Nicotinic Receptors Mediate the Release
of Amino Acid Neurotransmitters in
Cultured Cortical Neurons
E. López, C. Arce, S. Vicente, M.J. Oset-Gasque and
M.P. González
Nicotine stimulation of cortical neurons obtained from gestation day
19 rats provoked a dose-dependent release of aspartate, glutamate,
glycine and GABA, indicating a functional role for the nicotinic
receptor in this model. This release was exclusively Ca2+-dependent
(vesicular release) in the case of aspartate and dual (Ca2+dependent and Ca2+-independent) for glutamate, glycine and GABA.
Nicotine also raised the membrane potential and the intracellular
calcium concentration. These effects were specific, since they were
reversed by hexamethonium, an antagonist of the nicotinic receptor.
It was shown that L, N, and P/Q type Ca2+ channels are involved in
nicotine-mediated Ca2+ entry into cortical neurons. Evaluation of the
effects of nicotine on Ca2+ entry in isolated cells showed that 100%
of the cells responded to nicotine, although the intensity of the
response was variable: 63% of the neurons showed an increase in
intracellular Ca2+ of 152 ± 5 grey levels, 25% of 88 ± 12 grey levels
and 12% of 48 ± 1 grey levels. Tetrodotoxin, which blocks voltagedependent Na+ channels, completely reversed nicotine-induced
Ca2+ entry into single cells. This suggests that the Ca2+ increment is
mediated by opening of Ca2+ channels and not by the nicotinic
receptor.
nicotinic receptors in cerebral cortex through immunocytochemical techniques (Nakayama et al., 1995). The number of
these receptors seems to be diminished in neurological disorders
such as Parkinson’s and Alzheimer’s diseases (Araujo et al.,
1988; Schroder et al., 1995; Chesselli, 1997; Zamanim et al.,
1997) and schizophrenia (Miller et al., 1996). Pharmacological
and electrophysiological studies appear to suggest the existence
of different isoforms of the nicotinic receptor in the neocortex
and hippocampus (Wada et al., 1989, 1990; Seguela et al., 1993;
Lobron et al., 1995), although the exact location of these
isoforms is not known. This diversity of nicotinic receptors
in the CNS makes functional and pharmacological variations
possible (Papke et al., 1989; Luetje and Patrick, 1991). In
dopaminergic nigrostriatal axons nicotine induces dopamine
release after previous stimulation of glutamatergic neurons,
which release glutamate (García Muñoz et al., 1996). In
hippocampal synaptosomes release of noradrenaline may be
modulated by nicotinic receptors containing α3 and β4 subunits
(Clarke and Reuben, 1996). Furthermore, nicotine may induce
increases in glutamate, dopamine, serotonin and acetylcholine
release in the hippocampus, cerebellum, nucleous accumbens
and striatum (Thoth et al., 1992; Marshall et al., 1996, 1997;
Wilkie et al., 1996; Ferger and Kuschinsky, 1997). Conversely,
Izenwasser et al. found that in the striatum nicotine inhibits
[3H]dopamine uptake and induces its release (Izenwasser et
al., 1991). Moreover, Waniewski and Martin reported that the
stimulation of sympathetic ganglia with nicotinic or muscarinic
agonists provoked the release of [3H]taurine (Waniewski and
Martin, 1994). Although there is much data on the properties
and functional characteristics of the different cholinergic
receptors (AchR) in the CNS, most of these may be considered
presynaptic receptors since the action of nicotine has generally
been evaluated in terminal neurons (synaptosomes). However,
nicotinic receptors are distributed throughout the neurons. Few
investigations have centred on the action of AchRs on neurotransmitter release in neuronal preparations, although the effects
of nicotine in in vivo systems have also been studied (Thoth et
al., 1993). The aim of the present study was to investigate the
nicotinic receptor-mediated modulation of synaptic transmission
in cortical neurons. Our results suggest that in cortical neurons
in culture nicotine induces aspartate release by an exocytotic
pathway and glutamate, glycine and GA BA release by a dual
mechanism. It was also established that nicotine increased the
intracellular calcium concentration in all of the neurons
examined.
