 |
 Previous Article | Next Article 
The Journal of Neuroscience, August 15, 1998, 18(16):6230-6240
Timing of Dense-Core Vesicle Exocytosis Depends on the
Facilitation L-Type Ca Channel in Adrenal Chromaffin Cells
Abdeladim
Elhamdani1,
Zhuan
Zhou2, and
Cristina
R.
Artalejo1
1 Department of Pharmacology, Wayne State University,
School of Medicine, Detroit, Michigan 48201, and
2 Department of Biology, University of Science and
Technology of China, Hefei, Anhui 230027, China
 |
ABSTRACT |
Secretion from dense-core vesicles is reputedly much slower than
that from typical synaptic vesicles, possibly because of noncolocalization of Ca channels and release sites. We reinvestigated this question by measuring the kinetics of catecholamine release in
chromaffin cells from calf and adult bovines. Amperometric recording
from calf chromaffin cells stimulated by action potentials exhibited
two latencies of secretion that depended on both the frequency of
stimulation and the pathway of Ca entry. Short-latency responses (<25
msec delay; "strongly coupled") appeared at low (0.25 and 1 Hz) and
high (7 Hz) frequencies and were entirely dependent on recruitment of
"facilitation" L-type Ca channels as revealed by nisoldipine
blockade. Long-latency responses (>25 msec delay; "weakly
coupled") were more apparent at higher frequencies (7 Hz) and were
substantially reduced by toxins that blocked N- and P-type Ca channels.
Ca current recordings revealed that adult bovine chromaffin cells lack
facilitation channels; virtually all secretion was weakly coupled in
these cells. The mean delay of the strongly coupled signal was ~3
msec after the peak of the action potential (at 24°C), indicating
that dense-core vesicles can exhibit a rate of exocytosis approaching
that occurring in neurons. Although other explanations are possible,
these results are consistent with the idea that facilitation Ca
channels are colocalized with release sites in calf chromaffin cells.
Calculations based on a model incorporating this assumption suggest
that these channels must be within 13 nm of secretory sites to account
for such rapid exocytosis.
Key words:
dense-core vesicles exocytosis; facilitation L-type Ca
channels; adrenal chromaffin cells; colocalization of Ca channels and
release sites; amperometric recording; catecholamine release
| |
INTRODUCTION |
Neurotransmitter release from
presynaptic nerve terminals is extremely rapid. The time from spike
invasion of the presynaptic terminal to postsynaptic response may be
<1 msec at some synapses (Barrett and Stevens, 1972 ; Llinas et al.,
1981; Sabatini and Regehr, 1996). An important element contributing to
the speed of this event is the colocalization of Ca channels and
release sites at the active zone, minimizing the distance over which Ca ions must diffuse to reach the divalent cation receptors for secretion. In the nervous system, such colocalization has been shown on a number
of levels: direct morphological correlation between sites of Ca influx
and secretion (Robitaille, 1990; Haydon et al., 1994) as well as
biochemical association of specific Ca channel subtypes with protein
components of the exocytotic apparatus (Sheng et al., 1996).
Secretion by dense-core vesicles in both neurons and neuroendocrine
cells is generally considered to be much slower than that found at
typical excitatory neuronal synapses (Almers, 1990; Martin, 1994). At synapses that contain both small synaptic vesicles and peptide-containing large dense-core vesicles, higher-frequency stimulation is often necessary to evoke peptide secretion than that
required for small neurotransmitters (Whim and Lloyd, 1989). The slow
rates of secretion found for some cells containing dense-core vesicles
have been attributed in part to spatial separation between Ca channels
and sites of transmitter/hormone release, although there may be other
differences in the secretory machinery (Martin, 1994). For
example, in adult bovine chromaffin cells, detection of secreted
catecholamine by extracellular amperometric recording revealed a lag of
20-100 msec after stimulation (Chow et al., 1992, 1994, 1996). Such a
lag may reflect poor colocalization of Ca channels with the secretory
apparatus in this cell type (Chow et al., 1994, 1996; Zhou and Misler,
1995; Klingauf and Neher, 1997).
Despite these findings, there are indications that the relationship
between Ca channels and secretion is nonrandom in several cell types
containing dense-core vesicles. Insulin secretion is preferentially
coupled to L-type Ca channel activity in  -cells, suggesting that
these channels are closer to release sites than other types of Ca
channel (Bokvist et al., 1995). Rapid exocytosis in mouse adrenal
slices was also attributed to close association between Ca channels and
the secretory apparatus (Moser and Neher, 1997). Moreover, we
previously demonstrated selective coupling between a particular type of
Ca channel and secretion in calf adrenal chromaffin (AC) cells
(Artalejo et al., 1994). Using measurements of membrane capacitance as
an estimate of exocytosis, we found that Ca flowing through
facilitation L-type Ca channels was approximately five times more
efficiently coupled to secretion than Ca entering via N- and P-type Ca
channels (Artalejo et al., 1994). These results suggest that the
facilitation L-type Ca channel is close to the secretory apparatus in
these cells, but the limitations of capacitance recording precluded
precise resolution of rapid stimulus-secretion coupling kinetics.
To resolve this problem we have analyzed the kinetics of catecholamine
secretion from cells derived from young and adult bovines using more
sensitive amperometric techniques (Wightman et al., 1991; Chow et al.,
1992). We determined the contribution of different components of the Ca
current to the secretory responses in these two cell types. We found
that a major component of secretion in calf AC cells is strongly
coupled to the facilitation L-type Ca channel with average latencies of
<5 msec. Although other explanations are possible, these results can
be accounted for if facilitation Ca channels are in very close physical
apposition (<15 nm) to release sites in calf AC cells. By similar
reasoning, most Ca channels are remote from release sites in adult AC
cells.
