Synergism between toxin-
from Brazilian scorpion
Tityus serrulatus and veratridine in
chromaffin cells
Isaltino M.
Conceiçao1,2,
Ivo
Lebrun3,
María
Cano-Abad4,
Luis
Gandía4,
Jesús M.
Hernández-Guijo4,
Manuela G.
López4,
Mercedes
Villarroya4,
Aron
Jurkiewicz2, and
Antonio G.
García4,5
1 Laboratorio de
Farmacología and
3 Laboratorio de
Bioquímica e Biofísica, Instituto Butantan, 05503 São Paulo; 2 Departamento de
Farmacología, Escola Paulista de Medicina, 04034-970
São Paulo, Brazil;
4 Departamento de
Farmacología y Terapéutica, Facultad de Medicina,
Universidad Autónoma de Madrid, 28029 Madrid; and
5 Servicio de Farmacología
Clínica, Instituto de Gerontología, Hospital
Universitario de la Princesa, 28006 Madrid, Spain
 |
ABSTRACT |
Toxin-
(T
)
from the Brazilian scorpion Tityus
serrulatus venom caused a concentration- and
time-dependent increase in the release of norepinephrine and
epinephrine from bovine adrenal medullary chromaffin cells. T
was
~200-fold more potent than veratridine judged from
EC50 values, although the maximal
secretory efficacy of veratridine was 10-fold greater than that of T
(1.2 vs. 12 µg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca2+
concentration
([Ca2+]o),
when 30 µM veratridine plus 0.45 µM T
were used. T
(0.45 µM) doubled the basal uptake of
45Ca2+,
whereas veratridine (100 µM) tripled it. Again, a drastic synergism in enhancing Ca2+ entry was seen
when T
and veratridine were combined; this was particularly
pronounced at 5 mM
[Ca2+]o.
Veratridine induced oscillations of cytosolic
Ca2+ concentration
([Ca2+]i)
in single fura 2-loaded cells without elevation of basal levels. In
contrast, T
elevated basal
[Ca2+]i
levels, causing only small oscillations. When added together, T
and
veratridine elevated the basal levels of
[Ca2+]i
without causing large oscillations. T
shifted the current-voltage (I-V) curve for
Na+ channel current to the left.
The combination of T
with veratridine increased the shift of the
I-V curve to the left, resulting in a
greater recruitment of Na+
channels at more hyperpolarizing potentials. This led to enhanced and
more rapid accumulation of Na+ in
the cell, causing cell depolarization, the opening of voltage-dependent Ca2+ channels, and
Ca2+ entry and secretion.
sodium current; catecholamine release; calcium uptake; oscillations
of cytosolic calcium
 |
INTRODUCTION |
ALTHOUGH VOLTAGE-DEPENDENT
Na+ channels are present in
adrenal medullary chromaffin cells (16), their role in controlling the
physiological acetylcholine-mediated release of catecholamines (15) is
controversial. For example, the selective
Na+ channel blocker tetrodotoxin
(TTX) either does not block the nicotinic receptor-activated
catecholamine release response (11, 25) or decreases such a response
only partially and at low concentrations of nicotinic receptor agonists
(24). On the other hand, the activation of
Na+ channels by veratridine
provokes a secretory response in the perfused cat adrenal gland (27),
perfused guinea pig adrenal gland (20-22), and isolated bovine
adrenal chromaffin cells (26, 28). Veratridine is known to increase the
Na+ permeability of cells by
shifting the voltage dependence for activation of
Na+ channels toward more negative
values and by decreasing their rate of inactivation (35). This effect
leads to Na+ accumulation, cell
depolarization, opening of voltage-dependent Ca2+ channels, and
Ca2+ entry into bovine adrenal
chromaffin cells (30, 31). Although these experiments do not answer the
doubts regarding the possible role of
Na+ channels during the activation
of secretion under physiological conditions, they do suggest that the
activation of such channels can generate
Ca2+-dependent exocytotic
responses. In this sense, toxins acting on
Na+ channels are most useful for
an understanding of their function. Some of these toxins, purified from
Brazilian scorpion venoms, have not been previously tested in
chromaffin cells.
Despite the great number of scorpion species, only a few neurotoxins
are present in their secretions, particularly in those of the Buthidae
family. The neurotoxins are basic polypeptides with low molecular
weight (~7,000) composed of 40-70 amino acids (36). Scorpion
toxins are widely known to act on ionic channels, particularly
Na+ channels (9, 10, 13). Two
sites for scorpion toxins differing from TTX and veratridine sites have
been described in Na+ channels.
The so-called
-scorpion toxins bind at site
3 of Na+ channels,
causing a slowing of their inactivation.
-Scorpion toxins bind at
site 4, shifting the activation of
Na+ currents
(INa) toward
more negative potentials (9, 10, 13).
The Brazilian scorpion Tityus
serrulatus contains both
- and
-toxins (2).