Introduction
Although its physiological and psychological effects have long
suggested that nicotine exerts specific actions in the brain, the
identification of neuronal nicotinic receptors has only been
possible in the last few years due to the development of
molecular biology techniques [reviewed by McGehee and Rolen
(McGehee and Rolen, 1995)]. A variety of nicotinic acetylcholine
receptor (nAchR) complexes in the central nervous system
(CNS) are formed by a diverse array of subunits which confer
different pharmacological and physiological properties to the
receptors (Sargent, 1993; Rust et al., 1994). Although there is
little experimental evidence (Anano et al., 1991; Cooper et al.,
1991), the neuronal nAchR channel is assumed to be a pentamer
formed by α and β subunits, although many alternative α/β
stoichiometries may exist, and this could contribute to the
particular functionality of the nAchR channel. The expression of
specific nAchR subunits is important during brain development
since it inf luences neuronal excitability (Margiotta and Gurantz,
1989). Nicotinic AchRs are present on autonomic neurons and
adrenal chromaffin cells of the peripheral nervous system and
on many neurons in the CNS. Many of the properties of the
nAchR channels, such as ion selectivity and gating properties,
resemble those of muscle AchR. However, neuronal AchRs are
clearly distinct from muscle and are themselves diverse. The
most important role performed by cholinergic receptors in the
CNS is their participation in neuronal excitability modulation
(Aquilonius and Gillberg, 1990; Vernino et al., 1992) and in
neurotoxicity events (Slotkin et al., 1997). Cumulative evidence
from animal and human studies has indicated that nicotinic
systems play a major role in higher cognitive functions and
dysfunctions. Nakayama et al. were able to show the presence of
© Oxford University Press 2001. All rights reserved.
Instituto de Bioquímica (Centro Mixto CSIC-UCM), Facultad de
Farmacia, Ciudad Universitaria, 28040 Madrid, Spain
Materials and Methods
Materials
Fura 2A M and bis-[1,3-diethylthiobarbiturate]trimethine oxonol (bisoxonol) were obtained from Molecular Probes (Eugene, OR). Eagle’s
minimum essential medium (EMEM) was supplied by Bio-Whittaker and
Cerebral Cortex Feb 2001;11:158–163; 1047–3211/01/$4.00
foetal calf serum (FCS) and horse serum (HS) by Sera-Lab (Sussex, UK).
Nicotine, hexamethonium and muscimol came from Sigma (St Louis,
MA). The remaining chemicals were reactive grade products from Merck
(Darmstadt, Germany).
Cell Isolation and Culture
Brain neurons were obtained from foetal rat brains at gestation day 19
following the procedure described by Segal (Segal, 1983) with minor
modifications. Isolated neurons were suspended in EMEM containing
0.3 g/l glutamine, 0.6% glucose, 10% FCS, 10% HS, 100 U/ml penicillin,
100 µg/ml streptomycin, 40 µg/ml gentamycin and 5 µg/ml imipenem.
Cells, at a density of 2 × 106 cells/well, were plated onto plastic Petri
dishes treated with 10 µg/ml poly-L-lysine to aid attachment. The plates
were incubated in a humidified incubator in an atmosphere of 5%
CO2/95% air at 37°C. After 72 h the incubation medium was replaced
with fresh medium to which 10 µM cytosine arabinoside was added to
prevent overgrowth of contaminating glial cells. Cells were used after
10–15 days culture. Cell viability was checked by the trypan blue
exclusion method. Viability was routinely >95%. Cell purity was checked
by both cell staining with cresyl violet to identify neurons and with a
specific anti-GFAP antibody to identify glial cells.
Glial Contamination
After 10–15 days culture the cortical neurons were detached from the
culture plates with trypsin, as indicated below. Cells were the fixed (for
30 min) in 2% paraformaldehyde and washed in phosphate-buffered
saline (PBS) followed by treatment (1 h) with anti-rabbit GFAP antibody
(diluted 1/500). Cells were once again washed in PBS and treated with
anti-rabbit FITC-conjugated IgG at a dilution of 1/100 for 30 min and
identified by f low cytometry. Under these conditions the glial cells in the
cultures were estimated at 9 ± 3% of the total cell population (neural +
glial cells).
