From the
Bovine chromaffin cells cultured for 5 days in the presence of
depolarizing concentrations of K
Trans-synaptic induction (stimulation-secretion-synthesis
coupling) is an important regulatory process, allowing the neuron to
adapt to a strong stimulation by increasing the rate of synthesis of
proteins required for secretion(1) . Trans-synaptic induction
was initially observed for the biosynthetic enzymes of adrenal medulla,
tyrosine hydroxylase(2) , and dopamine
In adrenal medulla, the
catecholamine biosynthetic pathway involves the translocation of
dopamine from the cytosol to the chromaffin granule matrix. The uptake
is catalyzed by the vesicular monoamine transporter, a
H
What are the
consequences of an increased expression of the gene at the cellular
level? Does an increase of the monoamine transporter result in a
differential change in the composition of the secretory granule
membrane? To answer these questions, we analyzed cells cultured for 5
days in high K
For ultrastructural analysis, cell cultures grown on
collagen-coated glass coverslips were rinsed in phosphate-buffered
saline (PBS) and fixed for 1 h at room temperature in 2% glutaraldehyde
in phosphate buffer (0.1 M, pH 7.2). Cells were post-fixed for
1 h in 1% osmium tetroxide, dehydrated in ascending ethanol solutions,
and flat-embedded in Spurr epoxy resin (17). For morphometric analysis,
sections of randomly chosen areas were cut at uniform thickness (silver
interference color) from control and KCl-treated chromaffin cell
cultures. To eliminate variations due to differences in distributions
between the various cell compartments, entire sections were
photographed at a constant microscope magnification (
Chromaffin cells plated on collagen-coated 35-mm coverslips
at about 10
Bovine chromaffin cells were isolated and purified as
described (16). They were suspended at 10
The vesicular monoamine transporter was assayed by measuring
[
Catecholamines
were measured by the trihydroxyindole technique(21) . To
estimate the cellular catecholamine content, cells were scraped with
200 µl of 0.2% Triton X-100, the extract was precipitated by
addition of trichloroacetic acid (6% final concentration), and the
supernatant was assayed for catecholamines. Adrenaline and
noradrenaline were estimated by fluorometry after differential
oxidation(22) .
Cytochrome b
Cells (20-30
In contrast, the number of
[
The effect of a repolarizing period was also tested. Cells
were incubated in normal Locke medium for 45 min at 37 °C before
being challenged as above. This repolarization period is equivalent to
that used for the uptake of [
To identify the structures associated with
[
After 5 days in a medium containing depolarizing
concentrations of K
In K
K
It has already
been suggested that the protein composition of the secretory granule
membrane may change under certain conditions. After repeated reserpine
injections, the specific activity of membrane bound dopamine
What is the
physiological meaning of this regulation? It is classically proposed
that the rate-limiting step in catecholamine biogenesis is tyrosine
hydroxylation and that regulation of this pathway, by phosphorylation
of the enzyme or by increase of its rate of synthesis, occurs mainly at
this level. However, it has also been suggested that vesicular uptake
might be rate-limiting(41) . This hypothesis was formulated by
comparing in vitro measurements of the time required to fill
up rat chromaffin granules and in vivo estimates of the
maturation time obtained either in pulse experiments followed by
density gradient analysis or from the recovery of the normal granule
catecholamine content and granule density after insulin stimulation.
Under prolonged K
A more
quantitative estimate of the regulation of catecholamine biogenesis
might possibly be achieved by applying the metabolic network theory. In
this theory, regulation is not assumed to result from a unique step,
but to involve the whole pathway, each step being characterized by a
partial control coefficient(42) . The regulatory properties of
the vesicular monoamine transporter might be important in the early
steps of neurogenerative diseases, such as Parkinson's disease,
where the loss of the first neurons would be compensated by
overstimulation of those remaining and the induction of adaptative
processes, such as that described for the vesicular monoamine
transporter. This early phase would account for the threshold effect
reported in the development of the disease(43) .
Continuous
stimulation clearly decreased the number of granules and changed their
composition. Three different interpretations are possible, which might
not be mutually exclusive. First, the decrease might easily be
attributed to a dynamic state, since exocytosis was not blocked under
these conditions (model I). The newly synthesized vesicles would have
different properties. Alternatively, the conditions used might affect
granule biogenesis, decreasing the size of the vesicular pool and
thereby changing their composition (model II). Finally, it might also
be proposed that the decrease in the number of secretory granules
results from the phenotypic change associated with the culture in
elevated potassium (model III). Unsicker et al.(25) already reported the growth of neurite-like processes
and an increase of the noradrenaline to adrenaline ratio. In addition,
we have also noted a marked increase of synaptophysin and a decrease in
chromogranin A. These changes indicate a shift from a neuroendocrine to
a neuronal phenotype, which might be associated with a decrease of the
number of secretory granules.