| |
MATERIALS AND METHODS |
Cell culture
Both calf (average age, 10-12 weeks) and adult (age, >2 years)
chromaffin cells were prepared by digestion of bovine adrenal glands
obtained from local slaughterhouses. Cells were purified and cultured
using previously described methods (Artalejo et al., 1991a). 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 whole-cell recording. Our patch-clamp
techniques have been published previously (Artalejo et al., 1991a,b,
1992a); Axopatch 200 B (Axon Instruments, Foster City, CA) was used as the patch-clamp amplifier for these experiments. All experiments were
performed at room temperature (24°C). The standard patch-pipette solution contained (in mM): cesium glutamate 110, Cs-EGTA
0.1, HEPES 40, MgCl2 5, ATP 2, GTP 0.35, pH 7.2, with CsOH
(nucleotides 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 7.2. When
necessary, the following Ca channel antagonists were added to the
external solution to suppress individual Ca current components (Artalejo et al., 1994): nisoldipine (final 1 µM),
 -conotoxin-GVIA (-CgTx) (final 500 nM), or
-agatoxin-IVA (-Aga-Tx) (10 nM). In most
experiments a solution containing the drugs dissolved in the external
solution was applied directly to the cells via narrow-bore capillary
tubing placed within 50 mm of the cell surface. Perfusion rates were
computer-controlled, and complete bath exchange occurred in 100-200
msec. To assess the effects of "action potential-like" stimuli,
actual current clamp records were used as templates for voltage-clamp
command pulses (McCobb and Beam, 1991). Currents were sampled at a
frequency of 20 kHz.
Current-clamp recording. To evoke action potentials (APs),
cells maintained at  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. The eight-pole Bessel filter was set to a corner
frequency of 2 kHz for Vm recording and then sampled at 5 kHz. The pipette solution contained (in mM): potassium
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 7.2, with NaOH.
Carbon-fiber amperometry for catecholamine detection
Highly sensitive low-noise polypropylene-insulated carbon-fiber
electrodes (ProCFEs) (Zhou and Misler, 1996) 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). Briefly, a 7-mm-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 and 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 (Chow et al., 1992). The
beginning of the current spike was located where the leading edge of
the transient [which includes the "foot" signal when present (Chow
et al., 1992; Zhou et al., 1996)] exceeded the baseline current by two
times the SD of the baseline noise level. Only amperometric events
having 50-90% rise time faster than 3 msec were selected for these
histograms. Measurements are given as mean ± SEM.
| |
RESULTS |
Action potential waveforms recruit facilitation Ca channels in calf
but not in adult AC cells
The physiological stimulus for secretion in AC cells is the
neurotransmitter acetylcholine (ACh) released synaptically from splanchnic nerve terminals onto individual AC cells. ACh depolarizes AC
cells, giving rise to trains of APs (Douglas et al., 1967) that evoke
secretion (Kidokoro and Ritchie, 1980). To ascertain whether such
stimulus trains can recruit facilitation Ca currents in chromaffin
cells, we recorded APs in current-clamp mode and then played them back
as voltage-clamp commands [AP waveforms (APWs)]. As shown in Figure
1, trains of APWs at either 1 or 7 Hz
were able to recruit an extra component of inward Ca current in calf
(10-12 weeks old) chromaffin cells that was completely blocked by
nisoldipine (1 µM). Although 7 Hz stimulation exerted a
more rapid effect, additional 1 Hz trains eventually led to maximal
activation of facilitation current. These results indicate that
physiologically relevant stimulation of calf chromaffin cells activates
facilitation L-type channels and confirms our previous results with
square-wave depolarization (Artalejo et al., 1991a, 1992b). Nisoldipine
has no effect on Ca current in naive cells (i.e., not subject to large
predepolarizations or repetitive depolarizations in the physiological
range), suggesting that calf AC cells lack a "standard" L-type Ca
current. As described previously (Artalejo et al., 1994), the
nondihydropyridine-sensitive Ca current, comprising N- and P-type
components, was completely suppressed by the combination of
-conotoxin and -agatoxin (500 and 10 nM,
respectively).
View larger version (14K):
[in this window]
[in a new window]
|
Figure 1.
Action potential waveforms (APWs) recruit
facilitation L-current in AC cells from calf but not adult cattle. To
mimic Ca channel activation by depolarizing secretagogues such as ACh,
we recorded APs in current clamp and played them back as voltage-clamp
commands (APWs) in (A) calf and
(B) adult bovine AC cells. At
left, Ca currents elicited by single APWs
(a1) before and after a train of 20 APWs
delivered at 1 and 7 Hz in the same cell are shown; 4 min was allowed
for recovery between trains. The superimposed current traces are
(1) control Ca current and (2 and
3) Ca current at the end of the 1 or 7 Hz train. Note
that in A, APWs delivered at 1 Hz increased the Ca
current by 48%, whereas at 7 Hz the Ca current was increased by 98%;
in B no change was observed at either frequency,
indicating the absence of facilitation. At right, Ca
currents (b2) elicited by a single APW
(b1) before and after three consecutive trains
of 20 APWs at 7 Hz in the absence (1, 2) or presence of
nisoldipine (1 µM), alone (3) or
plus -CgTx (500 nM) + -AgaTx (10 nM)
(4); a 4 min interval was allowed for recovery
and application of antagonists. Nisoldipine selectively suppressed the
increased Ca current brought about by the 7 Hz stimulation in calf
(A) and decreased a component of Ca current
(presumed "standard" L-type) in adult (B);
the remaining current was almost completely suppressed by the addition
of -CgTx + -AgaTx.
|
|
When a similar APW train paradigm was applied to AC cells derived from
adult animals (>2 years old), no additional Ca current was observed at
either 1 or 7 Hz, indicating that facilitation Ca channels are absent
from these cells. However, a component of Ca current was inhibited by
nisoldipine (Fig. 1B, right panel), indicating that adult bovine chromaffin cells do have a "standard" (nonfacilitated) L-type Ca current. These results are summarized in
Figure 2. Facilitation Ca channels can
also be recruited by stimuli that raise cAMP levels in calf AC cells
(Artalejo et al., 1990; 1991a), but such treatments were ineffective in
augmenting Ca currents in adult AC cells [data not shown (also see
Engisch and Nowycky, 1996)].
View larger version (33K):
[in this window]
[in a new window]
|
Figure 2.
Statistical analysis of Ca currents in AC cells
from calf versus adult cattle: AP stimulation frequency and antagonist
effects. A, Effect of AP stimulation frequency on the
peak Ca current amplitude at the end of a train of 20 APWs at 1 or 7 Hz. Values are mean ± SEM; n is indicated in
parentheses above each bar. *, Significantly different
from the corresponding control at p < 0.0001. B, Pharmacological dissection of the Ca current
components, evoked by APWs at 7 Hz, shows that facilitation L-type
constitutes 42.32 ± 0.93% of the total Ca current in calf AC
cells, whereas standard L-type contributes 34.1 ± 2.53% to the
total current in adult AC cells.
|
|
Distinct role of facilitation Ca channels in catecholamine
secretion from calf AC cells
To determine whether facilitation Ca channels recruited by APs
contribute to catecholamine secretion in calf AC cells, we stimulated
the cells at 7 Hz with APs while recording catecholamine release
amperometrically using an extracellular carbon-fiber electrode (Fig.
