* Department of Physiology, Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Bunkyo-ku,
Tokyo 113, Japan
Ca2+-dependent exocytotic pathways in
mouse pancreatic cells were investigated using both
capacitance measurement and amperometric detection
of vesicular contents. Serotonin was preloaded into large dense-core vesicles for the amperometry. Exocytosis was induced by rapid elevation of cytosolic Ca2+
concentrations using caged-Ca2+ compounds. Capacitance measurement revealed two major components of
exocytosis, and only the slow component was accompanied by amperometric events reflecting quantal serotonin secretion. Moreover, the fast and slow exocytoses
induced the two forms of endocytosis that were reported to follow the exocytoses of small-clear and large
dense-core vesicles, respectively. Interestingly, we recorded two types of responses of quantal events: in the
type-1 response, most quantal events occurred with a
delay of 0.2 s and were rapidly exhausted with a time
constant of 1.7 s, while, in the type-2 response, quantal
events occurred with a delay of 2.5 s and were sustained. This suggests the existence of two pathways or
modes of the exocytosis involving large dense-core vesicles. Thus, we have revealed three exocytotic pathways
with divergent fusion kinetics in
cells, which provide a
new basis for the understanding of the physiology and
pathology of
cells.
Two types of secretory vesicles, small-clear vesicles
(SVs)1 and large dense-core vesicles (LVs), have
been identified in a wide variety of cells (Kelly,
1993 Two types of secretory vesicles have also been identified
in pancreatic In the present study Ca2+ and time dependencies of LV
exocytosis were examined using both capacitance measurement and amperometric detection of serotonin in Pancreatic islets were isolated from mice (ICR mice) older than 8 wk using collagenase and dispersed into single cells by low Ca2+ treatment as
described (Rorsman and Trube, 1986 Capacitance Measurement
Pancreatic Ca2+ Measurement and Photolysis of
Caged-Ca2+ Compounds
Dual-wavelength ratiometric fluorimetry was performed using a ratioable
long-wavelength Ca2+-indicator dye, BTC (Molecular Probes) as described (Kasai et al., 1996
In vivo calibration experiments of BTC were performed in whole-cell
clamped Amperometric Detection of Serotonin Secretion
For the real-time measurement of secretion of vesicular contents,
Statistical Analysis of the Quantal Events
A Ca2+ jump induced in a cell triggered a series of quantal events, which
we termed a "quantal response." Statistical analysis to determine the existence of two types of quantal responses induced by Ca2+ jumps was performed as follows. We assume that a quantal response is a set of random
samples from a quantal-event distribution between 0 and 30 s after the onset of Ca2+ jumps. Then, a probability density function, q(t), of the quantal-event distribution satisfies For this statistical analysis, all responses elicited by various magnitudes of
Ca2+ jumps were included (see Fig. 4), because the time courses of the occurrence of quantal events did not differ significantly (see Fig. 5). The
value of p was estimated to be 0.676 (± 0.012, SEM) from 779 events in
115 responses (see Fig. 4 A). A series of the parameters (N, FN, FN · pN,
AN, PN) was then obtained as (1, 24, 16, 21, 0.025), (2, 19, 8, 12, 0.09), (3, 13, 4, 8, 0.02), (4, 10, 2, 3, 0.34), (5, 6, 0.8, 3, 0.2), (6, 11, 1, 7, 0.5 × 10 Three Components of Capacitance Increases
Cultured mouse Ca2+ dependencies of the increases in membrane capacitance were systematically investigated as shown in Fig. 2.
No increase in capacitance was detected at [Ca2+]i <3 µM
(Fig. 2 A). The increases in capacitance induced at [Ca2+]i
of between 3 and 10 µM appeared as a single component,
while those induced at [Ca2+]i >10 µM often displayed the
fast and slow components (Figs. 1 B and 2 A). Ca2+ dependencies of the maximal rates of increase in capacitance are
plotted in Fig. 2, C and D. Circles and triangles represent the fast and slow components, respectively, for those data
for which the slow component could be separated from the
fast one. Changes in capacitance were normalized by the
total plasma membrane capacitance of each cell and expressed as percentage(s), because membrane capacitance
varied considerably among cells (see Materials and Methods). The rate of capacitance increase was saturated at [Ca2+]i >50 µM for both the fast and slow components.
The half-maximal rate was achieved at 23 µM and a Hill
coefficient of 2.8 in the slow component (Fig. 2 D). Maximum rates obtained from data for which capacitance increases appeared as a single component are represented
by squares in Fig. 2, C and D. These capacitance increases may involve both the fast and slow components (see below). Assuming that these rates preferentially reflect the
rate for the fast component, the half-maximal rate of that
component was calculated as 26 µM, with a Hill coefficient
of 3.2 (Fig. 2 C).
The slow component of the capacitance increase was decomposed into two phases in the cells where increases in
[Ca2+]i were long lasting (Figs. 1 C and 2 B). The early exponential phase (slow-1) showed a time constant of between 0.54 and 3 s (1.5 ± 0.4/s, mean ± SD, n = 17), suggesting depletion of a pool of vesicles. The late linear
phase (slow-2) occurred with a constant rate as long as
[Ca2+]i increases were sustained. The maximal rate of the
slow-2 component was therefore measured at 2 s after the
onset of Ca2+ jumps when the slow-1 component was almost
depleted. Fig. 2 E shows that the rate of the slow-2 component also saturated at high [Ca2+]i, and the half-maximal
rate was achieved at 18 µM with a Hill coefficient of 2.3.
