., 2004;
Krappmann et al., 2004). These
differences in the time course and level of the transcription factors may
contribute to the faster activation of the venom gland in female than in male
snakes (see Figs 6 and
7), and may also contribute to
the sexual dimorphism in activity and protein composition of Bothrops
jararaca venom reported by Menezes and colleagues
(Menezes et al., 2006).
Sex-based variation also occurs among bradykinin-potentiating peptides present
in the venom (Pimenta et al.,
2007). It is interesting to note that gene expression in the human
parotid gland is also gender dependent
(Srivastava et al., 2008).
In conclusion, we showed that noradrenaline released after venom removal
promotes the activation of transcription factors, by stimulating both
1- and β-adrenoceptors, and as a consequence changes
the synthesis of proteins of the venom gland, and that these proteins are
involved in activating the gland. Further studies are needed to identify the
proteins involved in venom gland activation. We also showed sex-based
differences in venom gland activation. It is important to point out that the
venom gland of Viperidae snake could be an attractive model to study
physiological regulation of protein synthesis in exocrine glands as, in
contrast to salivary glands, the venom gland can assume distinct quiescent and
activated stages. It is interesting to note that the sympathetic nervous
system is not involved in the synthesis of toxin proteins as seen in salivary
proteins of salivary glands.
LIST OF ABBREVIATIONS
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
|---|
Aksoy, M. O., Bin, W., Yang, Y., Yun-You, D. and Kelsen, S.
G. (2001). Nuclear factor-kB augments
β2-adrenergic receptor expression in human airway epithelial
cells. Am. J. Physiol. Lung Cell Mol. Physiol.
281,L1271
-L1278.
Amsterdam, A., Ohad, I. and Shramm, M. M.
(1969). Dynamic changes in the ultrastructure of acinar of the
rat parotid gland during the secretory cycle. J. Cell
Biol. 41,753
-773.
Ann, D. K. and Lin, H. (1997). Regulation of
salivary-gland-specific gene expression. Crit. Rev. Oral Biol.
Med. 8,244
-252.
Ann, D. K. and Lin, H. (1998). Transcriptional regulation of salivary proline-rich protein gene expression. Ann. NY Acad. Sci. 842,108 -114.[CrossRef][Medline]
Barka, T. (1965). Stimulation of DNA synthesis by isoproterenol in the salivary gland. Exp. Cell Res. 39,355 -364.[CrossRef][Medline]
Barka, T., Yagil, C., Van Der Noen, H. and Naito, Y. (1986). Induction of the synthesis of a specific protein in rat submandibular gland by isoproterenol. Lab. Invest. 54,165 -171.[Medline]
Belluomini, H. E. (1968). Extraction and quantities of venom obtained from some Brazilian snakes. In Venomous Animals and Their Venoms (ed. W. Buherl, V. Buckley and V. Deulofeu), pp. 97-117. New York: Academic Press.
Ben-Shaul, Y., Lifshitz, S. H. and Kochva, E. (1971). Ultrastructural aspects of secretion in the venom glands of Vipera palaestinae. In Toxins of Animal of Plants Origin (ed. A. De Vries and E. Kochva), pp.87 -105. London: Gordon and Breach.
Bradford, M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248 -254.[CrossRef][Medline]
Breno, M. C., Yamanouye, N., Prezoto, B. C., Lazari, M. F. M., Toffoletto, O. and Picarelli, Z. P. (1990). Maintenance of the snake Bothrops jararaca (Wied, 1824) in captivity. The Snake 22,126 -130.
Brown-Grant, K. (1961). Enlargement of salivary gland in mice treated with isopropylnoradrenaline. Nature 191,1076 -1078.[CrossRef]
Carneiro, S. M., Pinto, V. R., Jared, C., Lula, L. A. B. M., Faria, F. P. and Sesso, A. (1991). Morphometric studies on venom secretory cells from Bothrops jararacussu (jararacuçu) before and after venom extraction. Toxicon 29,569 -580.[Medline]
Chandrasekar, B., Marelli-Berg, F. M., Tone, M., Bysani, S.,
Prabhu, S. D. and Murray, D. R. (2004). β-Adrenergic
stimulation induces interleukin-18 expression via β2-AR, PI3K, Akt, IKK
and NF-B. Biochem. Biophys. Res. Commun.