3). We found that in two successive 70 sec stimulation periods, secretion in the second round, as quantitated
by cumulative amperometric spike amplitude, was very similar to that in
the first round, indicating that secretion shows no sign of exhaustion
under these conditions (Fig. 3A, a3).
When the second stimulation round was conducted in the presence of
nisoldipine, the rate and extent of catecholamine secretion were
significantly (>40%) depressed and showed a lag (Fig. 3A,
a2, a3; Table
1). These results suggest that
facilitation Ca channels contribute substantially to catecholamine release under these conditions. Despite the presence of sizeable Ca
currents in adult AC cells, the magnitude of secretion was only ~50%
of that exhibited by calf cells for comparable amounts of stimulation
(Figs. 2, 3B, b1,; Table 1), but
again remained undiminished in successive rounds of stimulation (Fig.
2B, b3). Nisoldipine did not
cause a significant lag in the onset of secretion but significantly
depressed catecholamine release in adult AC cells, indicating that
standard (nonfacilitated) L-type Ca channels are involved in secretion
in these cells (Fig. 3B, b2,
b3; Table 1).
View larger version (35K):
[in this window]
[in a new window]
|
Figure 3.
Distinct role of facilitation Ca channels in
catecholamine secretion from calf AC cells. A, Calf AC
cells were stimulated with trains of APs in current-clamp mode together
with amperometric detection of catecholamine secretion. Amperometric
spikes (Iamp) were generated in
response to trains of single APs (Vm) evoked by
short depolarizing current pulses at a frequency of 7 Hz
(a1). Sequential trains of 490 APs at 7 Hz
resulted in successive secretory episodes monitored as barrages of
amperometric spikes; a 6 min rest period separated each train. As
quantitated by the cumulative integral of the amperometric current
(a3), the second round (Control
2) was always slightly increased relative to the first
(Control 1), indicating that secretion shows no sign of
exhaustion under these conditions (average increase in the second round
was 12.8 ± 0.9%; n = 44). To determine
whether facilitation Ca channels contribute to catecholamine secretion
in calf AC cells, nisoldipine was added between trains. Secretion was
substantially reduced under these conditions
(a2, a3, 44% inhibition
compared with secretion in the first round). Inset
(a4) shows expanded time base for the first 10 sec of AP-stimulated secretion. When facilitation Ca channels are
recruited (Ctr1 and Ctr2), secretion
starts within the first second of stimulation, whereas after
nisoldipine (Nis) secretion only starts after a lag of
~9 sec. B, Adult AC cells treated under conditions
identical to those shown in A. Instead of enhancement, a
slight depression in cumulative secretion was seen in the second round
of stimulation (b3, Control 1, Control
2). When nisoldipine was applied before the second round of
stimulation, the signal was further depressed
(b2,
b3, 37% inhibition) indicating that
conventional L-type Ca channels contribute to secretion in these cells.
Inset (b4) shows that all three
types of Ca channels found in adult AC cells promote secretion, but
with an average lag of 6 sec; after nisoldipine the lag is ~9
sec.
|
|
View this table:
[in this window]
[in a new window]
|
Table 1.
Kinetic properties of catecholamine release in AC cells
from calf versus adult cattle: pharmacological dissection
|
|
A key role for facilitation Ca channels in the onset of the secretory
response also became apparent in these experiments. On recruitment of
these channels in calf AC cells, secretion started within 1 sec of the
beginning of the stimulus train. However, in nisoldipine-treated calf
AC cells, secretion only began after a lag of 9 sec (Fig. 3,
a3, a4). Similar results were
previously obtained when changes in membrane capacitance were used as
an assay of catecholamine secretion in calf AC cells (Artalejo et al.,
1994, their Fig. 2). A lag of 6-9 sec was also present in adult
AC cells and was not altered significantly by the presence of
nisoldipine (Fig. 3, b3, b4).
Thus the prompt onset of secretion in calf AC cells is dependent on the
recruitment of facilitation Ca channels.
Strong stimulus-secretion coupling is predominant in calf AC cells
but not in adult AC cells
We used amperometric latency histograms (Chow et al., 1992, 1994,
1996) to quantitate the delay between the peak of the preceding AP and
the onset of extracellular amperometric spikes in calf AC cells (Fig.
4A). Spikes that
occurred <25 msec after the action potential were operationally
classified as "strongly coupled," whereas those taking place >25
msec after the action potential were termed "weakly coupled." To
illustrate the coupling between APs and the amperometric spikes in calf
AC cells stimulated at 1 Hz, a representative record is shown in Figure
4B. Low-frequency stimulation gave rise to
amperometric spikes that were all (0.25 Hz) or almost all (1 Hz)
strongly coupled to the AP (Fig. 4C, c1, c2). The average delay
between AP and amperometric spike was ~3 msec at these frequencies.
When stimulation was increased to 7 Hz, a greater variation in
latencies was observed; in addition to the strongly coupled peak at 3 msec, a plateau component with delays of 25-143 msec was also found in
these histograms (Fig. 4C, c3). The decay time
constant ( ) of the strongly coupled peak in these histograms was
3-4 msec at all frequencies (Table 1). This value is used to compute
the approximate distance between facilitation Ca channels and release
sites (see below). To further test the idea that the strongly coupled
component of secretion is caused by the activity of Ca channels that
are preferentially associated with release sites, we performed
experiments in which the amount of Ca entering the cell during
stimulation was varied by changing external [Ca]. At high rates of
stimulation (7 Hz), reduction of external [Ca] from 2 to 0.25 mM selectively abolished the weakly coupled signals but had
no effect on the strongly coupled peak in latency histograms (data not
shown). One explanation for these results is that the lower amount of
Ca entering under these conditions fails to elicit secretion at distant
sites that contribute to the weakly coupled signal because of
cytoplasmic Ca buffering (Klingauf and Neher, 1997).
