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J Biol Chem, Vol. 274, Issue 27, 19095-19102, July 2, 1999
From the Department of Pharmacology, University of Alberta,
Edmonton, Alberta T6G 2H7, Canada
The fungal metabolite, brefeldin A (BFA), is
known to inhibit guanine nucleotide exchange on the ADP-ribosylating
factors that are involved in vesicle membrane trafficking. Here, we
investigated the action of BFA on Ca2+-regulated
exocytosis in single rat adrenal chromaffin cells. Incubation of
chromaffin cells with BFA (1 or 10 µM) for 2 h
effectively disrupted the Golgi membranes but did not affect the
pattern of catecholamine release triggered by high extracellular
K+, which was monitored with carbon fiber amperometry along
with cytosolic Ca2+ measurement. The BFA treatment,
however, increased the mean quantal size of catecholamine-containing
vesicles and the occurrence of amperometric events with a "foot" or
"stand alone" signal (which reflects sluggish or incomplete
dilation of the fusion pore). To examine whether BFA altered the
Ca2+-dependence of exocytosis, we employed the whole-cell
recording technique in conjunction with the capacitance measurement to
measure exocytosis evoked from the entire cell during voltage-gated
Ca2+ entry. Our results suggested that BFA treatment did
not alter either the initial rate of capacitance increase or the total
amount of capacitance increase. Therefore, in chromaffin cells, BFA
treatment affects Ca2+-regulated exocytosis predominantly
by increasing the quantal size and by slowing the fusion kinetics of
some vesicles.
The fungal metabolite brefeldin A
(BFA)1 has been reported to
disassemble the Golgi apparatus to form tubules, leading to the return
of the Golgi membrane to the endoplasmic reticulum (1, 2). This effect
of BFA is at least partially mediated via the inhibition of guanine
nucleotide exchange activity of a class of small monomeric GTP-binding
proteins called ADP-ribosylating factors (ARFs) (3, 4). In mammalian
cells, there are at least six forms of ARF proteins. Among the ARF
proteins, ARF1 is localized in the Golgi apparatus and is postulated to
be involved in the recruitment of cytosolic coat proteins to membranes
during transport vesicle formation (5). The functions of the other ARFs
are less well studied but may include regulation of phospholipase D (6,
7) and exocytosis (8).
Other than the disassembly of the Golgi apparatus, BFA has been
reported to inhibit peptide or protein secretion (9, 10) and in
vitro formation of synaptic vesicles (11). These actions were
postulated to be secondary to the effect of BFA on membrane trafficking. BFA has also been reported to have actions that may be
unrelated to membrane trafficking. For example,
Ca2+-dependent exocytosis in rat melanotropes
was inhibited by intracellular dialysis of BFA (10 µM),
and this acute effect could be mimicked by intracellular dialysis of
peptide fragment derived from residues 46-61 of ARF (12). In rat
intestinal smooth muscle cells, BFA (150 µM) reversibly
inhibited the voltage-gated Ca2+ current (13). In planar
lipid bilayer, BFA has also been reported to cause formation of small
(<15-picosiemens) cation channels (14).
Here we have employed carbon fiber amperometry to examine the effects
of BFA on quantal catecholamine release from rat chromaffin cells. We
have also measured [Ca2+]i and membrane
capacitance simultaneously to examine whether BFA affects
Ca2+-dependent exocytosis. Under the condition
that BFA had clearly disassembled the Golgi apparatus, we found that
BFA increased the quantal size of catecholamine release and
dramatically increased the proportion of "stand alone signals,"
which probably arise from slow release of catecholamine via fusion
pores that never dilate completely. However, BFA had no significant
effects on the Ca2+ dependence of exocytosis.
Chemicals--
BFA (Calbiochem-Novabiochem) was kept as stock
solution (10 mM) in ethanol and stored at Isolation and Primary Culture of Rat Adrenal Chromaffin
Cells--
Rat chromaffin cells were isolated and cultured as
described previously (15). Briefly, adrenal medullas were removed from male Sprague Dawley rats (200-250 g) killed with halothane in accordance with the standards of the Canadian Council on Animal Care.
The medullas were dissociated enzymatically in a modified Hanks'
solution containing collagenase type I (3.0 mg/ml), hyaluronidase type
I-S (2.4 mg/ml), and deoxyribonuclease type I (0.2 mg/ml) for 30 min at
37 °C. Single chromaffin cells were plated on glass coverslips
coated with either 1% gelatin and 1.6 mg/ml concanavalin A or 0.25 mg/ml poly-L-lysine. The cells were maintained in standard culture in Dulbecco's modified Eagle's medium supplemented with 10%
horse serum, 50 units/ml penicillin G, and 50 µg/ml streptomycin. All
electrochemical and electrophysiological recordings were performed at
room temperature (21-24 °C) on cells maintained in culture for 1-4 days.