The mean amplitude of the fast capacitance increases
was ~4.3 ± 1.7% (n = 108) of whole-cell capacitance in
those cells where peak Ca2+ concentrations were >10 µM,
while that of the slow-1 component was 16.2 ± 0.9% (n = 32). The amplitudes of the slow-2 component depended on
the time course of [Ca2+]i. The mean amplitude was 25.8 ± 6.6% (n = 12) in those cells where Ca2+ concentrations
were >10 µM for more than 10 s.
Quantal Secretion of Serotonin
To elucidate which of the two components represents exocytosis of LVs, we performed simultaneous capacitance
measurement and amperometric measurement of serotonin
in A Ca2+ jump induced in a cell triggered a sequence of
quantal events, which we refer to as a quantal response.
We noticed the existence of two types of quantal responses. In a type-1 response, quantal events occurred
within 1.5 s after the onset of a Ca2+ jump (Fig. 3, A and
B), whereas, in a type-2 response, quantal events were delayed (>1.5 s) and sustained (Fig. 3, C and D). The multiple occurrence of delayed events in type-2 responses supported the idea that both types of responses originated
from two distinct quantal-event distributions (see Materials and Methods). We assume that each quantal response
was a set of random samples from either a type-1 or a
type-2 distribution. These distributions were therefore estimated using responses involving multiple quantal events
to identify definitively the types of distribution from which
the quantal responses originated. Namely, the type-1 distribution (Fig. 4 B) was estimated from those responses
(type-1 response) involving at least five events within 1.5 s
after the onset of Ca2+ jumps, while the type-2 distribution
(Fig. 4 C) was estimated from those responses (type-2 response) with all of at least five events appearing more than
1.5 s after the onset of Ca2+ jumps. The estimated distributions were not affected to a significant degree if we used
responses involving no less than three events (open bars in
Fig. 4, B and C), or if we set the time threshold as 1.0 or 2.0 s
(data not shown). The distribution of the events for all responses (Fig. 4 A) apparently can be accounted for by the
sum of those for the two types of responses, consistent
with the idea that responses can be classified into two types.
For this study we used all responses elicited by various
magnitudes of Ca2+ jumps, because time courses of type-1
and type-2 distributions did not depend significantly on
[Ca2+]i (Fig. 5). Events involved in the type-1 and type-2
responses seem to contribute roughly equally to the distribution involving all responses (Fig. 4 A).
The estimated type-1 distribution showed a peak at 0.5 s
after the onset of Ca2+ jumps, and the major component
decayed rapidly with a time constant of 1.7 s (Fig. 4 B).
This indicated the existence of a pool of LVs (type-1 LV
pool) that fused with the membrane with a time constant
of 1.7 s. No quantal events were evoked at [Ca2+]i <3 µM
(data not shown), and the number of events per type-1 response was higher at greater [Ca2+]i (Fig. 5 A). Thus,
time and [Ca2+]i dependencies of the type-1 distribution
were consistent with those of the slow-1 component of
capacitance increase. Temporal correspondence of the
type-1 quantal response and capacitance increase was
found even in a single cell, where the time course of the
amperometric currents was roughly fitted with the time derivative of the slow component of capacitance increase
(Fig. 3 B). Amperometric currents appeared to contain a
nonquantal smooth component (Fig. 3 B), particularly in
those responses involving many events within a few seconds after the onset of Ca2+ jumps. This apparent nonquantal component was interpreted to be the sum of many
secretory events at sites distal to a recording carbon fiber,
because quantal events originating at distal sites should be
small and flat.
The estimated type-2 distribution (Fig. 4 C) peaked at
~2.5 s after the onset of Ca2+ jumps, and the frequency of
occurrence of quantal events decreased in parallel with the
decay in [Ca2+]i (Fig. 5 C). This indicates the existence of
another pool of LVs (type-2 LV pool) that fuse with the
membrane very slowly. The type-2 quantal secretion occurred with a time course similar to that of the slow-2
phase of capacitance increase. It should be noted that the
cells in which the type-2 response was recorded exhibited
amplitudes of capacitance increases in the slow-1 phase similar to those of cells in which the type-1 response was
recorded (Fig. 3); the mean amplitudes of the slow-1 component were 17.6 ± 1.0% (mean ± standard error, n = 10)
and 15.0 ± 1.6% (n = 12) in the cells showing type-1 and
type-2 responses, respectively. Thus, the type-1 pool appeared to exist with similar sizes in those cells that did not
exhibit the type-1 response (see Discussion).
Vesicles in type-1 and type-2 pools seemed to take up
similar amounts of serotonin. Peak amplitudes and total
charges of individual amperometric currents were not
significantly different between early events (<1.5 s) in the
typical type-1 responses and those in the typical type-2
responses. They were 2.24 ± 0.24 pA and 119 ± 24 fC
(mean ± standard error, n = 47), and 2.7 ± 0.7 pA and 163 ± 35 fC (n = 35), respectively.
Fast Exo- and Endocytosis
The properties of the fast exocytosis were investigated in
those cells where the fast component of exocytosis could
be distinguished from the slow one (Figs. 1 B, 2 B, and 6 A).
The fast exocytosis appeared after a delay and occurred
with a single exponential time course (Fig. 6 A). Both the
delay and the time constants depended on [Ca2+]i (Fig. 6,
B and C). The mean amplitude of the fast exocytosis was
4.3% of the total membrane area at [Ca2+]i >10 µM (Fig.