Toxin-
(T
), a
-toxin, is the main component of the venom,
showing the highest affinity for site 4 in the
Na+ channel. However, despite this
high affinity and selectivity (2, 3, 40), only a few isolated reports
have been made on its biological activity on
Na+ channels. The toxin induces
spontaneous electrical activity in nerve membranes of
Xenopus laevis (23) and neuroblastoma
cells (39) and shifts the current-voltage
(I-V) curve of
Na+ channel activation to more
negative potentials in both preparations (4, 40), with a decrease in
peak INa. In
skeletal muscle the toxin produces only a blockade of
INa (3),
suggesting that the effects of the toxin are tissue dependent. How this
dual response (activation and blockade of
Na+ channels) affects
neurotransmitter release is unknown.
T
was first isolated by Possani et al. (36) and subsequently by
others, who gave the toxin different names according to the method of
purification (28a, 37, 38). Recently, Carvalho et al. (7, 8) have
published a new method for the isolation of highly purified toxins from
T. serrulatus venom. Using this method
we have been able to isolate the toxin PII-4, which has 98%
NH2-terminal homology with T
(we refer to our purified toxin as T
throughout this report). The
aim of this study was to verify the effects of T
on the secretion of
catecholamines in bovine chromaffin cells, either alone or in
combination with the Na+ channel
agonist veratridine. Attempts to understand the observed secretory
effects were made by studying the actions of T
and veratridine,
either alone or in combination, on the
45Ca2+
uptake, the concentration of cytosolic
Ca2+
([Ca2+]i),
and the whole cell
INa in those
cells.
 |
MATERIALS AND METHODS |
Preparation of cells.
Bovine adrenal medullary chromaffin cells were isolated as previously
described (29) with some modifications (32, 33). For secretion
experiments, cells were plated on uncoated plastic culture wells
(24-well Costar plates) at a density of
106 cells/well containing 1 ml of
DMEM supplemented with 10% FCS, 10 µM cytosine arabinoside, 10 µM
fluorodeoxyuridine, 50 IU/ml penicillin, and 50 µg/ml streptomycin.
For
45Ca2+
uptake studies, cells were plated in 96-well Costar plates at a density
of 2 × 105 cells/well. For
[Ca2+]i
and patch-clamp studies, cells were plated on glass coverslips, coated
with poly-L-lysine, at a density
of 5 × 104 cells/ml.
Cultures were maintained for 2-6 days at 37°C in a water-saturated atmosphere with 5%
CO2. After 24 h, the medium was
replaced by 1 ml of serum-free fresh medium and subsequently changed
every 2 days. Trypan blue exclusion yielded cell viability values
>95%.
T
isolation.
T
isolation was performed using a method described by Carvalho et
al. (8) with minor modifications. Briefly, a gel-filtration chromatography-Sephadex G-50 (1.6 × 150 cm) column was
equilibrated, and the samples were eluted with 0.02 M of ammonium
bicarbonate (pH 8.0) at room temperature. The pool
II of peptides obtained by gel-filtration
chromatography were separated by reverse-phase HPLC using a
µC18 BondaPak column (Waters)
with a 25-80% linear gradient of 0.1% trifluoroacetic acid
solution and acetonitrile (90% in 0.1 trifluoroacetic acid solution)
in 45 min. The effluent was monitored by ultraviolet detection at 214 nm.
Catecholamine release.
Catecholamine release was studied in 24-well plates containing
106 cells/well. Determination of
the catecholamine content in the medium bathing the cells was carried
out in cells after 2-3 days in culture as previously described
(5). Briefly, after the cells were incubated with drugs or control
solution, a 500-µl sample was recovered. Norepinephrine and
epinephrine were separated by HPLC using a
µC18 BondaPak column with a
mobile phase consisting of (per liter) 6.9 g
NaH2PO4 · H2O,
80 mg EDTA, 250 mg 1-heptanesulfonic acid sodium salt, and 50 ml
methanol. Catecholamine releases were quantitated by measuring peak
heights or curve areas and expressed as micrograms per milliliter
released.
Measurements of
45Ca2+
uptake.
45Ca2+
uptake studies were carried out in cells after 2-3 days in
culture. Before the experiment, cells were washed twice with 0.5 ml
Krebs-HEPES solution of the following composition (mM): 140 NaCl, 5.9 KCl, 1.2 MgCl2, 1 CaCl2, 11 glucose, and 10 HEPES (pH 7.2) at 37°C.
45Ca2+
uptake into chromaffin cells was studied by incubating the cells at
37°C with
45CaCl2
at a final concentration of 5 µCi/ml in the presence of Krebs-HEPES
(basal uptake), T
, veratridine, or T
plus veratridine. This
incubation was carried out during different time periods, at the end of
which the test medium was rapidly aspirated and the uptake reaction was
ended by adding 0.5 ml of cold
Ca2+-free Krebs-HEPES containing
10 mM LaCl3. Finally, the cells
were washed five more times at 15-s intervals with 0.5 ml of
Ca2+-free Krebs-HEPES containing
10 mM LaCl3 and 2 mM EGTA.