Cytosolic [Ca2+]
Changes in intracellular calcium concentration, [Ca2+]i, were monitored
by Fura 2AM f luorescence. Six day cultured cells were detached from the
plates using tr ypsin (0.25% tr ypsin and 0.02% EDTA in Dulbecco’s
phosphate-buffered saline without calcium or magnesium) and washed
twice using 1 ml of a Krebs HEPES solution (Locke medium) containing
140 mM NaCl, 4.4 mM KCl, 2.5 mM CaCl2, 1.2 mM Mg(SO4)2, 1.2 mM
KH2PO4, 4.0 mM NaHCO3, 5.5 mM glucose, 0.58 mM ascorbic acid and
10 mM HEPES, adjusted to pH 7.5 and incubated with 5 µM Fura 2AM for
45 min at 37°C. Excessive dye was removed by washing the cells twice
with fresh Locke medium followed by suspension in this medium at
1 × 106 cells/ml. After Fura 2AM treatment, cell viability was checked as
indicated previously. Fluorescence (excitation wavelength 340/380 nm,
emission 510 nm) was monitored at 37°C in a well stirred cuvette
containing 1 ml of this suspension using a Perkin Elmer LS-50 spectrof luorimeter (slits 5 nm excitation, 10 nm emission). At the end of each
experiment 1% Triton X-100 was added to make the cells permeable and
permit the dye to gain access to the extracellular Ca2+ (2.5 mM). This Ca2+
concentration saturated the dye and provided a measure of the maximum
f luorescence signal (Fmax). To determine the minimum f luorescence
signal (Fmin), 20 mM Tris base was added to raise the pH above 8.2,
followed by 5 mM EGTA which reduced the Ca2+ to <1 nM. [Ca2+]i was
calculated with the Grynkiewicz equation (Grynkiewicz et al., 1985):
[Ca2+]i = Kd [(F – Fmin)/(Fmax – F)] × (SF2/SB2)
where F is the ratio between f luorescence values at 340 and 380 nm,
SF2 is the maximum f luorescence at 380 nm and SB2 is the minimum
f luorescence at 380 nm. The equilibrium dissociation constant (Kd) for
the complex [Ca2+]–Fura 2AM was of 224 nM.
Single Cell [Ca2+]i
Cells cultured on glass coverslips were loaded for 45 min with Fura 2AM
(5 µM) dissolved in Locke medium. After incubation at 37°C in the dark,
cells were washed with Locke medium and the coverlips placed under
a Nikon inverted stage microscope under continuous perfusion with
Locke medium. Light from a xenon lamp was filtered through two
different band-pass filters (340 or 380 nm) in the excitation path and
the specimen illuminated on the microscope stage by a dichroic mirror.
Excitation wavelengths of 340 and 380 nm were alternately applied to
the cells. The f luorescence emitted by the cells was passed through a
band-pass filter (510 nm) and video images were obtained using a cold
intensified camera. Ratios corresponding to 340/380 nm are given as
grey levels. Output from the camera was digitalized and stored in a
computerized imaging system (MiraCal). The reagents dissolved in Locke
medium were applied in the perfusion medium. When tetrodotoxin
(TTX) was used the order of nicotine and TTX applications was as follow.
First neurons were stimulated with 10 µM nicotine and images were taken
over 80 s. Then, cells were perfused for 10 min to permit recovery of the
nicotinic receptor. Subsequently, neurons were stimulated with 10 µM
nicotine plus 50 nM TTX and images taken for 80 s as before.
Membrane potential
Changes in the membrane potential of neurons were monitored using the
f luorescent dye bisoxonol. This is a lipophilic anion whose distribution
across the membrane is dependent upon the membrane potential. Thus,
an increase in bisoxonol f luorescence indicates that the membrane
has been depolarized, allowing more of this negatively charged dye
to enter the cells (Waggoner, 1979). Washed cells, as used for [Ca2+]i
determination, were suspended in Locke medium at a density of 5 × 105
cells. Neurons in suspension were incubated with 0.2 µM bisoxonol for
10–20 min and placed in a f luorimeter. Fluorescence was measured at an
excitation wavelength of 540 nm and emission wavelength of 565 nm
and monitored at 37°C in a well stirred cuvette using a Perkin Elmer
spectrof luorimeter. Drugs were added at the indicated concentrations.
Controls were performed using Locke medium in place of the drug.
Fluorescence intensity is reported in arbitrary units.
Amino Acid Secretion
High performance liquid chromatography (HPLC) of amino acids was
performed according to the methods described by Márquez et al.
(Márquez et al., 1986). Cells were washed twice at 10 min intervals with
1 ml of Locke medium. After removal of the medium cells were stimulated
for 15 min periods at 37°C with 0.5 ml of fresh Locke medium containing
the different secretagogues. The stimulating medium was then
withdrawn and cells ruptured by the addition of 0.5 ml of distilled water.
