Toxicology Letters 151 (2004) 163–170

Bovine chromaffin cell cultures as model to study

organophosporus neurotoxicity
E. Quesada, M.A. Sogorb, E. Vilanova, V. Carrera∗
a División de Toxicologı́a, Instituto de Bioingenierı́a, Universidad Miguel Hernández de Elche, Avda.
de la Universidad s/n. E-03202 Elche, Alicante, Spain

Received 5 January 2004; received in revised form 21 January 2004; accepted 28 January 2004
Dedicated to the late Christian Hodel

Available online 13 April 2004

Abstract

Based on the high level of phenyl valerate esterase activities, and in particular of neuropathy target esterase (NTE) found
in bovine adrenal medulla, chromaffin cells culture have been proposed as an alternative model for the study of organophos-
phorus neurotoxicity. Organophosphorus-induced polyneuropathy is a syndrome related to the inhibition and further modifi-
cation by organophosphorus compounds of NTE (a protein that displays phenyl valerate esterase activity resistant to mipafox
and sensitive to paraoxon). Total phenyl valerate esterase activities found in homogenate, particulate and soluble fractions of
bovine adrenal medulla were 5200 ± 35, 5000 ± 280 and 1700 ± 260 mU/g tissue, respectively. Cultured chromaffin cells
displayed a total hydrolysing activity of 41 ± 5 mU/106 cells. Homogenates of bovine adrenal medulla displayed only about
6% of activity sensitive to paraoxon. Most of the phenyl valerate esterase activity inhibited by mipafox (a neuropathy in-
ducing compound) was found in particulate fraction. Cultured chromaffin cells displayed kinetics of inhibition by mipafox
similar to the kinetics displayed by homogenates of bovine adrenal medulla. We conclude that NTE could be assayed in this
system by only using one inhibitor (mipafox) instead of two (paraoxon and mipafox). Also, the proposal is supported of us-
ing chromaffin cells as in vitro model for the study of the role of NTE and related esterases in organophosphorus-induced
polyneuropathy.
© 2004 Elsevier Ireland Ltd. All rights reserved.

Keywords: Carboxylesterases; Bovine adrenal medulla; Chromaffin cells; Neurotoxicity; Organophosphorus compounds; Mipafox; Paraoxon

1. Introduction cific carboxylesterase found in nervous system called

Certain organophosphorus compounds (OPs) in-
duce the so-called ‘organophosphorus-induced de-
layed polyneuropathy’. This syndrome is related to
inhibition and further modification (aging) of a spe-
∗ Corresponding author.
E-mail address: v.carrera@umh.es (V. Carrera).
‘neuropathy target esterase’ (NTE) (Clothier and
Johnson, 1980; Lotti, 1992; Lotti et al., 1993).
Bovine adrenal medulla displays high levels of car-
boxylesterase activities (phenyl valerate hydrolysing
activity), and particularly of neuropathy target es-
terase (Sogorb et al., 1994). Primary cultures of
bovine chromaffin cells also display high levels
of carboxylesterase activities, with an important

