|
|
|
|||||||
|
|||||||||
(Received for publication, September 9,
1994; and in revised form, December 12, 1994) From the
Previous investigations of the role of Ca
Secretion of cellular substances often occurs by exocytosis, a
process which involves the fusion of intracellular vesicles containing
hormones and/or neurotransmitters with the plasma
membrane(1, 2, 3, 4, 5, 6) .
The bovine adrenal chromaffin cell releases the catecholamine hormones
epinephrine and norepinephrine in this way. Although the details of the
exocytotic mechanism remain unclear at the molecular level, calcium
influx is known to be an essential trigger for the exocytotic process
in adrenal chromaffin and other cells(7, 8) .
Cytosolic free Ca Measurements of intracellular Ca The chromaffin cell is an excellent system to
probe the role of calcium in stimulus-secretion coupling because it has
been shown to undergo calcium-dependent catecholamine release (1, 3, 26) and has been extensively used a
model for neurosecretion(9, 27) . Many previous
studies of the relationship of these events have been undertaken in
chromaffin cell populations (1, 2, 3, 4, 28) and
perfused adrenal glands (29) . The results presented in this
paper show that catecholamine release, resolved at the level of single
cultured cells, correlates well with cytosolic free Ca
where Q = area under the current versus time trace for 60 s following secretagogue delivery (charge, in
coulombs), F = Faraday's constant (96,485
coulombs/equivalent), n = number of electrons passed in
reaction/mol (n = 2 for catecholamine), and m = total number of moles of catecholamine detected by
electrode.
Figure 1:
Repetitive deliveries of 60 mM K
Fig. 2shows both
Ca
Figure 2:
Concentration dependence of peak cytosolic
free Ca
Figure 3:
Dose response of peak cytosolic free
Ca
Figure 4:
Concentration dependence of peak cytosolic
free Ca
Figure 5:
Cytosolic free Ca
Figure 6:
Cytosolic free Ca
In the
absence of extracellular Ca
Figure 7:
The variety of catecholamine release and
cytosolic free Ca
In this work we have combined fluorescent detection of
cytosolic free Ca Transient exposure of a single cell to agents which
cause membrane depolarization lead to a concentration-dependent
increase in cytosolic free Ca With both DMPP and K Prolonged
opening of Na Caffeine induces catecholamine
secretion from perfused adrenal glands in both the presence and absence
of extracellular Ca In the absence of extracellular
Ca In sharp
contrast to caffeine, which was always able to induce catecholamine
secretion by initial release of an internal Ca When 2 mM Ca Simultaneous fluorescence detection of cytosolic free Ca
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5353-5359
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
at
Single Bovine Chromaffin Cells (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
in
stimulus-secretion coupling have been undertaken in populations of
adrenal chromaffin cells. In the present study, the simultaneous
detection of intracellular Ca, with the fluorescent
probe fura-2, and catecholamine release, using a carbon-fiber
microelectrode, are examined at single chromaffin cells in culture.
Results from classic depolarizing stimuli, high potassium (30-140
mM) and 1,1-dimethyl-4-phenylpiperazinium (3-50
µM), show a dependence of peak cytosolic Ca concentration and catecholamine release on secretagogue
concentration. Catecholamine release induced by transient high
K
stimulation increases logarithmically with
K concentration. Continuous exposure to veratridine
(50 µM) induces oscillations in intracellular
Ca and at higher concentrations (100 µM)
concomitant fluctuation of cytosolic Ca and
catecholamine secretion. Mobilization of both caffeine- and inositol
trisphosphate-sensitive intracellular Ca stores is
found to elicit secretion with or without extracellular
Ca. Caffeine-sensitive intracellular Ca stores can be depleted, refilled, and cause exocytosis in medium
without Ca. Single cell measurement of exocytosis and
the increase in cytosolic Ca induced by
bradykinin-activated intracellular stores reveal cell to cell
variability in exocytotic responses which is masked in populations of
cells. Taken together, these results show that exocytosis of
catecholamines can be induced by an increase in cytosolic
Ca either as a result of transmembrane entry or by
release of internal stores.
can be increased in two ways:
depolarizing stimuli can increase cytosolic Ca via
influx of extracellular Ca through calcium channels (9, 10) or, alternatively, release from intracellular
Ca stores can increase cytosolic free
Ca(11, 12) . The role of
intracellular Ca stores in the exocytotic process
remains controversial (3, 13, 14) . One
reason for this controversy is that measurements of catecholamine
release and Ca entry are often made in separate cell
preparations. Measurements in populations may conceal certain effects
due to heterogeneity within cell populations, as shown recently with
Ca/fura-2 measurements(15) . Thus, to further
define the role of Ca in exocytosis, it is necessary
that the elevation of cytosolic free Ca and
concomitant secretion be quantitated and simultaneously correlated at
single cells. in
single cells are possible with fluorescent
probes(16, 17) , but until recently measurements of
secretion were normally made in populations of cells (13, 18) . Release from single cells has been
indirectly monitored by examining the effects on cocultured cells (19) or by measurements of changes in whole cell
capacitance(20) . The direct measurement of secretion from
single cells with carbon-fiber microelectrodes has now been achieved,
enabling much higher resolution of individual vesicular secretion
events(21, 22, 23, 24) . The present
study employs fura-2 fluorescence (16) as a probe of cytosolic
free Ca and a carbon-fiber microelectrode, placed
adjacent to the cell, to monitor released catecholamine (21, 25) resolved at the individual vesicular level.
The fluorescent measurements give a measure of average changes in
cytosolic free Ca throughout the cell, whereas the
electrochemical signals record the individual exocytotic events which
occur at the region of the cell surface directly beneath the sensor
tip(24) . levels when classical depolarizing secretagogues, which cause
Ca influx through Ca channels, are
employed. In contrast, agents which liberate Ca from
caffeine-sensitive or IP
(
)-sensitive
stores(3, 14) show more variable responses from cell
to cell. These agents can induce exocytotic secretion in the absence of
extracellular Ca in some cells. Other cells do not
exhibit secretion even in the presence of extracellular Ca when cytosolic free Ca is elevated by release
of an intracellular store. The heterogeneity revealed in these studies
indicates that interpretation of the exocytotic mechanism requires
single cell measurements of cytosolic free Ca and
exocytotic release.
Chromaffin Cells and Solutions
Primary cultures
of bovine adrenal medullary cells were prepared from fresh
tissue(25) , enriched in epinephrine using a single-step
Renografin gradient(30) , and plated on glass coverslips
(Carolina Biological Supply, Burlington, NC) at a density of 6 
10
cells/35-mm diameter plate. All experiments were
performed at room temperature between days 3 and 8 of culture. For all
experiments the culture medium was replaced with Krebs-Ringer buffer
containing 145 mM NaCl, 5 mM KCl, 1.3 mM MgCl
, 1.2 mM NaHPO
,
10 mM glucose, and 20 mM HEPES. Either 2 mM CaCl or 0.2 mM EGTA (to give a free
extracellular Ca level < 10
M) (31) was added to achieve the desired
Ca content, and all solutions were adjusted to pH 7.4
with NaOH. All experiments were performed on the stage of an inverted
microscope (Axiovert 35, Zeiss, Thornwood, NY). When veratridine was
employed, a small volume of concentrated stock solution was added to
the plate at the indicated time. Other secretagogues were locally
applied for 3-5 s every 2 min via pressure ejection from glass
micropipettes using a Picospritzer (General Valve Corp., Fairfield,
NJ). When potassium was used as a secretagogue, the concentration of
NaCl in the pipette was reduced to maintain osmolarity.Electrochemical Measurement of
Secretion
Carbon-fiber microelectrodes were prepared by sealing
individual fibers (5-µm radius, Thornell P-55, Amoco Corp.,
Greenville, SC) into glass pipettes with epoxy (Epon 828 Resin and m-phenylenediamine hardener, MillerStephenson, Danbury, CT).
Electrodes were polished at a 45 ° angle on a micropipette beveller
(model BV-10, Sutter Instruments, Novato, CA) and then soaked in
2-propanol for at least 15 min before use(32) . Calibrations
were performed using a flow-injection apparatus with 50 µM epinephrine(24) . Amperometric measurements (E
= +650 mV versus sodium-saturated calomel electrode) employed an EI-400
potentiostat (Ensman Instrumentation, Bloomington, IN) in two-electrode
mode. The carbon-fiber working electrodes were positioned 1 µm away
from the cell with a piezoelectric driver (PCS-1000 Patch Clamp
Manipulator, Burleigh Instruments, Fishers, NY) as described
previously(22) . This arrangement has been shown to measure
secretion from a region extending 2 µm beyond the carbon-fiber
perimeter(24) . Amperometric electrode responses were low pass
filtered at 16.67 kHz, digitized using a PCM-2 A/D VCR Adaptor (Medical
Systems Corp., Greenvale, NY), and recorded on 1/2 inch videotape. For
analysis, the data was low-pass filtered at 25 Hz (Krohn-Hite 3750,
Avon, MA), digitized at 20 ms/point using a NIC-310 oscilloscope
(Nicolet Instrument Corp., Madison, WI), and imported into a personal
computer. Locally written software was used to determine the area under
the current versus time traces for 60 s following secretagogue
delivery. In this way the quantity of catecholamine released under the
electrode from a single exposure to a secretagogue was evaluated.
Charge can be related to the number of moles of catecholamine detected
with Faraday's law:
Fura-2 Calcium Measurements
Chromaffin cells were
incubated in Krebs-Ringer buffer containing 1 µM fura-2 AM
(stock solution dissolved in 20% Pluronic F-127 in MeSO),
0.1% bovine serum albumin, and 2 mM Ca for
30 min at room temperature. Culture plates were rinsed twice and
refilled with buffer with the desired Ca content.
Single cells were selected for fluorescence measurement using a
43-µm pinhole aperture with the EMPIX Photometer System
(Mississaugua, Canada). Cells were alternately excited at 340 and 380
nm, and light was collected through a 40 oil-immersion
objective (Fluar 40 X, Zeiss, Thornwood, NY) (31) . To reduce
photobleaching, a 0.5 neutral density filter was placed between the
excitation source and sample, and fluorescence was sampled every 250
ms. Since the presence of the microelectrode induced considerable
autofluorescence and some reflectance, it was necessary to correct the
measured fluorescence intensities. Data were not corrected for cell
autofluorescence because it could not be measured at the specific cells
used. Autofluorescence is significant; however, it was found to vary
from cell to cell, and its contribution to the measured signal leads to
an underestimate of cytosolic Ca concentrations. The
corrected fluorescence values were ratioed (F
/F
) and estimates of
intracellular Ca concentration were calculated using
a previously published method(16) . Errors for fluorescent and
electrochemical measurement are given as standard errors of the mean.Chemicals
Culture medium, Dulbecco's
modified Eagle's/Ham's F-12 medium, was obtained from Life
Technologies, Inc. Collagenase (Type I) for digestion of gland tissue
was obtained from Worthington Biochemical (Freehold, NJ). Renografin-60
was purchased from Squibb Diagnostics (New Brunswick, NJ). Fura-2 AM,
free acid, and Pluronic-F127 were obtained from Molecular Probes
(Eugene, OR). All other chemicals were obtained from Sigma, and
solutions were prepared with doubly distilled water.
Effect of Repetitive High K
Fig. 1shows the Ca Stimulations response (upper trace) and catecholamine release (lower trace) induced by 3-s delivery of 60 mM K repeated at 2-min intervals. A steady-state,
low cytosolic free Ca concentration and no exocytotic
events were exhibited at cells before the stimuli were applied. Upon
application of K, the fluorescent ratio from fura-2
rose rapidly, and current spikes from the exocytotic release of
catecholamine were observed in a parallel time course. After the
cytosolic free Ca reached a maximal concentration, a
slower decline in the fluorescent ratio commenced. At this time, the
frequency of exocytotic spikes decreased until cytosolic free
Ca fell below the threshold required to maintain
exocytosis and catecholamine spikes ceased. The shift in base line
observed after stimulation in the electrochemical traces is due to the
overlap of many catecholamine spikes.
to test reproducibility of a single chromaffin
cell. Every 2 min a 3-s application of 60 mM K was given to single chromaffin cells in medium with 2 mM Ca as indicated by the arrows.
Fluorescence of fura-2 (upper trace) was monitored
simultaneously with amperometric current from the oxidation of released
catecholamine (lower trace). The vertical axis applies to the fura-2 ratio trace, and the scale bar in
the bottom right corner quantitates oxidative current of catecholamine
release spikes. The inset shows the mean maximal cytosolic
free Ca concentration (open bars) and
release of catecholamine for 1 min following stimulation (solid
bars) normalized to the first stimulation as a function of order
of stimulation delivery (n = 5
cells).
influx and catecholamine release at a single cell
exposed to various concentrations of K. The mean total
charge due to catecholamine release detected from a single exposure to
140 mM K was 385 ± 36 pC
(corresponding to 2.0 ± 0.19 fmol of catecholamine) and the mean
apparent maximal cytosolic Ca was 330 ± 23
nM. The maximal free Ca and catecholamine
secretion were found to be dose dependent as application of 30 mM K elicited 39 ± 9.6% of the mean apparent
maximal cytosolic Ca response and only 4.2 ±
9.4% of the release of that from 140 mM K.
Transient delivery of 20 mM or 10 mM K, by pressure ejection, did not elicit
detectable secretion or changes in cytosolic free Ca from basal level. Pooled results of the maximal cytosolic
Ca and catecholamine release from six cells are
plotted versus log K in Fig. 3.
and catecholamine release from stimulation
with high K. A 3-s application of various
concentrations of K was given every 2 min to a single
chromaffin cell as indicated by the arrows. Fura-2 ratio
fluorescence (upper trace) was monitored simultaneously with
amperometric current from the oxidation of released catecholamine (lower trace).
and catecholamine release versus log
[K]. A, plot of peak cytosolic free
Ca concentration versus log
[K]. B, plot of catecholamine
release versus log [K]. A linear
regression is fit to the portion of the curve where K is sufficient to cause catecholamine release (r =
0.982). In all cases, the results were normalized to those obtained
with 140 mM K. Each data point is from
duplicate stimulations at six cells. Error bars are the
standard errors of the mean.
Concentration Dependence of DMPP-induced Changes
Fig. 4shows the concentration dependence of cytosolic
free Ca and catecholamine secretion for a single cell
exposed to DMPP, a nicotinic agonist known to trigger Ca influx. As with high K, increasing the
secretagogue concentration increased resultant cytosolic free
Ca and catecholamine responses. Delivery of 3
µM DMPP resulted in 48 ± 7.9% of the maximal
apparent cytosolic Ca response and 12 ± 9.6%
of the catecholamine release found with 50 µM DMPP (232
± 17 nM and 535 ± 50 pC (2.8 ± 0.26 fmol
of catecholamine), respectively). The durations of both cytosolic free
Ca elevation and catecholamine release from DMPP are
longer than those for K (45-80 s versus 25-60 s).
concentration and catecholamine release from
stimulation with DMPP. A 3-s application of various concentrations of
DMPP was given every 2 min to a single chromaffin cell as indicated by
the arrows. Fura-2 ratio fluorescence (upper trace)
was monitored simultaneously with amperometric current from the
oxidation of released catecholamine (lower trace). The inset shows the peak cytosolic free Ca concentration (open bars) and secretion of catecholamine
for 1 min following stimulation (solid bars) as a function of
DMPP concentration. The bars are the mean ± S.E.
normalized to the mean for the maximal dose (50 µM). Each bar represents data from duplicate stimulations at six
cells.
Responses to Veratridine
Preliminary experiments
showed that transient (3 s) exposure of cells to 100 µM veratridine did not effect cytosolic free Ca or
induce catecholamine release. Therefore, veratridine, an agent that
activates plasma membrane Na channels(18, 33) , was added to the entire
culture plate at the time indicated on the trace to give the desired
concentration. Fig. 5shows the results induced by veratridine
following a 3-s application of 60 mM K to
confirm viability of the cell. Veratridine (50 µM) was
found to cause oscillations in the internal Ca concentration (from apparent basal levels of 40 nM to
approximately 85 nM) which were insufficient to cause
significant catecholamine release (n = 7 cells).
Oscillations of free Ca were suppressed, and
intracellular Ca was returned to basal levels by
transient application of 10 µM tetrodotoxin (data not
shown). Upon increasing the veratridine concentration to 100
µM, Ca oscillations increased in
magnitude and frequency and catecholamine release began to parallel
this pattern in five of the eight cells (Fig. 5, inset). Two cells required 200 µM to cause
release to mimic the cytosolic free Ca transients.
After about 90 s of simultaneous oscillatory behavior, the cell
cytosolic free Ca and catecholamine release reached a
sustained elevated state. The final state of high activity continued
for several minutes. In one cell sequentially exposed to 50, 100, and
150 µM veratridine, an increase in the frequency of
Ca oscillations was noted with increased veratridine,
but exocytotic release never occurred. In this cell, the maximal
cytosolic free Ca concentrations during the
oscillations remained low.
oscillations and catecholamine release at a single cell due to exposure
to veratridine. A 3-s delivery of 60 mM K was
first given to test the viability of the cell before exposure to
veratridine. Veratridine was then added to the culture dish to give the
indicated concentrations during the times marked by the bars.
(Note the artifact created in both traces by activity during drug
addition.) Fura-2 fluorescence ratio (upper trace) was
monitored simultaneously with release of catecholamine (lower
trace). The inset is an expansion of the time period
directly following exposure to 100 µM veratridine.
Oscillations of cytosolic free Ca (upper
trace) and catecholamine release (lower trace) can be
seen to temporally coincide in this portion of the
trace.
Effects of Caffeine Delivery in the Presence and Absence
of Extracellular Ca
Caffeine pressure ejected
in the presence of extracellular Ca was found to
induce a rise in cytosolic free Ca and exocytotic
catecholamine release in single cells (Fig. 6A).
Regardless of the order of delivery, 40 mM caffeine always
elicited larger fura-2/Ca response and catecholamine
release than did 10 mM. The cytosolic free Ca responses induced by caffeine in Ca-containing
medium consisted of two phases: a rapid transient, attributed to
Ca expulsion from internal stores, and a longer
lasting plateau due to influx of extracellular
Ca(11) . The second phase was not apparent in
Ca-free medium. Results from repetitive stimulations
of caffeine remained consistent, with only slightly diminished peak
cytosolic free Ca values in five cells.
and
catecholamine responses to caffeine in the presence and absence of
external Ca. 10 mM and 40 mM caffeine was alternately applied to cells (A) in the
presence of 2 mM extracellular Ca (n = 5) and (B) in medium containing 0.2 mM EGTA (n = 7). Fura-2 fluorescence ratio (dashed lines) was monitored simultaneously with release of
catecholamine (solid lines). Under both conditions, 10
µM DMPP was delivered for 3 s before and after the
caffeine study in order to confirm cell viability. In the experiments
in medium containing 0.2 mM EGTA, the DMPP pipette solution
also contained 2 mM Ca so that fura-2
responses and release could be confirmed. This transient delivery was
sufficient to refill the depleted caffeine-sensitive stores in
experiments without extracellular
Ca.
(0.2 mM EGTA),
the first application of 10 mM or 40 mM caffeine
always elicited a fast increase in cytosolic free Ca and exocytotic release (Fig. 6B). Subsequent
stimulations of either concentration resulted in a smaller peak
cytosolic free Ca concentration and very little or no
release of catecholamine (n = 7 cells). A third
stimulation with caffeine could not elicit any cytosolic free
Ca rise or catecholamine release. A 3-s application
of 10 µM DMPP with 2 mM Ca after the caffeine applications verified that the exocytotic
machinery of the cell was still intact and served to refill
caffeine-sensitive stores as subsequent exposure to 10 mM caffeine showed restored cytosolic free Ca elevation with simultaneous catecholamine release (Fig. 6B).Heterogeneity of Responses from Bradykinin
Application
Individual cells were examined in media with and
without Ca to examine the effect of bradykinin, which
activates B-bradykinin receptors present on bovine
chromaffin cells and elevates intracellular IP levels which
induce a rise in cytosolic free
Ca(34, 35, 36) . Several
different patterns in the responses of cytosolic free Ca and catecholamine release were obtained in the present study (Fig. 7). In medium with 0.2 mM EGTA, 4 out of 15 cells
showed both cytosolic free Ca increase and robust
catecholamine release from delivery of 200 nM bradykinin (Fig. 7A). The release in these cases was often longer
in duration and larger in quantity (pC) compared to release induced by
60 mM K with 2 mM Ca at the same cell. Like caffeine, the cells which did exhibit
release only did so for the first exposure indicating that the
bradykinin-sensitive internal Ca store is also
quickly depleted. Of the cells that did not release catecholamine, one
had a long lasting, substantial rise in cytosolic free Ca (Fig. 7B). The remaining 10 cells showed neither
Ca influx nor secretion of catecholamine even though
cell viability was substantiated with high K deliveries before and after bradykinin (Fig. 7C).
When 2 mM Ca was present in the
extracellular media, 60% of the cells studied behaved as depicted in Fig. 7A.
responses to bradykinin in medium
containing 0.2 mM EGTA. A 5-s delivery of 200 nM bradykinin was given to 15 cells in the absence of extracellular
Ca. A, four of the cells studied resulted in
an increase of cytosolic free Ca, presumably from
IP-sensitive internal stores, and resultant exocytosis of
catecholamine. B, one of the cells studied showed a sustained
increased in cytosolic free Ca but did not cause
catecholamine release. The break in the traces indicates a 40-s pause. C, 10 of the cells did not induce
substantial cytosolic free Ca rise or exocytotic
release. For experiments in media with 2 mM Ca, results as in A (n = 5), B (n = 2), and C (n = 2) were obtained. Transient applications (3
s) of 60 mM K and 2 mM Ca were given before and after the bradykinin
study to ensure cell viability.
with electrochemical measurement of
catecholamine release at the single cell level to correlate responses
to various chemical agents. The methods employed leave the cell
membrane unperturbed thus providing a more physiological view of
biochemical changes induced in the cell by various secretagogues. The
microelectrode reports exocytotic events that occur in the region of
the cell membrane directly beneath it(24) . The use of fura-2
AM allows measurement of whole cell cytosolic Ca without the complication of washout of endogenous Ca buffers(37) , although, like all chelating fluorescence
probes, it may buffer the internal concentration changes that
occur(38, 39, 40) . The general picture that
emerges is that exocytotic secretion in each cell is tightly coupled to
an elevation of intracellular Ca. However, an
increase in intracellular Ca is not sufficient to
cause release; rather, the intracellular Ca concentration must exceed a threshold before release occurs. This
is the case whether Ca elevation is induced by
transmembrane entry or by mobilization of intracellular Ca stores. coupled with
catecholamine secretion by exocytosis. Both effects are more short
lived with elevated K, which causes direct
depolarization of the cell membrane, than with DMPP, which acts via the
nicotinic receptor. However, in both cases the results are consistent
with vesicular release triggered by entry of extracellular
Ca (41) via voltage-sensitive Ca channels(9) . Release and elevation of cytosolic
Ca remain quite similar with six repetitive exposures
to K, although the maximal free Ca concentration decreases slightly with stimulation number, perhaps
due to habituation of Ca channels(42) . at low concentrations, the
relative increases in cytosolic Ca are larger than
the relative release, consistent with observations made with
populations of chromaffin cells (4, 28) and support
the finding that a threshold Ca concentration is
necessary to trigger secretion. This is clearly seen when the
normalized responses are plotted versus the log K concentration, which is directly proportional to the degree of
membrane depolarization(43) . While secretion linearly
increases with membrane depolarization at concentrations above 30
mM, as found for dopamine release from
synaptosomes(44) , lower concentrations of K do not induce measurable secretion. In contrast, 30 mM K induces a significant rise in cytosolic
Ca while the two highest concentrations of
K tested induce comparable changes in
Ca. The sigmoidal curve is similar to that found for 
Ca uptake into brain synaptosomes
stimulated with K(43, 45) . When a
logarithmic plot of catecholamine release versus maximal
cytosolic free Ca is constructed from the pooled data
in Fig. 3, a third-order dependence on Ca is
found (slope = 3.06, r = 0.964). This supports
the view that multiple Ca ions act cooperatively at
the exocytotic trigger site (39) . Third-order dependence of
secretion on intracellular Ca also has been observed
in synaptosomes(46) , giant squid synapses(47) , and
via capacitance methods at chromaffin cells (20, 48) .
Thus, it appears that several aspects of stimulus-secretion coupling
are conserved in both endocrine and neuronal systems. channels by veratridine (33) causes influx of extracellular Ca and
may release Ca from internal stores, activating
Ca extrusion mechanisms including the
Na/Ca exchanger(18, 49) . The combined effects of this
long lasting activation result in oscillations of cytosolic
Ca concentration in chromaffin cells. However, as the
data show, oscillations of cytosolic Ca in bovine
chromaffin cells are only accompanied by exocytotic release once a
threshold Ca value is surpassed. Oscillating release
is seen at higher concentrations of veratridine (>50
µM) that temporally corresponds to the cytosolic
Ca oscillations. Recently, capacitance measurements
have revealed simultaneous exocytosis and Ca oscillations in rat gonadotropes(50) . Eventually, the
processes which lower cytosolic Ca are overwhelmed
and both responses remain elevated.(29) . This is in
contrast to the depolarizing agents and has lead to the concept that
mobilization of internal Ca stores can independently
induce exocytosis(29, 51) . Imaging studies have shown
that caffeine-sensitive internal stores of Ca are
homogeneously distributed throughout the cell, whereas the
Ca influx induced by depolarizing agents initially
occurs at the cellular membrane(9) . Thus, the spatially
averaged values obtained with caffeine more closely reflect the
concentration of Ca that exists at the release
sites(48, 52) . Rapid free diffusion of Ca in the cell cytosol is unlikely because of the presence of
immobile Ca buffers(38, 39, 40, 53) .
Since whole cell measurements of cytosolic Ca give an
average concentration which will not reflect localization regions of
high concentrations, these experiments with caffeine provide a more
direct measure of the Ca concentration threshold
necessary for exocytosis in intact single chromaffin cells. Caffeine,
in the absence of extracellular Ca, caused a rapid
increase of cytosolic Ca from its basal level (20
± 12 nM) (Fig. 6B). However,
catecholamine release did not occur until cytosolic Ca reaches an apparent concentration of 128 ± 27 nM (n = 5). ()This Ca threshold concentration is approximately 50% greater than that
found with the preceding exposure to DMPP and Ca at
the same cell, consistent with the spatial heterogeneity of
Ca concentration found immediately after delivery of
depolarizing agents (9) ., only the initial exposures to caffeine (10 or 40
mM) induced exocytosis of catecholamine because a majority of
the contents of the caffeine-sensitive Ca stores were
initially mobilized and thus remained
depleted(11, 54) . Restoration of the
caffeine-sensitive Ca store has been demonstrated by
prolonged incubation in Ca-containing
media(11, 12, 55) , but this work shows that
only a brief elevation (from 3 s, approximately 9 nl, of 10 µM DMPP and 2 mM Ca) in cytosolic free
Ca is required to refill the store sufficiently to
induce catecholamine release. The limited duration and quantity of
caffeine-induced catecholamine release in Ca-free
medium may explain the conflicting reports on this
topic(11, 13, 51, 55) . store,
bradykinin only could elicit secretion from 27% of cells in the absence
of extracellular Ca. This difference could be because
caffeine-releasable stores contain more free Ca than
those which are sensitive to bradykinin. Alternatively, since
bradykinin releases an IP-sensitive store that is near to
the nucleus(36) , while caffeine-sensitive stores are more
homogeneously
distributed(13, 52, 54, 56) , the
location of the Ca rise may also play a role. The
majority of cells exposed to bradykinin showed neither an increase in
cytosolic free Ca nor secretion of catecholamine,
perhaps due to the lack of B-bradykinin receptors or a
necessary second messenger, or simply that bradykinin-sensitive
Ca stores were empty. Failure to observe release,
even with the sustained increase in cytosolic free Ca seen in one cell, may be because the polarized location of the
Ca store was at a site distant from the electrode of
because the necessary Ca threshold was not achieved. was present in the
extracellular media, 60% of the cells showed an increase in cytosolic
free Ca accompanied by release, comparable to
previous work (36) . However, the total release from
populations of chromaffin cells induced by bradykinin in the presence
of external Ca was only 20% of that induced by
nicotine(34, 36) . The present study reveals that this
difference in secretion is due in part to the larger number of cells
that will secrete in response to nicotine exposure and not necessarily
that each single cell secretes more from nicotine stimulations. These
results show the possibility of misinterpreting whole population
measurements and reveal the benefits of single cell measurements when
studying agents with heterogeneous responses in cell populations. transients and electrochemical measurement of catecholamine
release allows the role of Ca in stimulus-secretion
coupling to be probed. These studies demonstrate the feasibility of
systematic investigations correlating cytosolic free Ca with exocytosis at the single cell level. Results using short or
long lasting depolarizing stimuli and agents that mobilize
Ca from caffeine- and IP-sensitive
internal stores show that different routes to Ca elevation usually lead to exocytosis. Further investigations
coupled with molecular biology could elucidate the mechanism of action
and specific Ca target activated during the short
delay between stimulus and vesicular release at the adrenal chromaffin
cell(23) .
), inositol trisphosphate; DMPP,
1,1-dimethyl-4-phenylpiperazinium.) values are lower than traditionally
accepted concentrations which culminate in
exocytosis(9, 37) . One of the reasons for this
discrepancy is the difficulty in Ca calibration on
the single-cell level; in situ cell lysis methods (using
digitonin or Triton-X) cannot be performed at single cells because
fura-2 leaks away from the measured region and Ca ionophores (ionomycin or A23187) do not fully equilibrate
Ca concentration resulting in an erroneously small
dynamic range between R
and R
(31) . In vitro calibration
methods were therefore employed in these experiments. However,
variability in cell autofluorescence, and the inability to determine it
for the specific fura-2-loaded cell, led to the underestimation of
Ca concentrations. Estimated correction for
autofluorescence yielded maximal cytosolic Ca concentrations from 2 to 5 times reported values.
Helpful discussions with Ricardo Borges are gratefully
acknowledged.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Ishizaki, M. Iigo, N. Yamamoto, and Y. Oka Different Modes of Gonadotropin-Releasing Hormone (GnRH) Release from Multiple GnRH Systems as Revealed by Radioimmunoassay Using Brain Slices of a Teleost, the Dwarf Gourami (Colisa lalia) Endocrinology, April 1, 2004; 145(4): 2092 - 2103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. P. Troyer and R. M. Wightman Temporal Separation of Vesicle Release from Vesicle Fusion during Exocytosis J. Biol. Chem., August 2, 2002; 277(32): 29101 - 29107. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. L. Mundorf, K. P. Troyer, S. E. Hochstetler, J. A. Near, and R. M. Wightman Vesicular Ca2+ Participates in the Catalysis of Exocytosis J. Biol. Chem., March 24, 2000; 275(13): 9136 - 9142. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Borges, E. R. Travis, S. E. Hochstetler, and R. M. Wightman Effects of External Osmotic Pressure on Vesicular Secretion from Bovine Adrenal Medullary Cells J. Biol. Chem., March 28, 1997; 272(13): 8325 - 8331. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |