Synergism between toxin-gamma 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
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Materials & Methods
Results
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References

Toxin-gamma (Tgamma ) 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. Tgamma 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 Tgamma (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 Tgamma were used. Tgamma (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 Tgamma 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, Tgamma elevated basal [Ca2+]i levels, causing only small oscillations. When added together, Tgamma and veratridine elevated the basal levels of [Ca2+]i without causing large oscillations. Tgamma shifted the current-voltage (I-V) curve for Na+ channel current to the left. The combination of Tgamma 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -scorpion toxins bind at site 3 of Na+ channels, causing a slowing of their inactivation. beta -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 alpha - and beta -toxins (2). Toxin-gamma (Tgamma ), a beta -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.

Tgamma 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 Tgamma (we refer to our purified toxin as Tgamma throughout this report). The aim of this study was to verify the effects of Tgamma 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 Tgamma 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
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Introduction
Materials & Methods
Results
Discussion
References

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%.

Tgamma isolation. Tgamma 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), Tgamma , veratridine, or Tgamma 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 MOmega ) 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
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Materials & Methods
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Tgamma 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 Tgamma 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 Tgamma 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-gamma (Tgamma ) 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 Tgamma (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 Tgamma 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 Tgamma 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 Tgamma . 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 Tgamma 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 Tgamma and veratridine. The secretory effects of Tgamma 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 Tgamma 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 Tgamma , 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 Tgamma 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 Tgamma , 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 Tgamma (0.45 µM), VTD (100 µM), Tgamma  + 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 Tgamma 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 Tgamma 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 Tgamma , 100 µM veratridine, or a combination of both toxins.


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Fig. 3.   A: catecholamine release induced by Tgamma 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 Tgamma (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 Tgamma 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.

Figure 3A shows the release of total catecholamines induced by Tgamma 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 Tgamma (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 Tgamma , 30 µM veratridine alone, or both. Basal catecholamine release was almost undetectable (Fig. 3B). The cells incubated with Tgamma 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 Tgamma or veratridine. The concentrations of Tgamma (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 Tgamma , 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 Tgamma , 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 Tgamma 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 Tgamma (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.

Combining Tgamma 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, Tgamma 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), Tgamma 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 Tgamma 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 Tgamma (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 Tgamma . Again, the association of Tgamma and veratridine caused a sharp synergism, which was particularly clear at the higher [Ca2+]o.


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Fig. 5.   Synergism between Tgamma 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 Tgamma (0.45 µM), VTD (100 µM), or Tgamma  + 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.

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 Tgamma 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 Tgamma 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, Tgamma 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, Tgamma 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 Tgamma 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 Tgamma 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 Tgamma . B: I-V curves of another cell before (control) and after 5 min of exposure to combined Tgamma (0.22 µM) and veratridine (30 µM). Insets: original current traces obtained at -40 mV test pulses.

Tgamma (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, Tgamma 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 Tgamma (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 Tgamma and veratridine on [Ca2+]i. The effect of toxin Tgamma 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 Tgamma 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 Tgamma and then to veratridine, still in presence of Tgamma . C: cell was first superfused with veratridine (bar at bottom) and then with Tgamma (bar at top). D: cell was simultaneously exposed to veratridine and Tgamma . Same [Ca2+]i pattern was observed in another 10 cells for veratridine alone, 5 cells for veratridine or Tgamma first and combination of both after, and 4 cells treated with Tgamma  + veratridine simultaneously.

Tgamma (0.45 µM) exhibited a pattern of [Ca2+]i changes quite different from that of veratridine (Fig. 7B). In contrast to veratridine, Tgamma 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 Tgamma 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 Tgamma 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 Tgamma . 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 omega -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 Tgamma and veratridine. In Fig. 8A, it is shown how the combination of nifedipine plus omega -conotoxin MVIIC reduces partially the increase in the [Ca2+]i evoked by Tgamma 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) + omega -conotoxin MVIIC (MVIIC, an N-, P-, and Q-type Ca2+ channel antagonist) on increase in [Ca2+]i induced by Tgamma (0.45 µM) + veratridine (30 µM). A: combination of Ca2+ channel antagonists was given once levels of [Ca2+]i had been increased by Tgamma  + VTD. B: toxins were given at same time as Tgamma  + VTD. Same pattern was observed in another 4 cells.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The results of this investigation prove that both veratridine and Tgamma 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 Tgamma , 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 Tgamma 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 Tgamma 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. Tgamma 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 Tgamma can be found.

The combined use of Tgamma 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 Tgamma 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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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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Am J Physiol Cell Physiol 274(6):C1745-C1754
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society




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