6 D). A very rapid decline in the capacitance followed the
fast exocytosis in 45% of the cells examined. In such cases,
the decline appeared to start after the capacitance increases reached a steady-state level, and it occurred with a single exponential time course. The time constant was also
Ca2+ dependent (Fig. 6 E). If endocytosis occurs with a delay (Thomas et al., 1994
Slow Endocytosis
Slow decreases in the capacitance, representing the slow
endocytosis, often followed the slow-2 exocytosis when
[Ca2+]i tended to decline (Fig. 7 A). The slow endocytosis
was often associated with stepwise decreases in capacitance (arrowheads in Fig. 7). The sizes of stepwise decreases ranged between 16 fF and 78 fF (Fig. 7 B); this indicates endocytosis of vesicles >0.8 µm, corresponding to
the phenomenon called "vacuolation" (Baker and Knight,
1981
We have investigated exocytosis in
Slow Exocytotic Pathways
We believe that the slow increases in membrane capacitance reflect exocytosis of LVs, first of all because they
were associated with secretion of serotonin that was reported to be loaded predominantly into LVs in The exocytosis that we studied at room temperature
mostly represents exocytosis of the fusion-ready vesicles
that have undergone the final ATP-dependent step (Südhof, 1995 Interestingly, we recorded two types of quantal responses, type-1 and type-2, with distinct quantal-event distributions (Fig. 4), indicating the existence of two LV
pools. The two LV pools can be accounted for by two LV
pathways with distinct time constants of fusion (Fig. 8 C).
The existence of the two pools might also be explained by
a sequential model (Fig. 8 B), in which LVs in the type-2
pool need to proceed to the type-1 pool before fusion. To
explain the existence of the two types of responses, however, we have to assume two types of sequential pathways
whose pools are filled with LVs to different degrees, otherwise we should have detected only a single type of response. The difference in the filling states of the pools
means that the rate constants of vesicle transitions between the pools are different among the two pathways. In
this sense, even considering the sequential mechanism, the
existence of two distinct LV pathways is required for understanding our data (Fig. 8 C). The two pathways represent either two independent pathways or two "modes" of
the same pathway where the modes are regulated by cellular metabolic states in We believe that the existence of both the type-1 and
type-2 pools of LVs in We have seen an apparent correspondence between the
two phases of slow capacitance increase and the two quantal responses, consistent with the vesicle hypothesis of secretion involving LVs. In more detail, however, the slow-1
phases of capacitance increases were similar in the cells
showing type-1 and type-2 responses (Fig. 3). This suggests
that a type-1 LV pool of a size similar to that in the cells
that exhibited the type-1 responses appeared to exist in
those cells that did not exhibit the type-1 responses. This
discrepancy can be accounted for by the following explanations. First, there is a regional variation in the distribution of LV pools within a single In the present study exocytosis was seldom induced at
[Ca2+]i <3 µM and did not saturate even at 10 µM (Figs. 2
and 5). This finding is consistent with the estimates obtained for permeabilized pancreatic Fast Exocytotic Pathway
The fast exocytosis likely represents exocytosis of SVs, because To compare kinetic features of the Ca2+-dependent exocytosis of SVs in ; De Camilli and Jahn, 1990). The properties of SVs
and LVs are best characterized in synaptic terminals, where
SVs contain classical neurotransmitters and LVs contain
neuropeptides. The two types of vesicles undergo exocytosis in response to distinct stimuli and are involved in different neuronal functions (Jan and Jan, 1982
; Andersson et al.,
1982
; Matteoli et al., 1988
; De Camilli and Jahn, 1990; Verhage et al., 1991
). In endocrine cells, hormones are primarily stored in LVs, while SVs are also present (Thomas-Reetz
and De Camilli, 1994). Moreover, even nonsecretory CHO
cells show massive exocytosis involving SVs and LVs
(Morimoto et al., 1995
; Ninomiya et al., 1996
; Coorssen et
al., 1996
). In general, SVs are generated by recycling between the plasma membrane and early endosomes (Kelly, 1993
), while LVs are generated in trans-Golgi networks.
The two types of vesicles also exhibit marked differences
in the time courses of exocytosis (Bruns and Jahn, 1995
).
Recent capacitance measurement in PC12 cells has suggested that SVs and LVs showed distinct time courses of fusion even when they were subjected to the same elevations
of cytosolic Ca2+ concentrations ([Ca2+]i) using caged-Ca2+
compounds (Kasai et al., 1996
; Ninomiya et al., 1997
).
cells (Thomas-Reetz and De Camilli, 1994),
where LVs and SVs contain primarily insulin and
-amino-
n-butyric acid (GABA), respectively. The existence of
multiple types of vesicles is a possible problem in studies
relying on capacitance measurement, because vesicle types
cannot readily be identified using this means (Gillis and
Misler, 1992
, 1993
; Ämmälä et al., 1993
, 1994
; Bokvist et al.,
1995
; Eliasson et al., 1996
; Renstroem et al., 1996
). A way
to resolve this problem has recently been developed. Vesicular secretion from single
cells can be recorded by
amperometric measurement using carbon fiber electrodes.
Insulin contained in LVs can be detected by ruthenium-coated carbon fiber electrodes (Kennedy et al., 1987
; Huang
et al., 1996
). Serotonin can be loaded into secretory granules by preincubating
cells with serotonin and can be detected by bare carbon fiber electrodes (Smith et al., 1995
;
Zhou and Misler, 1996
). Selective uptake of serotonin into
LVs has been detected using electron microscopic histochemistry (Jaimm-Etcheverry and Zieher, 1968
), autoradiography (Ekholm et al., 1971
), and subcellular fractionation (Hellman et al., 1972
). Secretion of serotonin could
be more effectively monitored than that of insulin and exhibited characteristics similar to those of insulin secretion
(Gylfe, 1978
; Smith et al., 1995
; Zhou and Misler, 1996
).