Staining of the Golgi Apparatus--
The Golgi stacks of living
chromaffin cells were stained by C6-NBD-ceramide, a
fluorescent lipid that has been demonstrated to label the Golgi
apparatus in living cells reliably (16). Briefly, rat chromaffin cells
were incubated with C6-NBD-ceramide (10 mmol/liter) in
Dulbecco's modified Eagle's medium containing 0.68 mg/ml bovine serum
albumin for 10 min at 37 °C. After washing off the
C6-NBD-ceramide, the cells were bathed in standard bath solution (see below) for 30 min at 37 °C. The stained cells were then observed under a fluorescent microscope via a 63× objective. For
BFA pretreatment, the cells were first incubated with 1 or 10 µM BFA for 2 h at 37 °C before staining with
C6-NBD-ceramide. Otherwise, BFA was applied to stained
cells acutely for the duration indicated.
Electrochemical Detection of Catecholamine
Release--
Amperometry with carbon fiber electrodes was performed on
single chromaffin cells to monitor real-time release of catecholamine molecules during exocytosis of vesicle (17). The fabrication of carbon
fiber electrodes (tip diameter of 7 µm) was as described in Refs. 18
and 19. During the recording, the tip of the carbon fiber electrode was
positioned such that it was touching the cell surface. A 700-mV
potential (D.C.) was applied to the carbon fiber electrode, using a
VA-10 Voltammeter (NPI Electronic GmbH, Tamm, Germany). The
amperometric current was filtered at 1 kHz, stored on videocassette
recorder tapes with a NeuroData PCM recorder (Neuro Data Corp., New
York, NY), and digitized at 10 kHz later. Cells were initially bathed
in the standard bath solution containing 150 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 8 mM glucose, and 10 mM Na-HEPES (pH 7.4). To trigger catecholamine release, cells were perfused with 50 mM KCl isotonic solution (the
[K+] in the standard bath solution was increased to 50 mM by decreasing equal [Na+]). Amperometric
events were analyzed with software developed by Drs. Robert H. Chow and
Zhuan Zhou (19).
Microfluorometry--
For combined fluorimetry and
electrochemical measurement, cells were incubated with 5 µM indo-1-AM in the standard bath solution for 30 min at
37 °C, followed by at least 15 min in dye-free solution at room
temperature (~23 °C) before [Ca2+]i
measurement. For combined fluorimetry and capacitance measurement, 100 µM of the pentapotassium salt of indo-1 (for [Ca2+]i elevations <3 µM) or
indo-1-FF (for [Ca2+]i elevations >3
µM) was included in the internal recording solution and
dialyzed into the cell through whole-cell pipettes. Fluorescence
signals were measured at 405 and 500 nm by photomultipliers, and the
ratio of fluorescence (405 nm/500 nm) was used to calculate [Ca2+]i according to the equation (20),
Electrophysiological Recording--
Membrane currents were
recorded with the whole-cell gigaseal method (23) using an EPC-7
amplifier. Pipettes were made from hematocrit glass (VWR Scientific,
London, Ontario, Canada), and the resistance was 2-3 megaohms after
being filled with pipette solution. In experiments where exocytosis was
triggered via short depolarizing voltage steps,
[Ca2+]i in the standard bath solution was
increased to 10 mM. In addition, tetradotoxin (0.5 µM) and apamin (0.4 µM) were included in
the bath solution to block Na+ currents and the small
conductance Ca2+-activated K+ currents,
respectively. The standard pipette solution contained 135 mM cesium aspartate, 10 mM tetraethylammonium
chloride, 20 mM HEPES, 3 mM MgCl2,
2 mM Na2-ATP, 0.3 mM GTP, and 0.1 mM indo-1 or indo-1-FF (pH 7.4).
To measure membrane capacitance changes ( Statistical Analysis--
Each treatment with BFA was compared
with control cells from the same batch(es) of culture. On each
experimental day, recordings were performed alternately on BFA-treated
and control cells. The number of cells as well as the number of
cultures pooled for each comparison are indicated in the figure
legends. All mean values are given as mean ± S.E. In order to
determine whether the BFA effect is statistically significant, a
Student's t test for two independent populations was
performed on data collected from BFA-treated cells and control cells of
the same batches. All t values with (p < 0.05) were considered statistically significant.
Disassembly of the Golgi Apparatus by BFA--
We have chosen to
examine the effects of BFA while the Golgi apparatus was disassembled.