We thank Dr. D. K. Apps (University of Edinburgh) for
the gift of the antibody against cytochrome b
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
ions show a decreased
number of secretory (chromaffin) granules per cell. These cells were
still capable of exocytosis. Their contents in catecholamine and
chromogranin A, components of the granule matrix, and cytochrome b
, a major protein of the granule membrane, were
decreased to 35, 30, and 50% of control cells, respectively. However,
in the same cells, the number of
[
H]dihydrotetrabenazine binding sites, a specific
ligand of the vesicular monoamine transporter, was increased to 180% of
controls. In situ uptake of noradrenaline in permeabilized
cells indicated that [
H]dihydrotetrabenazine
binding sites were associated with a functional vesicular monoamine
transporter. When analyzed by isopycnic centrifugation, these sites
cosedimented with catecholamine, chromogranin A, and cytochrome b
, in a peak with a density lighter than that
from controls. The composition of this peak suggests that it contains
incompletely matured secretory granules, with a 3-5-fold increase
in the vesicular monoamine transporter content of this membrane. This
increase might indicate that an adaptative process occurs which allows
a faster filling of the granules in continuously secreting cells.
-hydroxylase(3) , but it has been extended to proteins of
the chromaffin granule matrix, such as proenkephalin(4) . Strong
stimulation may therefore change the composition of the
``secretory mixture''(5) , presumably for
physiological purposes. These adaptative changes can be reproduced in vitro by stimulation with nicotinic agonists or by
depolarization of bovine chromaffin cells in
culture(6, 7, 8) .
/monoamine antiporter utilizing the
H
-electrochemical gradient generated by an
ATP-dependent proton pump of the V-type(9) . The same
transporter also operates in monoaminergic neurons, although two genes,
VMAT
and VMAT
(vesicular monoamine transporter
1 and 2)
(
)encoding this activity have been
recently described(10, 11) . In rats, VMAT
is expressed in adrenal medulla, whereas VMAT
is
present in the brain. On the other hand, both genes are expressed in
bovine adrenal medulla (12, 13) and from analysis of the
N-terminal extremity of the chromaffin granule protein VMAT
is the major gene(14) . Several ligands are specific for
the vesicular monoamine transporter, such as reserpine or
dihydrotetrabenazine ([
H]TBZOH)(15) .
These ligands are useful tools not only for biochemical experiments,
but also for physiological studies, including study of the regulation
of the expression of the vesicular monoamine transporter. After an
insulin shock, the number of [
H]TBZOH binding
sites of rat adrenal medulla increased for several days and was maximal
after 4-6 days(5) . A similar increase was observed in
vitro in bovine chromaffin cells in culture(16) . The
[
H]TBZOH binding site increase was maximal after
several days of culture in the presence of carbamylcholine or
depolarizing concentrations of potassium ions. The response involved
Ca
channels, and it was mimicked by forskolin and by
phorbol esters. Increase in the number of binding sites seemed likely
to involve transcriptional activation, since it was blocked by
actinomycin and cycloheximide. The hypothesis of a regulated expression
of the vesicular monoamine transporter was strongly supported by the
finding that K
depolarization of bovine chromaffin
cells increased the transcription of the VMAT
gene,
encoding the tetrabenazine-sensitive vesicular monoamine
transporter(12) . This was clearly seen on Northern blots, and
competitive polymerase chain reaction indicated a 3-fold increase in
VMAT
expression after a 6-h depolarization in 55 mM KCl. The concept of trans-synaptic induction might therefore be
extended to the vesicular monoamine transporter.
medium, which doubled their
[
H]TBZOH binding site content, and characterized
their secretory granules. We determined the subcellular distribution of
[
H]TBZOH binding sites in these cells, and we
investigated the functional meaning of this increase.
Electron Microscopy and Morphometry
3900), and
negatives were subsequently enlarged to give a final magnification of
8900. The number of secretory granules was counted and recorded
for each cell profile in each micrograph. Granules were classified as
either noradrenergic or adrenergic on the basis of their
ultrastructural appearance. Adrenergic granules were taken as those
with a larger diameter and lower uniform density of granule matrix,
whereas granules with very dense contents separated from the granule
membrane by an electron-lucent space were classified as
noradrenergic(18) . Each cell profile was then cut out on a
photocopy the same size as the micrograph and weighed, to provide a
relative estimate of the area. The actual surface area was determined
by comparison with the weight of a known area of photocopy paper, which
enabled granule density/100 µm
to be calculated.