Thus, amperometric measurement of serotonin secretion can be used to identify exocytosis involving LVs.
cells that were stimulated with rapid elevation of [Ca2+]i
using caged-Ca2+ compounds. Our data indicate the existence of three pathways of exocytosis in
cells, likely involving SVs and two pathways of LVs, and provide a new
basis for understanding Ca2+-dependent exocytosis in
cells and other cells.
Materials and Methods
Cell Preparation
). The cells were plated onto glass
coverslips (No. 00; Menzel Glass, Braunschweig, Germany) and maintained for 1-2 d in a humidified atmosphere containing 5% CO2 in DME-based tissue-culture medium (1.0 g/liter glucose) supplemented with 10%
FCS, 100 µU/ml penicillin, and 100 µg/ml streptomycin at 37°C. We chose
to study only those cells whose diameters were >10 µm, so that >90% of
the cells were expected to be
cells (Rorsman and Trube, 1986
).
cells were voltage clamped at
65 mV in the whole-cell
mode using a patch-clamp amplifier, AxoPatch (Axon Instruments, Foster
City, CA) at room temperature (20°-25°C). The standard external bathing
solution had the following composition (mM): 150 NaCl, 5 KCl, 1 MgCl2,
2 CaCl2, 10 Hepes, and 2.5 glucose at pH 7.4. Capacitance measurement
was performed as described (Kasai et al., 1996
). In brief, a 1-kHz sine-wave voltage command with a peak to peak amplitude of 40 mV was superimposed on the holding potential. Membrane capacitance was calculated
from 10 cycles of sine waves and sampled at 44 Hz in most experiments,
while it was calculated from one cycle of sine waves and sampled at 1 kHz
in some of them. A phase offset was obtained using a phase-tracking method (Joshi and Fernandez, 1988
), which was performed shortly before
inducing Ca2+ jumps and every 11 s thereafter. The internal solution had
the following composition (mM): 50 Cs-Hepes, 100 Cs-glutamate, 5 CsCl, 0.2 benzothiazole coumarin (BTC; Molecular Probes, Eugene, OR) at pH 7.2. In most experiments, 10 mM dimethoxynitrophenamine tetrasodium
salt (DM-nitrophen; Calbiochem-Novabiochem Corp., La Jolla, CA) was
added as a caged compound together with 0.5-4 mM CaCl2. In some experiments, 2-nitrophenyl O,O
-bis (2-aminoethyl) ethyleneglycol-N,N,N
,N
-tetraacetic acid tetrapotassium salt (NP-EGTA; Molecular Probes) was
used instead of DM-nitrophen in the presence of (mM): 2 MgCl2, 1 Na2ATP, and 0.2 GTP. The results were not significantly different from
those obtained using DM-nitrophen. The osmolarity of the internal solutions was ~320 mM after addition of these chemicals. Membrane capacitances of
cells ranged between 2.3 and 10.8 pF, and the mean value was
4.8 ± 1.4 pF (mean ± SD, n = 205). The mean access resistance was 9.6 ± 1.3 M
in our experiments.
). In brief, the dye was excited with light emitted
from a xenon lamp alternated rapidly between 430 and 480 nm (T.I.L.L. Photonics, Munich, Germany), and the emitted fluorescence was collected
using an objective lens, filtered through an LP520 (Olympus Corp., Tokyo, Japan), and detected using a photomultiplier (NT5783; Hamamatsu
Photonics, Hamamatsu, Japan). Photolysis of caged-Ca2+ compounds was
performed using either a mercury lamp (IX-RFC; Olympus Corp.) or a
xenon flash lamp (High-Tech Instruments, Salisbury, UK) as described
(Kasai et al., 1996
). Open circles in [Ca2+]i traces in Figs. 1 and 2 indicate
that the xenon flash lamp was used to generate Ca2+ jumps. Data obtained
using the xenon flash and mercury lamps were combined to generate the
histograms in Figs. 4 and 5.
Fig. 1.
Membrane capacitance changes evoked by Ca2+ jumps
in mouse cells. Capacitance changes recorded from three cells.
(A) The onset of the capacitance increases (Cm) preceded any
changes in current (I) and conductance (G). The fast exocytotic
component occurred with a delay and was followed by rapid endocytosis. (B) The slow exponential component of capacitance
increase after the fast exocytotic component. This slow component was not associated with any changes in current (I) or conductance (G). (C) A linear increase in capacitance after the slow
exponential component. This type of response frequently appeared when [Ca2+]i increases were sustained.
[View Larger Version of this Image (19K GIF file)]
Fig. 2.
Ca2+ dependence of the three components of capacitance increases in cells. (A) Typical capacitance traces recorded
from four different cells where [Ca2+]i was elevated to the levels
indicated on the left of each trace. Symbols denote the slope of
each of the two components of capacitance increases (see text).
(B) An example of a capacitance trace where increase in [Ca2+]i
was sustained and a linear slow phase of capacitance increase occurred. The slope of the linear component of capacitance increase was measured 2 s after the onset of Ca2+ jumps to minimize a contribution from the exponential component. (C-E) Ca2+
dependence of the rate of the fast component (fEX) and the two phases in the slow component of the increase in capacitance
(s1EX and s2EX). The ordinate represents the maximal slope
(rate) of each capacitance trace normalized by the total cell capacitance in each cell. Open small symbols represent data from
individual cells, and large filled symbols represent means of the
data obtained at certain intervals of [Ca2+]i increases. Vertical
bars in the filled symbols denote the SD for 3-12 data points.