To determine the experimental conditions under which BFA would cause
the disassembly of the Golgi apparatus, we employed a fluorescent
ceramide (C6-NBD-ceramide) that reliably labels the Golgi
membranes in living cells (16). In control chromaffin cells, the
fluorescence of C6-NBD-ceramide was always confined to a
small region adjacent to the nucleus. Acute application of BFA (10 µM) for 5-10 min at room temperature did not cause any
significant change in the pattern of staining. In contrast, in cells
pretreated with 1 or 10 µM BFA for 2 h (at
37 °C), the fluorescence became dispersed all over the cytoplasm
(except the nucleus). Thus, a 2-h incubation with BFA at 37 °C was
sufficient to disrupt the Golgi apparatus in most chromaffin cells
(>90%). Although BFA was removed from the cells during the time of
staining (40 min at 37 °C), the dispersed fluorescence persisted
throughout our observation (30 min, at room temperature). Thus, it is
likely that, at room temperature, the BFA-induced morphological changes in the Golgi stacks can persist for at least 1 h following BFA removal. In this study, we performed electrochemical and/or
electrophysiological experiments on cells treated with BFA under three
conditions. First, we applied BFA acutely during the experiment (3-5
min at room temperature) to examine whether BFA had any acute effects on quantal release or exocytosis before changes in the Golgi morphology could occur. Second, we pretreated the cells with BFA for 2 h (at
37 °C) and then performed measurements within 1 h (at room temperature) following BFA removal to examine the persistent effects of
BFA. Last, we performed experiments on cells continuously exposed to
BFA (2 h at 37 °C, followed by <1 h at room temperature) to investigate whether the continuous presence of BFA might have more
prominent effects.
BFA Treatment Increases the Quantal Size of Some
Catecholamine-containing Vesicles--
Fig.
1 shows a typical recording obtained from
a cell pretreated with 10 µM BFA for 2 h. The
resting [Ca2+]i in control cells was typically
between 50 and 300 nM, and all of the BFA treatments did
not affect this value in the vast majority of the cells (95-99%). For
all electrochemical experiments, we selected cells with resting
[Ca2+]i around 100 nM. We applied 50 mM K+ to depolarize the cell to trigger
voltage-gated Ca2+ entry and catecholamine secretion.
Following the perfusion of high extracellular [K+],
[Ca2+]i rose from the resting level (~100
nM) to the micromolar range. In the continuous presence of
high [K+], [Ca2+]i remained
elevated at a level of 300-500 nM. The
[Ca2+]i elevation was accompanied by amperometric
spikes that reflected quantal release of catecholamines (17). Similar
patterns of [Ca2+]i rise and amperometric signals
were observed in control cells (n = 28), cells acutely
exposed to 10 µM BFA (n = 6), cells pretreated with 1 (n = 8) or 10 µM BFA
(n = 7), and cells exposed continuously to BFA 10 µM (n = 6). The frequency and total
number of amperometric spikes recorded in the first few minutes varied among cells. Typically, during the first 2 min of stimulation with high
extracellular [K+], 100-400 amperometric spikes could be
recorded from an individual cell.
The similar pattern of [Ca2+]i rise and
amperometric signals in control and BFA-treated cells during
application of high [K+] suggested that BFA treatment had
no dramatic effect on the voltage-gated Ca2+ entry and the
triggering of catecholamine release. However, detailed analysis of
individual amperometric spikes suggested that BFA might have some major
actions on the quantal release of catecholamines. Fig.
2 plots the frequency histograms of
quantal charge (time integral of individual amperometric spike) in
control cells, and in cells pretreated with 1 or 10 µM
BFA. Note that in cells pretreated with 10 µM BFA, the
proportion of amperometric spikes with large quantal charge (>2 pC)
was increased. Concomitantly, the proportion of amperometric spikes
with small charge (<0.2 pC) was reduced. The effect of 10 µM BFA pretreatment could be clearly observed in the
cube-root transformation (charge1/3; Fig. 2B), or
the log transformation (log(charge); Fig. 2C), of the quantal charge
histogram. Both of these transformations are expected to convert the
quantal charge distribution into a normal distribution (26, 27). Note
that in comparison with the controls, the mean value of quantal
charge1/3 in cells pretreated with 10 µM BFA was
increased by ~1.2-fold (from 0.66 ± 0.01 to 0.78 ± 0.01 pC1/3), and the log (charge) was increased by 0.22 log unit
(from
Fig. 4 shows the amplitude, rise time
(the time between 50 and 90% of the peak amplitude), and the
half-width (duration of the amperometric signal at 50% of its peak
amplitude) of the control and BFA-pretreated cells. Note that in cells
pretreated with 10 µM BFA, there was an increase in the
proportion of amperometric spikes with large amplitude (e.g.
>300 pA; Fig. 4A), slow rise time (e.g. >3 ms;
Fig. 4B), and long half-width (e.g. >10 ms; Fig.
4C). Similar effects were also observed in cells exposed continuously to 10 µM BFA but not for cells acutely
exposed to 10 µM BFA (data not shown). As in Fig. 2, the
change in cells pretreated with 1 µM BFA was less
obvious. Overall, the patterns of change caused by BFA are consistent
with an increase in the amount of catecholamine released during an
average spike (quantum) and in the proportion of quanta with a slower
kinetics of release.