To measure the radioactivity retained, 0.5 ml of 10% TCA was added to
the cells, and they were scraped with a plastic pipette tip and
transferred to a scintillation minivial; then 3.5 ml of scintillation
fluid (Ready Micro, Beckman) were added, and the samples were counted
in a Packard beta counter. Results are expressed as counts per minute
(cpm), percentage of
45Ca2+,
or femtomoles per cell of
45Ca2+
plus
40Ca2+
taken up by cells.
Measurement of changes of
[Ca2+]i
in fura 2-loaded bovine chromaffin cells.
Chromaffin cells were loaded with fura 2 by incubating them with fura
2-AM (4 µM) for 60 min at 37°C in Krebs-HEPES solution (pH 7.4)
containing (in mM) 145 NaCl, 5.9 KCl, 1.2 MgCl2, 2.5 CaCl2, 10 Na-HEPES, and 10 glucose. The loading incubation was terminated by washing the coverslip
containing the attached cells several times with Krebs-HEPES. The cells
were then kept at 37°C in the incubator for 15-30 min.
The fluorescence of fura 2 in single cells was measured with the
photomultiplier-based system described by Almers and Neher (1), which
produces a spatially averaged measure of the
[Ca2+]i.
Fura 2 was excited with light alternating between 360 and 390 nm, using
a Nikon ×40 fluorite objective. Emitted light was transmitted
through a 425-nm dichroic mirror and 500- to 545-nm barrier filter
before being detected by the photomultiplier.
[Ca2+]i
was calculated from the ratios of the light emitted when the dye was
excited by the two alternating excitation wavelengths (18).
Measurements of whole cell currents through
Na+ channels.
Membrane currents were measured using the whole cell configuration of
the patch-clamp technique (19). Coverslips containing the cells were
placed in an experimental chamber mounted on the stage of a Nikon
Diaphot inverted microscope. The chamber was continuously perfused with
a control Tyrode solution containing (in mM) 137 NaCl, 1 MgCl2, 2 CaCl2, and 10 HEPES-NaOH (pH 7.4) at room temperature (22-25°C). Cells were internally dialyzed with a solution containing (in mM) 10 NaCl, 100 CsCl, 20 tetraethylammonium chloride, 5 MgATP, 14 EGTA, 20 HEPES-CsOH, and 0.3 NaGTP (pH 7.2).
Whole cell recordings were made with fire-polished glass electrodes
(resistance 2-5 M
) mounted on the head stage of a Dagan 8900 patch-clamp amplifier, allowing cancellation of capacitative transients
and compensation of series resistance. A Labmaster data acquisition and
analysis board and an IBM-compatible computer with pCLAMP software
(Axon Instruments, Foster City, CA) were used to acquire and analyze
the data.
Cells were clamped at
80 mV. Step depolarizations to different
test potentials from this holding potential lasted 20 ms and were
applied at 0.1 Hz. Leak and capacitative currents were subtracted by
using currents elicited by small hyperpolarizing pulses.
External solutions were exchanged by a fast superfusion device
consisting of a modified multibarreled pipette, the common outlet
of which was positioned 50-100 µm from the cell. Control and test solutions were changed with miniature solenoid
valves operated manually (Lee, Westbrook, CT). The flow rate
(0.2-0.5 ml/min) was regulated by gravity to achieve a
complete replacement of the fluid surrounding the cell in <1 s.
Statistical analysis.
Averaged data are means ± SE. The statistical significance of
differences between means was determined by the Student's
t-test for paired or grouped data.
Differences were considered significant at the level of
P < 0.05.
 |
RESULTS |
T
and veratridine cause a concentration-dependent
release of epinephrine and norepinephrine.
The cell populations used in these experiments
(106 cells/well) consisted of
70-80% epinephrine-storing cells and 20-30%
norepinephrine-storing cells (32). This proportion is reflected in the
spontaneous release of catecholamines into the Krebs-HEPES solution (1 mM Ca2+) bathing the cells,
i.e., 111 ± 13 ng/ml epinephrine and 36 ± 4 ng/ml
norepinephrine, after 1-min incubation of
106 cells
(n = 8 wells from 2 different
cultures). During incubation for 10 min, catecholamines recovered in
the medium rose to 158 ± 22 ng/ml epinephrine and 46 ± 7 ng/ml
norepinephrine (n = 36 wells from 9 different cultures).
Incubation of the cells for 10 min with increasing concentrations of
T
led to a progressive accumulation of catecholamines in the bathing
medium. At the threshold concentration of 4.5 nM, the total
catecholamine released amounted to 227 ± 68 ng/ml; a peak release
was obtained with a 100-fold higher concentration (1,199 ng/ml). An
approximate EC50 for the secretory
effects of T
was ~50 nM. Most of the catecholamines recovered in
the medium accounted for epinephrine, whose release also followed a
toxin concentration-dependent mode. The peak norepinephrine release was
at ~150 ng/ml, just ~10% of the total catecholamine peak (Fig. 1A).