Smooth lines are drawn according to the Hill equation with Hill
coefficients of 3.2, 2.8, and 2.3 in C, D, and E, respectively. The
dotted line in C is drawn according to Eq. 1 and predicted parameters in the text.
[View Larger Version of this Image (29K GIF file)]
Fig. 4.
Type-1 and type-2 distributions. (A) The distribution of
quantal events induced by Ca2+ jumps resulting in [Ca2+]i between 3 and 60 µM. (B) Estimated type-1 distribution obtained from the typical type-1 responses that contained at least N events (five and three for solid and open columns, respectively) within 1.5 s after the Ca2+ jumps. (C) Estimated type-2 distribution obtained from the typical type-2 responses that contained at least N
events (five and three for solid and open columns, respectively)
>1.5 s after the Ca2+ jumps. The width of the bin is set as 0.1 s until 0.5 s after the onset of Ca2+ jumps, when it is set as 0.5 s.
[View Larger Version of this Image (15K GIF file)]
Fig. 5.
Ca2+ dependence of type-1 and type-2 distributions.
(A-C and E-G) Estimated type-1 (A-C) and type-2 (E-G) distributions obtained from the typical type-1 and type-2 responses, respectively, induced by Ca2+ jumps resulting in [Ca2+]i between 3 and 20 µM (A and E), between 20 and 40 µM (B and F), and between 40 and 60 µM (C and G). (D and H) Superimposed traces
for time courses of [Ca2+]i, each trace corresponding to (A and
E), (B and F), and (C and G). Each point is an average for more
than five experiments.
[View Larger Version of this Image (36K GIF file)]
cells in the presence of caged compounds (Almers and Neher,
1985
; Kasai et al., 1996
). We prepared a set of 12 calibration solutions that
contained the basic internal solution and 10 mM of either caged or photolysed forms of DM-nitrophen or NP-EGTA, and the Ca2+ concentration
of these solutions was adjusted to either 0 mM, 15 µM, or 20 mM with
Ca2+ buffers and CaCl2. Fluorescence ratios were obtained a few min after
loading when the ratios stabilized. In our setup, Rmin, Rmax, and Kd
were
0.5, 2.3, and 109 µM for photolysed DM-nitrophen; 0.5, 1.9, and 107 µM
for DM-nitrophen; 0.47, 2.0, and 89 µM for photolysed NP-EGTA; and
0.5, 2.0, and 87 µM for NP-EGTA, respectively. We used these values for
photolysed caged-Ca2+ compounds to calculate peak [Ca2+]i (Kasai et al.,
1996
). We carried out all experimental procedures under yellow light illumination (FL40S-Y-F; National, Tokyo, Japan) to prevent photolysis of
caged-Ca2+ compounds.
cells
were maintained in the culture medium containing 0.5 mM serotonin and
0.5 mM 5-hydroxytryptophan for 4-8 h before recording. Discrete oxidative currents, "quantal events," each due to secretion of serotonin from individual LVs, were recorded using a patch-clamp amplifier (CE2400; Nihon Koden, Tokyo, Japan) and a carbon fiber electrode (Pro CFE; Axon
Instruments) with a diameter of ~5 µm to which a positive potential (650 mV) was applied (Ninomiya et al., 1997
). Amperometric currents were filtered at 0.1 kHz and sampled at 44 Hz (see Fig. 3). Artifacts of amperometry because of flash irradiation were subtracted using the trace from the
same cell at the second or third flash where no secretion was detected.
Forskolin (10 µM) was added to the bathing solution in most amperometric measurements.
Fig. 3.
Simultaneous measurement of membrane capacitance
and serotonin secretion. (A) A single component of capacitance
increase for which a quantal secretory event was not detected in
its early period. (B) An example of a type-1 response showing
multiple quantal events within 1.5 s from the onset of Ca2+ jumps.
(C and D) Two examples of type-2 responses where all of at least
five quantal events were detected >1.5 s after the Ca2+ jump. The
fast, slow-1, and slow-2 phases of capacitance increases are indicated by open circles, open triangles, and open diamonds, respectively.
[View Larger Version of this Image (27K GIF file)]
q(t)dt = 1. As a null hypothesis, we
claim that both types of quantal responses are samples from the same
quantal-event distribution. Consider a set of random FN quantal responses
that contain N quantal events. The null hypothesis predicts the number,
AN, of responses containing quantal events only between 2.5 and 30 s to be
FN · pN, where p represents the probability of the occurrence of quantal events between 2.5 and 30 s, namely, p =
q(t)dt. Using actual values of
FN, AN, and p, the hypothesis can be tested with a level of significance
5), (7, 4, 0.25, 2, 0.02), (8, 4, 0.2, 2, 0.01), (9, 4, 0.15, 1, 0.13), (10, 2, 0.04, 1, 0.04),
and (11, 5, 0.07, 4, 1.6 × 10
7). Thus, AN is always larger than FN · pN, and
the hypothesis is rejected at N = 1, 3, 6, 7, 8, 10, and 11 with PN < 0.05. This indicates that the quantal responses arise from at least two types of
quantal-event distributions, one involving fast events (type-1), and the
other delayed events (type-2).
