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The Journal of Neuroscience, September 1, 1999, 19(17):7375-7383
Ca2+-Dependent Activator Protein for Secretion Is
Critical for the Fusion of Dense-Core Vesicles with the Membrane in
Calf Adrenal Chromaffin Cells
Abdeladim
Elhamdani1,
Thomas F. J.
Martin2,
Judith A.
Kowalchyk2, and
Cristina R.
Artalejo1
1 Department of Pharmacology, Wayne State University
School of Medicine, Detroit, Michigan 48201, and
2 Department of Biochemistry, University of Wisconsin,
Madison, Wisconsin 53706
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ABSTRACT |
Calcium-dependent activator protein for secretion (CAPS) is a
neural/endocrine cell-specific protein that has been shown to function
at the Ca2+-dependent triggering step of dense-core
vesicle (DCV) exocytosis in permeabilized PC12 cells. To evaluate the
function of CAPS under physiological conditions, we introduced
affinity-purified anti-CAPS IgGs into calf adrenal chromaffin (AC)
cells via a patch pipette and tested the kinetics of catecholamine
secretion using both amperometric and membrane capacitance techniques.
The antibodies reacted with a single major ~145 kDa protein in AC
cells based on immunoblot analysis. AC cells stimulated with sequential
trains of action potentials at 7 Hz resulted in successive secretory episodes of equivalent magnitude. When either of two different anti-CAPS IgGs or their Fab fragments were present, a rapid and progressive inhibition of catecholamine release ensued to a maximum of
>80%. The effect was specific because preabsorption of IgGs with the
respective antigens ablated the inhibitory effect, and the IgGs had no
effect on Ca currents. CAPS immunoneutralization not only reduced the
number of amperometric spikes but markedly altered the kinetic
characteristics of the residual events. The remaining spikes were much
smaller (by 85%) and broader (by ~3.5-fold) than those in control
cells, suggesting that CAPS plays a role in determining release of
vesicle contents via the fusion pore. Anti-CAPS IgGs also slowed the
rate of the initial exocytotic capacitance burst, representing the
docked-and-primed vesicle pool, by ~90% but had no effect on the
kinetics of rapid endocytosis. These results suggest that CAPS is a key
component regulating the fusion of DCVs to the plasma membrane, and
possibly fusion pore dilation, in catecholamine secretion from AC cells.
Key words:
dense-core vesicles; exocytosis; CAPS; adrenal chromaffin
cells; fusion pore; amperometric recording; capacitance measurements; catecholamine release
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INTRODUCTION |
Much has been learned in recent
years about the molecular basis of exocytosis from small synaptic
vesicles (SSVs) in the nervous system. Several important components of
the SSV and presynaptic membrane have been cloned, and interactions
between them have been studied in some detail (for review, see Sudhof,
1995 ). Considerably less is known about exocytosis from dense-core
vesicles (DCVs) that store biogenic amines as well as neuropeptides.
Significant differences may exist in the characteristics of secretion
between these different types of vesicles (Martin, 1997). For example, although SSVs are released largely from well-defined active zones, DCVs
frequently appear to have a more diffuse pattern of release. Perhaps as
a result, secretion from DCVs is often, but not always, slower than
that from SSVs. Despite these kinetic differences, there are
substantial similarities between several components of the core
secretory machinery associated with SSVs and DCVs. For example, the
proteins synaptotagmin, synaptobrevin (VAMP), and rab 3a are all
present on both types of vesicle. In addition, secretion in either
system is sensitive to clostridial protease toxins that selectively
cleave various protein components of the secretory apparatus. These
findings suggest that regulated SSV and DCV exocytosis largely involves
a common set of proteins and mechanisms (for review, see Martin,
1997).
One approach to distinguishing between SSV and DCV secretion at the
molecular level is to identify proteins that may be critical to one
type of vesicle exocytosis but not the other. Calcium-dependent activator protein for secretion (CAPS) is a recently discovered protein
that has been suggested to be specific to DCV secretion (Walent et al.,
1992; Ann et al., 1997; Berwin et al., 1998; Tandon et al., 1998). Its
initial purification was based on the ability to reconstitute
catecholamine secretion from permeabilized PC12 cells (Walent et al.,
1992). Both permeabilized PC12 and adrenal chromaffin (AC) cells
secrete catecholamines by exocytosis when both ATP and
Ca2+ are exogenously supplied. The process
can be divided into two stages: an ATP-dependent "priming" step
that can take place in the absence of
Ca2+, and a
Ca2+-dependent "triggering" step for
which ATP is not required (Bittner and Holz, 1992; Hay and Martin,
1992). Extensive washing reduces secretion from permeabilized cells,
which can then be recovered by addition of exogenous concentrated rat
brain cytosol (Hay and Martin, 1992). Fractionation of the cytosol led
to the identification of three proteins that could reconstitute, in a
purified form, both the priming and triggering steps of secretion. Two
of the required proteins for the ATP-dependent priming step were
identified as components involved in phosphoinositide phosphorylation
(Hay and Martin, 1993), whereas the ~145 kDa CAPS was found to act at
the triggering (Ca2+-dependent) step
(Walent et al., 1992). Subsequent cloning of CAPS revealed that it is
homologous to the Caenorhabditis elegans gene unc-31 (Ann et
al., 1997). Loss-of-function mutations in this gene result in
pleiotropic nervous system abnormalities, suggesting that CAPS plays a
fundamental role in neurosecretion in these invertebrates (Avery et
al., 1993). Antibodies to CAPS are able to inhibit secretion in
permeabilized PC12 cells (Walent et al., 1992; Ann et al., 1997) and
selectively target catecholamine secretion in perforated rat brain
synaptosomes (Tandon et al., 1998). Anti-CAPS IgG blocks norepinephrine
secretion from PC12 cells and semi-intact synaptosomes but fails to
antagonize glutamate secretion in the latter preparation. These
findings indicate that CAPS plays a dedicated role in DCV but not SSV exocytosis.