Immunofluorescence
cells/coverslip were fixed for 30 min in
phosphate-buffered 4% formaldehyde (freshly prepared from p-formaldehyde), washed in PBS, and incubated for 30 min in
``blocking solution'' (5% BSA in PBS). Double
immunofluorescence was performed by incubating cultures simultaneously
for 2 h at room temperature with the two primary antisera: mouse
monoclonal anti-tyrosine hydroxylase (Euromedex, Strasbourg, France)
and a previously characterized (19) rabbit polyclonal antibody
prepared against a synthetic peptide named CAP14, residues
316-329 in the sequence of bovine chromogranin A. These were
diluted 1:500 and 1:1000, respectively, in PBS containing 1% BSA and
0.1% Triton X-100. After PBS washes, bound antibody was detected with
affinity-purified rhodamine-conjugated goat anti-mouse and
fluorescein-conjugated goat anti-rabbit immunoglobulins (Euromedex,
Strasbourg, France), diluted 1:200 in PBS/BSA. Coverslips were examined
with a Zeiss Microscope II equipped with Neofluor objectives and the
appropriate fluorescent barrier filters, and selected fields were
photographed on Ilford HP5 film.
Culture of Chromaffin Cells
cells/ml in
Dulbecco's modified Eagle's medium (Life Technologies,
Inc.), supplemented with 10% fetal calf serum (Life Technologies, Inc.)
containing 10 µM cytosine arabinoside, 10 µM 5-fluoro-2`-deoxyuridine, 50 µg/ml of streptomycin, and 50
units/ml of penicillin and plated at a density of 5
10
cells in 2-cm diameter wells or 5
10
cells in
5-cm diameter Petri dishes or 20-30
10
cells
in 75-cm
flasks. They were incubated for 2 days at 37
°C in a 5% CO
-saturated atmosphere. The culture medium
was then changed, and salts or drugs were added. Cells were harvested
by scraping in Locke medium or in 0.2% Triton X-100.
Biochemical Assays
H]TBZOH binding(20) . Samples from cell
homogenates or fractions of centrifugation gradients were incubated for
1 h with 3 nM [
H]TBZOH in 1 ml of Locke
medium containing 25 mM Hepes buffer (pH 8) at 25 °C, and
filtered on HAWP filters (Millipore). Nonspecific binding was obtained
by adding 3 µM TBZ to the incubation. Previous analyses (16) indicated a homogenous class of binding sites in control
and K
-depolarized cells with a K
of 16 nM and a B
value of 1
pmol/mg of protein for control cells homogenates.
in
cellular or subcellular fractions was estimated by semiquantitative
immunoblotting(16) , using an anti-cytochrome b
antiserum (generous gift from Dr. D. Apps), at 1:1500 dilution,
and radioiodinated protein A. The same approach was used to assay
intracellular or released chromogranin A, with an anti-chromogranin A
antiserum at 1:500 dilution detected by radioiodinated protein A or by
anti-rabbit immunoglobulin alkaline phosphatase conjugate.
Alternatively, subcellular fractions from the centrifugation gradients
were assayed for chromogranin A by enzyme-linked immunosorbent
assay(23) . This assay was linear up to 20 ng of chromogranin A.
Uptake and Release Experiments
Uptake and Release of
[
Chromaffin cells were
incubated for 30 min at 37 °C in Locke medium containing 2.5 mM CaClH]Noradrenaline
, 1.25 mM MgSO
, 0.5 mM ascorbic acid, and 1 mM Hepes, pH 7.4, and then for 45
min in 500 µl of the same medium to which 10 µM [
H]noradrenaline (0.3 µCi/ml) was added.
When the effects of TBZ and desipramine were tested, these drugs were
added during the uptake period at 3 and 4 µM concentration, respectively. Cells were washed four times for 10
min with Locke solution containing 2.5 mM CaCl
and
twice for 5 min with calcium-free Locke solution. Under the conditions
used, K
-depolarized and control cells contained the
same radioactivity. Cells were stimulated by suspension in 250 µl
of Locke solution, with or without 2.5 mM CaCl
,
containing 55 mM KCl or 20 µM nicotine. The
radioactivity in the cells and in the centrifuged extracellular fluids
was measured by liquid scintillation. Release of
[
H]noradrenaline was expressed as the percentage
of total radioactivity present in the cells prior to stimulation in
calcium containing media. Secretion after permeabilization by
streptolysin O was tested as described previously (24).
Release of Endogenous Catecholamines
After 5 days
in control or in K-containing medium, cells were
rapidly washed with 1 ml of Locke solution containing 1 mM MgCl
, 0.5 mM ascorbic acid, and 10
mM Hepes buffer, pH 7.4. They were either immediately
stimulated or preincubated for 45 min at 37 °C in 500 µl of
calcium containing Locke solution, before stimulation. Cells were
stimulated by resuspension in the wash medium containing either 55
mM KCl or 20 µM nicotine, in the presence or
absence of 2.5 mM CaCl
. Catecholamines were
assayed fluorometrically, in the external medium and in the cell
pellet. Release of catecholamines was expressed as the percentage of
the total catecholamine cell content.
