A .B. Harkins, A.P. Fox Brain Research 885 2000 231 –239
233
diluted to 100 mM in the external recording solution. For all of the prepulse experiments and many of the non-
prepulse experiments, nisoldipine or nitrendipine 1 mM was added to the external solutions to block any facilita-
21
tion L-type Ca current. Because there were no differ-
ences in any of the capacitance or current recordings for experiments conducted in the absence or presence of
dihydropyridine, all of the non-prepulse data were com- bined. Nisoldipine and nitrendipine Calbiochem, San
Diego, CA were stored as a 10 mM stock solution in ethanol at 08C and were diluted to 1 mM in the external
solution immediately prior to an experiment.
2.5. Analysis Because multiple stimulations result in variable amounts
of run down in the secretory response, the data reported here are largely from the initial train of depolarizations
applied to each cell. In many of the experiments Figs. 1, 2A and B, the only channel blocker employed was cesium
1
in the pipette to block K channels. As a result, these
21 1
current traces contained both Ca and Na
channel
1
currents. The Na channel component was completely
inactivated 8 ms after activation. Therefore, to measure the
21
Ca influx into cells, the current recordings were leak
subtracted and integrated after excluding the first 8 ms of each current trace. For the experiments that were per-
formed in channel blocking solutions, i.e. TEA, TTX and
21
cesium Figs. 2C and 3, the entire Ca current was
integrated after leak subtraction. For each cell, the sum of the integrals for five depolarizing steps provided the total
21
number of Ca ions that entered the cell. Statistical
analysis of the data are expressed as mean X6standard error of the mean S.E.M., and an independent Student’s
t-test was performed to test statistical significance.
Fig. 1. ATP inhibits granule release from adrenal chromaffin cells. A A representative capacitance trace is shown from three different groups of
cells under different stimulation conditions. Left panel shows the capaci- tance record from a cell stimulated in the absence of ATP Control while
3. Results
the middle pane shows the capacitance record from a cell stimulated in the presence of 100 mM ATP ATP. Each cell was stimulated with a
3.1. ATP inhibits secretion from adrenal chromaffin
train of five step depolarizations to 110 mV 50 ms pulse duration, 100 ms interpulse duration. The right panel shows the capacitance record of a
cells
cell stimulated in the presence of 100 mM ATP but preceded by depolarizing prepulses to 1100 mV lasting 100 ms 10 ms prior to the
In order to determine whether ATP can serve as an
test depolarization ATP1Prepulse. For each cell, the peak exocytotic
autoinhibitory neurotransmitter, ATP 100 mM was direct-
response was measured at the maximal change in the capacitance record
ly applied to adrenal chromaffin cells while monitoring
following the depolarizing stimulus dashed line at the top of each capacitance trace. The maximum rate of exocytosis was determined by
secretion with membrane capacitance measurements. ATP
finding the largest change in capacitance that occurred during any of the
reduced both the peak and the maximal rate of exocytosis
50 ms depolarizations. B plots currents elicited by the first depolariza-
from chromaffin cells when compared with control cells.
tion of a train, for the same three conditions plotted in panel A. Note that
1
Fig. 1A shows representative capacitance traces from each
the early current in each trace is Na current. The prepulse protocol
1
group of cells. Because there is run down in secretion
largely inhibited the Na current. C plots the average peak change in
membrane capacitance observed in the absence of ATP Control, n524,
when two sets of stimulations are applied to single cells
in the presence of ATP ATP, n520, and in the presence of ATP but
|30, see Fig. 2A, the control data, ATP data, and
with prepulse stimulation ATP1Prepulses, n515. ATP significantly
ATP1prepulse data presented in Fig. 1 are from the initial
reduced the average change in capacitance from 248 fF 632 fF, n524
stimulations of different cells. Fig. 1A shows the capaci-
under control conditions to 119 fF 620 fF, n520, P,0.01. Prepulses
tance trace obtained from three typical cells stimulated
in the presence of ATP restored the peak of the capacitance change to 216
1
fF 621, n515. All cells were recorded using the Na -based solution.