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Fig. 1.
Concentration-dependent secretory effects of toxin- (T ) and
veratridine (VTD) in bovine chromaffin cells. After a preequilibration
period at 37°C, cells in each well
(106) were incubated in
Krebs-HEPES solution containing 1 mM
Ca2+ with or without
concentrations of T (A) or VTD
(B) shown on
abscissas. After 10-min incubation,
different media were collected, and their total catecholamine (CA),
epinephrine (Epi), and norepinephrine (NE) contents were estimated as
shown in MATERIALS AND METHODS. In
each individual 24-well plate, each concentration of T or VTD was
studied in triplicate. Data are means ± SE of 9 wells from at least
3 different cell batches.
|
|
In molar terms, veratridine was much less potent than T
in enhancing
the release of catecholamines into the medium bathing the cells (Fig.
1B). Thus the threshold
concentration that enhanced the rate of spontaneous catecholamine
output was ~10 µM, a concentration 200-fold higher than that of
T
. Another difference was that saturation was not reached even at
the concentration of 100 µM; higher concentrations of veratridine
displayed problems of solubility. Hence an
EC50 could not be estimated with a
minimum of reliability. From Fig. 1 (cf.
A and
B), an estimation of 200- to
1,000-fold difference of potency emerges between T
and veratridine.
As for the differential release of norepinephrine and epinephrine, the
same comment applies: over 85-90% accounts for the latter and
10-15% for the former amine.
Time course of secretory effects of T
and
veratridine.
The secretory effects of T
and veratridine followed a slow time
course (Fig.
2A) that
sharply contrasts with that of high K+ concentrations (see below). The
release of catecholamines induced by 0.45 µM T
rose from 0.7 µg/ml after 1-min incubation to 1.9 µg/ml at 10 min. Extension of
the incubation time to 30 min did not further enhance the accumulation
of catecholamines in the medium. Although veratridine released a
greater amount of catecholamines than T
, it is worth noticing that
its time course was similar. Thus secretion increased sharply from 1.2 µg/ml at 1 min to 12.2 µg/ml catecholamines at 10 min. Again,
secretion did not augment with the 30-min longer incubation period.
Some additivity of the secretory effects of T
and veratridine was
observed when both of them were added together (Fig.
2A). It is interesting that the
basal catecholamine output in the absence of toxins increased little
from 1 to 10 and 30 min of cell incubation in Krebs-HEPES (Fig. 2,
A and
B).

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Fig. 2.
Time course of secretory effects of T , VTD, and high
K+ in chromaffin cells. Protocols
were similar to those in Fig. 1. Cells were incubated in Krebs-HEPES
solution containing 1 mM Ca2+ with
or without T (0.45 µM), VTD (100 µM), T + VTD
(A), or high
K+ (70 mM,
B) for times indicated on
abscissas. Basal secretion was taken
as that obtained without latter substances. Data are means ± SE of
9 wells from at least 3 experiments performed with different batches of
cells.
|
|
For comparative purposes, the time course of the secretory effects of a
high K+ concentration (70 mM
K+, low
Na+, 1 mM
Ca2+) were also studied (Fig.
2B). The release of catecholamines
10 s after K+ exposure was 0.65 µg/ml and after 1 min was 2.1 µg/ml. These values rose to 5 and 7 µg/ml catecholamines 10 and 30 min after K+ exposure. Thus, with high
K+, the secretory response did not
reach saturation even after 30 min. The time courses followed by the
release of epinephrine and norepinephrine were similar to that of total
catecholamines.
Ca2+
dependence of secretory actions of T
and veratridine.
The experiments shown in Fig. 3 were done
to explore the relationship between the extracellular
Ca2+ concentration
([Ca2+]o)
and the size of the secretory responses to T
or veratridine treatment. After preequilibration of the cells in a Krebs-HEPES medium
containing increasing
[Ca2+]o
(0.5, 1, 2, or 5 mM), they were incubated for a further 10-min period
in the same medium containing 0.45 µM T
, 100 µM veratridine, or
a combination of both toxins.

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Fig. 3.
A: catecholamine release induced by
T and VTD at increasing concentrations of extracellular
Ca2+
([Ca2+]o).
Cells were preincubated in Krebs-HEPES solution containing increasing
concentrations of
[Ca2+]o
(abscissa) with or without T
(0.45 µM), VTD (100 µM), or their combination. Data are means ± SE of 9 wells from at least 3 different cell batches.
* P < 0.05, ** P < 0.01 compared with
basal secretion. B: synergism between
T and VTD on release of catecholamines at high
[Ca2+]o.
Chromaffin cells were exposed to normal Krebs-HEPES solution containing
5 mM Ca2+, for 10 min, in absence
(basal release) or presence of toxins at indicated concentrations. Data
are means ± SE of 8 wells from 2 experiments performed in
quadruplicate with 2 different cell batches.
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Figure 3A shows the release of total
catecholamines induced by T
and veratridine, alone or in
combination, at increasing [Ca2+]o.