BFA Treatment Alters the Fusion Kinetics of Some
Catecholamine-containing Vesicles--
Consistent with previous
studies in chromaffin cells and other catecholamine-secreting cells
(28-31), some of the amperometric spikes we recorded are preceded by a
"foot" signal (Fig. 5A), reflecting transmitter leakage through an early fusion pore that does
not dilate immediately. In control cells, the occurrence of the foot
signals was 19 ± 2% of all amperometric events. Interestingly, BFA treatment increased the occurrence of the foot signals (summarized in Fig. 5B). This increase is statistically significant in
cells exposed to 10 µM BFA acutely, continuously, or in
pretreatment. Previous electrochemical studies also demonstrated the
existence of stand alone signals in amperometry (Fig. 5C),
which represent openings of fusion pores that never dilate but flicker
before closing (19, 30, 31). These stand alone signals typically have a
rapid rise time and have rapid current fluctuations that clearly exceed
the base-line noise. Except for the absence of a following spike, they
are similar to foot signals in both temporal and amplitude
characteristics. Under normal conditions, the stand alone signals are
rarely observed in chromaffin cells (0.8 ± 0.3% of all
amperometric signals in our control cells; also see Ref. 30). BFA
treatments (10 µM; pretreatment or continuous treatment), however, significantly increased the frequency of the stand alone signals (Fig. 5D).
BFA Treatment Has Little or No Effect on the Ca2+
Dependence of Exocytosis--
The results above suggested that BFA
caused a change in the quantal size as well as the kinetics of
catecholamine release in some vesicles. We further examined whether BFA
affected the Ca2+ dependence of exocytosis. In this series
of experiments, single chromaffin cells were whole-cell voltage-clamped
at
We first examined Ca2+-dependent exocytosis in
cells pretreated with 1 µM BFA, a treatment that clearly
disrupted the Golgi apparatus but caused no significant increase in
quantal size. Fig. 7 summarizes the
relationship between [Ca2+]i and exocytosis in
control cells and cells pretreated with 1 µM BFA. Since
chromaffin cells vary in size (3.0-8.2 pF), both the
Cm jump and the total Cm rise
were normalized by cell surface area (initial Cm)
and expressed as percentage increase in cell capacitance. The
relationship between exocytosis and the peak amplitude of the
[Ca2+]i transient was plotted in Fig. 7,
A and B. Since exocytosis was triggered by
Ca2+ entry via voltage-gated Ca2+ channels, our
measurements of average cytosolic [Ca2+] may
underestimate the local [Ca2+] near the vesicles.
Therefore, we also plotted the relationship between exocytosis and
Ca2+ entry during the depolarization in Fig. 7,
C and D. The Ca2+ entry during
depolarization was estimated by the time integral of
ICa and normalized to individual cell volume. In
this series of experiments, 57 control cells and 32 cells pretreated
with 1 µM BFA (from 15 batches) were analyzed. These
cells were selected according to the following criteria. First, only
cells with basal [Ca2+]i < 300 nM
were analyzed. Second, to reduce error in [Ca2+]i
measurement, cells with [Ca2+]i that did not fall
into the dynamic range of indo-1 (>3 µM) or indo-1 FF
(<3 µM) were not included in the analysis. Third, in
cells where ICa appeared to be contaminated by
incomplete suppression of small conductance Ca2+-activated
K+ current (34), only the amplitude of the
[Ca2+]i transient was included in the analysis.
As shown in Fig. 7, A and B, at peak
[Ca2+]i of <3 µM, there was little
difference in the Ca2+ dependence of exocytosis between the
control cells and the BFA-treated cells. For
[Ca2+]i of >3 µM, a slightly
larger Cm jump and total Cm rise
were observed in BFA-treated cells than in control cells (but not
statistically different). A similar result was observed in Fig. 7,
C and D. For Ca2+ entry of <0.6
mmol/liter, Cm jump or total Cm rise was similar in both groups of cells. At Ca2+ entry of
>0.6 mmol/liter, however, a slightly larger total
Cm rise was observed in BFA-treated cells (but not
statistically different).
Our amperometry experiments (e.g. Fig. 1) have shown that
during continuous perfusion of high [K+], BFA-treated
cells could release catecholamine for many minutes. This suggested that
BFA might not have dramatic effects on the mobilization of vesicle in
the time course of many minutes. However, the amperometry method only
monitors catecholamine secretion from a fraction of the cell surface,
and previous studies in chromaffin cells (35) have also suggested that
the sites of catecholamine release might be unevenly distributed on the
cell surface. Therefore, it is hard to rely on amperometry to get a
quantitative comparison of secretion from different groups of cells. To
further examine whether BFA affected vesicle mobilization, we measured
the cumulative increase in capacitance during a train of depolarization
steps. In this set of experiments, the cell was held at
We have shown earlier that a continuous exposure of 10 µM
BFA not only disrupted the Golgi apparatus but also increased the mean
quantal charge in chromaffin cells (Fig. 3). To examine whether such a
BFA treatment can affect Ca2+-dependent
exocytosis, we repeated the train of depolarization experiments
(similar to Fig. 8, A and B) on cells
continuously exposed to 10 µM BFA. Fig. 8, E
and F, shows that even in cells treated continuously with 10 µM BFA, there was no statistically significant change in
the Ca2+ dependence of the cumulative exocytosis or the
initial rate of exocytosis. These results further confirm that BFA
treatment has little effect on Ca2+-dependent exocytosis.