Although studies on permeabilized preparations strongly suggest that
CAPS is required for DCV exocytosis, they lack the kinetic resolution
that can be obtained with electrophysiological analysis of intact
cells. To further our understanding of the role of CAPS in secretion,
we turned to patch-clamped calf AC cells. Secretion from these cells
have been well characterized and can be recorded with millisecond
resolution using either capacitance or amperometric techniques (Neher
and Marty, 1982; Wightman et al., 1991; Chow et al., 1992; Artalejo et
al., 1994; Elhamdani et al., 1998). We previously showed by using
capacitance measurements that catecholamine secretion in these cells is
preferentially coupled to a particular type of L-type Ca channel termed
the facilitation Ca channel (Artalejo et al., 1994). More recent
experiments using the high-resolution amperometric technique revealed
that catecholamine secretion consists of two kinetic components
(Elhamdani et al., 1998). Surprisingly, secretion elicited by
activation of facilitation Ca channels is remarkably rapid (delay of
~3 msec after the depolarization; termed "strongly coupled"
secretion), approaching the speeds characteristic of synaptic
transmission (Elhamdani et al., 1998). We attributed strongly coupled
secretion to colocalization of facilitation Ca channels with DCV
release sites. Slower secretion (delay of >25 msec or weakly coupled)
is also observed, probably attributable to Ca channels that are not
colocalized with the release apparatus (Klingauf and Neher, 1997;
Elhamdani et al., 1998). Capacitance recordings also reveal multiple
phases of secretion with an initial "exocytotic burst," manifest as
a very high initial rate of secretion, being caused by an already
docked-and-primed "release-ready" pool of DCVs preceding a slower
phase that may reflect secretion of newly recruited vesicles (Parsons
et al., 1995). This kinetic diversity, along with the opportunity to
analyze other parameters revealed by electrochemical analysis, such as
the shape of unitary amperometric spikes, permits us in the present
study to examine the effects of intracellular antagonism of CAPS on
several aspects of catecholamine secretion in detail. The results
suggest that CAPS plays a role at the very final step in DCV exocytosis
where fusion of the vesicle with the membrane surface takes place.
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MATERIALS AND METHODS |
Cell culture
Bovine calf (average age 10-12 weeks) chromaffin cells were
prepared by digestion of adrenal glands obtained from local
slaughterhouses. Cells were purified and cultured using previously
described methods (Artalejo et al., 1991). Cells plated at a density of
3 × 105 cells on collagen-coated
35-mm-diameter dishes were used in all studies, within 1 week of plating.
Electrophysiology
Conventional patch-clamp current and capacitance
recording. Our patch-clamp techniques have been published
previously (Artalejo et al., 1995); an Axopatch 200 B (Axon
Instruments, Foster City, CA) was used as the patch-clamp amplifier
throughout these experiments. Capacitance was measured by a computer
program using a phase-tracking technique. A standard protocol of ten 50 msec depolarizations from a holding potential of  90 mV to +10 mV,
each pulse preceded by a prepulse to +120 mV to recruit facilitation Ca
channels, was used to evoke secretion. After the secretory phase, rapid endocytosis (RE) manifests as a decrease in capacitance; both the rate
and extent of RE were measured, as well as the rate and extent of
exocytosis and the magnitude of Ca current. All experiments were
performed at room temperature (24°C). The standard patch pipette
solution contained (in mM): Cs-glutamate 110, Cs-EGTA 0.1, HEPES 40, MgCl2 5, ATP 2, GTP 0.35, pH 7.2, with
CsOH (nucleotides and various anti-CAPS IgGs as indicated in the figure
legends were added fresh to the stock salt solution just before the
experiment). The external solution consisted of (in mM):
CaCl2 2, TEA-Cl 150, HEPES 10, glucose 10, MgCl2 1, and 1 µM tetrodotoxin, pH
7.3.
Current-clamp recording. To evoke APs, cells
maintained at a Vm of 70 to 90 mV, through
application of a holding current of 1 to 4 pA, were stimulated by
depolarizing currents (+10 to +20 pA) of 20 msec (Elhamdani et al.,
1998). An eight-pole Bessel filter was set to a corner frequency of 2 kHz for Vm recording, then sampled at 5 kHz. The
pipette solution contained (in mM): K-glutamate
100, K-EGTA 0.1, NaCl 12, HEPES 30, MgCl2 5, ATP
2, GTP 0.35, pH 7.2, with KOH. The external solution consisted of (in
mM): NaCl 140, glucose 10, HEPES 10, MgCl2 1, KCl 5.5, CaCl2 2, pH 7.3, with NaOH.