In Situ Vesicular Uptake of
[
Chromaffin cells were
washed with 1 ml of Locke solution containing 1 mM MgClH]Noradrenaline
, 0.5 mM ascorbic acid, 10 mM
Hepes buffer, pH 7.4. They were permeabilized by incubation with
streptolysin O (4 units/well) in 200 µl of 150 mM potassium glutamate, 0.2% BSA, 4 mM ATP, 3 mM
MgSO
, and 5 mM nitrilotriacetic acid, pH 7.2, for
3 min and washed twice with 500 µl of medium without streptolysin
O. [
H]Noradrenaline uptake was measured in 500
µl of the same medium containing 0.5 mM ascorbic acid, 1
mM pargyline, 10 µM
[
H]noradrenaline (0.15-0.6 µCi/ml), at
25 °C. To avoid interference from endogenous monoamines, the
[
H]noradrenaline concentration was adjusted with
unlabeled noradrenaline to 10 µM, a concentration which is
saturating for the vesicular monoamine transporter. Moreover, the
concentration of endogenous catecholamines in the external medium after
permeabilization was the same in K
-treated and control
cells. Nonspecific uptake was measured by addition of 3 µM TBZ to the incubation medium. The uptake was stopped by washing
three times with 1 ml of the permeabilization medium. Cells were
scraped in 200 µl of 0.2% Triton X-100 and the radioactivity of
aliquots measured by liquid scintillation.
Subcellular Fractionation of Chromaffin Cells
10
cells) were
cultured in 75-cm
flasks or 5-cm Petri dishes. They were
harvested by scraping in 5 ml of 5 mM EDTA, 20 mM Hepes buffer, pH 7.4, containing 10.5% sucrose and 2 units/ml
leupeptin (buffer A). This homogenate was centrifuged for 10 min at 800
g, and the resulting pellet was resuspended in 0.5 ml
of the same buffer, homogenized manually in a glass-Teflon homogenizer,
and again centrifuged under the same conditions. The pooled
supernatants (postnuclear supernatant) were centrifuged at 200,000
g for 80 min. The pellet was resuspended in 0.6 ml of
buffer A, and 0.5 ml of the suspension was layered onto 4.5 ml of a
linear sucrose gradient (23-55%) in the same EDTA/leupeptin/Hepes
buffer. The tubes were centrifuged for 3 h at 4 °C at 240,000
g in a SW-65 Beckman rotor. Fractions (400 µl)
were collected from the bottom of the tubes.
Morphological Changes Induced by K
As described by Unsicker et
al.(25) , long term depolarization of bovine
chromaffin cells induces flattening of the cells and the extension of
neurite-like processes. The difference between control and depolarized
cells was clearly seen after 5 days of culture on both plastic dishes
or collagen-coated coverslips. At the ultrastructural level (Fig. 1), dense secretory vesicles (chromaffin granules) were
observed in both depolarized and control cells. Two types of granules
were present in both cultures, small dense noradrenaline-containing
granules in noradrenergic cells and larger lighter
adrenaline-containing granules(18) . In the
KDepolarization
-depolarized cultures, 37% of the cells were
estimated to be of the noradrenergic type, whereas control cultures
contained only 16% of noradrenergic cells (). Because the
number of cells was the same in the two types of culture, this change
was attributed to phenotype plasticity and not to differential
survival(25) . K
-cultured cells were more
vacuolar and clearly contained less secretory granules than control
ones. The granules of the K
-cultured cells often
appeared to have an electron-dense core within a larger vesicle. The
number of granules per unit surface area was determined under both
culture conditions. K
depolarization resulted in a
decrease in the number of granules to about 30% of the controls, in
both cell types. Long term stimulation by K
depolarization therefore decreases the number of chromaffin
granules, presumably as a result of continuous exocytosis, but without
completely depleting the cell of its secretory granules.
Figure 1:
Electron micrographs of control (a) and KCl-treated (b) bovine chromaffin cell
cultures. Both show cells with secretory granules more typical of
adrenergic chromaffin cells, although some granules with apparently
condensed matrices are present in the treated cells. Note the dramatic
reduction in granule density in treated cells. Bar represents
1 µm.
An
immunofluorescence study using antibodies against chromogranin A, a
component of chromaffin granule matrix, confirmed the decrease in the
number of mature secretory granules (Fig. 2). Almost all cells in
treated cultures showed a dramatic reduction in labeling for
chromogranin A (compare Fig. 2, b and d),
although a faint punctate labeling could still be detected. Less than
5% of the cells, which also had undergone less obvious morphological
changes, appeared to be much less affected. The two cell cultures were
also labeled for cytosolic tyrosine hydroxylase (Fig. 2, a and c), used as a marker of trans-synaptic
induction(2, 8) . Compared with control cultures (Fig. 2a), the intensity of tyrosine hydroxylase
labeling in treated cultures (Fig. 2c) was increased in
all cells, except those which were still labeled fairly strongly for
chromogranin A, and this diffuse cytoplasmic labeling filled both
perikarya and cell extensions. Thus, the intensity of tyrosine
hydroxylase labeling in treated cultures was negatively correlated with
that for chromogranin A.