with five depolarizations to 110 mV. The peak of the
234 A
Fig. 2. ATP inhibits catecholamine secretion when elicited with two stimulations in the same cell. Each cell was stimulated twice with identical trains of depolarizations applied 5 min apart. A top, plots the capacitance response from a representative cell that was stimulated twice, while A middle, plots the
current elicited by the first depolarization of each train. A bottom, plots the capacitance response of seven cells stimulated the first and second time under control conditions. The peak exocytotic response from the second stimulation was normalized to the first stimulation. On average, the second control
1
response was 71 66, n57, P,0.005 of the first control response. These cells were recorded with the Na -based solution. B top, plots capacitance, while the middle panel plots current from cells stimulated first in the presence of ATP and then in the absence of ATP. B bottom, shows the average
capacitance response of 13 cells stimulated first in the presence of ATP followed by a second stimulation after ATP had been washed away for 5 min. On average, the second control response was 58 623, n513, P,0.05 larger than the first response observed in the presence of ATP. These cells were
1
recorded with the Na -based solution. C The P antagonist Reactive Blue-2 RB-2, 100 mM, blocked the inhibition of secretion produced by ATP. C
2Y
top, plots capacitance, while the middle panel plots current from cells stimulated first in the presence of ATP and RB-2 and then in the presence of RB-2 alone. C bottom, shows the average capacitance response from six cells. The first stimulation in the presence of RB-2 and ATP elicited a larger peak
secretory response than the second stimulation in the presence of RB-2 alone. On average, the second stimulation was 49 69.7, n56, P,0.001 of the first stimulation. The TEA based solution was used for the experiments shown in C.
capacitance trace was measured as indicated by the upper ATP-treated cell, the peak of the capacitance trace was 112
dashed line. Although it is possible that release is not fF and the maximal rate of exocytosis was 534 fF s. These
constant during each 50 ms depolarization, we calculate data suggest that ATP can act on purinergic autoreceptors
the maximal rate of exocytosis by dividing the largest to inhibit evoked release.
capacitance change observed during a single depolarization The inhibition produced by ATP was similar but not
by the 50 ms pulse duration of the depolarization. For the identical throughout each depolarization in the train. In the
control cell in the absence of ATP, left panel, the peak of first depolarization, ATP inhibited secretion from 5568.3
the capacitance trace was 281 fF and the maximal rate of fF n524 to 29.365.7 fF n517 while for the fifth
exocytosis was 1124 fF s. The middle panel Fig. 1A stimulation, secretion was reduced from 13.663.0 fF n5
shows the capacitance trace from a different cell that was 24 to 9.662.2 fF n517. In the first depolarization, ATP
21 6
6
exposed to ATP for 5 s prior to stimulation using a inhibited Ca
influx from 25.1310 ions 62.6310
6 6
protocol identical to that described for control. For this ions, n524 to 14.6310
ions 61.2310 ions, n520
A .B. Harkins, A.P. Fox Brain Research 885 2000 231 –239
235
of ATP is mediated entirely by block of I or whether
Ca
there is a secondary effect of ATP on the secretory machinery, we used 100 ms prepulses to 1100 mV to
relieve the inhibition of I . These prepulses are not
Ca
expected to directly affect the secretory machinery. Nor
21
should the prepulses alter Ca -influx as the depolariza-
21
tions reach or exceed the equilibrium potential for Ca .
21
Prepulses relieved ATP mediated inhibition of the Ca channel current, and largely reversed ATP’s inhibitory
effect on exocytosis. Fig. 1A right panel shows a capacitance trace from a cell exposed to ATP for 5 s prior
to stimulation. In this experiment, prepulses were applied to reverse the ATP-mediated inhibition of I . The peak of
Ca
the capacitance trace was 207 fF and the maximal rate of exocytosis was 1112 fF s.
Fig. 1B plots currents elicited by the first depolarization of a train, from three representative cells under ‘Control’,
‘ATP’ and ‘ATP1Prepulse’ conditions shown in Fig. 1A.