Carbon-fiber amperometry for catecholamine detection
Highly sensitive low-noise polypropylene-insulated carbon-fiber
electrodes were used for electrochemical monitoring of quantal release
of catecholamines from single AC cells as described previously (Wightman et al., 1991; Chow et al., 1992; Elhamdani et al., 1998). Briefly, a 7-µm-diameter carbon fiber (Amoco Performance Products, Greenville, SC) was inserted into a polypropylene pipette (Continental Lab Products). The tip was then heated and pulled with a homemade CFE puller. The ProCFE was prepared for recording by inserting it into a micromanipulator and cutting the tip with a microscissor. To
obtain electrical contact between the carbon fiber and the Ag wire
input to the head stage of the amplifier, the shank of the ProCFE was
back-filled with mercury. Each ProCFE was then used in a maximum of one
to three cells to ensure the highest possible sensitivity. The tip of
the electrode was closely apposed to the cell surface to minimize the
diffusion distance from release sites. The amperometric current
(Iamp), generated by oxidation of
catecholamines at the exposed tip of the CFE, was measured using an
Axopatch 200A amplifier, operated in the voltage-clamp mode at a
holding potential of +780 mV. Amperometric signals were low-pass-filtered at 1 kHz, then sampled at 2 kHz by an Axobasic system. The data were collected and then analyzed by computer using
IGOR software (WaveMetrics, Lake Oswego, OR). Latencies (defined as the
time from the peak of the AP to the beginning of the current spike)
were analyzed using latency histograms (Elhamdani et al., 1998). The
beginning of the current spike was located where the leading edge of
the transient (which includes the "foot" signal when present)
exceeded the baseline current by two times the SD of the baseline noise
level. Measurements are given as means ± SEM.
Preparation of anti-CAPS antibodies. Antisera
(referred to as bCAPS and FP5 antibody, respectively) were generated by
immunization of rabbits with recombinant CAPS proteins: a full-length
protein (bCAPS) produced by baculovirus-encoded expression in insect
cells (Ann et al., 1997) or a truncated protein (FP5 corresponding to amino acids 264-364) produced as a glutathione
S-transferase fusion protein in E. coli. IgGs for
patch-clamp studies were purified by chromatography on protein
A-Sepharose. IgG fractions depleted for CAPS-neutralizing antibodies
were prepared by affinity chromatography on the FP5 protein coupled to
Sepharose 4B by CNBr activation.
Analysis of CAPS expression in chromaffin cells.
Extracts of calf AC cells, PC12 cells, and rat1a fibroblasts were
prepared by sonication in a lysis buffer containing (in
mM): 50 Tris-HCl, pH 7.4, 2 EDTA, 1 EGTA, 5 2-mercaptoethanol, 0.5 PMSF, and 0.01 leupeptin. After sonication,
aliquots were taken for protein assay, and the extracts were
immediately solubilized in SDS sample buffer. Samples (100 µg) were
separated by SDS-7.5% PAGE, and proteins were transferred to
nitrocellulose and subjected to immunoblotting procedures as described
previously (Artalejo et al., 1995). AC cells for immunocytochemical
analysis were also prepared as before (Artalejo et al., 1995). After
incubation in primary IgG (1 µg/ml), coverslips were washed and
incubated for 30 min in biotin-coupled goat anti-rabbit IgG (0.2 µg/ml) followed by 10 min in Cy3-coupled streptavidin (0.1 mg/ml).
Fluorescence was viewed in a Noran Odyssey confocal microscope.
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RESULTS |
CAPS is an abundant protein in calf AC cells
Previous studies showed that CAPS expression was restricted to
neural and secretory endocrine tissues in the rat and is also present
in adult bovine AC cells (Ann et al., 1997). To verify that the calf AC
cells used in the present study also contain CAPS and that the
antibodies used are specific, we performed immunoanalysis of these
cells. As shown in Figure
1A, two anti-CAPS IgGs
used in the present study react with a major ~145 kDa protein in
immunoblots of total calf AC cell protein; rat PC12 cells were also
positive but rat fibroblasts were negative, in line with previous
studies showing a limited tissue distribution for this protein (Ann et al., 1997). Immunoreactivity was completely blocked by preabsorption of
IgGs with the respective antigens (data not shown). The same antibodies
stained calf AC cells in situ (Fig. 1B),
confirming that CAPS is an abundant protein in these cells.
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Figure 1.
CAPS is an abundant protein in calf AC cells.
A, Immunoblots showing the reactivity of the two CAPS
IgGs used in the present study with calf AC cell (lanes
1), PC12 cell (lanes 2), and rat 1a fibroblast
(lanes 3) cell extracts. Each lane contains 100 µg of
total cell protein. Both anti-CAPS-FP5 and anti-CAPS peptide IgGs were
used at 1 µg/ml. Detection was with peroxidase-coupled secondary
antibodies and ECL. Note the presence of the major ~145 kDa species
in lanes 1 and 2 but its absence in
lanes 3. B, Immunofluorescence of
anti-CAPS-FP5 IgG with calf AC cells. Fixed calf AC cells on coverslips
were incubated with anti-CAPS IgG (1 µg/ml), then with biotin-labeled
goat anti-rabbit secondary antibodies followed by streptavidin-Cy3
label. The cells were visualized in a Noran Odyssey confocal microscope
of which a typical section is shown (top panel).
Scale bar, 25 µm. Only faint background signal was observed when
staining was performed with preabsorbed antibody (lower
panel).
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Anti-CAPS IgGs progressively and specifically inhibit
catecholamine secretion
To ascertain the effects of CAPS antagonism on secretion, we
introduced two anti-CAPS IgG preparations or their controls into calf
AC cells via the patch pipette and recorded secretion using an
extracellular carbon-fiber electrode. As described previously (Elhamdani et al., 1998), when such cells are stimulated using sequential trains of action potentials delivered at a frequency of 7 Hz, rates of secretion as quantitated by cumulative amperometric current are similar in successive periods (Fig.
2A). Introduction of
anti-CAPS IgG (anti-bCAPS raised against recombinant CAPS generated in
insect cells; 1.5 mg/ml) resulted in a progressive decline in the rate
and magnitude of secretion from these cells (Fig. 2B). After 8 min the cumulative amperometric spike
amplitude was reduced by ~67%, and by 14 min the reduction had
increased to 86%, presumably reflecting the time course of IgG
diffusion into the cell from the pipette, and did not decline further.