0378-4274/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2004.01.019
164 E. Quesada et al. / Toxicology Letters 151 (2004) 163–170
proportion of NTE (Sogorb et al., 1996). This system
has been proposed as in vitro model for the study of
organophosphorus delayed neurotoxicity.
NTE has been operatively defined as the phenyl
valerate esterase activity resistant to the OP paraoxon
and sensitive to the OP mipafox. Classically, the assay
of NTE requires the incubation of the enzyme in pres-
ence of paraoxon, in order to determine the so-called
‘B-activity’ (phenyl valerate hydrolysing activity re-
sistant to paraoxon), and in presence of mipafox
plus paraoxon, in order to determine the so-called
‘C-activity’ (phenyl valerate hydrolysing activity, re-
sistant to paraoxon, Johnson, 1969, 1977). NTE is
calculated as the difference between B- and C-activity
(Johnson, 1982; Barril et al., 1989; Vilanova et al.,
1993; Carrera et al., 1994).
Paraoxon is a ‘non-neuropathic’ OP, and its role in
the classical NTE assay was to inhibit esterases with-
out relevance for the induction of neuropathy. How-
ever, studies in peripheral nerves have demonstrated
that this compound inhibits esterases in a reversible
way (Barril and Vilanova, 1997). Therefore, the al-
leged capability of this compound to discriminate
esterases should be questioned.
The present work deals with the kinetics of inhibi-
tion by paraoxon and mipafox of the phenyl valerate
esterases in bovine adrenal medulla and in chromaffin
cells cultures. We conclude that it is possible to assay
NTE activity in bovine chromaffin cells using only
mipafox.
2. Materials and methods
2.1. Reagents
Sigma Chemicals (Madrid, Spain) supplied diethyl
p-nitrophenyl phosphate (paraoxon). Phenyl valer-
ate and N,N -diisopropyl diamidophosphorofluoridate
(mipafox) were provided by Lark Enterprises (Web-
ster, MA, USA). All others reagents (analytical grade)
were obtained from local commercial sources.
For the enzymatic assays in cell cultures a modi-
fied Krebs buffer was used with the following compo-
sition: 134 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4 ,
15 mM HEPES, 11 mM glucose and 100 mg/l ascorbic
acid. In all cases the pH of the solution was adjusted
to 7.4.
Paraoxon and mipafox stock solutions (10 mM)
were prepared in acetone and Tris/citrate pH 6, re-
spectively. They were diluted with buffer to yield the
appropriate concentration immediately prior to use.
The substrate phenyl valerate was prepared from a
stock solution (473 mM) in N,N-dimethyl formamide
and diluted 1/30 (v/v) immediately before use in
water or in Krebs buffer for the cell assays.
2.2. Tissue sampling and preparation of subcellular
fractions
Fresh bovine adrenal glands were obtained from the
Alicante Municipal Slaughterhouse within 20–30 min
of death of the animals and transported to the labora-
tory immersed in standard buffer (4 ◦ C) for no longer
than 30 min. The glands were cleaned of superfi-
cial fat, and the adrenal cortex was dissected. The
adrenal medulla was homogenised using a Polytron
homogeniser (Kinematica AG) at 100 mg tissue/ml in
standard buffer.
The crude homogenate was centrifuged at 760 × g
for 10 min (4 ◦ C). The supernatant was recovered
and made up to the original volume and designated
‘sample H’; it constituted the starting material for
subsequent subfractioning. Particulate fraction (P)
was the pellet resulting from the centrifugation at
100,000 × g for 60 min (4 ◦ C) of sample H, and sol-
uble fraction (S) was the supernatant resulting from
this centrifugation. P and S fractions were made up
to the same H sample volume.
2.3. Enzymatic assays of esterase activity in bovine
adrenal medulla fractions
The kinetic studies were performed incubating
the tissue with different concentrations of paraoxon
or mipafox, according to a previously published
protocol (Sogorb et al., 1994). In brief, 100 ␮l of
inhibitor at the appropriate concentration were incu-
bated during 30 min at 37 ◦ C with 1 ml of solution
containing 2 mg of fresh tissue. Afterwards, 1 ml of
solution substrate (phenyl valerate) prepared from a