The concentration of amino acids was determined by reversed phase
HPLC using pre-column derivation with dansyl chloride and UV detection
at 254 nm. Integration of peaks was achieved using a Sprectraphysis
integrator. Peaks were quantified by comparison with those obtained
using simultaneously prepared amino acid standards. Separation of dansyl
derivatives was performed using a 5 µM Spherisorb-ODS-2 column (15 ×
0.46 cm).
Proteins were identified according to Bradford (Bradford, 1976).
Results were expressed as nmol neurotransmitter/mg protein/well or as
the percentage of amino acids released into the incubation medium with
respect to the total amino acid content (incubation medium + cells).
Data Presentation
Data are presented as the means of three or four separate experiments
performed on different cell cultures. Each experiment was performed
in duplicate using different batches of cells. Student’s t-test was used to
statistically compare data.
Results
A dose-dependent release of the amino acids aspartate, glutamate, glycine and GA BA was shown when cortical neurons
were stimulated with nicotine. The effect of nicotine was first
observed at a nicotine dose of 50 µM. Glycine was released in
greatest quantity (Fig. 1). Measurement of secretion mediated
by nicotine in a medium with and without external calcium
revealed that aspartate release was exclusively Ca2+-dependent
(vesicular release), while the release of the remaining amino
acids occurred through Ca2+-dependent and Ca2+-independent
processes. The order of Ca2+-dependent release of these amino
acids at a nicotine concentration of 200 µM was: aspartate ¾
Cerebral Cortex Feb 2001, V 11 N 2 159
Figure 1. Effect of different nicotine concentrations on amino acid neurotransmitter secretion in cortical neurons in culture. Basal is spontaneous release in the absence of nicotine,
results without stimulation. Results are given as means ± SEM of two separate experiments, from cells of different cultures, each one performed in triplicate. Statistical significance
is with respect to basal amino acids release. NS, not significant; ***, P < 0.001.
glycine > glutamate > GABA. The Ca2+-dependent release of all of
these amino acids was higher than that observed in calcium-free
medium, except in the case of GABA, which was preferentially
released by a Ca2+-independent process (Fig. 2).
The nicotinic acetylcholine receptor antagonist hexamethonium completely blocked the release of these amino acid neurotransmitters evoked by nicotine, which implies that the effect
may be attributed to activation of the nicotinic receptor (Fig. 3).
Nicotine also increased both the membrane potential, measured as arbitrary f luorescence units, and intracellular calcium
levels in a dose-dependent manner. Both effects were inhibited
by hexamethonium (Fig. 4A,B).
Release of the four amino acid neurotransmitters evoked by
200 µM nicotine was inhibited by verapamil, an antagonist of L
type Ca2+ channels; ω-conotoxin GVIA, an antagonist of N type
Ca2+ channels, inhibited the release of all the amino acid neurotransmitters with the exception of aspartate. Further, ω-agatoxin
GIVA, which blocks P/Q type Ca2+ channels, inhibited the
release of aspartate, glycine and GABA, but not of glutamate
(Fig. 5).
When the nicotine-mediated increments in intracellular calcium levels were measured in single cells it was observed that all
of the neurons responded to nicotine, although these responses
varied depending on the cell. Increases in intracellular Ca2+
levels of 152 ± 5 grey levels were recorded in 63% of the cortical
neurons, of 90 ± 10 in 25% and of 48 ± 1 in 12% (Fig. 6).
TTX, which blocks voltage-dependent Na+ channels, completely reversed the Ca2+ entry mediated by nicotine in 100% of
the cells under study (Fig. 7).
Discussion
Despite abundant data on AchR diversity in the CNS, there is still
little evidence for classical nicotinic synaptic transmission. The
results presented here clearly demonstrate that when cortical
neurons are stimulated with concentrations of nicotine from 50
to 200 µM there is a dose-dependent release of aspartate, glutamate, glycine and GABA, which is exclusively Ca2+-dependent
(exocytotic release) or dual (Ca2+-dependent and Ca2+-independent), depending on the amino acid. The nicotine concentration
needed to induce amino acid release appears high compared
with the findings of electrophysiological studies (McGehee et
160 Amino Acid Neurotransmitter Release • E. López et al.
Figure 2. Effect of 200 µM nicotine on amino acid neurotransmitter release measured
in a medium with (total release) and without external Ca2+ (Ca2+-independent
release). Ca2+-dependent amino acid release is the difference between total release
and release found in the absence of calcium in the medium. All results are given after
subtracting the correspondent basal values. *, statistical significance with respect to
the corresponding basal values (with and without external calcium). •, statistical
significance between release measured in the presence and absence of external
calcium. •• or **, P < 0.01, ••• or ***, P < 0.001.