Results
cells were whole-cell clamped with a
pipette containing a caged-Ca2+ compound and a Ca2+ indicator, BTC. The membrane capacitance of
cells was
continuously monitored, and rapid stepwise elevation of
cytosolic Ca2+ concentrations (Ca2+ jump) was induced
using photolysis of the caged-Ca2+ compound. Increases in
membrane capacitance, evoked by the Ca2+ jumps, exhibited two components as in other endocrine cells (Fig. 1, A
and B) (Thomas et al., 1993
; Neher and Zucker, 1993
; Kasai et al., 1996
). The fast increase in capacitance occurred
after a short delay and was not preceded by any change in
membrane current or conductance (Fig. 1 A). There was
a rapid decrease in capacitance after the first increase in
45% of the cells (Fig. 1, A and B). The kinetic properties
of the fast increase and decrease in capacitance will be analyzed in detail below. The increase in capacitance continued as long as [Ca2+]i remained high (Fig. 1 C), and the slow
component was decomposed further into an early exponential phase (phase-1) and a late linear phase (phase-2).
cells stimulated with a caged-Ca2+ compound.
cells
were preincubated with serotonin for 4-8 h. We frequently
recorded transient oxidative currents (quantal events) representing fusion of a single LV containing serotonin after Ca2+ jumps (Figs. 3 and 4) (Smith et al., 1995
; Zhou and
Misler, 1996
). However, no quantal event was detected
during the fast component of capacitance increase in the
examples shown in Fig. 3 (open circles). This can also be
seen in the latency histogram (Fig. 4 A), where only four
out of 779 quantal events were detected within 0.2 s after
the onset of Ca2+ jumps at any [Ca2+]i. This suggests that
the fast component does not reflect exocytosis of LVs.
Even when the capacitance increase appeared as a single component, serotonin secretion was not detected at the
beginning of the increase (Fig. 3 A). Thus, the apparent
single component was considered to involve both the fast
and slow exocytotic processes.
; Heinemann et al., 1994
; Kasai et al.,
1996
), the amplitudes of exocytosis and endocytosis can be
quantified (Kasai et al., 1996
). The amplitudes of the endocytosis (fEN) quantified as the decrease in capacitance
during the fast endocytosis tended to correlate with those
of the preceding fast exocytosis (fEX) in the same cell
(Fig. 6 F). This suggests the coupling between the fast exocytosis and endocytosis (Kasai et al., 1996
).
Fig. 6.
Fast exo- and endocytosis. (A) Time courses of the fast
exo- and endocytosis. The dashed curve is an exponential fit of the data. (B-D) Ca2+ dependence of the delays (B), time constants
(C), and amplitudes (D) of the fast exocytosis. Smooth dashed
curves in B and D are drawn according to Eq. 1 and parameters
in the text. (E) Ca2+ dependence of the time constants of the fast
endocytosis. (F) Correlation between the amplitudes of the fast
exocytosis and endocytosis. The solid line indicates the relationship fEX = fEN. The correlation coefficient is 0.449 (P < 0.025).
The width of the bin is set as 0.1 s until 0.5 s after the onset of
Ca2+ jumps, when it is set as 0.5 s.
[View Larger Version of this Image (28K GIF file)]
; Back et al., 1993
; Knoll et al., 1992
; Kasai et al., 1996
).
The stepwise decreases could also be identified as negative
spikes in the time derivative of the capacitance traces (Fig.
7 A). Interestingly, the slow-1 phase of exocytosis was followed by a transient decrease in the capacitance in seven
cells where [Ca2+]i was >30 µM (Fig. 7 C). The endocytosis after the slow-1 phase was also associated with the stepwise decreases in membrane capacitance. Thus, both slow-1
and slow-2 phases of exocytosis triggered endocytosis with
vacuolation, but the onsets of the endocytoses were different. These data are consistent in suggesting that distinct
pools of LVs caused two phases of exocytosis and subsequent slow endocytosis.
Fig. 7.
Slow endocytosis. (A) An example of the slow endocytosis that occurred after the slow-2 phase of capacitance increase.
Arrows indicate the stepwise decreases in capacitance, which appear as negative spikes in the time derivative of capacitance (Cm/dt). (B) Amplitude distribution of the stepwise capacitance decreases, representing endocytosis of large vesicles. Diameters of
the vesicles are calculated assuming a spherical shape of the vesicles and a membrane capacitance of 1 µF/cm2. (C) An example of
the slow endocytosis that occurred immediately after the slow-1
phase of capacitance increase.
[View Larger Version of this Image (17K GIF file)]
Discussion
cells by simultaneously using capacitance measurement and amperometric detection of vesicular contents. Three lines of evidence
indicated the existence of three exocytotic pathways in
cells. First, the increase in membrane capacitance induced by Ca2+ jumps exhibited two major components, a
fast and a slow component, and the latter was further decomposed into two phases (Fig. 8 A). Second, quantal
events of serotonin secretion occurred with two distinct
time distributions, each of which corresponds to one of the
two phases of slow capacitance increase. Finally, three forms of endocytosis followed the three components of
exocytosis. We will discuss below the validity and implications of the multiple exocytotic pathways in
cells (Fig. 8,
B and C).
Fig. 8.
Exocytotic events induced by Ca2+ jumps in a pancreatic cell. (A) Three phases (a, b, and c) in the increase in membrane capacitance induced by Ca2+ jumps. (B) Sequential model.
This model contains only one fusion step with a rate constant (a)
preceded by two sequential steps with rate constants (b and c).