Similar results were obtained when the antibody was allowed to diffuse into a resting cell for 14 min before any stimulation; when these cells
were then stimulated at 7 Hz, secretion was still inhibited by
84.5 ± 6.8% (n = 9 cells) compared with control
cells with no antibody. When stimulation was performed at a lower
frequency (1 Hz), >95% inhibition of secretion was observed in
anti-CAPS IgG-treated cells after 14 min (data not shown).
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Figure 2.
Anti-CAPS IgGs progressively
inhibit catecholamine secretion from calf AC cells. The effect of
intracellular anti-CAPS IgG (bCAPS antibody raised against recombinant
CAPS generated in insect cells; 1.5 mg/ml) on secretion was tested with
amperometry. A calf AC cell was stimulated by three successive trains
of 490 APs at 7 Hz (bottom traces); 5-6 min separated
each train. Amperometric spikes were recorded with an extracellular
carbon fiber electrode (top traces) as described in
Materials and Methods. A, Under control conditions
(either no IgG or preimmune IgG in the pipette), secretion in the third
round (14 min) was not significantly different from the first (2 min)
or second (8 min) rounds, indicating that depletion of releasable
vesicles did not occur (a1-a3). Graph on
right (a4) represents cumulative
amperometric spike current as a function of time during the three
recording periods. B, Cell loaded with anti-CAPS IgG:
quantal release during the first series of APs (2 min,
b1) was not significantly different from control, but
thereafter (b2, b3) a progressive inhibition of
secretion ensued, such that the total quantal release during the
third round (14 min) was reduced by 86%, as determined by
reduction in cumulative amperometric spike current
(b4), compared with secretion in the first round.
C, Anti-CAPS IgG reduces the total number of
amperometric events by 80%.
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To test for the specificity of this effect, we used a second anti-CAPS
IgG (anti-FP5 antibody raised against a CAPS fusion protein) and
compared the response of the IgG alone with that of IgG that had been
preabsorbed with an excess of the antigen (Fig.
3). The anti-FP5 antibody (1.5 mg/ml)
also reduced secretion by ~80% after 14 min when stimulation was
conducted at 7 Hz (Fig. 3B), whereas preabsorbed (Fig.
3C) or preimmune (Table 1)
IgGs were without discernible effect. None of the IgG preparations influenced the magnitude of the Ca current, indicating that the reduction in secretion was not caused by alteration in the amount of
Ca2+ entering the cell during stimulation.
As a further negative control for the FP5 antibody, we used
"immune" serum that was depleted of neutralizing antibody by
passage through an affinity column of the antigen; such antibodies were
also ineffective in blocking secretion. These results are summarized in
Table 1.
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Figure 3.
Anti-CAPS IgGs specifically inhibit catecholamine
secretion. Continuous amperometric recordings, using a stimulation
paradigm identical to that in Figure 2, from AC cells in which
(A) no protein, (B)
anti-CAPS IgG (FP5 antibody raised against a CAPS fusion protein; 1.5 mg/ml), or (C) anti-CAPS IgG (1.5 mg/ml)
preabsorbed with an approximate fourfold molar excess of FP5 were
included in the patch pipette. Note in A and
C that both rounds of exocytosis occur with similar
quantal release, whereas in B the second round of
release is strongly blocked after the antibody has diffused into the
cell (in the first round, secretion is normal because insufficient
antibody has diffused into the cell). Similar results were also
obtained with anti-bCAPS IgG (Table 1). From n = 46 treated cells, anti-CAPS IgG blocks the total charge of the
amperometric spikes by 83.6 ± 4.1%. From n = 7 treated cells, anti-FP5 Fab blocks the total charge of the
amperometric spikes by 82.4 ± 5.4%.
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We have shown previously that secretion of catecholamines from calf AC
cells occurs in two kinetically distinct phases that we term strongly
coupled and weakly coupled (Elhamdani et al., 1998). This distinction
is based on the analysis of amperometric latency histograms that depict
the temporal relationship between the evoked action potential and the
resultant extracellular amperometric spike (Chow et al., 1992).
Accordingly, during stimulation at 7 Hz we found that many amperometric
events occurred <5 msec after the action potential, forming an early
strongly coupled peak in histograms (Fig.
4A). This early peak is
abolished by the Ca channel antagonist nisoldipine, suggesting that it
is caused entirely by the action of facilitation L-type Ca channels in
these cells. By contrast, weakly coupled secretion (Fig.
4A) occurs with a significant delay (>25 msec) after
the action potential and contributes a plateau phase in these
histograms that is relatively resistant to nisoldipine (Elhamdani et
al., 1998). As shown in Figure 4B, anti-CAPS IgG
inhibited both phases of secretion after 14 min of intracellular
dialysis. No inhibition of either peak was obtained with either the
preabsorbed (Fig. 4C) or preimmune (Table 1) IgGs,
indicating that the effect was specific.
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Figure 4.
Anti-CAPS blocks both strongly and
weakly coupled secretion. To determine the contribution of CAPS to the
different kinetic components of secretion in calf AC cells (Elhamdani
et al., 1998), we analyzed latency histograms from amperometric
experiments. These histograms reflect the delay in the amperometric
spike after the action potential. Two successive stimulation periods of
7 Hz (980 APs) were performed in cells loaded with
(A) no addition, (B)
anti-CAPS IgG at 14 min, or (C) preabsorbed CAPS
antibody. In control cells, or those loaded with preabsorbed antibody,
46% of the events had a latency <25 msec, whereas 54% were
>25 msec. In B, the strongly coupled component is
reduced by ~89%, whereas the weakly coupled component is reduced by
~59%.