al., 1995). However, it is much lower than the concentrations
used (1 mM or higher) in studies in which amino acid release
was measured directly (Thoth et al., 1993). Besides its effects on
amino acid neurotransmitter release, nicotine also induced
membrane depolarization and intracellular Ca2+ increases. All
these effects were blocked by hexamethonium, an antagonist of
the nicotinic receptor, indicating their specificity. Intracellular
calcium increases mediated by this cholinergic receptor were
also shown by Mulle et al. in rat CNS neurons (Mulle et al., 1992).
Further, Letz et al. observed that the stimulation of cholinergic
receptors on pinealocytes may cause membrane depolarization
and activation of L type Ca2+ channels (Letz et al., 1997).
Figure 3. Effect of 200 µM hexamethonium on the amino acid release evoked by 50
µM nicotine. Results are means of two separate experiments, with different cultures,
each one performed in duplicate. Values are given after subtracting the basal values.
***, P < 0.001.
Figure 4. Effect of nicotine on: (A) membrane potential, measured as arbitrary
fluorescence units (as indicated in Materials and Methods), in the presence and
absence of 200 µM hexamethonium; (B) increase in intracellular Ca2+, measured in the
absence and presence of 200 µM hexamethonium. The basal intracellular calcium
concentration was 141 ± 20 nM. Results are means of two or three separate
experiments, with cells of different cultures, each one performed in duplicate or
triplicate.
The data presented not only demonstrate the presence of
nicotinic receptors in cultured cortical neurons, but also indicate
their specificity and functionality. These findings are in accordance with those obtained by Vidal and Changeus, who reported
an increase in the negative wave of field potentials ref lecting
increased excitability of cortical neurons when neocortical slices
were treated with acetylcholine or dimethylphenyl piperazinium
(Vidal and Changeus, 1989).
In the present study nicotine was also able to induce the
release of amino acid neurotransmitters in a calcium-free medium
(Ca2+-independent release). This Ca2+-independent amino acid
release may be attributable to the reverse action of amino acid
transporters, given that the nicotinic receptor is a Na+-permeable
channel and, when open, the resulting increased intracellular
Na+ level is a condition required to activate amino acid
transporters.
Figure 5. Effect of calcium channel antagonists on amino acid neurotransmitter release
evoked by 200 µM nicotine. *, statistical significance between amino acid release
evoked by 200 µM nicotine in the absence and presence of calcium channel
antagonists. Results are means ± SEM of two separate experiments from different
cultured cells, each one performed in duplicate. Statistical significances are given
between intracellular Ca2+ increments induced by nicotine or nicotine with the
corresponding Ca2+ channel antagonist. NS, not significant; *, P > 0.05; ***, P >
0.001.
Figure 6. Whole neuron recordings of intracellular calcium increments evoked by
10 µM nicotine in single cells. Traces show representative examples of typical
F340/F380 ratio responses (grey levels) produced in a single neuron after stimulation
with the indicated agent. Measurements were performed in medium with external
calcium. The insert indicates the percentage of cells with similar behaviour. The number
of neurons analysed was 35 from three different cultures.
Our data seem to indicate that different Ca2+ channels might
be involved in release of the different amino acid neurotransmitters evoked by nicotine, since blockers of L, N and P/Q type
Ca2+ channels inhibited release of the inhibitory amino acid
neurotransmitters (glycine and GABA). However, N type Ca2+
channel blockers did not affect aspartate release and P/Q type
Ca2+ channel blockers did not affect release of glutamate. These
data would appear to suggest that opening of one Ca2+ channel or
another is important in secretion of the different amino acid
neurotransmitters mediated by nicotine.
The fact that nicotine-stimulated cortical neurons release
Cerebral Cortex Feb 2001, V 11 N 2 161
Figure 7. Whole neuron recording of intracellular calcium increments evoked by 10 µM
nicotine in the absence or presence of 1 µM tetrodotoxin. Traces are representative
examples of typical F340/F380 ratio responses, expressed as grey levels, produced in a
single neuron after stimulation with the indicated agents, as indicated in Materials and
Methods. Measurements were performed in medium with external calcium. The
number of neurons analysed was 15 from two different cultures.
excitatory (aspartate and glutamate) and inhibitory (glycine and
GA BA) amino acids would seem to indicate that nicotinic
receptors are able to modulate excitatory and inhibitory synapses in cortical neurons.