Figure 2:
Double immunofluorescent labeling of
control (a, b) and KCl-treated cultures (c, d) for
tyrosine hydroxylase (a, c) and chromogranin A (b,
d). Note that cells have an apparently rounder morphology in
control compared with treated cultures. Tyrosine hydroxylase
fluorescence appears to be augmented in treated cultures, whereas
chromogranin A fluorescence is dramatically reduced in the vast
majority of cells in treated cultures. A small number, less than 5% of
all chromaffin cells, retain relatively high level of labeling with
this antibody (d) in treated cultures. Bar represents
25 µm.
Biochemical Analysis of
K
In parallel to the
morphometric study, a biochemical analysis of the catecholamine content
of the two types of culture was performed. The designation of the two
cell types as adrenergic and noradrenergic cells was confirmed by the
ratio of the two catecholamines (). K-depolarized Cells
depolarization decreased the catecholamine content to 35% of the
controls. In addition, immunological assays were used to estimate the
cellular contents of chromogranin A and cytochrome b
, which are major components of the matrix and
of the membrane of chromaffin granules, respectively(26) .
K
depolarization induced a marked decrease of the
content not only of chromogranin A, but also of cytochrome b
().
H]TBZOH binding sites specifically associated
with the vesicular monoamine transporter in the same cultures was shown
to increase to 180% of controls after long term depolarization ().
Secretory Capacity of K
To characterize the functional aspects of
the granules, the secretory capacity of these cells was analyzed in
various ways. First, K-depolarized
Chromaffin Cells
-depolarized and control cells
were charged with [
H]noradrenaline in a
nondepolarizing Na
-containing medium and challenged
with 20 µM nicotine in the presence of Ca
ions. [
H]Noradrenaline uptake was
desipramine-sensitive, indicating that it occurred through the cell
membrane transporters. It was also tetrabenazine-sensitive, since 3
µM TBZ decreased the cellular
[
H]noradrenaline content by 30-50% in
control cells and by 60% in K
-depolarized ones, thus
showing that the tritiated catecholamine accumulated in the vesicular
compartment, through the vesicular transporter. The conditions of
uptake (substrate concentration, uptake incubation time) were adjusted
so that the same radioactivity accumulated in both cultures. Under
these conditions, the secretory capacity of K
-cultured
cells, defined as the percentage of tritiated noradrenaline released,
was similar to that of controls (Fig. 3A). Secretion in
these cells was Ca
-dependent and it occurred through
the vesicular compartment, since it was inhibited when
[
H]noradrenaline was taken up in the presence of
3 µM TBZ. The release of
[
H]noradrenaline was also tested after
permeabilization of the charged cells by the bacterial toxin
streptolysin O(27) . When such cells were challenged with 20
µM Ca
,
[
H]noradrenaline release was identical in
K
-cultured and in control cells (data not shown),
confirming that release occurred from the vesicular pool (and not the
cytoplasm), by an unaffected process.
Figure 3:
Secretion of catecholamine by chromaffin
cells cultured for 5 days in control (C) or high potassium (K) medium. A, [H]noradrenaline
release. Control (C) or K
-depolarized (K) cells were incubated for 45 min in the presence of
[
H]noradrenaline and then challenged for 15 min
at 37 °C by 20 µM nicotine in the presence (black
boxes) or absence (white boxes) of 2.5 mM
CaCl
. Released [
H]noradrenaline is
expressed as percentage of total radioactivity present in the cell
prior to stimulation. Results are means (± S.E.) of six
independent experiments. B, release of endogenous
catecholamine by control (C) or K
-depolarized (K) cells. After resuspension in Locke solution, cells were
immediately stimulated with 20 µM nicotine for 10 min at
37 °C, in the presence (black boxes) or absence (white
boxes) of 2.5 mM CaCl
. Results are means of
five independent experiments. C, release of endogenous
catecholamine by control (C) or K
-depolarized (K) cells, preincubated in nondepolarizing medium. After
resuspension in Locke medium, cells were incubated for 45 min at 37
°C before stimulation. Results are means (±S.E.) of four
independent experiments.
In the above approach,
[H]noradrenaline uptake required repolarization
of cells, since the plasma membrane transporter utilizes the
Na
electrochemical gradient as a driving force. The
secretory capacity of long term K
-treated cells was
also analyzed by following the release of endogenous catecholamines by
spectrofluorometry. After rapid transfer to
Ca
-containing Locke medium and stimulation by 20
µM nicotine, K
-cultured and control cells
were found to release 2.6 ± 0.2 and 14 ± 1.4 of nmol
catecholamine/well, respectively, in a Ca
-dependent
nicotine-stimulated manner. Expressed as percentages of cellular
catecholamines, the corresponding figures are 9 ± 0.2 and 17
± 1.0%, taking into account the lower amine content of the
depolarized cells (Fig. 3B). This result suggests some
desensitization of the secretory pathway, which could account for the
presence of a stock of secretory granules in continuously stimulated
cells.