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CAPS antagonism affects the time course of quantal
catecholamine secretion
When stimulation was performed at 7 Hz even in the presence of
high anti-CAPS IgG concentrations (2 mg/ml), inhibition of secretion
was incomplete after 14 min. We investigated the kinetic properties of
the residual amperometric spikes to assess whether they differed from
those in control cells. Four distinctive parameters were analyzed in
the absence and presence of anti-CAPS IgG (Figs. 5, 6):
maximum spike amplitude, total charge (or quantal content, Q) including the "foot" (Chow et al., 1992) when
present, rise time (RT), and half-width
(HW). The spike amplitude and spike charge histograms
reflect the amount and distribution of catecholamine that is released
per quantal event (for review, see Travis and Wightman, 1998). In
control cells, amplitude histograms formed a broad distribution with a
mean value of 127.6 pA, and only 32.7% of the spikes had an amplitude
<40 pA (Fig. 5A, a1, and legend). Amplitude-charge distributions were well-fit with a linear relationship having a slope of 99 pA/pC (Fig. 5A, a2),
indicating that spike charge is directly proportional to amplitude, as
seen in the representative amperometric spikes in Figure 5A,
a3. By contrast, cells loaded with anti-CAPS IgG exhibited
only low amplitude spikes, with ~88% of the events <40 pA (mean
24.5 pA) (Fig. 5B, b1, and legend). The linear
regression fit of the spike amplitude versus charge plot gave a slope
of only 46 pA/pC. Thus anti-CAPS antibodies reduced spike amplitude to
one-sixth the control value but only halved the quantal content.
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Figure 5.
Effect of CAPS antagonism on the kinetics
of individual amperometric spikes: amplitude and charge histograms.
A, Control cells. a1, Amplitude histogram
(bin of 20 pA) for spikes obtained during stimulation of two cells with
980 APs. A broad distribution is apparent with a mean value of 127.6 pA. a2, Distribution of spike amplitude versus charge
(Q) fitted by linear regression
(Y = A + BX;
B = 99 pA/pC). a3, Representative
current spike obtained from the two cells analyzed in
a1. Their amplitude and charge from left
to right was 13.5 pA/0.042 pC, 21 pA/0.06 pC, 142 pA/0.316 pC, 225 pA/1.31pC, 415 pA/1.52 pC, and 774 pA/2.047 pC. The
average values (n = 10 cells) for amplitude,
charge, and amplitude/charge were 142.6 ± 25 pA, 1.21 ± 0.17 pC, and 96 ± 5.9 pA/pC, respectively. B,
Anti-CAPS IgG-treated cells. Data were collected from two
representative cells dialyzed with anti-CAPS IgG (anti-FP5) for 14 min
to achieve maximum blockade of secretion and then stimulated with 980 APs. b1, Amplitude histograms show that 88% of the
events are <40 pA (average 24.5 pA). Anti-CAPS IgG reduces the
amplitude of the spike by approximately sixfold. b2,
Distribution of spike amplitude versus charge gives a much reduced
slope of 46 pA/pC compared with control. b3,
Representative amperometric spikes illustrate their typical reduced
magnitude compared with controls (a3). Their amplitude
and charge from left to right were 5.5 pA/0.071 pC, 7 pA/0.105 pC, 6.5 pA/0.199 pC, 19.5 pA/0.464 pC, 18.5 pA/0.445 pC, and 23 pA/0.375 pC. Note the different scale between
top and bottom traces. The
average values (n = 15 cells) for amplitude,
charge, and amplitude/charge were 23.3 ± 1.4 pA, 0.53 ± 0.04 pC, and 33.1 ± 3.7 pA/pC, respectively. From
n = 7 cells treated with anti-FP5 Fab, the average
values were 31.3 ± 6.4 pA, 0.66 ± 0.13 pC, and 34.3 ± 5.8 pA/pC, respectively. C, Superimposition of two
amperometric spikes, from a control cell (note small foot preceding a
very fast spike) versus an anti-CAPS IgG-treated cell (sustained
flickering foot), to illustrate the kinetic differences.
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Figure 6.
Effect of CAPS antagonism on the kinetics of
individual spikes: rise time (RT) and half-width
(HW) analyses. Time course of quantal
catecholamine release in the absence (A) and
presence (B) of anti-CAPS IgG. A,
a1, a2, Histograms of the rise time and
half-width of spikes obtained in the same control cells as depicted in
Figure 5A. The average RT is 0.98 msec, and the HW is
5.3 msec. a3, Representative spike on fast time base,
selected from Figure 5A (a3, *) is used to illustrate
how the RT and HW are generated. RT is the period from 50 to 90% of
the maximum spike amplitude [RT = T
(90%) T(50%)]. HW is the time
corresponding to the width of the amperometric spike at 50% of its
maximum amplitude. The charge (Q =  idt) is the
integral of the amperometric current corresponding to the gray
area between the baseline (dashed line) and the
amperometric spike. From n = 10 control cells, the
average values for RT and HW were 1.18 ± 0.08 and 6.43 ± 0.29 msec, respectively. B, Histograms of the RT
(b1) and HW (b2) of spikes obtained in
the same anti-CAPS IgG-treated cells as depicted in Figure
5B. The average value for RT is 4.3 msec and for HW is
22 msec, both approximately four times longer than control.
b3, A typical amperometric spike, selected from Figure
5B (b3, *) to explain, as in Figure
6A, how the analysis has been performed. From
n = 15 antibody-treated cells, the average values
for RT and HW were 4.23 ± 0.39 and 21 ± 1.6 msec,
respectively. From n = 7 cells treated with
anti-FP5 Fab, the average values were 4.2 ± 0.3 and 22.1 ± 1.5 msec, respectively.