An observation that warrants particular attention was the high
release of aspartate produced when cortical neurons were
stimulated with high concentrations of nicotine. This might
indicate that in this part of the brain the toxic effect attributed
to overexcitability produced by nicotine may be due not only
to glutamate release but also to aspartate release that, like
glutamate, is able to bind to NMDA receptors. The high release of
glycine mediated by nicotine is also of note. Glycine release was
some four times higher than GABA release. As both these amino
acids are inhibitory, it may be considered that in nicotinestimulated cortical neurons glycine may mediate inhibition or
modulate the response of the NMDA receptor. This matter
requires further study.
It may be inferred from the study of Ca2+ entry mediated
by nicotine in single neurons that most cortical neurons have
nicotinic receptors, although the possibility that Ca2+ entry could
be mediated by excitator y amino acids released by nicotine
should not be discarded. The increment in intracellular Ca2+
mediated by nicotine could be due to Ca2+ entry through: (i)
voltage-dependent Ca2+ channels, since nicotine induced membrane depolarization, and/or (ii) Ca2+ entry through the nicotinic
receptors, since, according to some authors, neuronal AchRs
show significant permeability to Ca2+ (Adams and Nulter, 1992;
Vernino et al., 1992, 1994). This Ca2+ permeation is sufficient
to activate Ca2+-dependent cellular processes. However, in the
presence of TTX, which completely blocked the Ca2+ entry
mediated by 10 µM nicotine, the intracellular Ca2+ increment
evoked by nicotine appeared to be mediated by the opening of
Ca2+ channels and not by the nicotinic receptor. In this case the
nicotinic receptors in cortical neurons seems to be impermeable to Ca2+ ions. These results agree with those of Lena and
Changeux, which showed that the depolarizing effect of
nicotine in thalamic neurons appears to be mediated through
entry of calcium through voltage-sensitive calcium channels
(Lena and Changeux, 1997). This is not surprising given the two
major functional properties of neuronal nAChRs established by
Vernino et al. (Vernino et al., 1992). These authors described
nAChRs which show substantial permeability to Ca2+ and a
162 Amino Acid Neurotransmitter Release • E. López et al.
further population of receptors which do not appear to permit
Ca2+ movement. These differences may be attributed to the high
degree of heterogeneity of neuronal nAChRs brought about by
the combination of different subunits to give rise to many
structural and functional variants of this neuronal receptor
(Boulter et al., 1987; Duvoisin et al., 1989; Couturier et al.,
1990).
Herrero et al. recently demonstrated that the Ca2+ channel
blockers used in the present study also block α7 and α3β4 AchRs
in chromaffin cells (Herrero et al., 1999), giving rise to the possibility that the present effect could be due to blockade of the
nAchR. However, our results indicate that the effect of the toxins
is mediated by Ca2+ channel blockade given that (i) the toxins
show different sensitivity in inhibiting release of the different
amino acid neurotransmitters and (ii) TTX, a voltage dependent
Na+ channel blocker, completely abolished the Ca2+ entry mediated by 10 µM nicotine.
It may be concluded that: (i) cortical neurons contain functional nicotinic receptors since when stimulated with nicotine
these neurons release aspartate, glutamate, glycine and GABA;
(ii) the mechanism by which nicotine induces amino acid release
is exocytotic or dual, depending on the amino acid; (iii) the
effect of nicotine is specific since it is blocked by hexamethonium; (iv) L, N and P/Q type Ca2+ channels are involved in the
nicotine effect.
Notes
This work was supported by CA ICYT grants PM98-0121 and CA M
08.8/0012/1998. E.L. is the recipient of fellowships from the Ministerio de
Educación y Ciencia. S.V. is the recipient of a fellowship from UC. We
thank M. García Mauriño for helping us with culture preparation.
Address correspondence to M.P. González, Instituto de Bioquímica
(Centro Mixto CSIC-UCM), Facultad de Farmacia, Ciudad Universitaria,
28040 Madrid, Spain. Email: pilarg@ eucmax.sim.ucm.es
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