319,304
-311.[CrossRef][Medline]
Chippaux, J. P., Williams, V. and White, J. (1991). Snake venom variability: methods of study, results and interpretation. Toxicon 29,1279 -1303.[Medline]
De Lucca, F. L., Haddad, A., Kochva, E., Rothschild, A. M. and Valeri, V. (1974). Protein synthesis and morphological changes in the secretory epithelium of the venom gland of Crotalus durissus terrificus at different times after manual extraction of venom. Toxicon 12,361 -369.[Medline]
Fujioka, S., Niu, J., Schmidt, C., Sclabas, G. M., Peng, B.,
Uwagawa, T., Li, Z., Evans, D. B., Abbruzzese, J. L. and Chiao, P. L.
(2004). NF-B and AP-1 connection: Mechanims of
NF-B-dependent regulation of AP-1 activity. Mol. Cell.
Biol. 24,7806
-7819.
García-Saínz, J. A. and
Alcántara-Hernández, R. (1996).
1- and β-adrenoceptor activation increase c-fos
expression in isolated guinea pig hepatocytes. Pharmacol.
Commun. 7,107
-113.
García-Saínz, J. A.,
Alcántara-Hernández, R. and Vázquez-Prado, J.
(1998). 1-Adrenoceptor subtype activation increases
proto-oncogene mRNA levels. Role of protein kinase C. Eur. J.
Pharmacol. 342,311
-317.[CrossRef][Medline]
Gonçalves, L. R. C., Yamanouye, N., Nuñez-Burgos, G. B., Furtado, M. F. D., Britto, L. R. G. and Nicolau, J. (1997). Detection of calcium-binding proteins in venom and Duvernoy's glands of South American snakes and their secretions. Comp. Biochem. Physiol. 118C,207 -211.
Im, Y. B., Won, J. S., Suh, H. W., Huh, S. O., Kim, Y. H. and Song, D. K. (1998). Differential effects of adrenaline and noradrenaline on the hepatic expression of immediate early gene in mice. J. Auton. Pharmacol. 18,149 -155.[CrossRef][Medline]
Iwaki, K., Sukhatma, V. P., Shubeita, H. E. and Chien, K. R.
(1990). Alpha- and beta-adrenergic stimulation induces distinct
patterns of immediate early gene expression in neonatal rat myocardial cells.
fos/jun expression is associated with sarcomere assembly: Egr-1 induction is
primarily an alpha 1-mediated response. J. Biol. Chem.
265,13809
-13817.
Jamieson, J. D. and Palade, G. E. (1967a).
Intracellular transport of secretory proteins in the pancreatic exocrine cell.
I. Role of the peripheral elements of the Golgi complex. J. Cell
Biol. 34,577
-596.
Jamieson, J. D. and Palade, G. E. (1967b).
Intracellular transport of secretory proteins in the pancreatic exocrine cell.
II. Transport to condensing vacuoles and zymogen granules. J. Cell
Biol. 34,597
-615.
Johnson, D. A. (1988). Changes in rat parotid saliva protein composition following chronic reserpine treatment and their relation to inanition. Arch. Oral Biol. 33,463 -466.[CrossRef][Medline]
Karin, M. (1995). The regulation of AP-1 activity by mitogen-activated protein kinases. J. Biol. Chem. 2816483 -16486.
Kerchove, C. M., Carneiro, S. M., Markus, R. P. and Yamanouye,
N. (2004). Stimulation of the -adrenoceptor triggers
the venom production cycle in the venom gland of Bothrops jararaca.J. Exp. Biol. 207,411
-416.
Kerchove, C. M., Luna, M. S. A., Zablith, M. B., Smaili, S. S. and Yamanouye. N. (2008). Alpha-adrenoceptor triggers snake venom production cycle in secretory cells by activating phosphatidylinositol 4,5-bisphosphate hydrolysis and ERK signaling pathway. Comp. Biochem. Physiol A 150,431 -437.[CrossRef][Medline]
Kochva, E. (1960). A quantitative study of
venom secretion by Vipera palaestinae. Am. J. Trop. Med.
Hyg. 9,381
-390.
Kochva, E. (1978). Oral glands of the reptilia. In Biology of the Reptilia 8b (ed. C. Gans), pp.43 -161. London: Academic Press.