Consistent with studies in other cell types (1, 2), our staining
with the fluorescent lipid, C6-NBD-ceramide indicates that
a 2-h treatment with BFA (1 or 10 µM) effectively
disrupted the Golgi apparatus in most chromaffin cells. This effect in
chromaffin cells is not rapidly reversible and persists for >1 h at
room temperature. The breakdown of the stacks of Golgi membrane has been postulated to lead to blockade of vesicle trafficking, and inhibition of "constitutive" secretion of membrane protein (1). However, our amperometry experiments demonstrated that BFA treatment did not affect the pattern of catecholamine release evoked by high
[K+]. In both control cells and BFA-treated cells,
amperometric spikes elicited by continuous high [K+]
perfusion persisted for at least 5 min and up to 30 min in a few cells.
These results suggest that the mobilization of catecholamine-containing vesicles in chromaffin cells does not significantly involve any BFA-sensitive compartments. Nevertheless, analysis of the quantal characteristics of catecholamine-containing vesicles showed that BFA
pretreatment or continuous treatment for 2 h tended to increase the quantal size of some vesicles. This trend was clear for the higher
BFA concentration (10 µM), which almost doubled the mean quantal size (Figs. 2 and 3). This change in the catecholamine content
in single vesicles may be due to an alteration in the concentration of
catecholamine and/or the vesicle volume. BFA is known to inhibit the
formation of coated vesicles, hence causing a misregulation of vesicle
budding. Thus, it is possible that the budding of some catecholamine
vesicles either at the plasma membrane or at internal membranes is
misregulated by the BFA treatment, and this in turn leads to the
formation of larger vesicles. Alternatively, fusion between
catecholamine-containing vesicles may occur when BFA disrupts the
membrane trafficking pathway. In theory, an increase in the average
size of vesicles may be detected as a larger increase in capacitance
for identical Ca2+ entry during activation of voltage-gated
Ca2+ channels (i.e. if the same number of
vesicles is triggered to undergo exocytosis). However, in cells
continuously treated with 10 µM BFA for 2 h (the
experimental condition that caused the largest increase in quantal size
of catecholamine vesicles), we detected no significant change in either
the cumulative exocytosis or the initial rate of exocytosis (Fig. 8,
E and F). If the increase in quantal charge
indeed came from larger vesicles, it is possible that the number of
vesicles readily releasable by activation of voltage-gated
Ca2+ channels was also decreased. Finally, although BFA is
not known to affect amine transporters, the possibility that there is
increased accumulation of catecholamine molecules in some vesicles
after BFA treatment cannot be completely ruled out here. Indeed,
significant increase in the quantal size in some amine-secreting cells
has been reported (36).
In this study, either pretreatment or continuous exposure of 10 µM BFA results in an increase in the number of
amperometric events with preceding foot and stand alone signals,
suggesting that 10 µM BFA affects the fusion kinetics of
some vesicles. Both the foot and stand alone signals represent a
delayed or aborted dilation of the fusion pore. Acute application of 10 µM BFA significantly increased the frequency of events
with foot but not with stand alone signals. This suggests that the
onset of the effect of BFA on foot signals is more rapid than that on
the stand alone signals. However, the effects on both types of signals
persisted after the termination of BFA pretreatment (1 or 10 µM). These persistent effects of BFA may be mediated by
changes in proteins (37, 38) or the lipid composition of the vesicle.
Consistent with the notion of slow dilation of the fusion pore, BFA
pretreatment (10 µM) also increased the proportion of
quantal spikes with longer rise time (Fig. 4). Hence, it is reasonable
to suggest that after the disruption of the Golgi apparatus by a 2-h
treatment of 10 µM BFA, specific protein(s) or lipid(s)
that are required for complete dilation of the fusion pore may fail to
be incorporated in some vesicles.
Earlier studies have shown that BFA could rapidly and reversibly
inhibit constitutive secretion of membrane proteins or peptides (1). In
pituitary melanotropes (12), intracellular dialysis of BFA has been
shown to reduce the initial rate of
Ca2+-dependent exocytosis. In the present
study, the BFA-treated cells with the disrupted Golgi apparatus could
release catecholamine for at least 5 min and even up to 20-30 min in
some cases. In addition, our capacitance experiments show that BFA (1 µM pretreatment or 10 µM continuous
treatment for 2 h) had no inhibitory effects on either the initial
rate of exocytosis or the amount of calcium-regulated exocytosis during
single depolarization or a train of depolarizations. Thus, BFA
treatment has no significant inhibition on the exocytosis of the
readily releasable vesicle or on vesicle mobilization.