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The fact that spike amplitudes were small even for quite large charge
events is reflective of the fact that most spikes were broader in
anti-CAPS IgG-treated cells, as is readily apparent in the
representative examples shown in Figures 5B, b3,
and 6C. This was quantitated by measuring the rise time and
half-width of spikes in control versus fully anti-CAPS-inhibited cells.
In control cells the mean rise time and half-width were 1.18 ± 0.08 msec and 6.43 ± 0.29 msec (n = 10),
respectively (Fig. 6A, a1, a2).
These values are quite comparable with those found previously in adult
bovine AC cells stimulated with high
[K+] (Pihel et al., 1996). Anti-CAPS
slowed the rise time and prolonged the half-width to values of 4.3 and
22 msec (n = 15), respectively (Fig.
6B, b1, b2). In summary, these
kinetic results indicate that the residual quantal events seen in
CAPS-inhibited cells had different characteristics than those of
control cells. Such kinetic alterations in spike morphology suggest
that CAPS plays a role at the final stage of
Ca2+-triggering secretion, possibly at the
level of the fusion process itself (see Discussion).
CAPS antagonism reduces the exocytotic burst but does not affect
the rate of rapid endocytosis
An alternative method of estimating secretion in single AC cells
is that of membrane capacitance measurements. It is well established
that secretion in AC cells is accompanied by an increase in membrane
capacitance, and we have shown that this can be achieved through
physiological stimulation of cells with short depolarizing stimuli
(Artalejo et al., 1994, 1995). Apart from special circumstances, there
is generally good agreement between the results obtained by capacitance
and those simultaneously gathered by amperometry (Haller et al., 1998).
In AC cells the initial exocytotic capacitance burst represents the
release of already docked-and-primed granules because it can be
elicited by a rise in Ca2+ in the absence
of ATP (Parsons et al., 1995). If CAPS plays a postpriming role in
exocytosis during the Ca2+-triggering
step, then CAPS antagonism should slow the rate of the initial
exocytotic burst. Indeed, introduction of anti-CAPS IgG (anti-FP5) into
AC cells progressively slowed the maximal rate of exocytosis from
934.8 ± 29.7 fF/sec to 80.1 ± 4.1 fF/sec (n = 18) after 14 min (Fig. 7). It could be
argued that repetitive stimulation in the presence of anti-CAPS IgGs
had depleted the release-ready vesicular pool under these conditions
and that the subsequent reduction in exocytotic rate might reflect an
inhibitory effect of anti-CAPS IgGs on docking and/or priming of new
DCVs. To control for this possibility, we introduced anti-CAPS IgGs into calf AC cells and waited 14 min before any stimulation. Under these conditions the initial capacitance burst was still inhibited 91 ± 1.4% (n = 12 cells) compared with control
cells, suggesting that CAPS acts at a postpriming step in
exocytosis.
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|
Figure 7.
CAPS plays a central role in exocytosis but is not
involved in rapid endocytosis. Time course of CAPS antagonism on
membrane capacitance measurements (Cm).
Continuous recordings after formation of whole-cell configuration
from a cell loaded with anti-CAPS IgG (anti-FP5, 1.5 mg/ml).
Secretion was elicited by a train of depolarizations at the times
indicated above the capacitance traces. The Ca current elicited by the
first test depolarization is plotted below the
Cm trace. CAPS antagonism inhibited exocytosis
without affecting endocytosis. A, Initial rates of
exocytosis during the first exocytotic burst were not significantly
different from control, reflecting the possibility that insufficient
antibody has diffused into the cell at this time. The rate of
exocytosis was obtained by measuring the steepest upward slope of the
Cm. Each panel shows records at a
fast time base to reveal in detail the amplitude of the first four
Cm jumps and therefore the rate of secretion
during the first second of the train ("exocytotic burst"). During
the four sequential trains of depolarization, the magnitude and rate of
secretion were inhibited by 88 and 95%, respectively. Slower time base
records (insets) show that rapid endocytosis is not
significantly different from control.
|
|
We have demonstrated previously that membrane capacitance reproducibly
declines back to baseline when stimulation is terminated in calf AC
cells (Artalejo et al., 1995, 1996). We termed this membrane retrieval
mechanism RE to distinguish it from other endocytotic processes
in the cell (Artalejo et al., 1995). Quantitation of RE kinetics
indicated that they remained resistant to CAPS immunoneutralization despite the large reduction in exocytotic rate (Fig. 7). In anti-CAPS IgG-treated cells, a  value of 10.4 ± 0.9 sec
(n = 18) for the fast-2 component of RE [ of the
exponential that fit the decline of Cm on
termination of secretion (Artalejo et al., 1995)] was found, not
significantly different from that in control cells with a similar
magnitude of exocytosis ( of 10.9 ± 0.6 sec; n = 25). The percentages of membrane retrieval were 101.6 ± 2.7 and
100.9 ± 1.1, respectively, for the same groups of cells. These results suggest that CAPS plays a central role in the last
Ca2+-triggering step of exocytosis but is
not involved in endocytosis in calf AC cells.
| |
DISCUSSION |
The exocytotic machinery is a complex assemblage of proteins whose
coordinate function ensures correct targeting and fusion of secretory
vesicles with the plasma membrane. A further layer of complexity is
introduced in regulated secretion because of the requirement for a
triggering step that usually requires a rise in intracellular
Ca2+. Several proteins associated with
SSVs and/or DCVs exhibit Ca2+ sensitivity,
so there may not be a single "Ca2+
sensor" for exocytosis but several factors at different steps of
secretion requiring the presence of optimal
[Ca2+]. Thus, although synaptotagmin I
has been suggested as the primary Ca2+
sensor for secretion at many nerve terminals (Sudhof, 1995), asynchronous secretion still occurs in the absence of this protein (Geppert et al., 1994). In this study we show that another putative Ca2+-binding protein associated with the
secretory apparatus is essential for DCV exocytosis in calf AC cells.