Kochva, E. (1987). The origin of snakes and evolution of the venom apparatus. Toxicon 25, 65-106.[Medline]
Kochva, E. and Gans, C. (1970). Salivary glands of snakes. Clin. Toxicol. 3, 363-387.[Medline]
Krappmann, D., Wegener, E., Sunami, Y., Esen, M., Thiel, A.,
Mordmuller, B. and Scheidereit, C. (2004). The IB
kinase complex and NFB act as master regulators of
lipopolysaccharide-induced gene expression and control subordinate activation
of AP-1. Mol. Cell. Biol.
24,6488
-6500.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[CrossRef][Medline]
Lymperopoulos, A., Karkoulias, G., Koch, W. J. and Flordellis,
C. S. (2006). 2-Adrenergic receptor subtype-specific
activation of NF-B in PC12 cells. Neurosci.
Lett. 402,210
-215.[CrossRef][Medline]
Mackessy, S. P. (1991). Morphology and ultrastructure of the venom gland of the Northen pacific rattlesnake Crotalus viridis oreganus. J. Morphol. 208,109 -128.[CrossRef]
Martinez, J. R., Adelstein, E., Quissell, D. O. and Barbero, G. (1975a). The chronically reserpinized rat as a possible model for cystic fibrosis. I. Submaxillary gland morphology and ultrastructure. Pediatr. Res. 9, 463-469.[Medline]
Martinez, J. R., Adshead, P. C., Quissell, D. O. and Barbero, G. (1975b). The chronically reserpinized rat as a possible model for cystic fibrosis. II. Composition and cilioinhibitory effects of submaxillary saliva. Pediatr. Res. 9, 470-475.[Medline]
McBride, K. and Nemer, M. (1998). The
C-terminal domain of c-fos is required for activation of an AP-1 site specific
for jun-fos heterodimers. Mol. Cell. Biol.
18,5073
-5081.
Mehansho, H. and Carlson, D. M. (1983).
Induction of protein and glycoprotein synthesis in rat submandibular glands by
isoproterenol. J. Biol. Chem.
258,6616
-6620.
Meier, J. (1986). Individual and age-dependent variations in the venom of the fer-de-lance (Bothrops atrox). Toxicon 24,41 -46.[Medline]
Meldrum, D. R., Cleveland, J. C., Jr, Sheridan, B. C., Rowland,.
R. T., Selzman, C. H., Banerjee, A. and Harken, A. H. (1997).
-adrenergic activation of myocardial NFkB during hemorrhage.
J. Surgical Res. 69,268
-276.[CrossRef][Medline]
Menezes, M. C., Furtado, M. F., Travaglia-Cardoso, S. R., Camargo, A. C. M. and Serrano, S. M. T. (2006). Sex-related individual variantion of snake venom proteome among eighteen Bothrops jararaca siblings. Toxicon 47,304 -312.[Medline]
Monteiro, R. Q., Dutra, D. L., Machado, O. L., Carlini, C. R., Guimarães, J. A., Bon, C. and Zingali, R. B. (1998a). Bothrops jararaca snakes produce several bothrojaracin isoforms following an individual pattern. Comp. Biochem. Physiol. B 120,791 -798.[CrossRef][Medline]
Monteiro, R. Q., Yamanouye, N., Carlini, C. R., Guimarães, J. A., Bon, C. and Zingali, R. B. (1998b). Variability of bothrojaracin isoforms and other venom principle in individual jararaca (Bothrops jararaca) snakes maintained under seasonally invariant conditions. Toxicon 36,153 -163.[Medline]
Nunes-Burgos, G. B., Gonçalves, L. R. C., Furtado, M. F. D., Fernandes, W. and Nicolau, J. (1993). Alteration of the protein composition of Bothrops jararaca venom and venom gland by isoproterenol treatment. Int. J. Biochem. 25,1491 -1496.[CrossRef]
Okasaki, M., Hu, Z., Fujinaga, M. and Hoffman, B. B. (1994). Alpha-1 adrenergic receptor-induced c-fos gene expression in rat aorta and cultured vascular smooth muscle cells. J. Clin. Invest. 94,210 -218.[Medline]
Oron, U. and Bdolah, A. (1973). Regulation of
protein synthesis in venom gland of viperid snakes. J. Cell
Biol. 56,177
-190.