View larger version (18K):
[in this window]
[in a new window]
|
Figure 4.
Strong stimulus-secretion coupling is predominant
in calf AC cells but not in adult AC cells. To quantitate the delay
between the onset of stimulation and detection of amperometric spikes
from calf and adult AC cells, we analyzed latency histograms.
A, In such histograms, "coupling" is defined as the
degree of coincidence between APs and the subsequent amperometric
spike(s). The time from the peak of the AP (vertical dashed
line) to the beginning of a current spike (indicated here by
arrows 1 and 2) is termed latency, and
these are collected and displayed as latency histograms (Chow et al.,
1992). B, Representative amperometric spikes
(Iamp) generated from a calf AC cell
in response to trains of single APs (Vm) evoked at 1 Hz illustrate the coupling between APs (bottom trace)
and amperometric spikes (top trace). C,
Latency histograms of multiple events collected from calf AC cells
stimulated by single APs applied at 0.25 Hz
(c1), 1 Hz (c2), or 7 Hz
(c3), respectively. Filled bars
represent "strongly coupled" signals with latency <25 msec,
whereas open bars represent "weakly coupled" signals
(latency 25-143 msec). At 0.25 Hz, nearly all amperometric spikes
(97%) are strongly coupled; at 1 Hz, 83% are strongly coupled,
whereas the remaining events form a dispersed tail. At 7 Hz, 40% of
events are strongly coupled, whereas the other 60% are weakly coupled.
D, Latency histograms from adult AC cells stimulated
under conditions identical to those shown in C. No
events were apparent at 0.25 Hz (d1), and only a
plateau component of latencies from 0 to 143 msec was observed at 1 and
7 Hz (d2,
d3). Only 4 and 14% of the total
events had a latency <25 msec at 1 and 7 Hz, respectively. Latency
histograms in C and D were determined
from different cells stimulated by similar number of APs at each
frequency, of which only 200 msec is shown 450 APs
(c1, d1, 0.25 Hz), 490 APs
(c2, d2, 1 Hz), and 490 APs
(c3, d3, 7 Hz). Only
amperometric events having 50-90% rise time faster than 3 msec were
selected for these histograms.
|
|
Latency histograms in AC cells from adult cattle revealed no strongly
coupled peak component at any stimulation frequency (Fig.
4D). Instead, a plateau of delay values stretching
from 0 to 143 msec was observed. Although some events with latencies <25 msec were found (Fig. 4D, d2), they
comprised only ~14% of the total signal (Table 1) and thus may
correspond to a "randomly colocalized" component of secretion in
this cell type, as suggested in previous studies (Chow et al., 1994;
Zhou and Misler, 1995; Klingauf and Neher, 1997). The behavior of the
two cell types at 0.25 Hz was particularly striking: in calf cells,
substantial rapid secretion occurred but no secretion could be evoked
from adult cells (Fig. 4, compare
c1,d1).
Strong stimulus-secretion coupling is caused by activation of
facilitation L-type Ca channels
To examine the role of different Ca channels in the patterns of
secretion observed above, we performed amperometric latency histogram
analyses in the presence of toxins that differentially target either
L-type (nisoldipine) or non-L-type (-conotoxin + -agatoxin) Ca
channels (Fig. 5, Table 1). In calf AC
cells, nisoldipine eliminated the strongly coupled peak of secretion at
all frequencies but only attenuated the appearance of the weakly coupled plateau at 7 Hz. By contrast, -conotoxin plus -agatoxin did not affect the short-latency peak at any frequency, but strongly depressed (77%) the weakly coupled plateau seen at 7 Hz. These results
clearly suggest that facilitation L-type Ca channels are responsible
for strong stimulus-secretion coupling in calf AC cells, whereas N-
and P-type channels contribute substantially to delayed secretion. In
AC cells from adults, nisoldipine or the toxin combination simply
reduced secretion at all latencies to virtually the same extent,
indicating that all three channel types play approximately equivalent
roles in secretion from these cells. These results are summarized in
Table 1.
View larger version (21K):
[in this window]
[in a new window]
|
Figure 5.
Strong stimulus-secretion coupling is
attributable to activation of facilitation L-type Ca channels. To
determine the proximity of different types of Ca channels to release
sites and their contribution to secretion at different frequencies, we
analyzed latency histograms from amperometric experiments in the
presence of antagonists that selectively block L-type (nisoldipine) or
N-type [-Conotoxin-GVIA (-CgTx)] and P-type [-Agatoxin-IVA
(-AgaTx)] Ca channels. A, Nisoldipine eliminates the
strongly coupled peak of secretion at all frequencies in calf AC cells.
Latency histograms were derived from the amperometric spikes that
resulted from two successive stimulation periods of 1 Hz
(a1, a2, 490 APs each) or 7 Hz (a3, a4, 490 APs each) in
the absence (Control, a1,
a3) or presence of nisoldipine
(Nisoldipine, a2,
a4). At 7 Hz, nisoldipine suppressed 85% of events
with latencies <25 msec and 30% of those with latencies >25 msec
after the AP (49% of total events). In parallel experiments
(a5, >490 APs), the combination of -CgTx and
-AgaTx had little effect on the short-latency responses (reduced
14% of events <25 msec) but eliminated 77% of events from the weakly
coupled plateau (53% of total events). B, Latency
histograms derived from adult AC cells using an experimental protocol
similar to that shown in A: 1 Hz
(b1, b2, 490 APs), 7 Hz
(b3, b4, 490 APs)
stimulation, each set from the same cell, in the absence
(Control, a1 and
a3) or presence of nisoldipine
(Nisoldipine, b2,
b4), or 7 Hz (b5) in the
presence of -CgTx + -AgaTx. At 7 Hz, in comparison with the
control, 30 and 69% of total events were suppressed by nisoldipine or
the combination of toxins, respectively.