Introduction of anti-CAPS antibodies into these cells resulted in a
dramatic decline in total catecholamine secretion as determined by both
amperometric and capacitance recordings. To our knowledge, this is the
first report of antibody effects on secretion visualized with the
amperometric technique. The effects of anti-CAPS IgGs were specific and
selective because neither Ca currents or APs nor RE, another
Ca2+-dependent event in the calf AC cell
secretory cycle (Artalejo et al., 1995, 1996), were modified, and
preabsorption of anti-CAPS IgGs with their antigens abolished the
inhibitory effect. In addition, it is unlikely that the antibodies
inhibited secretion merely as a result of cross-linking DCVs because
Fab fragments exerted an effect similar to that of intact IgGs.
Moreover, we have also shown that introduction of IgGs to DCV surface
proteins that are thought not to be involved directly in exocytosis
(e.g., cytochrome b561) had no effect on secretion, suggesting that
steric hindrance of DCV integration with the secretory machinery
cannot explain the inhibitory effects of anti-CAPS IgGs.
Consideration of both in situ and in vitro data
has led to the elaboration of a multistep model for exocytosis (Martin,
1997). In brief, vesicles are first recruited from a tethered pool to an anchored state at the plasma membrane. The subsequent docked stage,
defined morphologically in AC cells as that fraction of DCVs in close
apposition to the plasma membrane, may correspond biochemically to the
formation of the SNARE [soluble N-ethylmaleimide-sensitive fusion (NSF) attachment protein (SNAP) receptor] complex. This is
followed by a priming step, which is MgATP dependent and may involve
NSF-dependent disassembly of the SNARE complex. Finally, docked-and-primed vesicles require only triggering by elevation of
cytoplasmic Ca2+ to fuse with the plasma
membrane. Previously, CAPS was found to play a role at the latter
Ca2+-dependent triggering step in
permeabilized PC12 cells, not at any antecedent step in this sequence
(Hay and Martin, 1992; Ann et al., 1997; Tandon et al., 1998). Our
results are consistent with a role for CAPS at a
Ca2+-dependent triggering step in DCV
secretion, rather than at an earlier vesicle recruitment, docking, or
priming step, because of the effects of anti-CAPS IgGs on the shape of
residual amperometric spikes and the rate of the initial exocytotic
burst. If CAPS were playing a role at a step before fusion, such as
recruitment or docking, then removing CAPS would be expected to affect
only the total number of amperometric spikes observed (i.e., a reduced number of docked and primed vesicles) but not their unitary
characteristics. Similarly, in capacitance experiments we would expect
a decline in the magnitude of the initial exocytotic burst but not in
its rate if CAPS were only involved in a step before
Ca2+-dependent triggering and fusion. In
fact, we found effects of anti-CAPS IgGs on both the shape of
individual amperometric spikes and the rate of the capacitance burst.
Detailed analysis of amperometric recordings revealed that several
parameters were altered by CAPS antibodies. It was clear that although
anti-CAPS IgGs severely reduced the total number of amperometric
spikes, some secretion remained, especially at high-frequency
stimulation. Although the spared secretion seen at 7 Hz might suggest
the existence of a CAPS-independent population of DCVs, this does not
appear to be the case, because stimulation at 1 Hz in the presence of
anti-CAPS IgGs did lead to a virtually complete block in secretion.
Moreover, the remaining spikes in anti-CAPS-treated cells had
substantially lower amplitudes and were much broader than spikes from
untreated cells, suggesting that the antibodies did not "select" a
minor population of spikes present under normal conditions. Altered
spike parameters are thought to reflect the rate of diffusion of
catecholamine from the DCV into the extracellular space, rather than
any subsequent event (for review, see Travis and Wightman, 1998),
although release events occurring far from the electrode tip could
result in broad, low-amplitude spikes caused by diffusional effects
(Schroeder et al., 1992). This explanation is unlikely to apply in our
studies because the amperometric electrode was always close to the cell surface, and broad spikes like those seen in anti-CAPS IgG-treated cells were never seen in control recordings. Instances of
spike-broadening of a much smaller magnitude were observed previously
in pharmacological experiments on adult bovine AC cells and were
attributed to an altered rate of catecholamine dissociation from the
vesicular core (Jankowski et al., 1994; Wightman et al., 1995).
In the present case it is difficult to see how a
cytoplasmic/peripheral DCV surface protein like CAPS could alter
intravesicular chemistry to effect the much larger changes in spike
shape seen here. A more attractive explanation is that spike broadening
is caused by changes in fusion pore kinetics (Alvarez de Toledo, 1993).
Fusion pores have been seen in various cells, including AC cells
(Albillos et al., 1997), and exhibit a continuum of conductances (for
review, see Monck and Fernandez, 1996). Moreover, fusion pore dilation
has been reported to be Ca2+ dependent
(Hartmann and Lindau, 1995), implying that a
Ca2+ sensor is involved in the process. If
CAPS plays a role in governing fusion pore opening, perhaps by
stabilizing the DCV membrane interface, then its progressive
immunoneutralization may gradually hinder pore dilation, leading to a
slower and inefficient exit of catecholamines. Fusion pores can also
flicker; thus spike broadening could involve repeated flickering to a
constant pore diameter rather than reduction in size of an open pore
(Rosenboom and Lindau, 1994). As with a narrower fusion pore diameter,
release of DCV contents is thought to be slower through a flickering
pore (Alvarez de Toledo et al., 1993). Further studies of fusion pore
kinetics in calf AC cells may help to resolve this problem.