Pimenta, D., Prezoto, B. C., Konno, K., Melo, R. L., Furtado, M. F., Camargo, A. C. M. and Serrano, S. M. T. (2007). Mass spectrometric analysis of individual variability of Bothrops jararaca venom peptide fraction: evidence for sex-based variation among the bradykinin-potentiating peptides. Rapid Commun. Mass Spectrom. 21,1034 -1042.[CrossRef][Medline]
Rong, Y. and Baudry, M. (1996). Seizure activity results in a rapid induction of nuclear factor-kB in adult but not juvenile rat limbic structures. J. Neurochem. 67,662 -668.[Medline]
Rotenberg, D., Bamberger, E. S. and Kochva, E. (1971). Studies of ribonucleic and synthesis in the venom glands of Vipera palaestinae (Ophidia, Reptilia). Biochem. J. 121,609 -612.[Medline]
Schneyer, C. A. (1962). Salivary gland changes
after isoproterenol-induced enlargement. Am. J.
Physiol. 203,232
-236.
Secor, S. M. and Nagy, K. (1994). Bioenergetic correlates of foraging mode for the snakes Crotalus cerastes and Masticophis flagellum. Ecology 75,1600 -1614.[CrossRef]
Selye, H., Veilleux, R. and Cantin, M. (1961).
Excessive stimulation of salivary gland growth by isoproterenol.
Science 133,44
-45.
Sheng, M., Thompson, M. A. and Greenberg, M. E.
(1991). CREB: a Ca2+-regulated transcription factor
phosphorylated by calmodulin-dependent kinases.
Science 252,1427
-1430.
Shilling, K., Luk, D., Morgan, J. I. and Curran, T.
(1991). Regulation of a fos-lacZ fusion gene: a paradigm for
quantitative analysis of stimulus-transcription coupling. Proc.
Natl. Acad. Sci. USA 88,5665
-5669.
Srivastava, A., Wang, J., Zhou, H., Melvin, J. E. and Wong, D. T. (2008). Age and gender related differences in human parotid gland gene expression. Arch. Oral Biol. 53,1058 -1070.[CrossRef][Medline]
Staal, F. J., Roederer, M. and Herzenberg, L. A.
(1990). Intracellular thiols regulate activation of nuclear
factor kappa B and transcription of human immunodeficiency virus.
Proc. Natl. Acad. Sci. USA
87,9943
-9947.
Taimor, G., Schlüter, K. D., Best, P., Helmig, S. and
Piper, H. M. (2004). Transcription activator protein 1
mediates - but not β-adrenergic hypertrophic growth responses in
adult cardiomyocytes. Am. J. Physiol. Heart Circ.
Physiol. 286,H2369
-H2375.
Ten Hagen, K. G., Balys, M. M., Tabak, L. A. and Melvin, J.
E. (2002). Analysis of isoproterenol-induced changes in
parotid gland gene expression. Physiol. Genomics
8, 107-114.
Thonberg, H., Zhang, S. J., Tvrdik, P., Jacobsson, A. and
Nedergaard, J. (1994). Norepinephrine utilizes
1- and β-adrenoceptors synergistically to maximally
induce c-fos expression in brown adipocytes. J. Biol.
Chem. 269,33179
-33186.
Vugman, I. and Hand, A. R. (1995). Quantitative immunocytochemical study of secretory protein expression in parotid glands of rats chronically treated with isoproterenol. Microsc. Res. Tech. 31,106 -117.[CrossRef][Medline]
Watson, E., Dowd, F., Jacobson, K. and Horwitz, H.
(1984). Reserpinization: effects on parotid gland function.
J. Dent. Res. 63,82
-86.
Woon, P. Y., Jeyaseelan, K. and Thiyagarajah, P. (1993). Adrenergic regulation of RNA synthesis in the rat parotid gland. Biochem. Pharmacol. 45,1395 -1401.[CrossRef][Medline]
Yamanouye, N., Britto, L. R. G., Carneiro, S. M. and Markus, R. P. (1997). Control of venom production and secretion by sympathetic outflow in the snake Bothrops jararaca. J. Exp. Biol. 200,2547 -2556.[Abstract]
Yamanouye, N., Carneiro, S. M., Scrivano, C. N. and Markus, R. P. (2000). Characterization of β-adrenocptors responsible for venom production in the venom gland of the snake (Bothrops jararaca). Life Sci. 67,217 -226.[CrossRef][Medline]
Yamanouye, N., Kerchove, C. M., Moura-Da-Silva, A. M., Carneiro, S. M. and Markus, R. P. (2007). Long-term primary culture of secretory cells of Bothrops jararaca venom gland for venom production in vitro. Nat. Protoc. 1,2763 -2766.[CrossRef]
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