1
Please note that Na currents were unblocked, variable
1
between cells, and that prepulses largely inhibited the Na
21
current. On average, ATP reduced the total Ca influx
6 6
under control conditions from 100.3310 ions 69.2310
6 6
ions, n524 to 69.4310 ions 65.6310
ions, n520, P,0.01, a 31 reduction. Depolarizing prepulses re-
21 6
6
stored the total Ca influx to 105.2310 ions 65.7310
ions, n514 in the continued presence of ATP. Therefore, prepulse stimulation in the presence of ATP relieved the
21
Fig. 3. Preconditioned
media inhibited
secretion. Densely
plated
inhibition of the Ca channel current. Fig. 1C summarizes
chromaffin cells were induced to secrete by a 5 min incubation in the
the average peak exocytotic response for each group of
depolarizing TEA-based solution, after which the ‘Preconditioned Media’
cells. On average, ATP reduced the peak exocytotic
was collected and applied to voltage-clamped cells. A shows representa-
response by 52 compared to control conditions Fig. 1C,
tive capacitance traces from a cell stimulated twice, first in the presence
a statistically significant response P,0.01. Prepulses in
of Preconditioned Media left panel and then in the Control solution right panel. B plots the I
recorded during the first depolarizations of
the presence of ATP restored the peak exocytotic response
Ca
each train. C plots average capacitance data from nine cells exposed to
to 87 of control cells Fig. 1C, a value not statistically
Preconditioned Media. The peak exocytotic response from the second
different than control. ATP also reduced the maximal rate
stimulation was normalized to the first stimulation. The Control response
of exocytosis from 1308 fF s 6162, n524 under control
was 188 639, n59, P,0.05 of that obtained in the presence of
conditions to 666 fF s 694, n520, P,0.01 in the
Preconditioned Media. Data for all nine cells were obtained in TEA-based solution.
presence of ATP, a 49 reduction. Prepulses in the presence of ATP restored the maximal rate of secretion to
1115 fF s 6120, n515, 85 of control cells. Thus, prepulse stimulation completely relieved the inhibition of
21
while for the fifth stimulation, Ca influx was reduced
I , and largely reversed the peak secretory response and
Ca 6
6 6
from 15.9310 ions 61.3310 ions, n524 to 12.9310 maximal rate of exocytosis. These data suggest that ATP
6
ions 61.1310 ions, n520. acts on purinergic autoreceptors to primarily inhibit secre-
21
tion through a Ca channel-dependent mechanism.
3.2. The inhibition of secretion by ATP is relieved by prepulses
3.3. ATP inhibits secretion when both control and ATP responses are elicited in the same cell
21
A wide variety of neurotransmitters can inhibit Ca channel current, and in many cases the inhibition is
Because secretion can run down as a function of time thought to be mediated by bg subunits of G-proteins [13].
within a single cell, the data shown in Fig. 1 was obtained This inhibitory interaction, which is voltage-dependent, is
by stimulating individual cells once, in either the absence relieved by prepulses to strongly depolarizing potentials
or presence of ATP, and then analyzing each cell in-
21
[5]. Previous studies have shown that ATP-mediated Ca dependently. A more compelling case for the inhibitory
current inhibition in chromaffin cells is relieved by pre- action of ATP can be made by comparing secretion in the
pulses [10,21]. To determine whether the inhibitory effect absence or presence of ATP in the same cell. Fig. 2A top
236 A
shows capacitance data from a representative cell stimu- collected and immediately applied to a chromaffin cell.
lated with two trains of depolarizations, separated by 5 Each cell was stimulated twice, first in the presence of
min, both in the absence of ATP control. Fig. 2A Preconditioned Media, then in its absence Control. Fig.
middle shows the currents elicited by the first depolariza- 3A illustrates that the Preconditioned Media inhibited
tion of each train. The bottom panel Fig. 1A, summarizes secretion. Representative capacitance traces were recorded
capacitance data from seven cells. The peak change in from a single cell in the presence left panel and absence
capacitance of the second stimulus was reduced to 71 of right panel of the Preconditioned Media. Fig. 3B shows
the first stimulus. On average, total I was reduced 8
that I was inhibited by the Preconditioned Media. Fig.
