232 A
receptors [29], and opioid receptors [1,10,29,42]. Addition- mm in diameter positioned directly adjacent to the cell.
ally, exogenously applied ATP activates P purinergic
The outlet pipe was fed by six separate reservoirs equally
2Y
receptors which inhibit I via a G-protein-coupled path-
pressurized for similar flow rates |70 ml min and
Ca
way [1,10,12,21]. Chromaffin cell secretory granules con- controlled by valves operated and timed with the data
tain ATP at millimolar concentrations [45] leading to the acquisition software. The exchange time for switching
possibility that autoactivation of endogenous purinergic between two solutions was ,150 ms. The input rate of the
receptors regulates release. perfusion system was matched with the outflow rate of a
In this study, we sought to determine whether ATP peristaltic pump to maintain a constant bath volume in
could modulate secretion in chromaffin cells. Capacitance order to reduce artifactual capacitance changes.
measurements were employed to measure secretion in response to stimulation. We show that, as previously
2.2. Preconditioned media described [1,10,12,21], ATP significantly reduces I
Most
Ca.
importantly, ATP reduces release by .50. Stimulation of To determine whether chromaffin cells released suffi-
chromaffin cells which results in the release of endogenous cient ATP to activate autoinhibition, cells were plated at
ATP suppresses secretion in these cells. The inhibition high density |10-fold that of experimental dishes. Just
mediated by ATP was blocked by Reactive Blue-2 sug- prior to use, a dish of cells was incubated for 5 min in 1 ml
gesting the involvement of P receptors. Large prepulses,
of the TEA solution described above which induced
2Y
which relieve the inhibition of I , largely reverse the depolarization and thus large amounts of secretion. The
Ca
inhibition of secretion. Plots of secretion as a function of preconditioned solution was removed and immediately
21
Ca influx are not significantly different in the absence of
applied to voltage-clamped chromaffin cells. ATP or presence of ATP and prepulses. Our results
indicate that the primary mechanism by which ATP acts to 2.3. Capacitance recordings
inhibit secretion from chromaffin cells is by inhibiting I .
Ca
ATP autoinhibition may provide an important regulatory Capacitance measurements were made with the phase
mechanism for catecholamine release in chromaffin cells. tracking technique [27] in which a 60 mV peak to peak sine
wave was superimposed on a holding potential of 280 mV [36]. The combination of the sine wave and the holding
2. Materials and methods potential was chosen to provide a good signal to noise ratio
1 21
without activating voltage-dependent Na or Ca
chan- 2.1. Cell culture and recording configuration
nels. Details of the technique can be found in Harkins and Fox [23]. The data were collected at a 500 ms sampling
Bovine chromaffin cells were prepared from adrenal rate and filtered at 2 kHz. All current records were
glands of animals approximately 18-weeks-old obtained compensated for series resistance and whole-cell capaci-
from a local abattoir. The cell isolation procedure has been tance. Single pulse current data were leak subtracted by an
described previously [2,10]. Experiments were performed average of 10 hyperpolarizing sweeps. Junction potentials
24–96 h after preparation of the chromaffin cells. of |29 mV were not subtracted from the data. Experiments
Chromaffin cells were washed with an external solution were carried out at room temperature 22–248C.
that contained in mM: 135 NaCl, 2 KCl, 1 MgCl , 5
2
CaCl , 12 HEPES, and 10 glucose pH57.3, osmolality 2.4. Stimulation protocol
2
|295 mOsm. In some experiments, recordings were made in an external TEA solution which contained in mM: 140
Five minutes after the whole-cell configuration was TEA, 10 glucose, 10 HEPES, 0.0001 TTX, and 5 CaCl
obtained, each cell was stimulated with a train of five step
2
pH57.3, osmolality |300 mOsm. No differences in depolarizations to 110 mV lasting 50 ms with a 100 ms
21
secretion or total Ca influx were observed between
interpulse duration. For some experiments, two trains of experiments carried out in the Na-based or TEA-based
depolarizations were applied to a single cell 5 min apart. external solutions. Sylgard-coated electrodes were filled
For the cells recorded in the TEA external solution, the with an internal solution that contained in mM: 120
cells were depolarized to 120 mV rather than 110 mV CsAsp, 5 MgCl , 0.1 EGTA, 40 HEPES, 2 ATP, and 0.3
since the peak of I was shifted in the TEA solution. In
2 Ca
GTP pH57.3, osmolality |310 mOsm. A single cell was some experiments, prepulse depolarizations of 100 ms
patch clamped in the whole-cell configuration [22] with an duration to 1100 mV were given 10 ms prior to the test
Axopatch 1C amplifier Axon, Foster City, CA modified depolarization. For ATP application, the cell was perfused
for capacitance measurements. After formation of a giga- for 5–10 s prior to and during the stimulating train of
ohm seal, the cell was continuously perfused by an depolarizations with ATP containing external solution.
external solution supplied by an Adams and List Wes- Within 5 s of the last depolarization of the train, the
tbury, NY DAD 12 perfusion system. This perfusion solution was switched back to the external control solution.
system consisted of an outlet pipe quartz capillary, 100 ATP was prepared as a 10 mM stock solution in water and
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