|
|
Facilitation channels are within 13 nm of release sites in calf
chromaffin cells
There are several possible explanations for the close relationship
between facilitation Ca channels and catecholamine secretion (see
Discussion). One possibility, favored by us, is that these data reflect
colocalization of these channels and secretory sites. Klingauf and
Neher (1997) elaborated a model based on amperometric histograms
similar to those used here that allowed them to calculate the average
distance between Ca channels and secretory sites in adult bovine AC
cells. The main assumption made in this model is that release-ready
granules are randomly distributed within a regularly spaced grid of Ca
channels with a constant interchannel distance. Klingauf and Neher
(1997) determined that most vesicles were located at substantial
distances from Ca channels but that a very minor fraction (~8%) were
randomly colocalized with Ca channels. We have used this model and
their data to derive an empirical function describing the algebraic
relationship between the average distance ( ) from Ca channels to
release sites and the decay time constant () of the amperometric
latency histogram (Fig. 6). In addition
to points derived from the data of Klingauf and Neher (1997), we use a
third point estimated from studies on synaptic release (Adler et al.,
1991; Lando and Zucker, 1994; Roberts, 1994). The distance between Ca
channels and release sites at synapses has been estimated to be ~10
nm, and fast release is accomplished within 1-2 msec (Sabatini and
Regehr, 1996). After graphing the appropriate function, we then
determined by interpolation that a value of 3.1 msec (derived from
Table 1, line 1) yields a value of 13 nm for the average distance
between facilitation Ca channels and release sites in calf AC cells. A
variable in the Klingauf-Neher model is the magnitude of the Ca
current. Larger Ca currents would lead to a reduction in the estimated
average distance between Ca channels and release sites. In adult AC
cells, Klingauf and Neher (1997) used a value of 500 pA, which is about half the average Ca current through facilitation channels found in the
present study (~1000 pA; 927.1 ± 55.73; n = 51)
(Fig. 2). With their assumptions that (1) all Ca channels are
evenly/randomly distributed and (2) all Ca channels have a similar
conductance, for a given whole-cell Ca current
(ICa) the following relationship can be
derived: = 300 × (500/ICa)1/2, where is in
nanometers and ICa is in picoamperes. According to this relationship, increasing the Ca current from 500 to 1000 pA in
the Klingauf-Neher model would reduce values by ~30% (e.g., from 300 to 212 nm for the bulk of the noncolocalized vesicles and from
30 to 21.2 nm for the randomly colocalized population). The value
for the noncolocalized population is still much larger than the 13 nm
distance between facilitation Ca channels and release sites calculated
for calf AC cells (Fig. 6). Thus the latter value cannot be explained
by the large facilitation Ca current of ~1000 pA found in the present
study and must be caused by another factor, such as the colocalization
suggested here.
View larger version (22K):
[in this window]
[in a new window]
|
Figure 6.
Estimated distance between facilitation Ca
channels and the adjacent release site. To estimate from the decay
time constant () of an amperometric latency histogram
(ALH), an empirical function was obtained by a
standard optimum fit procedure of data describing the relationship
between Ca channels and secretory sites in adult AC cells (Klingauf and
Neher, 1997). In that model, two points ( = 8.7 msec, = 30 nm; = 25 msec, = 300 nm) were calculated from a specific set
of conditions prevalent in those cells ( ). To derive the empirical
function, we added a third point ( = 10 nm, = 1 msec;  )
derived from work on synaptic terminals (Adler et al., 1991; Lando and
Zucker, 1994; Roberts, 1994; Sabatini and Regehr, 1996). An optimum fit
with the three points ( = 1, = 10; = 8.7 msec, = 30 nm;
= 25 msec, = 300 nm) results in an empirical formula log = 0.94 + 0.063 . The resultant equation was plotted, and the known value of 3.1 msec derived from the latency histogram at 0.25 Hz (Table
1) was used to determine the corresponding value of 13 nm by
interpolation ( ). The triangles show modified data
points of Klingauf and Neher (1997), corrected for 1000 pA Ca current
( ) (see Results for details).
|
|
| |
DISCUSSION |
Previous analyses of stimulus-secretion coupling in
neuroendocrine cells led to the supposition that the kinetics of
secretion from dense-core vesicles is generally much slower than that
from small synaptic vesicles in neurons. The results presented here show that dense-core vesicles can exhibit rates of secretion quite comparable to those found in neurons and that this depends on the
proximity of these vesicles to a specific subtype of Ca channel. We
hypothesized previously that facilitation L-type Ca channels are
colocalized with sites of exocytosis in calf AC cells, based on
capacitance measurements in the presence of specific antagonists of the
different Ca channels present in these cells (Artalejo et al., 1994).
In the present work we demonstrate that APWs are able to recruit these
same facilitation Ca channels in a manner indistinguishable from that
seen previously with single, large, square-wave depolarizing prepulses
or pulse trains in the physiological range (Artalejo et al., 1991a,b).
Consequently, facilitation Ca channels do become active under
physiologically relevant conditions, because acetylcholine leads to
trains of action potentials in these cells (Douglas et al., 1967). We
then used amperometry to define the delay or latency between the peak
of the evoked AP and the onset of catecholamine secretion from
dense-core vesicles. Both strong and weak coupling between AP
stimulation and secretion was found in calf AC cells, based on analysis
of amperometric spike latency histograms. Strong coupling, defined as a
delay of <25 msec between the AP and the amperometric spike,
manifested as a peak at all stimulation frequencies, whereas weak
coupling (delay >25 msec) was substantial only at higher frequencies
and comprised a plateau of values stretching to ~143 msec.
Nisoldipine was able to selectively abolish the strongly coupled signal
at all stimulation frequencies, indicating that this peak in the latency histogram is mediated by facilitation L-type Ca channels. Because the latency is a measure of the coupling between ports of Ca
entry and release sites, these results support our previous contention
that facilitation L-type Ca channels are located closer to release
sites than other types of Ca channel found in calf AC cells (Artalejo
et al., 1994). Consistent with this scenario, weakly coupled, but not
strongly coupled, stimulus-secretion responses were dependent on Ca
influx via N- and P-type Ca channels because these long-latency signals
were reduced by the combination of -conotoxin and -agatoxin.
