Stimulation of the -adrenoceptor triggers the venom production cycle in the venom gland of Bothrops jararaca
1 Laboratório de Farmacologia, Instituto Butantan, Av. Vital Brazil,
1500, 05503-900, São Paulo, Brazil
2 Laboratório de Biologia Celular, Instituto Butantan, Av. Vital
Brazil, 1500, 05503-900, São Paulo, Brazil
3 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 30 October 2003
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Summary |
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Key words: -adrenoceptor, desensitization, venom production, venom gland, Bothrops jararaca, snake
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Introduction |
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After a bite or manual extraction of venom, the amount of venom inside the
lumen decreases and the secretory epithelium undergoes morphological and
biochemical changes. The epithelial cells change their shape from cuboid to
columnar, the cisternae of the rough endoplasmic reticulum expand, and venom
is synthesized. The maximal synthetic activity of the secretory cells and the
highest mRNA concentration are observed 48 days after manual
extraction. After that, the synthetic activity decreases and the venom is
gradually accumulated in the gland lumen, while the epithelium returns to a
quiescent stage (Ben-Shaul et al.,
1971; Rotenberg et al.,
1971
; Oron and Bdollah,
1973
; De Lucca et al.,
1974
; Kochva,
1978
; Carneiro et al.,
1991
; Yamanouye et al.,
1997
). A complete venom production cycle lasts around 3050
days.
The mechanisms that control the regulation of venom synthesis and secretion
are not well understood. The noradrenenergic innervation of the venom gland
appears essential for the venom production cycle because depletion of
catecholamines by administration of reserpine blocks the cycle
(Yamanouye et al., 1997). Both
- and ß-adrenoceptors seem involved, as both the
-adrenoceptor agonist phenylephrine and the ß-adrenoceptor agonist
isoprenaline reverted many of the morphological changes in the venom gland
seen after administration of reserpine
(Yamanouye et al., 1997
).
The aim of the current study was to investigate the role of the
-adrenoceptor during the venom production cycle in the Bothrops
jararaca venom gland. Here, by functional studies using microphysiometry,
we demonstrate that the
-adrenoceptor present in the snake venom gland
has low sensitivity to noradrenaline and phenylephrine and undergoes a
long-term desensitization after stimulation. We also show that stimulation of
the
-adrenoceptor is essential for the onset of venom production.
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Materials and methods |
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Drugs
L-phenylephrine hydrochloride, ()-arterenol bitartrate,
()-propranolol hydrochloride, crystalline reserpine, hyaluronidase,
antibiotics and antimycotic powder and 0.4% Trypan Blue solution were
purchased from Sigma Chemical Co. (St Louis, MO, USA). Collagenase was
purchased from Worthington Biochem. Co. (Lakewood, NJ, USA). Sodium
pentobarbital was purchased from Cristália (São Paulo, Brasil).
DMEM and foetal bovine serum were purchased from Gibco (Rockville, MD, USA).
Other chemicals were of analytical or reagent grade, and were purchased from
commercial suppliers.
Preparation of dispersed cells
Preparation of dispersed cells was based on the protocol described by
Yamanouye et al. (2000) with
some modifications. Briefly, snakes were anesthetized with sodium
pentobarbital (30 mg kg1, s.c.), decapitated and the venom
glands removed. The venom glands were freed from connective tissue and venom,
and were cut into slices of 250 µm (McIlwain Tissue Chopper
Brinkmann). Cells were dispersed in an orbital shaker for 90 min at room
temperature in KrebsHepes solution (composition in mmol
l1: NaCl 120; KCl 4; MgSO4 1.2;
KH2PO4 1.2; Hepes 15 and glucose 10; pH 7.4) containing
collagenase (3 U mg1 of wet tissue), hyaluronidase (3.5 U
mg1 wet tissue) and antibiotics and antimycotic (100 i.u.