stock, as described in Section 2.1, was added, and
the incubation at 37 ◦ C was continued for another
30 min. The reaction was stopped adding 1 ml of
2% SDS-0.25 mg 4-aminoantypirine/ml in standard
buffer. The complex formed between the released
 E. Quesada et al. / Toxicology Letters 151 (2004) 163–170
phenol and 4-aminoantypirine was further reduced
with 0.5 ml of 0.4% potassium ferrocyanide in wa-
ter. The released phenol was quantified recording the
absorbance at 510 nm ( = 13.900 M−1 cm−1 ).
2.4. Chromaffin cell isolation and culture
Chromaffin cells were isolated from bovine adrenal
glands by collagenase digestion (Ballesta et al., 1989)
and cultured as was described previously (Kumakura
et al., 1986; Nakata et al., 1990). Chromaffin cells
were further separated from debris and erythrocytes
by centrifugation on Percoll gradients, as described
by Almazan et al. (1984) and Gomis et al. (1994).
Cells were maintained in monolayer cultures us-
ing Dulbecco’s modified Eagle’s medium supple-
mented with 10% fetal calf serum, 10 ␮M cytosine
arabinoside, 10 ␮M 5-fluoro-2 -deoxyuridine to pre-
vent fibroblast proliferation, 50 IU/ml penicillin and
50 ␮g/ml streptomycin. Cells were cultured in 96-well
plates (Costar) at a density of 100,000 cells per well
and were maintained at 37 ◦ C in a humidified incubator
under atmosphere of 95% air and 5% CO2 . Cells were
used between the second and third day after plating.
2.5. Enzymatic assays in cultured chromaffin cells
The kinetic studies were performed incubating the
tissue with different concentrations of paraoxon or
mipafox according to a previously published protocol
(Sogorb et al., 1996). The Biomek 1000 workstation
(Beckman Instrument, Madrid, Spain) was employed
throughout the process, including the photometric
measurement. The procedure started by washing cells
three times with Krebs solution pH 7.4. Afterwards,
the cells were incubated for 30 min at 37 ◦ C with
100 ␮l of modified Krebs solution, containing mipafox
at the appropriate concentration. Then, the inhibitor
was removed form the wells. One hundred microlitres
of substrate solution (phenyl valerate) in Krebs were
added, and incubation at 37 ◦ C was continued for
another 30 min. Reaction was stopped, and colour
was developed using 100 ␮l of 2% SDS-0.25 mg
4-aminoantypirine/ml and 50 ␮l of 1% potassium fer-
rocyanide. Finally after 20 min, the absorbance was
read at 510 nm. The resulting phenol concentration
was calculated comparing with standard curves ob-
tained simultaneously in each experiment. One mil-
165
liunit (1 mU) of enzyme corresponds to the amount
of enzyme catalysing the release of 1 nmol phenol in
1 min at 37 ◦ C.
2.6. Computation
The results of inhibitions at different paraoxon or
mipafox concentrations were analysed with mathemat-
ical models involving two exponential (sensitive com-
ponent) and one independent (resistant component)
terms, using the Sigma Plot 5.0 software. The model
equations employed were:
Activity (%)
E
= 100 × = A1 e−k1 It + A2 e−k2 It + Ar
Eo
If the time is fixed, then the model equation can be
reduced to:
Activity (%) = A1 e−k1 I + A2 e−k2 I + Ar
where E is activity at the time t when a concentration
I of inhibitor was used; Eo is the control activity in
the absence of inhibitor; A1 and A2 are the amplitudes
or proportions of the sensitive components 1 and 2; k1
and k2 are the exponential constants of each compo-
nent and Ar is the amplitude of the inhibitor-resistant
component. The I50 of each component was calculated
as I50 = ln 2/ki .
The data were fitted to the model using the following
constraints: A1 , A2 , Ar , k1 , k2 > 0 and A1 +A2 +Ar =
100.
2.7. Protein determination
The protein determinations were carried out accord-
ing to Bradford (1976), adapted to use with the Biomek
1000 workstation. Bovine serum albumin was used as
the reference standard protein.
3. Results
3.1. Esterase levels in adrenal medulla fractions
The adrenal medulla homogenate (H) showed
5200 ± 35 mU/g tissue (mean ± S.D., n = 4) of es-
terase activity. Similar to previous studies (Sogorb
166 E. Quesada et al. / Toxicology Letters 151 (2004) 163–170