H]noradrenaline.
Under these conditions, 3 ± 0.1 and 9 ± 1.5 nmol of
catecholamine/well were specifically released by
K
-cultured and control cells; however, the percentages
were similar, corresponding to 14.5 ± 0.8 and 16 ± 1.4%,
respectively (Fig. 3C). This might be because
desensitization is rapidly reversible, and all secretory granules are
functional, secretion being proportional to the number of granules.
Vesicular [
[H]Noradrenaline
Uptake by K
-depolarized Chromaffin
Cells
H]TBZOH binding to the vesicular
monoamine transporter occurs even when the transporter has been
uncoupled from the H
electrochemical
gradient(15) . Binding of this ligand has been observed using
detergent-solubilized transporter(28) . The increase of binding
in K
-depolarized cells might have been due to proteins
present on non vesicular structures, such as the endoplasmic reticulum,
which are unable to generate a H
-electrochemical
gradient, or to inactive proteins present in the endosomal network. To
test the functionality of the transporter, cells were permeabilized
with streptolysin O(27) , and vesicular ATP-dependent
[
H]noradrenaline uptake was measured in situ (Fig. 4). The accumulation of
[
H]noradrenaline was insensitive to 1 µM desipramine, showing that the plasma membrane transporter was
by-passed, but was blocked by 3 µM TBZ, indicating a
vesicular uptake. The experiment was performed at a saturating
noradrenaline concentration, 10 µM, to measure the
transporter concentration and to avoid any interference with residual
endogenous monoamines. In three experiments similar to that of Fig. 4, the initial rate of uptake was increased in the
K
-depolarized cells by a factor of 1.7 ± 0.3,
which was similar to the increase of [
H]TBZOH
binding sites. It should be noted that in K
-treated
cells the uptake leveled more rapidly than in control ones, indicating
a lower plateau value consistent with a lower number of granules in the
treated cells.
Figure 4:
Vesicular uptake of
[H]noradrenaline in permeabilized cells.
Chromaffin cells were permeabilized by incubation with the bacterial
toxin streptolysin O. Vesicular uptake was then measured in situ by addition of [
H]noradrenaline and ATP.
Nonspecific uptake was estimated in the presence of 3 µM TBZ and was subtracted. Data are means (±S.E.) of two
independent experiments in which measurements were performed in
quadruplicate.
Subcellular Fractionation of
K
To determine the
localization of [-depolarized Cells
H]TBZOH binding sites, the
cellular extracts were fractionated by isopycnic centrifugation on
linear sucrose gradients (23-55%), and the profiles of
[
H]TBZOH binding sites were determined for
K
-depolarized and control cells. Three representative
experiments are shown in Fig. 5. In both types of culture, a main
peak of [
H]TBZOH binding sites was observed. In
control cells, the peak was centered in the dense part of the gradient
at 45% (1.6 M) sucrose. In more than 15 experiments,
K
-depolarized cells were characterized by a larger
peak, the ratio of peak areas reflecting the cellular increase in
[
H]TBZOH binding. The peak from depolarized cells
also had a lighter equilibration density, and it was often
asymmetrical, being broader on the lighter side. In nine different
experiments, the fraction of sites equilibrating at a density lighter
than 43% sucrose in control and in depolarized cells was 33 ± 10
and 62 ± 13%, respectively. The asymmetry of the broad peak of
K
-cultured cells was not due to size heterogeneity,
since this peak was not resolved when the force and the duration of the
centrifugation were decreased.
Figure 5:
Analysis of [H]TBZOH
binding sites by isopycnic centrifugation on linear sucrose gradients.
Resuspended high speed pellets from control (
) or 5-day
depolarized (
) cells were layered onto a 23-55%
(0.7-2 M) sucrose gradient and centrifuged for 3 h at
240,000
g. The fractions were analyzed for specific
[
H]TBZOH binding. The results of three
independent experiments are shown.