ml1 penicillin, 100 µg ml1 streptomycin
and 0.25 µg ml1 amphotericin B, respectively). After
dispersion, the cells were washed with Dulbecco's Modified Eagle Medium (DMEM)
containing sodium bicarbonate (40 mmol l1), antibiotics and
antimycotic, filtered through a nylon mesh, then washed again and resuspended
in DMEM containing 10% foetal bovine serum. All procedures were done under
sterile conditions. Viable cells were counted in the presence of 0.4% Trypan
Blue and 2x106 cells well1 were plated in a
24-well plate. The dispersed cells were cultured at 30°C in a humidified
incubator (5% CO2) for up to 2 days.
Measurement of extracellular acidification rate
Energy metabolism in living cells is tightly coupled to cellular ATP usage,
so that any events that perturb cellular ATP levels, such receptor activation
and initiation of signal transduction, will result in a change in acid
excretion. Changes in the rate of cellular acid secretion have been used as a
real-time indicator for changes in functional activity upon receptor
stimulation in a wide variety of cells
(Hafner, 2000), and have been
used to characterize G protein-coupled receptor
(Pihlavisto and Scheinin,
1999
; MacLennan et al.,
2000
; Meloy et al.,
2001
).
Extracellular acidification rates were measured using a four-channel Cytosensor Microphysiometer system (Molecular Devices, Menlo Park, CA, USA). Dispersed cells were suspended in a 30% agarose solution and 0.51.0x106 cells were placed in a capsule cup. The capsule cups were loaded into the sensor chamber and the chambers were perfused at a flow of 100 µl min1 with bicarbonate-free DMEM (containing 40 mmol l1 NaCl and 0.57 mmol l1 ascorbic acid) at 30°C. The cells were allowed to equilibrate for at least 45 min before exposure to agonists. The pump cycle time was 3 min, which included a 40 s pump-off period during which the acidification rate was measured. Drugs were diluted and perfused through a second fluid path to the sensor chamber. Cells were exposed to agonists for 1 min and a 30 min wash period was employed between successive agonist exposures. This stimulation protocol was found to induce reproducible responses in preliminary experiments. The rate of extracellular acidification was calculated by the Cytosoft Program (Molecular Devices Corporation, Sunnyvale, CA, USA). Changes in the rate of acidification were calculated as the difference between the maximum effect after agonist addition and the measurement taken immediately before agonist addition (basal acidification rate). Baseline acidification rates were normalized to 100% and changes due to agonist exposure were calculated as percent increases over normalized baseline.
Design of experiments
Response of venom gland cells to -adrenoceptor agonists
Venom glands were removed from snakes from which no venom had been
extracted for at least 40 days in order to have cells in a quiescent stage.
Dispersed cells were placed in a Cytosensor system for the measurement of
extracellular acidification rate. Doseresponse curves were constructed
for phenylephrine and noradrenaline. These experiments were done in the
presence of 104 mol l1 propranolol to
block ß-adrenoceptors present in the venom gland (Yamanouye et al.,
1997,
2000
). This dose of
propranolol completely blocks the response of isoprenaline, a
ß-adrenoceptor agonist, in this preparation.
-adrenoceptor sensitivity throughout the venom production cycle
We used snakes that had fasted for 40 days, and from which no venom was
extracted during this period. After that, we extracted the venom and waited a
variable period (<5 min, and 4, 7, 15 or 30 days) until the snake was
killed and cells harvested from the venom gland. A control group was treated
similarly except that at the time of sacrifice no venom had been extracted for
at least 40 days. Dispersed cells were placed in the Cytosensor system and
doseresponse curves to phenylephrine were constructed.
Effect of in vitro stimulation of -adrenoceptors in the venom gland on the sensitivity of these receptors
Quiescent cells were harvested from snakes from which no venom had been
extracted for at least 40 days. The dispersed cells were incubated for 5 min
with a high concentration of noradrenaline (104 mol
l1), kept at 30°C for 24 h, and placed in the Cytosensor
system for the construction of doseresponse curves to
phenylephrine.