This model is inconsistent with our data for
cells, first because
quantal serotonin secretion was only associated with the slow capacitance increase, suggesting a separate SV pathway, and second
because the two types of responses of quantal events were detected during the slow capacitance increase, indicating the existence of two LV pathways (see text). (C) Multi-pathway model.
Three pathways, one SV and two LV pathways, undergo exocytosis with time constants reflected by a, b, and c. The two LV pathways either represent two independent pathways or two modes of
the same pathway where the modes are regulated by cellular metabolic states.
[View Larger Version of this Image (19K GIF file)]
cells
(Jaimm-Etcheverry and Zieher, 1968
; Ekholm et al., 1971
;
Hellman et al., 1972
). Although in some neurons monoamines are also believed to be stored in small dense-core vesicles (Bruns and Jahn, 1995
; Bauerfeind et al., 1995
), the existence of such vesicles in endocrine cells has not been
reported. Second, the slow exocytosis was followed by a
slow decline in the capacitance with large stepwise reductions, the phenomenon termed vacuolation, which is reported to follow LV exocytosis (Fig. 6) (Baker and Knight,
1981
; Back et al., 1993
; Knoll et al., 1992
).
), because it did not depend to any great extent
on cytosolic ATP concentration; we performed most of the
experiments in the absence of ATP (see Materials and
Methods) (Renstroem et al., 1996
). The first Ca2+ jumps
induced exocytosis of most of the fusion-ready vesicles, while the second Ca2+ jumps induced little exocytosis in
our preparations, unlike those reported to occur in synaptic terminals (Hsu et al., 1996
). Thus, the first Ca2+ jumps
exhausted the fusion-ready vesicles. Assuming the mean diameter of LVs to be 250 nm and a membrane capacitance of 1 µF/cm2, the mean amplitude of the slow component of capacitance increase (2.1 ± 0.3 pF) gives a lower
limit of the number of fusion-ready LVs as 950-1,250 per
cell. Since the native pancreatic
cells possess ~13,000 insulin granules (Dean, 1973
), at least 7-9% of the granules
appear to be fusion-ready in
cells.
cells.
cells is important for
cell functions for the following two reasons. First, other secretory
cells exhibit either a type-1 or a type-2 pool; the type-1
pool is the major pool in adrenal chromaffin cells exhibiting rapid secretion of catecholamine (Ninomiya et al., 1997
),
whereas the type-2 pool is predominant in PC12 cells (Ninomiya et al., 1997
) and likely predominant in exocrine
acinar cells exhibiting slow secretion of zymogen granules
(Ito et al., 1997
). Second, the
cells are known to exhibit
biphasic patterns of insulin secretion during application of
glucose or other stimuli (Cerasi and Luft, 1967
; Curry et al.,
1968
; Wollheim and Sharp, 1981
; Hagopian et al., 1993
). It
can be speculated that, after the fast exocytosis of type-1
pool LVs in
cells, a substantial portion of type-2 pool LVs remains available for the late phase of secretion.
Thus, the two pools of LVs may contribute to the biphasic
secretion of insulin, in addition to other mechanisms underlying the biphasic secretion of insulin that have already
been proposed (Wollheim and Sharp, 1981
). The time
courses of the slow-2 phase of LV exocytosis in the present
study (>10 s) appeared to be faster than that of the late
phase of insulin secretion in vivo (>5 min) (Wollheim and Sharp, 1981
). This may be due to the fact that secretion
was triggered by sustained increases in [Ca2+]i in this
study, while it was triggered intermittently by Ca2+ spikes
in vivo (Bergesten, 1995
). Selective deficit of the fast pulsatile secretion of insulin has been reported as an early
symptom with non-insulin-dependent diabetes (O'Rahilly et al., 1988
). Our data suggest the new possibility that
selective impairment of the type-1 LV exocytosis and/or
selective depletion of the type-1 LV pool underlie the
pathogenesis of diabetes.
cell, and the type-1 response can be recorded only from a part of the surface
area of the cell. Indeed, polarized localization of LVs has
been observed using quinacrine in cultured mouse
cells
(Bokvist et al., 1995
), and a carbon fiber electrode can
sense secretory substances present within only a few micrometers from the electrodes (Schroeder et al., 1994
).
Second, serotonin uptake into vesicles of the two LV pools
might be variable among
cells.
cells using electrical
shock (Jones et al., 1985
), digitonin (Tamagawa et al., 1985
),
or streptolysin-O (Okazaki et al., 1994
). This finding, however, is not in line with those obtained in microfluorimetric
studies, where insulin secretion and increases in [Ca2+]i
were simultaneously measured, and where insulin secretion occurred at [Ca2+]i <1 µM (Smith et al., 1995
; Bergesten, 1995
). However, it has been reported that there are
[Ca2+]i hot spots in
cells (Theler et al., 1992
; Bokvist et al., 1995
), and that [Ca2+]i in the hot spots can be underestimated using the high affinity Ca2+ indicators (Ito et al., 1997
)
that have been used in previous studies (Smith et al., 1995
;
Bergesten, 1995
). Hence, local [Ca2+]i increases around
secretory vesicles can be larger than reported, and more
precise [Ca2+]i measurements using low affinity Ca2+ indicators and tomographic imaging techniques are necessary
to determine the precise Ca2+ dependence of exocytosis
during physiological stimulation. Recently, it has been
pointed out that exocytosis of LVs in exocrine acinar cells
requires larger increases in [Ca2+]i than hitherto considered (Ito et al., 1997
).