Table 1
Total esterase activity in homogenate (H), particulate (P) and soluble (S) fractions of bovine adrenal medulla
Fraction Tissue (mU/g) Protein (mU/g)

H 5200 ± 35 (n = 4) (100%) 118 ± 10 (n = 2)

P 5000 ± 280 (n = 9) (96%) (75%)a 440 ± 94 (n = 7)
S 1700 ± 260 (n = 4) (32%) (25%)a 79 ± 12 (n = 4)
The mean ±S.D. for n independent experiments performed by duplicate is indicated. Samples contained 2 mg/ml of tissue were preincubated
with buffer for 30 min at 37 ◦ C. This was followed by incubation with substrate under the same conditions.
a Percent over the sum P + S.

et al., 1994), most esterase activity was found in the
P fraction (96%), although significant activity was
also noted in the S fraction (32%). Table 1 shows
the esterase activity values in bovine adrenal medulla
fractions.
3.2. Fixed time inhibition curve of bovine adrenal
medulla fractions
Fixed time (30 min) paraoxon and mipafox
inhibition curves were obtained using 20 different
concentrations (between 0 and 1000 ␮M). The kinetic
inhibition behaviour is presented in Fig. 1. Table 2
shows the parameters deduced when data were fitted
to the two-exponential model.
In the homogenate (H) paraoxon inhibition curve, a
highly paraoxon-sensitive (I50 = 0.5 ␮M) component
was observed that represents only 6% of total activity.
A second, much less sensitive component with 35%
amplitude and I50 = 206 ␮M was detected (35%).
This component may be considered as almost resis-
tant, compared to the first component. A completely
paraoxon resistant component with 58% amplitude
was also noted. Similar results were observed in the P
fraction.
The soluble fraction (S) represents only a low pro-
portion of the total activity. However, in this case,
the highly paraoxon-sensitive component (I50 =
0.14 ␮M) was found in a higher proportion (38%)
than in the P fraction. Another sensitive component
was detected (30% amplitude) with I50 = 51 ␮M.
Finally, only 32% of S fraction may be considered
as a completely resistant component. Therefore, we
conclude that the S fraction, although being only a
low proportion of the total activity, contains a high
proportion (38%) of highly paraoxon-sensitive ac-
tivity, while in P only 5% is highly sensitive. Thus,
most of the activity in the H and P fractions is
resistant or only minimally sensitive to paraoxon
(Table 2).

Table 2
Parameters deduced from the fit of the experimental data presented in Fig. 1 to mathematical models
Fraction Component Mipafox Paraoxon
Ai (%) ki (␮M−1 ) I50 (␮M) Ai (%) ki (␮M−1 ) I50 (␮M)
H 1 46 0.0616 11 6 1.0664 0.7
2 27 0.0084 82 35 0.0033 206
r 27 – – 58 – –
P (75%)a 1 70 0.11 6.3 5 1.388 0.5
2 15 0.008 85.8 46 0.034 205
r 15 – – 49 – –
S (25%)a 1 46 4.96 0.14 38 4.96 0.14
2 27 0.0135 51 51 0.0135 51
r 27 – – 32 – –
Samples were incubated with the corresponding inhibitor for 30 min at 37 ◦ C. This was followed by the incubation with substrate in the
same conditions. Experimental data were fitted to the mathematical model presented in Section 2. Ai represents the amplitude of each
sensitive component while Ar represents the amplitude of the resistant component. ki are the exponential constants of each sensitive
component. I50 were calculated in all cases as ln 2/ki .
a Percent over the sum P + S.
 E. Quesada et al. / Toxicology Letters 151 (2004) 163–170 167

PARAOXON MIPAFOX

100
H H

80
% Activity

60

40

20

100
P P

80
% Activity

60

40

20

100
S S

80
% Activity

60

40

20

0
0 200 400 600 800 1000 0 200 400 600 800 1000

Paraoxon concentration (µM) Mipafox concentration (µM)

Fig. 1. Paraoxon and mipafox inhibition curves of esterase activities in homogenate (H), particulate (P) and soluble (S) fractions of
bovine adrenal medulla. Samples were incubated with several inhibitor concentrations for 30 min at 37 ◦ C. Afterwards, esterase activity
was assayed using phenyl valerate as was described in Section 2. Control activities were similar to values displayed in Table 1. Results
were expressed as percentage of controls and were fitted to the exponential models presented in Section 2. The parameters of the fit are
displayed in Table 2. Each point was assayed by triplicate. Other independent experiments displayed similar results.

Data obtained from the inhibition curves with mi-
pafox (Fig. 1, right) were also fitted with a model of
two (sensitive plus resistant) components (Table 2).
In H, about half of total activity (46%) is a high
sensitive component (I50 = 11 ␮M); 27% of activ-
ity is less sensitive (I50 = 82 ␮M) and only a low
proportion (27%) could be considered as resistant.
Most mipafox-sensitive activity is associated with
membranes, 75% of this activity is recovered in the P
fraction in which 85% is sensitive to mipafox. In the
soluble fraction also about half of the activity is sen-
sitive to mipafox, but the S fraction represents only a
lower proportion of total activity.
3.3. Time-dependent inhibition by mipafox
The time-dependent esterase inhibition of the P frac-
tion in bovine adrenal medulla was studied at sev-
eral mipafox concentrations (Fig. 2). When the model
equations were fitted to the kinetic data (Table 3) the
curves followed a two-exponential behaviour with a
resistant component.
3.4. Mipafox inhibition of cultured chromaffin cells
Fig. 3 shows the fixed time (30 min) mipafox inhibi-
tion curve for esterase activity when bovine chromaffin
168 E. Quesada et al. / Toxicology Letters 151 (2004) 163–170