Ca Ca
6 6
from 142310 ions 62.7310 ions for the first stimula- 3C shows average data from nine cells demonstrating that
6 6
tion to 131310 ions 62.3310
ions for the second the contents of chromaffin cell secretory granules could
stimulation. When the first stimulation was delivered in the function to autoinhibit secretion. When the first stimulation
presence of ATP and the second in the absence of ATP, the was delivered in the presence of the Preconditioned Media
change in capacitance increased 58 from stimulus 1 to and the second in the control solution, the change in peak
stimulus 2 Fig. 2B. Total I increased 54 from 693
capacitance increased 88 from stimulus 1 to stimulus 2
Ca 6
6 6
Fig. 3C. Total I increased 24 from 142310
ions 10 ions 66.0310 ions for the first stimulation in the
Ca 6
6 6
69.5310 ions for the first stimulation in the Pre-
presence of ATP to 106310 ions 69.5310 ions for the
6 6
conditioned Media to 176310 ions 610.5310 ions for second stimulation in the absence of ATP. Thus, the data
the second stimulation in the control solution. shown in Fig. 2A and B strongly suggest that ATP
inhibited the secretory response in chromaffin cells and
21
3.6. ATP inhibits secretion via a Ca channel-
that the inhibition was due to a direct inhibition of the dependent mechanism
calcium current. Although the prepulse data are good evidence that ATP
3.4. ATP acts through a P receptor to inhibit secretion
21 2 y
acts via a Ca channel-dependent mechanism, further
evidence was available from the change in capacitance and Both the trypanocidal drug suramin and the anthra-
21
total Ca influx data for each cell. Fig. 4 shows a plot of
quinone-sulfonic acid derivative Reactive Blue-2 RB-2 the maximal secretory response elicited as a function of
have been used as P purinergic receptor antagonists. Both
2
drugs possess only limited selectivity for the receptor [11,20]. Between the two antagonists, RB-2 exhibits a
somewhat higher degree of selectivity for the P receptor
2Y
than does suramin although this may vary between species and tissue type so we tested this putative P
receptor
2Y
antagonist on the ATP-mediated inhibition in chromaffin cells. RB-2 100 mM prevented the inhibition of secretion
associated with ATP application Fig. 2C. This group of cells was stimulated twice, both times in the presence of
RB-2. The initial stimulation with ATP and RB-2 pro- duced a larger secretory response than the second stimula-
tion without ATP which is likely caused by run down. The peak change in capacitance of the second stimulation was
reduced to 49 of the first stimulation, and total I
was
Ca 6
6
increased 3 from 275310 ions 628.5310 ions for
6 6
the first stimulation to 283310 ions 630.0310 ions for the second stimulation. Please note that this set of
experiments was carried out in a TEA-based solution which suppressed the Na1 current and which may have
accelerated the run down of secretion.
21
Fig. 4. ATP inhibits secretion via inhibition of Ca channel current. For
each cell, the sum of the integrated current for the five depolarizations
21
3.5. Release of endogenous ATP from chromaffin cells
number of Ca ions is plotted as a function of the maximal change in
inhibits secretion
membrane capacitance. The scatter plot shows all of the cells from the cells stimulated in the absence of ATP h and in the presence of ATP
with prepulses s. The prepulse protocol is identical to that used in Fig.
Plates of chromaffin cells were exposed to depolarizing
3. Both data sets were fit by a linear regression. The slope of the
solutions see Materials and methods, which resulted in
regression fit was 2.1 for the ‘absence of ATP’ and 1.8 for ‘ATP1
the release of a large number of secretory granules. The
prepulses’. The similarity of the two fitting functions used to fit the data
media bathing these cells Preconditioned Media was
suggests that there was no significant difference between the data sets.
A .B. Harkins, A.P. Fox Brain Research 885 2000 231 –239
237
21
total Ca influx from the first stimulation of each cell for
cells [43], PC12 cells [30], and endothelial cells [44]. the control condition squares and the cells treated with
These apparently conflicting results are explained by the ATP and prepulses circles. No segregation between the
fact that cells express different classes of purinergic two groups of cells is apparent and they all appear to
receptors. For instance, chromaffin cells express both
21
follow the same Ca influx versus secretion relationship.
ionotropic P and metabotropic P purinergic re-
2X 2Y
This analysis suggests that if there is an additional effect of ceptors [8,40]. P
receptors are ligand activated ion
2X 21
21
ATP on secretion, other than of Ca channel inhibition,
channels that are highly permeable to Ca whereas P
2Y
the effect is relatively small. receptors are G-protein-coupled receptors [4]. Thus, the
actions of ATP vary depending on the purinergic re- ceptors activated. For example, ATP, acting through
21
4. Discussion Ca