In summary, our study suggests that different vesicle cycling patterns
exist for constitutive secretion and regulated secretion. The
disruption of the Golgi apparatus by BFA may hinder the constitutive secretory pathway but not the regulated one. The mechanisms underlying the more subtle effects of BFA treatment on the quantal size and release kinetics of catecholamine remain to be elucidated.
We thank Drs. Andy Lee and Amy Tse for
discussions and Robert Chow and Zhuan Zhou for the analysis program of
amperometric signals.
*
This work was supported by Canadian Medical Research Council
(MRC) Grant MT-12070 and by the Alberta Heritage Foundation for Medical
Research (AHFMR).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The abbreviations used are:
BFA, brefeldin A;
ARF, ADP-ribosylating factor;
C6-NBD-ceramide, 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)sphingosine.
Brefeldin A Increases the Quantal Size and Alters the Kinetics of
Catecholamine Release from Rat Adrenal Chromaffin Cells*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C.
Immediately before use, BFA was diluted to 1 or 10 µM
with Dulbecco's modified Eagle's medium supplemented with 10% horse
serum (Life Technologies, Inc.). C6-NBD-ceramide and indo-1
were purchased from Molecular Probes, Inc. (Eugene, OR). Indo-1FF was
purchased from Teflabs (Austin, TX). Collagenase, deoxyribonuclease,
hyaluronidase, and concanavalin A were purchased from Sigma.
where all three calibration constants,
Rmin, Rmax, and
K* were determined in situ for both indo-1 and
indo-1-FF by dialyzing various solutions of known Ca2+
concentration into cells as described previously (21, 22). The values
for Rmin, Rmax, and
K* were 0.27, 2.82, and 2.54 µM for indo-1 and
0.21, 1.49, and 29.4 µM for indo-1-FF.
![[<UP>Ca</UP><SUP><UP>2+</UP></SUP>]<SUB>i</SUB>=K<SUP>*</SUP>(R−R<SUB><UP>min</UP></SUB>)<UP>/</UP>(R<SUB><UP>max</UP></SUB><IT>−R</IT>)](/content/vol274/issue27/fulltext/19095/img001.gif)
(Eq. 1)
Cm),
which reflected the addition of plasma membrane and hence exocytosis at
high temporal resolution, we employed a software-based phase-sensitive detector (Pulse Control) (24) developed by Drs. Jack Herrington and
Richard Bookman (University of Miami, Miami, FL). A 30-mV, 822-Hz
sinusoidal wave was added to the holding potential (90 mV D.C.). The
resulting current signal at two orthogonal phase angles was analyzed to
generate an output for changes in cell capacitance
(Cm) and another output, G, which
reflects changes in membrane conductance, electrode seal, and series
resistance. To reduce contamination in the capacitance signal due to
the gating of Na+ channels (25), all capacitance recordings
were subtracted with the capacity transient in response to a 5-ms
depolarization delivered to the same cell.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
[Ca2+]i transient and
amperometric events triggered by depolarization. A,
application of high [K+] solution (50 mM)
elevated the [Ca2+]i. B,
[Ca2+]i elevation was accompanied by amperometric
current spikes, which reflect exocytosis of catecholamine-containing
vesicles. In the continued presence of KCl solution,
[Ca2+]i remained ~0.4 µM, and
amperometric current spikes persisted for at least 5 min. The recording
shown here is from a cell pretreated with 10 µM BFA for
2 h at 37 °C. This pattern of response is representative of
both control cells and cells exposed to BFA (acutely, pretreated or
continuously). The cell was loaded with indo-1-AM as the
Ca2+ indicator.
0.63 ± 0.02 to 0.41 ± 0.02). These correspond to
a ~1.7-fold increase in mean quantal charge after 10 µM
BFA pretreatment. Fig. 3 summarizes the
mean values of quantal charge1/3 of control cells and cells
subjected to different BFA treatments. Note that in comparison with the
controls, pretreatment or continuous exposure of 10 µM
BFA increased the mean of quantal charge1/3 by more than
1.2-fold. However, acute application of 10 µM BFA had no
significant effect in the mean quantal charge. These results suggest
that prolonged BFA treatment is needed to alter the quantal charge in
chromaffin cells. The similarity between the BFA-pretreated cells and
the continuously treated cells suggests that at room temperature, the
BFA-induced increase in quantal charge cannot be reversed in 1 h.
This effect of BFA also appeared to be
concentration-dependent. In cells pretreated with 1 µM BFA (Fig. 2), there was an increase in the proportion
of spikes with large total charge, but neither the cube root (0.67 ± 0.01 pC1/3) nor the log (0.62 ± 0.02) of quantal
charge showed any significant difference from those in control
cells.
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Fig. 2.
BFA treatment increases the quantal size of
some vesicles in chromaffin cells. A, the
quantal charge of single catecholamine-containing vesicles in control
and BFA-pretreated cells. The quantal charge was estimated from the
time integral of individual amperometric events. Note that in cells
pretreated with 10 µM BFA, the proportion of amperometric
events with bigger charge (>2 pC) was increased, but the proportion of
events with smaller charge (<0.2 pC) was reduced. B and
C plot the cube root of charge and log transformation of
charge, respectively. Pretreatment with 10 µM BFA caused
a clear shift in the distribution of the quantal charge. The total
number of amperometric events in this analysis was 793 for control (8 cells), 800 for 1 µM BFA pretreatment (8 cells), and 685 for 10 µM BFA pretreatment (7 cells). Between 60 and 130 nonoverlapping amperometric events from each cell were analyzed. The
cells came from three batches of culture.
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Fig. 3.
The BFA effect on quantal size of vesicles
depends on the concentration and duration of treatment. For each
BFA treatment, the mean value of cube roots of quantal charge is
normalized to that of control cells from the same batch(es). The
asterisks denote BFA treatments that exhibit significant
increase in mean quantal charge when compared with control
(p < 0.05). The number of cell cultures used in each
comparison is as follows: acute treatment of 10 µM BFA
(1), pretreatment with 1 µM BFA (2), pretreatment with 10 µM BFA (2), continuous treatment with 10 µM
BFA (1). The number of cells for each comparison (control:treatment) is
as follows: acute treatment of 10 µM BFA (7:6),
pretreatment with 1 µM BFA (8:8), pretreatment with 10 µM BFA (8:7), and continuous treatment with 10 µM BFA (5:6).
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Fig. 4.
The peak amplitude and kinetic features of
the amperometric current. A, the peak amplitude;
B, the rise time (the time between 50 and 90% of the peak
amplitude); C, the half-width (duration of the signal at
50% of its peak amplitude) of the amperometric signals from control
and BFA-pretreated cells. The pretreatment with 10 µM BFA
clearly resulted in more events with large amplitudes (e.g.
>300 pA), slow rise time (e.g. >3 ms), and long half-width
(e.g. >10 ms). The amperometric signals were low
pass-filtered at 1 kHz. Therefore, rise time of <1 ms and half-width
of <2 ms were not included in the histograms in B and
C. The events with a rise time of <1 ms were 68.2% in
control cells, 70.7% in 1 µM BFA-pretreated cells, and
62.3% in 10 µM BFA-pretreated cells. The events with
half-width of <2 ms were 24.2% in control cells, 26.8% in 1 µM BFA-pretreated cells, and 25.1% in 10 µM BFA-pretreated cells. The numbers of events, cells,
and batches of cultures are identical to those described in Fig.
2.
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Fig. 5.
BFA treatment alters the fusion kinetics of
some vesicles. A, example of an amperometric event with
a preceding foot signal, which reflects catecholamine leakage through a
narrow fusion pore before its complete dilation. B, the
proportion of amperometric events with a preceding foot signal was
normalized to that of control from the same cell batch(es). The
asterisks denote BFA treatments that cause significant
increase on the frequency of foot signals when compared with their
corresponding controls. C, example of a stand alone signal,
which reflects incomplete catecholamine release through a flickering
fusion pore that does not dilate. Note that the stand alone signal is
similar to a foot signal in rise time, duration, and amplitude, but it
is not followed by a complete amperometric event. D, the
proportion of stand alone signals is normalized to that of control from
the same cell batch. The asterisks denote BFA treatments
that cause significant increase in the occurrence of stand alone
signals. In B and D, the data came from the same
batch(es) of cultures as described in Fig. 3. The numbers of cells for
each comparison (control:treatment) are also as described in Fig.
3.
90 mV (D.C.). Exocytosis was triggered by a 250- or 500-ms
depolarization to +10 mV to stimulate extracellular Ca2+
entry via voltage-gated Ca2+ channels. The rise in
cell-averaged [Ca2+]i was measured by the
ratiometric fluorescent indicator indo-1 or indo-1-FF, while the
triggered exocytosis was monitored as changes in membrane capacitance.
Fig. 6 shows a typical experiment in
control cells. During the depolarization, Ca2+ current
(ICa; peak amplitude, ~250 pA) was activated,
and it was accompanied by a rise in [Ca2+]i and
an increase in membrane capacitance (Cm), which
reflects exocytosis. Following the termination of the voltage step,
[Ca2+]i decayed slowly and remained above 1 µM for >3 s. Meanwhile, the membrane capacitance
continued to increase slowly for ~300 ms and then started to decline.
Note that the gradual decrease in Cm actually
overshot the original resting value. This pattern of
Cm decrease has been interpreted as endocytosis
of the "excessive retrieval" type (32, 33) and is typically
observed following a large [Ca2+]i elevation. In
our analysis, we used both the capacitance increase during the
depolarizing pulse (referred to as the "Cm jump") and the "total Cm rise" (the sum of
Cm jump and any Cm rise following
the depolarizing pulse) as indices of exocytosis evoked by a single
depolarization (Fig. 5). Similar experiments were repeated on cells
treated with BFA.