Stimulation of the -adrenoceptor and cellular machinery involved in venom production
Snakes were injected with reserpine (20 mg kg1 body mass,
s.c.) to deplete endogenous catecholamine stores. Venom was extracted 24 h
later, and venom extraction was immediately followed by an injection of
phenylephrine (100 mg kg1 body mass, s.c.) or vehicle. On
the day of venom extraction and the subsequent 13 days, snakes continued to
receive injections with reserpine (5 mg kg1
day1, s.c.). 1 day after the last injection, the snakes were
anesthetized and killed and their venom glands removed. The venom glands were
prepared for electron microscopy as described by Carneiro et al.
(1991). Ultrathin sections (70
nm) were analyzed using an LEO 906E transmission electron microscope.
Data analysis and statistics
Doseresponse curves were fitted through a non-linear regression and
pD2 values (logEC50) were calculated using the
curve-fitting program Graph-Pad PRISM 3.0 (GraphPad Software, San Diego, CA,
USA). The results are expressed as means ± S.E.M. of at
least 3 independent experiments with 512 replicates each. Statistical
significance (P<0.05) was assessed using Student's t-test
to compare two values, or analysis of variance (ANOVA) followed by the
NewmanKeuls test for multiple comparisons.
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Results |
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Noradrenaline and phenylephrine increased the rate of extracellular acidification in a dose-dependent manner in dispersed cells in the quiescent stage of the venom production cycle (Fig. 1). The pD2 values were 4.77±0.09 (N=7) and 3.75±0.07 (N=11), respectively. The pD2 values for phenylephrine in absence or presence of propranolol were not significantly different (data not shown).
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-adrenoceptor sensitivity throughout the venom production cycle
In this experiment we measured the effect of the -adrenoceptor
agonist phenylephrine on venom gland cells that were harvested from the snake
at various times after venom extraction. The pD2 fell from
3.75±0.07 (N=11) in quiescent cells to 3.27±0.02
(N=6) immediately after venom extraction (within 5 min,
P<0.05, Fig. 2),
and remained reduced for at least 15 days: day 4, 3.32±0.04
(N=6); day 7, 3.31±0.03 (N=5) and day 15,
3.29±0.04 (N=6), P<0.05). 30 days after venom
extraction, pD2 had returned to quiescent cells values
(3.64±0.04, N=6).
|
Effect of in vitro stimulation of -adrenoceptors in the venom gland on the sensitivity of these receptors
We hypothesized that the long-term desensitization might be due to
stimulation of the -adrenoceptor by noradrenaline released during venom
extraction. To test if administration of noradrenaline desensitizes the
-adrenoceptors in the venom gland, quiescent cells were dispersed,
incubated for 5 min with a high concentration of noradrenaline
(104 mol l1), and kept at 30°C for the
next 24 h. This treatment displaced the doseresponse curve for
phenylephrine to the right (P<0.05,
Fig. 3). The pD2
values in cells stimulated with noradrenaline (3.20±0.02, N=6)
was close to the value seen in cells harvested soon after extraction of venom
(3.27±0.02, N=6).
|
Stimulation of the -adrenoceptor and cellular machinery involved in venom production
After venom extraction, the secretory cells increase in size and assume a
columnar shape, the rough endoplasmic reticulum intracisternal spaces expand,
many secretory vesicles appear near apical membrane and the Golgi apparatus
become well developed (Yamanouye et al.,
1997). In snakes that were treated with reserpine for 15 days,
beginning the day before the extraction of venom, the cisternae of the rough
endoplasmic reticulum were narrow and parallel and the Golgi apparatus
appeared quiescent (Fig. 4A; Yamanouye et al., 1997
). In a
previous study we show that chronic treatment with phenylephrine (100 mg
kg1 body mass, s.c., 10 days after venom extraction)
reverses the effect of reserpine on morphology of venom gland cells
(Yamanouye et al., 1997
).