100

80
% Activity

60
1.5 µM

40
5 µM
15 µM
20 75 µM

0
0 20 40 60 80 100 120

Time (min)

Fig. 2. Time-progressive inhibition of the phenyl valerate esterase activity by mipafox in particulate (P) fraction of bovine adrenal medulla.
Samples of the P fraction of bovine adrenal medulla were incubated with mipafox at the displayed concentrations during the indicated
time. Phenyl valerate esterase activity was assayed as described in Section 2. Results were expressed as percentage of the controls (samples
incubated during 30 min without mipafox).

cells were cultured, as indicated in Section 2. Mipafox nent could be discriminated. The sensitive component
inhibition data were analysed with the model equa- represents an 80% activity (I50 = 20 ␮M) while the
tions, and the curves followed exponential behaviour resistant component represented the remaining 20%.
(Table 4). Only one sensitive and one resistant compo- Table 5 shows the phenyl valerate esterase and
NTE activities in bovine chromaffin cell cultures.
Table 3
Parameters deduced from the fit of the experimental data presented
in Fig. 2 to mathematical models
Mipafox Component Ai (%) ki (␮M−1 ) I50 (␮M)
100
concentration
(␮M)
80
1.5 1 11 0.787 0.9
% Activity

2 88 0.0039 178
60
r – – –
5 1 16 1.512 0.5 40
2 71 0.012 58
r 13 – – 20

15 1 34 1.324 0.5
0
2 46 0.028 25
0 200 400 600 800 1000
r 20 – –
Mipafox concentration (µM)
75 1 58 655.374 0.001
2 25 0.064 11 Fig. 3. Curve of the inhibition of the phenyl valerate esterase
r 17 – – activities by mipafox in cultured chromaffin cells. Cells were
Chromaffin cells were incubated with mipafox for 30 min at 37 ◦ C. incubated with the displayed mipafox concentrations during 30 min
This was followed by the incubation with substrate in the same at 37 ◦ C. Then, the inhibitor was removed and phenyl valerate
conditions. Experimental data were fitted to the mathematical esterase activity assayed as was indicated in Section 2. Results were
model presented in Section 2 with one sensitive component plus expressed as percentage of the control (0 ␮M mipafox). Control
another resistant. Ai and Ar represent the amplitudes of the sen- cells displayed an activity of 41 ± 5 mU/106 cells. Each point was
sitive component and resistant component, respectively. ki are the assayed by quadruplicate. Results were fitted to a mathematical
exponential constants of the sensitive component, which I50 was model displayed in Section 2 with two components (one sensitive
calculated as ln 2/ki . plus one resistant). Results of the fits are displayed in Table 4.
 E. Quesada et al. / Toxicology Letters 151 (2004) 163–170
Table 4
Parameters resulting from fitting the data of the inhibition of phenyl
valerate esterase activities by mipafox in cultured chromaffin cells
(Fig. 3) to exponential model
i Ai (%) ki (␮M−1 ) I50 (␮M)
1 80 0.034 21
r 20 – –
The chromaffin cells were preincubated with mipafox for 30 min
at 37 ◦ C and after this, incubated with the substrate for 30 min
at 37 ◦ C. Ai represents the amplitude of each component, ki is
the exponential constant of each component. I50 was calculated as
ln 2/ki . Components 1 is the sensitive components and component
r is the resistant component usually called ‘C-activity’.
Table 5
Phenyl valerate esterase and NTE activities in bovine chromaffin
cell cultures
Activity mU/106
A 22 ± 2
B 15 ± 3
C 2 ± 0.7
NTE 11 ± 3
The mean values ± S.D. for n = 4 independent experiments per-
formed by duplicate is indicated. A: total esterase activity; B: es-
terase activity resistant to 40 ␮M of paraoxon (30 min at 37 ◦ C);
C: esterase activity resistant to 40 ␮M of paraoxon + 250 ␮M of
mipafox (30 min at 37 ◦ C); NTE = B − C.
NTE activity was calculated under standard con-
ditions as the difference between activity resistant
to 40 ␮M of paraoxon (B) and activity resistant to
40 ␮M of paraoxon plus 250 ␮M of mipafox (C).
NTE activity (11 ± 3 mU/106 cells) represents 50%
of total phenyl valerate esterase activity (A) and the
73% of B-activity. The Student’s t-test showed that
the differences between B and NTE activities were