Synaptic-like microvesicles are
present in endocrine cells(29, 30) , and in bovine
chromaffin cells, they have been reported to contain
catecholamines(31) , though this view is not consistent with
results obtained in rat pheochromocytoma cells(32) . To test for
[H]TBZOH binding sites on these vesicles, high
speed pellets were analyzed by centrifugation on a 18% sucrose buffer
layer on the top of a 22-35% linear sucrose gradient. Variable
amounts of [
H]TBZOH binding sites were found in
the 27-30% sucrose fractions, containing synaptophysin, the
marker of synaptic-like microvesicles (data not shown). This density is
consistent with that given by O'Grady et al. (30); however, cosedimentation of synaptophysin and
[
H]TBZOH binding sites is difficult to interpret,
since membranes from osmotically shocked chromaffin granules also
sedimented in this region. Therefore, although the presence of
[
H]TBZOH binding sites on synaptic-like
microvesicles cannot be entirely ruled out, in control and in
K
-depolarized cells the major fraction of these sites
(80%) is associated with structures equilibrating at heavier densities
(>35% sucrose). It is of interest to note that synaptophysin
immunoreactivity was clearly increased in the sucrose density gradient
fractions from K
-cultured cells (data not shown).
H]TBZOH binding sites, the distributions of
catecholamines, chromogranin A, and cytochrome b
were analyzed on the 23-55% linear sucrose gradients (Fig. 6). In control (A) and in
K
-cultured (B) cells,
[
H]TBZOH comigrated not only with cytochrome b
, a marker of the chromaffin granule membrane,
but with the secretion products, catecholamine and chromogranin A, thus
indicating that [
H]TBZOH binding sites were
present on secretory granules.
Figure 6:
Subcellular fractionation of control and
K-depolarized cells. The resuspended high speed pellet
from control (A) or 5-day depolarized (B) cells was
analyzed by centrifugation on a 23-55% (0.7-2 M)
sucrose gradient for 3 h at 240,000
g. The fractions
were analyzed for [
H]TBZOH binding sites
(
), catecholamine (
), chromogranin A (
) and
cytochrome b
(
). The experiment was
repeated more than 12 times with similar results. For
[
H]TBZOH binding, one unit is 100 fmol and for
catecholamine, one unit is 10 nmol.
Comparison of the distributions of
the markers catecholamine, chromogranin A, and cytochrome b indicated that K
depolarization induced two reproducible changes. First, the
distribution was slightly shifted toward lighter densities. In four
different experiments, the fraction of the three markers equilibrating
at a density lighter than 45% sucrose was 42 ± 15%
(catecholamine), 31 ± 14% (chromogranin A) and 30 ± 10%
(cytochrome b
) in control cells, and in
depolarized cells, this fraction increased to 67 ± 18%
(catecholamine), 46 ± 16% (chromogranin A), and 65 ± 20%
(cytochrome b
). Second, the intensity of the
peaks, equilibrating between 35 and 55% sucrose, was markedly
decreased. For catecholamine and for chromogranin A, the sums of the
peak fractions in the treated cells were, respectively, 33 ± 6%
and 29 ± 2% (n = 4) of those in control cells.
For cytochrome b
, a decrease was also observed,
but it was less marked; cytochrome b
values were
43 ± 6% of controls. In the same cultures, potassium
depolarization increased [
H]TBZOH binding to 146
± 8% of control cells. Therefore, treated cells were
characterized by secretory granules with higher transporter to marker
ratios. The ratios for transporter to catecholamine, chromogranin A,
and cytochrome b
were increased by factors of
4.4, 5, and 3.4, respectively.
ions, chromaffin cells still
contain secretory granules. This contention is supported by three
different types of data: (i) morphologically, the depolarized cells
contained subcellular structures identifiable as chromaffin granules.
As in control cells, noradrenergic granules could be distinguished from
adrenergic ones and the morphological differences were supported by
biochemical data (); (ii) functionally, the
K
-cultured cells released catecholamines (Fig. 3) and chromogranin A (data not shown) from a vesicular
pool in a Ca
-dependent manner; (iii) subcellular
fractionation experiments also indicated the presence of chromaffin
granules in these cells, characterized by the colocalization of
catecholamines, chromogranin A, cytochrome b
,
and [
H]TBZOH binding sites (Fig. 6). The
finding that the rate of vesicular
[
H]noradrenaline uptake in permeabilized cells is
increased by a factor similar to that observed for
[
H]TBZOH binding sites is consistent with the
view that the binding sites are primarily associated with chromaffin
granules.
-depolarized cells, the four markers,
catecholamines, chromogranin A, cytochrome b
,
and [
H]TBZOH binding sites, equilibrated at a
density slightly lighter than in control cells, and the major
difference between the two profiles was observed between 35 and 40%
sucrose. The vesicles containing these markers differ certainly from
rough endoplasmic reticulum and Golgi vesicles which equilibrate below
35% sucrose (33) and from synaptic-like microvesicles which
equilibrate in 28% sucrose fractions(30) . If
[
H]TBZOH binding sites are present on synaptic
like microvesicles, these sites are certainly a minor population,
representing less than 20% of the total. The lighter density of the
granules from K
-depolarized cells suggests the
presence of newly synthesized, immature granules in the stimulated
cells. Such immature granules have been characterized in PC12 cells
(34). Their membrane was described as more often irregularly shaped and
loosely surrounding the core, in contrast to mature secretory granules,
the membrane of which surrounds the core more uniformly. Such images
were frequently present in K
-depolarized cells (see Fig. 1). It is thus possible that maturation of the chromaffin
granule involves a membrane retrieval step occurring after
catecholamine uptake, which would concentrate the matrix material and
act as a late sorting
step(34, 35, 36, 37) .