Considering that
-adrenoceptors undergo a long-term desensitization we
hypothesized that their stimulation can trigger the venom production cycle. In
this study, a single injection of phenylephrine (100 mg kg1
body mass, s.c.) given immediately after the extraction of venom, reversed the
effect of reserpine: the morphology of venom gland cells after this treatment
(Fig. 4B) seemed similar to
that of untreated cells. This corroborates our hypothesis about the importance
of the
-adrenoceptors at the onset of the venom production cycle.
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Discussion |
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Although the sensitivity of the -adrenoceptor in the venom gland is
low, it may have an important role because the amount of noradrenaline in the
venom gland is high (approximately 3 ng mg1 wet tissue;
Yamanouye et al., 1997
), much
higher than in the salivary glands, trachea and heart of the rat or salivary
glands and brain of the mouse (De Avellar
and Markus, 1990
; Murai et
al., 1995
). We have previously shown that the ß-adrenoceptor
in the venom gland also has a low sensitivity to ß-adrenoceptor ligands
(Yamanouye et al., 2000
).
Our study shows that the -adrenoceptor became desensitized
immediately after the extraction of venom. Desensitization also occurred when
venom gland cells were incubated for 5 min with 104 mol
l1 noradrenaline, which suggests that the
-adrenoceptor desensitization after venom extraction is due to release
of noradrenaline in the venom gland. Whether this rapid desensitization was
due to uncoupling or to internalization of the receptor is not clear. We have
previously shown that ß-adrenoceptors in the venom gland become
desensitized by extraction of venom
(Yamanouye et al., 2000
),
suggesting that this desensitization is due to uncoupling. The
-adrenoceptor remained desensitized for at least 15 days. The long time
necessary for recovery of the response suggests that a downregulation process
occurred. However, sensitivity was restored 30 days after the extraction of
venom. Since both the venom production cycle and
-adrenoceptor
resensitization take around 30 days, it seems that the machinery necessary for
initiating a new cycle is only ready when the gland is full of venom and the
secretory cells have entered the quiescent stage.
We have previously shown that the effect of reserpine on morphology of the
venom gland can be blocked with a chronic treatment of phenylephrine (100 mg
kg1 body mass day, s.c., for 10 days). The current finding
that a single dose of phenylephrine of 100 mg kg1 body mass,
given immediately after venom extraction, is as effective as chronic treatment
supports our idea that release of noradrenaline in the venom gland is
especially pronounced immediately after the extraction of venom or biting. In
previous work we have shown that the stimulation of ß-adrenoceptor
increases cAMP production in quiescent but not in activated cells, showing its
relevance to starting the venom production cycle
(Yamanouye et al., 2000).
Therefore, taking all these data together, we conclude that noradrenergic
innervation in the Bothrops jararaca venom gland is crucial to
trigger the venom production and secretion machinery.
In conclusion, when the venom gland is full of venom, the secretory cells are in the quiescent state. Biting or manual extraction of venom triggers the production of new venom, which involves a change in the shape of the cells of the secretory epithelium from cuboid to columnar, expansion of the cisternae of the rough endoplasmic reticulum, and the production of secretory vesicles by the Golgi apparatus. To start this cycle, the presence of noradrenaline is essential: no venom is produced in the presence of reserpine. However, our data show that it is sufficient to have noradrenaline present only in the beginning of the venom production cycle, and that the noradrenergic innervation is not essential once the venom production is on the way. Our data also suggest that extraction of venom induces the release of noradrenaline, which indicates that noradrenaline is a causative rather than a permissive factor in the start of the venom production cycle. Knowledge of the dynamic of the venom gland will be helpful for understanding cellular mechanisms involved in venom production and secretion, and would aid the future production of venom in culture. Moreover, this peculiar exocrine gland is an attractive preparation for the investigation of cellular mechanisms of protein synthesis and secretion.
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Acknowledgments |
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