not significant.
4. Discussion
Neuropathy target esterase is the target of
the so-called ‘organophosphorus-induced delayed
polyneuropathy’. It is accepted that ‘non-neuropathic’
OPs (i.e. paraoxon) do not inhibit NTE. Therefore,
NTE is typically assayed as the phenyl valerate es-
terase activity resistant to paraoxon and sensitive to
mipafox. This operative definition implies that two
169
inhibitors are necessary for the assay of this activity:
the enzyme is incubated in presence of paraoxon and
in presence of paraoxon plus mipafox, and NTE is
calculated as the difference of phenyl valerate activity
between these two conditions.
Certain findings have suggested to change this
methodology. First, reports showed a reversibility of
the inhibition of esterases by paraoxon in certain ner-
vous tissues (Barril and Vilanova, 1997) and, second,
reports showed interferences between paraoxon and
phenyl valerate, both interacting with the active centre
of the enzymes (Estevez, 2003). These two findings
together suggest that paraoxon might not be a good
choice for discriminating between NTE and other
esterases.
Former studies have reported that bovine adrenal
medulla is a tissue with high levels of phenyl valerate
esterase activities, and in particular of NTE (Sogorb
et al., 1994). Since chromaffin cells in culture display
a similar distribution of esterases, this system is pro-
posed as possible in vitro model for the study of NTE
(Sogorb et al., 1996). It will require detailed charac-
terisation of the NTE and related esterases in these
tissues.
The present study has found that homogenates and
fractions of bovine adrenal medulla displayed only
6% of total activity sensitive to paraoxon. Most of
the phenyl valerate esterases inhibitable by the neu-
ropathic OP mipafox were found in particulate frac-
tion. Around 85% of total activity exhibited by this
tissue was considered as sensitive to mipafox. This
sensitive activity was found in two components (70%
amplitude with I50 = 6 ␮M and 15% amplitude with
I50 = 86 ␮M). The total percentage of activity sensi-
tive to mipafox was similar in chromaffin cells in cul-
ture (80%). However, in this case it was not possible
to discriminate two components.
Due to the low proportion of the activity sensitive
to paraoxon these data suggest that in these tissues
it might be possible to discriminate NTE using only
mipafox as inhibitor. In this case NTE should be de-
fined as phenyl valerate esterase activity sensitive to
mipafox. According to this definition, and since NTE
was previously reported to be in the microsomal frac-
tion of bovine adrenal medulla (Sogorb et al., 1994),
it should represent around 80% of total phenyl valer-
ate esterase activity in this tissue (Fig. 1). This implies
a magnitude of around 4000 mU of NTE activity per
170 E. Quesada et al. / Toxicology Letters 151 (2004) 163–170
gram tissue. Previous studies in bovine adrenal mi-
crosomes reported an activity of 3800 mU of phenyl
valerate esterase, resistant to 40 ␮M paraoxon and sen-
sitive to 250 ␮M mipafox (Sogorb et al., 1994). There-
fore, this supports the proposal to assay NTE activity
in membrane fraction of bovine adrenal medulla using
only mipafox.
In addition, there were no statistically significant
differences (Student’s t-test) between B and NTE ac-
tivity of chromaffin cultured cells when these activi-
ties were assayed using standard conditions (Table 5).
It supports the proposal to assay NTE activity using
only the mipafox inhibitior.
In essence, we have presented evidence that it is pos-
sible to assay NTE activity in microsomes of bovine
adrenal medulla using only one inhibitor. It gives ad-
ditional weight to chromaffin cells as an in vitro model
for the study of the physiological role of NTE and re-
lated esterases.
Acknowledgements
This study was supported by FIS grant 1100/00. E.
Quesada is enjoying a fellowship from Spanish Min-
istry of Education and Culture. We thank L. Rojo for
her technical help.
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