cells possess both SVs and LVs, and the fast exocytosis was not accompanied by secretion of serotonin. In
addition, some of the kinetic properties of the fast exocytosis are similar to those of the fast exocytosis in PC12 cells,
including a delay in the occurrence of exocytosis (Figs. 1 A
and 6 A) and a tendency for exocytosis to be canceled out
by the following rapid endocytosis (Fig. 6 F) that is Ca2+
dependent (Fig. 6, B and C). The fast exocytosis in PC12
cells has been recently demonstrated to represent exocytosis of SVs (Kasai et al., 1996
; Ninomiya et al., 1997
). Our
data, however, cannot entirely rule out the possibility that
the fast exocytotic pathway in
cells involves secretory
vesicles other than SVs, e.g., LVs that did not take up serotonin. The exocytosis of SVs may have a secretory function; cells of one
cell line,
-TC6, secrete GABA upon
high glucose stimulation (Gaskins et al., 1995
). Physiological effects of GABA secretion on glucagon-secreting cells
were previously proposed (Rorsman et al., 1989
; Sorenson et al., 1991
; Gilon et al., 1991
). SV exocytosis may have
functions other than secretion, such as membrane remodeling and resealing (Bi et al., 1995
) or transport of molecules in the plasma membrane (Kiss et al., 1994
).
cells and those in neurons, the rate constants and delays of the fast exocytosis were fitted to a
model used to explain those of the exocytosis in a nerve
terminal (Heidelberger et al., 1994
). We postulate cooperative binding of four Ca ions, because the Hill coefficient
of the Ca2+ dependence of the fast exocytotic component
was 3.2.
(1)
where V0 to V4 represent vesicles whose Ca2+ binding sites
are occupied by zero to four Ca ions, F represents a fused
vesicle, k is the rate constant of Ca2+ binding, C is the concentration of Ca2+, l is the rate constant of Ca2+ dissociation, and a is the rate constant of fusion. A parameter c is
used to account for the cooperative Ca2+ binding (Heidelberger et al., 1994) and is fixed at 0.2. We estimated the
rate constant of fusion, a, to be 100/s from the maximum
rate of exocytosis at high [Ca2+]i. Then, the delay in the
exocytosis was chiefly attributed to k, and the Ca2+ sensitivity to k and l. A satisfactory fit of the data was obtained by setting k = 4 × 106/s/M and l = 300/s (dashed line in
Figs. 2 C and 6, B and C). Thus, the SVs of
cells exhibit a
10 times smaller fusion rate (a) than the SVs of synaptic
vesicles.
Another important characteristic of SV exocytosis in cells is its Ca2+ dependence, which is similar to that of LV
exocytosis in the same cells. This suggests that SV exocytosis was induced by elevation of [Ca2+]i in a similar way to
LV exocytosis. Capacitance measurement has been widely
used for elucidating the regulatory mechanisms of exocytosis in
cells that are subjected to depolarization (Gillis
and Misler, 1992
, 1993
; Ämmälä et al., 1993
, 1994
; Bokvist
et al., 1995
; Eliasson et al., 1996
; Renstroem et al., 1996
).
In such experiments, the contribution of the fast exocytosis to the capacitance increase cannot be precisely estimated because the exact increase of [Ca2+]i in the vicinity
of SVs is not known. Assuming, however, that [Ca2+]i increases to 5 µM in the vicinity of vesicles (Bokvist et al., 1995
) for 200 ms, the above model predicts exocytosis of
16% of the fusion-ready SVs, which amounts to a 33 fF increase in membrane capacitance. Hence, the capacitance
increases induced by depolarization should be interpreted
with caution, since they may substantially involve SV as well
as LV exocytosis. In contrast, the capacitance increases induced by Ca2+ jumps offer the possibility of investigating
exocytosis of each type of vesicle with distinct fusion rate
constants (Fig. 8, A and C).
The capacitance changes induced by Ca2+ jumps (Fig. 8 A)
have often been interpreted using a single-pathway sequential model (Fig. 8 B) for endocrine cells (Neher and
Zucker, 1993; Thomas et al., 1993
). The sequential model
postulates only one type of vesicle and one final fusion
step. Previous studies in chromaffin and PC12 cells, however, indicated the existence of two Ca2+-dependent exocytotic pathways, involving SVs and LVs, with distinct fusion rate constants (Ninomiya et al., 1997
). In the present study we have further suggested the existence of two LV
pathways with distinct fusion time constants and distributions in
cells (Fig. 8 C). Thus, the involvement of multiple exocytotic pathways needs to be considered to interpret the changes in membrane capacitance in secretory cells.
Pancreatic
cells offer a good system to elucidate morphological bases of multiple exocytotic pathways and to
reveal molecular mechanisms underlying divergent fusion kinetics in secretory cells.
Received for publication 10 December 1996 and in revised form 30 April 1997.
1. Abbreviations used in this paper: BTC, benzothiazole coumarin; [Ca2+]i, cytosolic Ca2+ concentration; DM-nitrophen, dimethoxynitrophenamine tetrasodium salt; GABA,We thank K. Yasuda (Third Department of Internal Medicine) for helpful discussion, and Y. Ninomiya, K. Ito, and T. Kishimoto (Department of Physiology) for technical assistance.
This work was supported by grants-in-aid from the Japanese Ministry of Education, Science, and Culture, a grant from the Takeda Foundation, a research grant from the Human Frontier Science Program, Research for the Future of the Japan Society for the Promotion of Science, and Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation. N. Takahashi is a research fellow of the Japan Society for the Promotion of Science.
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