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Fig. 6.
Simultaneous measurements of voltage-gated
Ca2+ current (ICa),
[Ca2+]i and changes in membrane capacitance
( Cm) in a cell pretreated with
BFA (1 µM). The cell was
voltage-clamped at 90 mV (D.C.) and depolarized to +10 mV for 500 ms.
Activation of ICa was accompanied by a transient
[Ca2+]i elevation and an increase in membrane
capacitance, reflecting exocytosis. Note that the increase in
Cm during the voltage step (denoted by the
difference between arrows a and b) is
referred as the Cm jump here. Shortly after the
termination of the voltage step, [Ca2+]i remained
elevated, and the membrane capacitance continued to increase slightly
(denoted by the difference between arrows b and
c). Therefore, in this study, the total
Cm rise (denoted by the difference between
arrows a and c) was measured as the
sum of the Cm jump during the depolarization and the
subsequent Cm rise. Following exocytosis, the
membrane capacitance gradually decreased and eventually fell below its
initial value, reflecting endocytosis of the "excessive retrieval"
type.
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Fig. 7.
The Ca2+ dependence of exocytosis
is not affected by BFA treatment. A and B,
plots of Cm jump and total Cm
rise versus peak [Ca2+]i in control
cells (solid circles) and BFA (1 µM)-pretreated cells (open
circles). Values of Cm jump and total
Cm rise were normalized to initial membrane
capacitance of individual cells. C and D, plots
of Cm jump and total Cm rise
versus Ca2+ entry. Ca2+ entry was
estimated by the time integral of ICa and
normalized to the volume of individual cells (same cells as in
A and B). Data were collected from 15 cultures.
Each data point is the average from at least six cells, and the number
of experiments for each point is shown in brackets.
90 mV (D.C.). A train of 15 depolarization steps (held at +10 mV for 50 ms in each
step) was delivered at 4 Hz. An example of such an experiment on a cell
pretreated with 1 µM BFA was shown in Fig.
8, A and B. In this
cell, the train of depolarization steps triggered a [Ca2+]i rise as well as increases in
Cm. Fig. 8C plots the cumulative
exocytosis versus the peak [Ca2+]i in
both the control cells and cells pretreated with 1 µM
BFA. Note that at comparable peak [Ca2+]i, no
significant difference could be detected between the two groups. We
also examined whether BFA affected the initial rate of exocytosis by
measuring the rate of Cm jump during the first 50-ms
depolarization (Fig. 8D). Since the Ca2+ entry
during the brief depolarization (50 ms) might not be distributed uniformly throughout the cell volume, we have normalized the
Ca2+ entry (time integral of ICa) to
the cell surface area (initial cell membrane capacitance) instead of
the cell volume. Fig. 8D shows that at comparable
Ca2+ entry, the initial rate of exocytosis was not
significantly affected by BFA. Thus, 1 µM BFA
pretreatment had no significant effect on either the cumulative
Ca2+-dependent exocytosis or the initial rate
of Ca2+-dependent exocytosis in chromaffin
cells.
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Fig. 8.
BFA treatment does not affect vesicle
mobilization or the initial rate of exocytosis. A,
[Ca2+]i elevation and Cm
increment triggered by a train of 15 depolarizations (50 ms, 4 Hz). The
cell was held at 90 mV and pretreated with BFA (1 µM).
B, Ca2+ current recorded during the first
depolarization. C, plot of the cumulative
Cm rise versus peak
[Ca2+]i in controls (solid
symbols) and cells pretreated with 1 µM BFA
(open symbols). The Cm rise
was normalized to the initial membrane capacitance of individual cells.
No significant difference was observed between the average exocytic
response of the controls (solid symbols) and the
BFA-treated cells (open symbols). D,
plot of the initial rate of exocytosis
(Cm/t) versus
Ca2+ entry in controls (solid
symbols) and cells pretreated with 1 µM BFA
(open symbols). The initial rate of exocytosis
was estimated by the Cm jump during the first 50-ms
depolarization. Ca2+ entry was estimated by the time
integral of ICa and normalized to the cell
surface of individual cells (initial cell membrane capacitance).
E, plot of the cumulative Cm rise
versus peak [Ca2+]i in controls
(solid symbols) and cells continuously treated
with 10 µM BFA (open symbols). For
the BFA-treated cells, the average exocytic response is not
significantly different from that of the controls. F,
continuous treatment of 10 µM BFA did not cause any
significant change in the Ca2+ dependence of the initial
rate of exocytosis (Cm/t). The data
in C and D are from two batches of cell cultures,
and the data in E and F are from two other
batches. The number of cells for each data point is shown in
brackets.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
An AHFMR and MRC Scholar. To whom correspondence should be
addressed: Dept. of Pharmacology, 9-70 Medical Science Bldg.,
University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.:
780-492-3876; Fax: 780-492-4325; E-mail: Fred.Tse{at}ualberta.ca.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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