K
-depolarized cells might therefore be an interesting
model to study the biogenesis of secretory granules.
-depolarized cells contain less secretory
granules. Quantitative analysis of chromaffin granules isolated from
K
-depolarized cells indicated that catecholamine,
chromogranin A, and cytochrome b
were decreased
to 33, 29, and 43% of control values, respectively, whereas
[
H]TBZOH binding sites were increased to 146%.
The decrease of the granule matrix material, catecholamine and
chromogranin A, associated with an increase of
[
H]TBZOH binding sites might suggest the presence
of incompletely filled newly formed granules. However, this hypothesis
is unlikely for two reasons: (i) cytochrome b
,
which like the vesicular transporter is a membrane protein, is clearly
decreased rather than increased. Although the regulation of this
protein has not been studied at the molecular level, physiological
experiments suggested that the synthesis of this protein is not
regulated under our experimental conditions(5) ; this result
suggests also that proteins from the chromaffin granule membrane were
not quantitatively recycled after exocytosis; (ii) catecholamine and
chromogranin A are decreased to similar levels, though their transport
into secretory vesicles is an independent process, occurring at
different steps in the maturation of the granule; chromogranin A
originates from the Golgi apparatus and is an early component, whereas
catecholamine is a late component, which is taken up from the
cytoplasm. Finally, the morphometric data, indicating that in
depolarized cells the number of granules was decreased to 30% of
controls, strongly support the view that the granules from these cells
contain their normal quantity of matrix components and that their
membrane is enriched in vesicular monoamine transporter. The enrichment
of the membrane in vesicular monoamine transporter can be calculated by
normalizing the [
H]TBZOH binding site increase to
the markers content. Depending upon the marker chosen, the enrichment
ranges from 3.4 (normalized to cytochrome b
) to
5 (normalized to chromogranin A). Therefore, it can be concluded that
chromaffin cells adapt to prolonged secretion by changing the
composition of their secretory vesicles membrane.
-hydroxylase was increased to 67% above control level, whereas in
the same animals, the pool of chromaffin granule membrane stayed
constant, as shown by morphometric analysis(38) . In treated
animals, the size of the granules was reduced and this effect was
compensated by a slight increase in the number of granules. However,
this study is difficult to interpret, because dopamine
-hydroxylase exists in both a membrane bound and a soluble form,
and the relationship between these two forms is not clearly elucidated.
More recently, a different concept has been proposed by Mahata et
al.(39, 40) , who reported a differential
mRNA regulation for the vesicular monoamine transporter and the matrix
peptide NPY. Short term neurogenic stimulation induced a 4-fold
increase of the NPY mRNA level in rat adrenal medulla without changing
that of the vesicular monoamine transporter VMAT
,
suggesting that the synthesis of proteins of the matrix, but not of the
membrane, was regulated. In bovine chromaffin cells, K
depolarization for 2 days induced an increase in the expression
of VMAT
RNA(12) , which was reproducible. The
difference between the results of Mahata et al.(39) and the present data might be explained by the fact
that the former ones were obtained on rat adrenal medulla, where
VMAT
is the only expressed gene(10) , whereas bovine
chromaffin cells used in the latter study expressed predominantly
VMAT
. In bovine K
-depolarized cells,
VMAT
is not induced in contrast to
VMAT
.
(
)Alternatively, the difference
might result from the use of more vigorous stimulation conditions in
the present work (5 days of continuous depolarization in culture versus insulin shock in vivo). After an insulin
shock, there is an increase in the number of
[
H]TBZOH binding sites, visible 96 h after the
shock, but which is not significant after 24 h(5) , at the time
chosen by Mahata et al.(39) .
stimulation, catecholamine synthesis
is increased as a result of the induction of tyrosine
hydroxylase(8) , and the number of chromaffin granule is
decreased (see Fig. 2). Since the cytosolic catecholamine
concentration of control cells is estimated to be in the 10-20
µM range (41), the vesicular monoamine transporter is
already operating at its maximal rate, and its regulation would be
physiologically relevant. Accordingly, the relative enrichment of the
granule membrane in monoamine transporter might indicate an adaptative
process, allowing a faster filling of the granules.
Table: Morphological and biochemical characterization
of K-depolarized cells
]quinolizine);
[
H]TBZOH,
[2-
H]dihydrotetrabenazine; PBS, phosphate
buffered saline; BSA, bovine serum albumin.
.
We are indebted to Dr. C. Tougard (Collège de France, Paris) for
preliminary morphological studies and to Dr. F. Darchen for fruitful
discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.