There are other possible explanations of the efficiency with which
facilitation Ca channels elicit secretion in calf AC cells. For
example, such channels could be clustered, thus providing a locus for
large local increases in submembrane Ca that could trigger with greater
efficiency the secretion of DCVs that happened to be close by. Although
this is evidently a possibility, our previous single-channel data
suggested that clusters of facilitation Ca channels do not exist in
calf AC cells (Artalejo et al., 1990, 1991b). Moreover, large local
changes in Ca attributable to influx at a hypothetical Ca channel
cluster would be expected to spread laterally and cause release of
vesicles at some distance from the cluster. Such events should appear
as a "tail" in amperometry histograms, but at low stimulation
frequencies no tail was observed (Fig. 3B,C). Yet another
possibility is that intracellular Ca buffering may differ (i.e., be
weaker) in the region around facilitation Ca channels compared with
other parts of the sub-plasma membrane domain. Once again, in this
model one would anticipate that the spread of Ca from facilitation
channels would be significant and give rise to a tail in latency
histograms. Moreover, it has been suggested that the sustained increase
in [Ca]i and the mobile buffers should be similar for all
three types of channels (Nowycky and Pinter, 1993; Zhou and Neher,
1993). Although we favor the colocalization model, further studies will be necessary to demonstrate a molecular link between facilitation channels and the secretory apparatus.
In their extensive analysis of secretion from adult bovine AC cells,
Neher's group (Chow et al., 1992, 1994, 1996) concluded that there was
little evidence for coupling of Ca channels with release sites, and
similar conclusions were reached in adult rat AC cells (Zhou and
Misler, 1995). With the assumption that latencies reflect distances,
recent modeling data suggested that the average distance between most
Ca channels and release sites is 200-300 nm in these cells (Klingauf
and Neher, 1997), assuming these elements to be symmetrically
distributed. A small (<10%) fraction of Ca channels was proposed to
be located in a stochastic manner closer to release sites (~30 nm),
to explain an asymmetry in latency histograms biased toward shorter
events [also see Zhou and Misler (1995)]. Our results with adult AC
cells are largely in agreement with those of Chow et al. (1992, 1994,
1996). We found a wide range of latencies with only ~10% of events
showing a delay of <25 msec; no distinct early peak appeared in
amperometric latency histograms, because of the absence of facilitation
L-type Ca channels in these cells. Instead, a standard L-type Ca
current that was not increased by large prepulses, repetitive
depolarization in the physiological range, or cAMP but was sensitive to
nisoldipine is present. Pharmacological suppression of either this
current or other types of Ca channels in these cells did not affect a particular part of the latency histogram spectrum, suggesting that all
types of Ca channels are located at roughly the same distance from
secretory sites. By contrast, a "random colocalization" hypothesis
cannot account for either the much larger component of strongly coupled
secretion (comprising ~97% of the total secretion at 0.25 Hz) (Fig.
4) seen here in calf AC cells or its selective abolition by
nisoldipine. With the model elaborated by Klingauf and Neher (1997), we
were able to estimate the average distance between facilitation Ca
channels and release sites in calf AC cells using our experimentally
determined decay time constant of 3.1 msec for the strongly coupled
component (Fig. 6). This analysis yielded a value of 13 nm, indicating
an association of molecular dimensions between this type of Ca channel
and the exocytotic apparatus in these cells.
The present data imply that the Ca channel complement of bovine
chromaffin cells changes during development and thus explains several
discrepancies that have appeared in the literature. Some studies have
described a form of voltage-dependent facilitation of Ca current in
bovine chromaffin cells that apparently is caused by relief of a tonic
G-protein-mediated inhibition of non-L-type Ca channels (Doupnik and
Pun, 1994; Albillos et al., 1996), a phenomenon well
known in neuronal preparations (for review, see Dolphin, 1996). Because
these studies were all conducted on AC cells derived from adult cattle,
it would not have been possible to detect the facilitation L-type Ca
channel, thus negating the claim by some authors that they have
"disproved" the existence of the facilitation L-type Ca channel in
bovine chromaffin cells (Albillos et al., 1996). The
facilitation L-type Ca channel in calf AC cells is regulated, in fact,
by a phosphorylation event and not by G-proteins. Voltage-dependent
facilitation is blocked in the presence of kinase inhibitors, is
augmented by phosphatase inhibitors, and can be prevented by inclusion
of the catalytic subunit of protein phosphatase 2A in the patch pipette
(Artalejo et al., 1992b). Introduction of agents that modify
G-protein function into calf AC cells (GDPS and GTP S) have no
effect on the recruitment of facilitation Ca current (Artalejo et al.,
1995; and our unpublished results). Voltage-dependent facilitation of
L-type Ca channels has been seen in a number of neuronal preparations
(for review, see Dolphin, 1996). How closely related such channels will
be at the molecular level remains to be determined. It is not
inherently surprising that cells derived from animals of different ages
express different types of Ca channels. Developmental regulation of Ca channel expression has been observed in a number of cell types (McCobb
et al., 1989; Scholz and Miller, 1995). Moreover, previous studies on
AC cells have shown that other responses, such as to nerve growth
factor, are strongly developmentally regulated (Naujocks et al.,
1982). Because AC cells derived from adult cattle possess a
"standard" L-type Ca current (i.e., one not recruited by
depolarizing stimuli), it will be interesting to determine whether the
facilitation L-type channel is replaced by the product of a separate
gene or whether some regulatory feature that alters the behavior of the same channel is lost during aging.
Our results suggest that secretion from neuroendocrine cells has a
component that approaches the rate of events occurring in nerve
terminals. The latency of ~3 msec for the strongly coupled component
is the shortest delay reported for a neuroendocrine cell. Because our
experiments were all conducted at 24°C, and it is known that
secretion from chromaffin cells is very temperature sensitive (Pihel et
al., 1996), it is likely that at 37°C strongly coupled secretion
could occur in substantially <3 msec. These values are comparable with
those found for transmitter release at some nerve terminals using small
synaptic vesicles. For example, in Retzius neurons of the leech,
release of serotonin from such vesicles exhibited a latency of 4.6 msec
(Bruns and Jahn, 1995). In the latter study, the latency of serotonin
secretion from large dense-core vesicles was 16 msec; however, it was
not clear whether this reflected differences in the underlying fusion
reactions, distance from Ca entry sites, or even differences in the
rate of release of transmitter from the matrix of the different types of vesicle. Why would calf AC cells possess such a rapid exocytosis mechanism? We speculate that facilitation Ca channels may be critical to the "fight-or-flight" response. The speed of catecholamine secretion into the bloodstream determines, in part, the rapidity with
which the organism can prepare for escape in times of stress. The
recruitment of facilitation Ca channels by repetitive action potentials
ensures that they will dominate the secretory response during strong
synaptic activation, providing a type of "tuning" mechanism. It is
entirely possible that the developmental regulation of L-type Ca
channels described here, namely facilitation channels giving way to
standard channels, relates to the strength of the fight-or-flight
response in animals of different ages and may have an evolutionary
origin. The rapidity of the response in younger animals, still in their
reproductive prime, maximizes their chances of escape from danger.