Corroborative evidence that CAPS is involved at a late step in DCV
exocytosis but is not involved in the vesicle recovery process known as
RE became apparent in capacitance measurements. Several studies have
shown that secretion from AC cells is characterized by an early fast
rise in Cm followed by a slower more sustained phase (Parsons et al., 1995). The early phase is resistant to ATP
removal and hence may represent docked and primed DCVs that simply
require an elevation in Ca2+ to fuse with
the membrane and might correspond to the Ca-dependent but
ATP-independent secretion seen in permeabilized cells. In the present
experiments, anti-CAPS IgGs dramatically reduced the rate of the
initial exocytotic burst, consistent with a role for CAPS at the final
Ca2+-triggering step. Because it is
difficult to determine in these experiments whether CAPS affects both
priming and triggering, we conducted experiments in which no
stimulation was conducted during antibody loading in an attempt to
preserve the docked and primed vesicle pool. The reduction in
exocytotic release rate after 14 min was similar to that seen in cells
that underwent previous stimulation, again suggesting that CAPS exerts
an effect at the triggering rather than at the vesicle recruitment
stage. There is increasing evidence that secretion from AC cell DCVs can occur by a "kiss-and-run" mechanism where full
incorporation of the vesicle membrane into the plasma membrane does not
take place. After fusion and release of catecholamine, the "empty" vesicle is retracted intact by a dynamin-dependent rapid endocytotic mechanism (Albillos et al., 1997; Artalejo et al., 1998). Within this
scenario it is likely that CAPS function is linked to the fusion but
not the fission process, because CAPS neutralization had no effect on
the kinetics of RE.
The precise mechanism of CAPS action is still unknown. CAPS has several
domains with weak homology to other proteins (Ann et al., 1997), but it
is unclear which domains of CAPS are essential for its function. CAPS
binds Ca2+ with low affinity (Ann et al.,
1997), and the protein also binds to PtdIns(4,5)P2, which has been
implicated in secretory events in several systems (Loyet et al., 1998).
In permeabilized PC12 cells, the production of PtdIns(4,5)P2 seems to
be essential at the priming step of exocytosis because two proteins
involved at this step are PtdIns4P 5-kinase and PtdIns transfer
protein, which together may synthesize vesicular PtdIns(4,5)P2 (Martin,
1997). The conformational change brought about by PtdIns(4,5)P2 binding may permit CAPS to penetrate the lipid bilayer and enable the protein
to mediate increased contacts between the fusion partner membranes.
Ca2+ appears to switch the phospholipid
specificity of CAPS: it binds to PtdIns(4,5)P2 in the absence of
Ca2+, but in the range of 10-100
µM Ca2+, it exhibits a
preference for phosphatidylcholine/phosphatidylethanolamine-containing liposomes (Loyet et al., 1998). Ca2+
influx may trigger the fusion process at least in part by changing the
phospholipid-binding properties of CAPS. CAPS has a pleckstrin homology
domain that has a preference for binding to PtdIns(4,5)P2, and this may
well be the locus of interaction of the protein with the phospholipid
bilayer (Loyet et al., 1998). However, it is likely that there are
other protein-protein interactions that define the binding specificity
of CAPS with some precision. If the CAPS dimer interacts with both the
DCV surface and the inner face of the plasmalemma, the association
between the two membranes may be stabilized; this may act as an
essential prelude to the formation of the fusion pore through which
catecholamines escape from the vesicle. A central question that remains
is to what extent CAPS represents a crucial divalent cation receptor
for secretion from DCVs. Other divalent cation binding proteins such as
synaptotagmin are present in chromaffin cell DCVs (Trifaro et al.,
1989). The Ca2+-dependent actin-severing
protein scinderin is also thought to play an important role in
catecholamine secretion (Rodriguez Del Castillo et al., 1990). It may
be that, as with SSVs, several Ca2+
binding proteins must be activated simultaneously to effect optimal secretion. Further studies similar to those described here will be
needed to define the relative importance of these different species in
secretion by DCVs from intact AC cells.
| |
FOOTNOTES |
Received Feb. 26, 1999; revised June 18, 1999; accepted June 23, 1999.
This work was supported by Public Health Service Grants NIH-DK46928
(C.R.A.), NSF-IBN-9604849 (C.R.A.), DGICYT P.M.95-0035 (C.R.A.),
NIH-DK40428 (T.F.J.M.), and NIH-DK25861 (T.F.J.M.). We thank Drs. Clive
Palfrey and Rodrigo Andrade for helpful criticism of this manuscript.
We also thank Dr. Bruce Porter for the production and purification of
the antibodies.
Correspondence should be addressed to Cristina R. Artalejo, Department
of Pharmacology, Wayne State University, School of Medicine, 540 E. Canfield Avenue, Detroit, MI 48201.
| |
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R. N. Grishanin, V. A. Klenchin, K. M. Loyet, J. A. Kowalchyk, K. Ann, and T. F. J. Martin
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T. K. Bratanova-Tochkova, H. Cheng, S. Daniel, S. Gunawardana, Y.-J. Liu, J. Mulvaney-Musa, T. Schermerhorn, S. G. Straub, H. Yajima, and G. W.G. Sharp
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Diabetes,
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[Abstract]
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A. G. Teschemacher and E. P. Seward
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A. Elhamdani, M. E. Brown, C. R. Artalejo, and H. C. Palfrey
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M. Rupnik, M. Kreft, S. K. Sikdar, S. Grilc, R. Romih, G. Zupancic, T. F. J. Martin, and R. Zorec
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TOP
ABSTRACT
INTRODUCTION