A dual effect was seen when the toxins were used alone, i.e., an
increased secretion from 0.5 to 1 mM
Ca2+, followed by a decline at 2 and 5 mM Ca2+. Such a decrease
turned into a drastic increase at all
[Ca2+]o
when both toxins were associated. Thus combined T
(0.45 µM) plus
veratridine (100 µM) increased the release of catecholamines from
0.94 to 15.3 µg/ml in 2 mM Ca2+
(P < 0.05) and from 0.75 to 17.9 µg/ml in 5 mM Ca2+
(P < 0.01).
Because the synergism between the two toxins became more apparent at
the higher
[Ca2+]o,
it was likely that, with a lower concentration of veratridine (30 µM)
and the higher
[Ca2+]o
(5 mM), a more clear-cut synergism could be shown. The experiment of
Fig. 3B proves that this prediction
was true. After equilibration, cells were incubated for 10 min in
normal Krebs-HEPES solution (basal release) or in solutions containing
0.45 µM T
, 30 µM veratridine alone, or both. Basal catecholamine
release was almost undetectable (Fig.
3B). The cells incubated with T
released 1.5 µg/ml catecholamines, whereas those incubated with
veratridine secreted 2 µg/ml of the amines. Secretion rose
dramatically to 14.2 µg/ml of catecholamines in the cells incubated
with the two toxins (P < 0.001).
Time course of
45Ca2+
entry into chromaffin cells exposed to T
or
veratridine.
The concentrations of T
(0.45 µM) and veratridine (100 µM) used
in most studies of secretion were also selected to analyze their
effects on the
45Ca2+
entry into chromaffin cells. The protocol in these experiments was
similar to that followed in the secretion studies. After a 10-min
preequilibration period at 37°C in Krebs-HEPES solution containing
1 mM Ca2+, cells were incubated
for 1, 10, or 30 min in the same solution containing 5 µCi/ml
45Ca2+
in the absence (basal
45Ca2+
uptake) or in the presence of T
, veratridine, or both compounds together.
Figure 4 shows that, after 1-min incubation
in Krebs-HEPES, the basal
45Ca2+
taken up by cells amounted to 892 cpm; this value rose to 1,846 and
2,066 cpm after 10- or 30-min incubation in
45Ca2+-containing
solution. In the presence of T
, the
45Ca2+
entry increased modestly in 1 min to 1,203 cpm, in 10 min to 2,065 cpm,
and in 30 min to 2,583 cpm. Veratridine caused a greater 45Ca2+
entry increase, particularly after 10 (2,707 cpm) and 30 min (3,640 cpm) of incubation (P < 0.001 compared with basal).

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Fig. 4.
Time course of augmentation by T and VTD of
45Ca2+
uptake into chromaffin cells. Experiments were performed in conditions
similar to those used for time course of catecholamine release shown in
Fig. 2. Cells were subjected to a preequilibration 10-min incubation
period, followed by incubations in solutions containing
45Ca2+
in absence (basal uptake) or presence of T (0.45 µM), VTD (100 µM), or combination of both, during times shown on
abscissa. In
A, total
45Ca2+
taken up by cells as actual cpm found in cells of a single microwell (2 × 105) is shown. In
B, net
45Ca2+
taken up by cells was calculated by subtracting basal
45Ca2+
at each time point from total
45Ca2+
taken up by cells in presence of toxins. Data are means ± SE of 3 experiments performed in triplicate.
*** P < 0.001 with respect to
basal
45Ca2+
uptake at indicated time.
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Combining T
and veratridine led to a sharp potentiation of
45Ca2+
uptake that was particularly clear after 1-min exposure (Fig. 4A). It is interesting that, when
added separately, T
and veratridine did not significantly raise the
entry of
45Ca2+
after 1-min exposure, but, when combined,
45Ca2+
taken up by cells rose significantly from 892 to 2,369 cpm
(P < 0.001). If subtraction was made
of the basal
45Ca2+
(Fig. 4B), T
alone caused a net
45Ca2+
entry of 314 cpm and veratridine of 381 cpm. However, the combination of both toxins induced a net
45Ca2+
uptake of 1,477 cpm into the cells after 1 min of incubation.
45Ca2+
entry into cells bathed with increasing concentrations of
[Ca2+]o
with or without T
and veratridine exposure.
Figure 5 shows the results of
45Ca2+
uptake into cells that were incubated in Krebs-HEPES solutions
containing increasing concentrations of
Ca2+ (0.5, 1, 2, or 5 mM). Because
all solutions had the same amount of
45Ca2+,
after 10 min, the cells incubated in Krebs-HEPES (basal) retained decreasing amounts of Ca2+ when
measured as cpm. Thus
45Ca2+
uptake went from 2,359 cpm in 0.5 mM
[Ca2+]o
to ~784 cpm for 2 × 105
cells in 5 mM
[Ca2+]o.
In the presence of T
(0.45 µM) or veratridine (100 µM), an increased uptake of
45Ca2+
was observed at all
[Ca2+]o
tested compared with the
45Ca2+
uptake taken up by cells in basal conditions. Veratridine was slightly
more effective than T
. Again, the association of T
and
veratridine caused a sharp synergism, which was particularly clear at
the higher
[Ca2+]o.

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Fig. 5.
Synergism between T and VTD in enhancing
Ca2+ entry into chromaffin cells
at increasing
[Ca2+]o
(abscissas). After preequilibration
(10 min in Krebs-HEPES containing increasing
[Ca2+]o),
cells were incubated with
45Ca2+
(5 µCi/ml) in corresponding
[Ca2+]o,
for 10 min either in absence (basal
Ca2+ uptake) or in presence of
T (0.45 µM), VTD (100 µM), or T + VTD.
A: actual cpm
(ordinate), taken up and retained by
cells at each
[Ca2+]o.
B: total
Ca2+
(45Ca2+ + 40Ca2+)
taken up by cells at each
[Ca2+]o,
expressed as fmol/cell (ordinate).
C: net total
Ca2+
(45Ca2+ + 40Ca2+)
taken up by cells at each
[Ca2+]o,
expressed as fmol/cell (ordinate).
Data are means ± SE of 3 experiments performed in triplicate.
*** P < 0.001 with respect to
basal
45Ca2+
uptake.
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When correction of the specific activity of
45Ca2+
was made, at the different
[Ca2+]o,
the actual total Ca2+
(45Ca2+ + 40Ca2+)
taken up by the cells could be measured (Fig.
5B). Again, the synergism between
T
and veratridine was better seen at the higher [Ca2+]o.
For example, at 5 mM Ca2+, cells
took up as much as 15.6 fmol/cell of
Ca2+ compared with 6.4 fmol/cell
taken up by cells exposed to veratridine alone or 3.07 fmol/cell by
cells exposed to T
alone. A plot of the net
Ca2+ taken up by cells, after
subtraction for the basal Ca2+, is
shown in Fig. 5C. At 2 mM
[Ca2+]o,
T
produced a net increase in
Ca2+ uptake of 1.01 fmol/cell and
veratridine of 2.84 fmol/cell. Both toxins combined increased
Ca2+ uptake by 6.64 fmol/cell. The
synergism was better seen at 5 mM
[Ca2+]o.
Again, T
enhanced Ca2+ uptake
by 1.38 fmol/cell and veratridine by 3.3 fmol/cell; when combined, the
two compounds enhanced Ca2+ uptake
by as much as 12.6 fmol/cell.
Effects of T
and veratridine on
INa.
The effects of the toxins on Na+
channels were studied directly using the whole cell configuration of
the patch-clamp technique. Chromaffin cells were voltage clamped at
80 mV, and cells were superfused continuously with an
extracellular solution containing 137 mM
Na+, suitable to isolate the
INa through
Na+ channels, generated by
increasing 20-ms voltage-depolarizing steps applied at 10-s intervals
(see MATERIALS AND METHODS). Figure 6 shows control
I-V curves in the range of
60
to +60 mV. The threshold for activation of the current was at
30
mV, peak current was
0 mV, and the apparent reversal potential
was +30 mV. Peak current at 0 mV averaged 511 ± 56 pA
(n = 17 cells).

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Fig. 6.
Effects of T alone or in combination with VTD on
Na+ currents in chromaffin cells.
Cells were voltage clamped at 80 mV. Depolarizing 20-ms test
pulses to different voltages were applied at 10-s intervals to obtain
current-voltage relationships (I-V
curves). A:
I-V curves obtained before and after 5 min in presence of 0.22 µM T . B:
I-V curves of another cell before
(control) and after 5 min of exposure to combined T (0.22 µM) and
veratridine (30 µM). Insets:
original current traces obtained at 40 mV test pulses.
|
|
T
(0.22 µM) had a dual effect on
INa, i.e., a
blockade of peak current and a shift to the left of the
I-V curve. In the experiment shown in
Fig. 6A, T
augmented
INa at
40
mV from 50 to 300 pA (inset);
however, at 0 mV, the current was blocked by 70%. Data sets from 10 cells show averages of 290.2 ± 30.8% increase at
30 mV and
62.9 ± 6.9% blockade at 0 mV. Combined T
(0.22 µM) and
veratridine (30 µM) produced an exaggeration of the effects on the
I-V curve (Fig.
6B). Thus a more significant current
was seen at
60 mV (~250 pA); at
30 mV,
INa augmented to
as much as 400 pA; control current was almost undetectable (see
inset). At 0 mV, the blockade
amounted to 80%. Veratridine itself (30 µM) caused no apparent
shifts of the I-V curve; neither
compound blocked the peak current (not shown).
Effect of T
and veratridine on
[Ca2+]i.
The effect of toxin T
and veratridine on
[Ca2+]i
was studied in single fura 2-loaded cells. Figure
7 shows four representative recordings of
cells treated with the toxins alone or in combination. Figure
7A shows the typical large
oscillations of
[Ca2+]i
induced by veratridine (30, 31). Usually, the oscillations started with
a delay of 1-3 min after exposure of the cells to veratridine;
they were initially small (0.1-0.5 µM) and infrequent and
gradually became more frequent and of higher amplitude (up to 2 µM
Ca2+). The oscillations lasted
for periods of 30- to 60-min exposure to veratridine and continued for
at least 10-20 min after the toxin washout (not shown). The basal
level of
[Ca2+]i
was increased little by veratridine; the oscillations were usually
initiated from the baseline.

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Fig. 7.
Effect of T and VTD alone or in combination on
[Ca2+]i
in fura 2-loaded cells. Toxins were superfused onto a selected cell
during time period shown by horizontal bars at
bottom and
top of each trace.
A: typical continuous oscillations of
[Ca2+]i
induced by veratridine. B: cell was
exposed first to T and then to veratridine, still in presence of
T . C: cell was first
superfused with veratridine (bar at
bottom) and then with T (bar at
top).
D: cell was simultaneously exposed to
veratridine and T . Same
[Ca2+]i
pattern was observed in another 10 cells for veratridine alone, 5 cells
for veratridine or T first and combination of both after, and 4 cells treated with T + veratridine simultaneously.
|
|
T
(0.45 µM) exhibited a pattern of
[Ca2+]i
changes quite different from that of veratridine (Fig.
7B). In contrast to veratridine, T
increased the basal level of
[Ca2+]i
1-2 min after its addition. This elevation of
[Ca2+]i
reached a peak after 5 min and then declined to nearly basal levels in
another 5-min period. The
[Ca2+]i
followed an oscillatory pattern, with oscillations having a magnitude
substantially lower (0.3-0.5 µM) than those seen with veratridine. The addition of veratridine (30 µM) 15 min after T
did not change the pattern of small oscillations.
In the cell shown in Fig. 7C,
veratridine was added first, and 15 min later the cell was superfused
with both T
and veratridine. The most outstanding feature in this
experiment was the abolition of the
[Ca2+]i
oscillations generated by veratridine, 1-2 min after adding T
.
In Fig. 7D, a cell exposed from the
beginning and simultaneously to both toxins is shown. A rapid increase
in the
[Ca2+]i
to a peak of 1.6 µM followed by a decline to basal levels was observed.
We also performed experiments using the
Ca2+ channel blockers nifedipine
(L type) and
-conotoxin MVIIC (N, P, and Q type), at 3 µM
concentration, to determine the participation of voltage-dependent Ca2+ channels in the increase in
the
[Ca2+]i
induced by T
and veratridine. In Fig.
8A, it is
shown how the combination of nifedipine plus
-conotoxin MVIIC
reduces partially the increase in the
[Ca2+]i
evoked by T
and veratridine given in combination. However, because
the increase in
[Ca2+]i
induced by these toxins inactivates rather rapidly (see Fig. 7D), it was difficult to see a
clear-cut effect of the Ca2+
channel antagonists. In Fig. 8B, the
Ca2+ channel blockers were given
together with the Na+ channel
toxins from the beginning. In this case, no increase in the
[Ca2+]i
was seen, indicating that Ca2+
entry induced by these toxins is through voltage-dependent
Ca2+ channels.

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Fig. 8.
Effect of 3 µM nifedipine (NIFE; an L-type
Ca+ channel antagonist) + -conotoxin MVIIC (MVIIC, an N-, P-, and Q-type
Ca2+ channel antagonist) on
increase in
[Ca2+]i
induced by T (0.45 µM) + veratridine (30 µM).
A: combination of
Ca2+ channel antagonists was given
once levels of
[Ca2+]i
had been increased by T + VTD. B:
toxins were given at same time as T + VTD. Same pattern was observed
in another 4 cells.
|
|
 |
DISCUSSION |
The results of this investigation prove that both veratridine and T
promote the release of catecholamines from bovine adrenal medullary
chromaffin cells. Such secretory effects are indistinctly exerted on
norepinephrine-containing as well as on epinephrine-containing cells.
That veratridine is capable of releasing adrenal medullary catecholamines is a well-established fact (see introduction for references). The secretory effects of T
, however, are more dubious. There is only indirect evidence suggesting that its contractile actions
in the rat vas deferens are likely due to the neurogenic release of ATP
and/or norepinephrine (12), but these secretory signals
triggered individually by veratridine or T
are not the most relevant
finding in this study. The most striking observation was the drastic
synergism on Ca2+ entry and
secretion when both agents were combined, an interaction that was
particularly exaggerated at the higher
[Ca2+]o
used.
In a previous report, we proposed the following mechanism to explain
the oscillatory pattern of the changes in
[Ca2+]i
induced by veratridine (30): 1) the
plasma membrane depolarizes slowly due to a decrease in
K+ conductance through
[Ca2+]i-dependent
K+ channels, perhaps secondary to
a decrease in
[Ca2+]i;
2) the threshold for activation of
Na+ channels (decreased by
veratridine) is reached, producing further depolarization and the
recruitment of Ca2+ channels, and
inactivation of both Ca2+ channels
and veratridine-poisoned Na+
channels is slow; and 3) the
K+ conductance increases, due to
activation of Ca2+-dependent
K+ channels by the increased
[Ca2+]i,
and the membrane repolarizes. Thus the contribution of the Na+ channels seems essential for
the generation of the
[Ca2+]i
oscillations and also probably for the activation of catecholamine release.
When T
and veratridine were combined, there was a more rapid
transient increase in the
[Ca2+]i;
the initial increase was followed by a slow decay of the
[Ca2+]i.
This effect was much more rapid than the
[Ca2+]i
increases caused by each toxin individually (Fig. 7) and may be
explained on the basis of the complementary synergistic effects of the
toxins also found when studying
INa. T
makes
Na+ channels readily available for
opening at more hyperpolarizing potentials, and veratridine delays
their inactivation (see introduction for references). Thus, in the
conditions used here to study Ca2+
entry and secretion, more Na+
channels surely open at potentials near the resting membrane potential
of bovine chromaffin cells (
50 to
60 mV; Refs. 6, 16) and
remain open longer (35). That this might be the case is supported by
the marked shift to the left of the
I-V curves for
INa, which is
even further enhanced when both toxins were combined (Fig. 6). Now the
question arises as to why this synergism is observed better at the
higher
[Ca2+]o.
In a recent study, we observed that the oscillations of
[Ca2+]i,
induced by veratridine in fura 2-loaded bovine chromaffin cells, were
unexpectedly decreased with elevation of the
[Ca2+]o
(30). In addition, the cytotoxic effects of veratridine in these same
cells were prevented in high
[Ca2+]o
(31). Because both oscillations of
[Ca2+]i
and cell death were strictly dependent on
[Ca2+]o
(with
[Ca2+]o
deprivation,
[Ca2+]i
oscillations and cell damage caused by veratridine ceased), it was
expected that, in high
[Ca2+]o,
those effects would increase. The unexpected decrease in veratridine effects of high Ca2+ can be
explained in light of the well-established "membrane-stabilizing" actions of divalent cations. Frankenhaeuser and Hodgkin (17) first
described Ca2+ as having the
property of strongly screening negative surface charges associated to
plasmalemmal lipids of excitable cells. High
[Ca2+]o
can therefore limit the oscillations of the membrane potential induced
by veratridine as well as the opening of voltage-dependent Ca2+ channels following the
depolarizing phase of each oscillation (30). In high
[Ca2+]o,
the cell becomes less excitable, and stronger depolarizing stimuli are
therefore required to cause responses similar to those obtained in
normal
[Ca2+]o.
We believe that it is in this frame that an explanation for the
synergistic effects of veratridine and T
can be found.
The combined use of T
and veratridine opens new pathways for
studying the role of Na+ channels
in the control of exocytosis in chromaffin cells under physiological
conditions. The poor effects of TTX on the nicotinic receptor-mediated
secretory response in cultured cells (11) or intact glands (27) cast
doubts on the physiological significance of
Na+ channels in controlling the
release of catecholamines in the adrenal gland.
Na+-dependent action potentials
are certainly triggered by acetylcholine application to rat (24), mouse
(34), and bovine chromaffin cells (16). However, it is uncertain what
their role might be in recruiting
Ca2+ channels. In addition,
Ca2+-dependent action potentials
can be triggered in the absence of Na+ or presence of TTX. Are these
action potentials sufficient to regulate secretion in the adrenal
medulla? Do Na+ channels control
the electrical activity of chromaffin cells only under certain
stressful conditions? Are there two types of Na+ channels in chromaffin cells,
with low and high sensitivity to TTX? We expect that the combined use
of T
and veratridine will help to find answers for some of these
questions.
 |
ACKNOWLEDGEMENTS |
We thank M. C. Molinos for typing the manuscript and Ricardo de
Pascual for the preparation of excellent bovine adrenal medullary chromaffin cells. We also thank Román Olivares for the HPLC
analysis of epinephrine and norepinephrine.
 |
FOOTNOTES |
This study was supported by grants from Dirección General de
Investigación Científica y Técnica (PB94-0150)
and Fundación Ramón Areces (Spain) to A. G. García
and grants from Fundação de Amparo a Pesquisa do Estado de
São Paolo (Brazil) to A. Jurkiewicz. M. Cano-Abad is a fellow of
Formación de Personal Investigador, and J. M. Hernández-Guijo is a fellow of Comunidad Autónoma de Madrid
(Spain).
This study was performed under the established collaboration of
Universidad Autónoma de Madrid and Escola Paulista de Medicina.
Address for reprint requests: A. G. García, Dept. de
Farmacología, Facultad de Medicina, Universidad Autónoma
de Madrid, c/o Arzobispo Morcillo 4, 28029 Madrid, Spain.
Received 18 November 1997; accepted in final form 10 February
1998.
 |
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