As the speed of the response declines in older animals, it may
render them more susceptible to predatory cull. If this speculation is
correct, then the type of colocalization described here may not be a
mere curiosity of calf AC cells. Recent results on mouse chromaffin
cells in slice preparations also yielded evidence for "rapid
exocytosis" (Moser and Neher, 1997) that was interpreted as support
for colocalization, although the type of Ca channels involved was not
defined in that study. Rat (Hollins and Ikeda, 1996) and human (A. Elhamdani and C. R. Artalejo, unpublished results) AC cells
possess facilitation Ca channels and thus may also exhibit the same
type of strong stimulus-secretion coupling described here.
| |
FOOTNOTES |
Received March 10, 1998; revised May 7, 1998; accepted June 8, 1998.
This work was supported by grants from National Institutes of Health
(C.R.A.), National Science Foundation (C.R.A.) Grant DGICYT
PM95-0035 (C.R.A.), and Human Science Frontiers Foundation (A.E.). C.R.A. is a Sloan Fellow. We thank Yanfang Hu for expert preparation of ProCFE. We thank Drs. T. F. J. Thomas, Martin, E. Neher, H. C. Palfrey, and O. D. Uchitel for their helpful
criticisms on this manuscript.
Correspondence should be addressed to Dr. Cristina R. Artalejo,
Department of Pharmacology, Wayne State University, School of Medicine,
540 E. Canfield Avenue, Detroit, MI 48202.
| |
REFERENCES |
-
Adler EM,
Augustine GJ,
Duffy SN,
Charlton MP
(1991)
Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse.
J Neurosci
11:1496-1507[Abstract].
-
Albillos A,
Gandia L,
Michelana P,
Gilabert J-A,
del Valle M,
Carbone E,
Garcia A
(1996)
The mechanism of calcium channel facilitation in bovine chromaffin cells.
J Physiol (Lond)
494:687-695[ISI].
-
Almers W
(1990)
Exocytosis.
Annu Rev Physiol
52:607-624[ISI][Medline].
-
Artalejo CR,
Ariano MA,
Perlman RL,
Fox AP
(1990)
D1 Dopamine receptors activate facilitation Ca channels in chromaffin cells via a cAMP/protein kinase A mechanism.
Nature
348:239-242[Medline].
-
Artalejo CR,
Dahmer MK,
Perlman RL,
Fox AP
(1991a)
Facilitation of Ca current in bovine chromaffin cells is due to recruitment of a second type of whole-cell current with novel properties.
J Physiol (Lond)
432:681-707[Abstract/Free Full Text].
-
Artalejo CR,
Mogul DJ,
Perlman RL,
Fox AP
(1991b)
Bovine chromaffin cells exhibit three types of calcium channels: facilitation, induced by large pre-depolarizations or repetitive activity is due to the increased opening probability of a 27 pS channel.
J Physiol (Lond)
444:213-240[Abstract/Free Full Text].
-
Artalejo CR,
Perlman RL,
Fox AP
(1992a)
, -Conotoxin GVIA blocks a Ca2+ current in bovine chromaffin cells that is not of the "classic" N type.
Neuron
8:85-95[ISI][Medline].
-
Artalejo CR,
Perlman RL,
Fox AP
(1992b)
Voltage-dependent phosphorylation may recruit Ca2+ current facilitation in chromaffin cells.
Nature
358:63-66[Medline].
-
Artalejo CR,
Adams ME,
Fox AP
(1994)
Three types of Ca channel trigger secretion with different efficacies in chromaffin cells.
Nature
367:72-76[Medline].
-
Artalejo CR,
Henley J,
McNiven M,
Palfrey HC
(1995)
Rapid endocytosis coupled to exocytosis in adrenal chromaffin cells involves Ca2+, GTP and dynamin but not clathrin.
Proc Natl Acad Sci USA
92:8328-8332[Abstract/Free Full Text].
-
Barrett EF,
Stevens CS
(1972)
The kinetics of transmitter release at the frog neuromuscular junction.
J Physiol (Lond)
227:691-708[Abstract/Free Full Text].
-
Bokvist K,
Eliasson L,
Ammala C,
Renstrom E,
Rorsman P
(1995)
Colocalization of L-type Ca channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic -cells.
EMBO J
15:50-57.
-
Bruns D,
Jahn R
(1995)
Real-time measurement of transmitter release from single synaptic vesicles.
Nature
37:62-65.
-
Chow RH,
von Ruden L,
Neher E
(1992)
Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells.
Nature
356:60-63[Medline].
-
Chow RH,
Klingauf J,
Neher E
(1994)
Time course of Ca concentration triggering exocytosis in neuroendocrine cells.
Proc Natl Acad Sci USA
91:12765-12769[Abstract/Free Full Text].
-
Chow RH,
Klingauf J,
Heinemann C,
Zucker RS,
Neher E
(1996)
Mechanisms determining the time course of secretion in neuroendocrine cells.
Neuron
16:369-376[ISI][Medline].
-
Dolphin AC
(1996)
Calcium current facilitation in excitable cells.
Trends Neurosci
19:35-43[ISI][Medline].
-
Douglas WW,
Kanno T,
Sampson SR
(1967)
Influence of the ionic environment on the membrane potential of adrenal chromaffin cells and on the depolarizing effect of acetylcholine.
J Physiol (Lond)
191:107-121[Abstract/Free Full Text].
-
Doupnik CA,
Pun RYK
(1994)
G-protein activation mediates prepulse facilitation of Ca channel currents in bovine chromaffin cells.
J Membr Biol
140:47-56[ISI][Medline].
-
Engisch KL,
Nowycky MC
(1996)
Calcium dependence of large dense-core vesicle exocytosis evoked by Ca influx in bovine adrenal chromaffin cells.
J Neurosci
16:1359-1369[Abstract/Free Full Text].
-
Haydon PG,
Henderson E,
Stanley EF
(1994)
Localization of individual calcium channels at the release face of a presynaptic nerve terminal.
Neuron
13:1275-1280[ISI][Medline].
-
Hollins B,
Ikeda SR
(1996)
Inward currents underlying action potentials in rat chromaffin cells.
J Neurophysiol
76:1195-1211[Abstract/Free Full Text].
-
Kidokoro Y,
Ritchie AK
(1980)
, Chromaffin cell action potentials and their possible role in adrenaline secretion from rat adrenal medulla.
Nature
278:63-65.
-
Klingauf J,
Neher E
(1997)
Modeling buffered Ca diffusion near the membrane: implications for secretion in neuroendocrine cells.
Biophys J
72:674-690[ISI][Medline].
-
Lando L,
Zucker RS
(1994)
Ca2+ cooperativity in neurosecretion measured using photolabile Ca2+ chelators.
J Neurophysiol
72:825-831[Abstract/Free Full Text].
-
Llinas R,
Steinberg IZ,
Walton K
(1981)
Relationship between presynaptic Ca current and postsynaptic potential in squid giant synapse.
Biophys J
33:323-351[Abstract/Free Full Text].
-
Martin TFJ
(1994)
The molecular machinery for fast and slow neurosecretion.
Curr Opin Neurobiol
4:626-632[Medline].
-
McCobb DP,
Beam KG
(1991)
Action potential waveform voltage-clamp commands reveal striking differences in calcium entry via low- and high-voltage-activated calcium channels.
Neuron
7:119-127[ISI][Medline].
-
McCobb DP,
Best PM,
Beam KG
(1989)
Development alters the expression of calcium channels in chick motoneurons.
Neuron
2:1633-1643[ISI][Medline].
-
Moser T,
Neher E
(1997)
Rapid exocytosis in single chromaffin cells recorded from mouse adrenal slices.
J Neurosci
17:2314-2323[Abstract/Free Full Text].
-
Naujocks KW,
Korsching S,
Rohrer H,
Thoenen H
(1982)
Nerve growth factor-mediated induction of tyrosine hydroxylase and of neurite outgrowth in cultures of bovine adrenal chromaffin cells: dependence on developmental stage.
Dev Biol
92:365-379[ISI][Medline].
-
Nowycky MC,
Pinter MJ
(1993)
Time courses of calcium-bound buffers following calcium influx in a model cell.
Biophys J
64:77-91[Abstract/Free Full Text].
-
Pihel K,
Travis ER,
Borges R,
Wightman RM
(1996)
Exocytotic release from individual granules exhibits similar properties at mast and chromaffin cells.
Biophys J
71:1633-1640[Abstract/Free Full Text].
-
Roberts WM
(1994)
Localization of calcium signals by a mobile calcium buffer in frog saccular hair cells.
J Neurosci
14:3246-3252[Abstract].
-
Robitaille R,
Adler EM,
Charlton MP
(1990)
Strategic location of Ca channels at transmitter release sites of frog neuromuscular synapses.
Neuron
6:773-779.
-
Sabatini BL,
Regehr WG
(1996)
Timing of neurotransmission at fast synapses in the mammalian brain.
Nature
384:170-172[Medline].
-
Scholz KP,
Miller RJ
(1995)
Developmental changes in presynaptic Ca channels coupled to glutamate release in cultured rat hippocampal neurons.
J Neurosci
15:4612-4617[Abstract].
-
Sheng ZH,
Rettig J,
Cook T,
Catterall WA
(1996)
Calcium-dependent interaction of N-type Ca channels with the synaptic core complex.
Nature
379:451-454[Medline].
-
Whim MD,
Lloyd PE
(1989)
Frequency-dependent release of peptide cotransmitters from identified cholinergic motor neurons in Aplysia.
Proc Natl Acad Sci USA
86:9034-9038[Abstract/Free Full Text].
-
Wightman RM,
Jankowski JA,
Kennedy RT,
Kawagoe KT,
Schroeder TJ,
Leszczyszyn DJ,
Near JA,
Diliberto EJ,
Viveros OH
(1991)
Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells.
Proc Natl Acad Sci USA
88:10754-10758[Abstract/Free Full Text].
-
Zhou Z,
Misler S
(1995)
Action potential-induced quantal secretion of catecholamines from rat adrenal chromaffin cells.
J Biol Chem
270:3498-3505[Abstract/Free Full Text].
-
Zhou Z,
Misler S
(1996)
Amperometric detection of quantal secretion from patch-clamped rat pancreatic -cells.
J Biol Chem
271:270-277[Abstract/Free Full Text].
-
Zhou Z,
Neher E
(1993)
Mobile and immobile Ca2+ buffers in bovine adrenal chromaffin cells.
J Physiol (Lond)
469:245-273[Abstract/Free Full Text].
-
Zhou Z,
Misler S,
Chow RH
(1996)
Rapid fluctuations in transmitter release from single vesicles in bovine adrenal chromaffin cells.
Biophys J
70:1543-1552[Abstract/Free Full Text].
Copyright © 1998 Society for Neuroscience 0270-6474/98/18166230-11$05.00/0
This article has been cited by other articles:
|
|
|
|
 
H.-P. Huang, S.-R. Wang, W. Yao, C. Zhang, Y. Zhou, X.-W. Chen, B. Zhang, W. Xiong, L.-Y. Wang, L.-H. Zheng, et al.
Long latency of evoked quantal transmitter release from somata of locus coeruleus neurons in rat pontine slices
PNAS,
January 23, 2007;
104(4):
1401 - 1406.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
I. Dudanova, S. Sedej, M. Ahmad, H. Masius, V. Sargsyan, W. Zhang, D. Riedel, F. Angenstein, D. Schild, M. Rupnik, et al.
Important Contribution of {alpha}-Neurexins to Ca2+-Triggered Exocytosis of Secretory Granules
J. Neurosci.,
October 11, 2006;
26(41):
10599 - 10613.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. G. Garcia, A. M. Garcia-De-Diego, L. Gandia, R. Borges, and J. Garcia-Sancho
Calcium signaling and exocytosis in adrenal chromaffin cells.
Physiol Rev,
October 1, 2006;
86(4):
1093 - 1131.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. Arroyo, J. Fuentealba, N. Sevane-Fernandez, M. Aldea, A. G. Garcia, and A. Albillos
Amperometric Study of the Kinetics of Exocytosis in Mouse Adrenal Slice Chromaffin Cells: Physiological and Methodological Insights
J Neurophysiol,
September 1, 2006;
96(3):
1196 - 1202.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction
