(Received for publication, September 9, 1994; and in revised form, December 12, 1994)
From the
Previous investigations of the role of Ca 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.
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 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.
Measurements of intracellular Ca 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) .
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 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.
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 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).
Fig. 2shows both
Ca 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.
Figure 2:
Concentration dependence of peak cytosolic
free Ca 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).
Figure 3:
Dose response of peak cytosolic free
Ca 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.
Figure 4:
Concentration dependence of peak cytosolic
free Ca 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.
Figure 5:
Cytosolic free Ca
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.
Figure 6:
Cytosolic free Ca 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
.
In the
absence of 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).
Figure 7:
The variety of catecholamine release and
cytosolic free Ca 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.
In this work we have combined fluorescent detection of
cytosolic free Ca 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.
Transient exposure of a single cell to agents which
cause membrane depolarization lead to a concentration-dependent
increase in cytosolic free Ca 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) .
With both DMPP and K 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.
Prolonged
opening of Na 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.
Caffeine induces catecholamine
secretion from perfused adrenal glands in both the presence and absence
of extracellular Ca(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) .
In the absence of extracellular
Ca, 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) .
In sharp
contrast to caffeine, which was always able to induce catecholamine
secretion by initial release of an internal Ca 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.
When 2 mM Ca 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.
Simultaneous fluorescence detection of cytosolic free Ca 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) .