Stimulation of cardiac L-type calcium channels
by extracellular ATP
Qi-Yi
Liu and
Robert L.
Rosenberg
Department of Pharmacology, University of North Carolina at Chapel
Hill, Chapel Hill, North Carolina 27599-7365
 |
ABSTRACT |
The co-release of ATP with norepinephrine from sympathetic
nerve terminals in the heart may augment adrenergic stimulation of
cardiac Ca2+ channel activity. To test for a possible
direct effect of extracellular ATP on L-type Ca2+ channels,
single channels were reconstituted from porcine sarcolemma into planar
lipid bilayers so that intracellular signaling pathways could be
controlled. Extracellular ATP (2-100 µM) increased the open
probability of the reconstituted channels, with a maximal increase of
~2.6-fold and an EC50 of 3.9 µM. The increase in open probability was due to an increase in channel availability and a
decrease in channel inactivation rate. Other nucleotides displayed a
rank order of effectiveness of ATP >
,
-methylene-ATP > 2-methylthio-ATP > UTP > adenosine
5'-O-(3-thiotriphosphate) >> ADP; adenosine had no effect.
Several antagonists of P2 receptors had no impact on the ATP-dependent
increase in open probability, indicating that receptor activation was
not required. These results suggest that extracellular ATP and other
nucleotides can stimulate the activity of cardiac L-type
Ca2+ channels via a direct interaction with the channels.
reconstitution; planar lipid bilayers; adenosine; nucleotide; P2
receptor
 |
INTRODUCTION |
ATP IS PRESENT IN
MILLIMOLAR concentrations in the cytosol of all cells and can be
stored at high concentrations in secretory granules and vesicles of
many cell types, including platelets, adrenal chromaffin cells, mast
cells, sympathetic neurons, and other neuronal cells (8).
Both the cytosolic and packaged pools of ATP can be released under
specific circumstances; ATP is released with norepinephrine from
sympathetic neurons and is considered a cotransmitter
(33), and cytosolic ATP can be released from cardiac
myocytes and red blood cells under hypoxic conditions (11). The extracellular concentration of ATP reaches
micromolar levels after release from intracellular pools, and local
concentrations may reach even higher levels under pathological
conditions such as hypoxia, ischemia, and aggregation of
platelets (12).
ATP has complex actions in the heart because of multiple types of
purinergic receptors (3); multiple intracellular signaling pathways involving several G proteins and different effectors (14); metabolism of ATP by ubiquitous ectonucleotidases to
other active compounds such as ADP, AMP, and adenosine
(12); and the presence of multiple cell types including
endothelial cells, vascular smooth muscle cells, and cardiac myocytes
that differentially respond to and release vasoactive compounds. For
example, ATP causes both vasoconstriction and vasodilation of cardiac
blood vessels. Vasoconstriction is primarily due to direct activation of vascular smooth muscle P2 receptors by ATP and the subsequent increase in intracellular Ca2+, whereas the vasorelaxation
is largely due to the activation of vascular endothelial cells by ATP
and the subsequent release of vasorelaxant compounds such as
prostacyclin and nitric oxide (8). Similarly,
extracellular ATP produces multiple complex effects on cardiac
myocytes. ATP has negative inotropic effects on ferret ventricular
myocytes due to decreases in Ca2+ current, and the effects
have been attributed to activation of P2Y receptors (23,
25). On the other hand, extracellular ATP has clear positive
inotropic effects on cardiac tissue and isolated cardiac myocytes from
rat and guinea pig (5, 18, 29), especially after exclusion
of the negative inotropic effects of adenosine (20).
There is solid evidence that the positive inotropic effect of
extracellular ATP is due, at least in part, to an increase in L-type
Ca2+ channel activity. Such an increase would lead to
increased Ca2+ influx, increased Ca2+ release
from the sarcoplasmic reticulum, and increased strength of cardiac
contraction. In whole cell voltage clamp of frog or rat ventricular
myocytes, extracellular ATP or adenosine
5'-O-(3-thiotriphosphate) (ATP
S) increases the magnitude
of the L-type Ca2+ channel current (1, 28, 30,
31). The increased influx of Ca2+ through L-type
Ca2+ channels, combined with increased
Ca2+-dependent Ca2+ release from the
sarcoplasmic reticulum, is largely responsible for increased
intracellular Ca2+ concentrations (2,
5-7).
The mechanisms via which extracellular ATP increases L-type
Ca2+ channel activity are not completely known.
Extracellular ATP
S activates receptors that are coupled to
stimulatory G protein (Gs) (28), and
the resulting stimulation of adenylyl cyclase increases the
intracellular concentration of cAMP (22), leading to
activation of protein kinase A and increased L-type Ca2+
channel activity (21). In addition, direct
interactions between activated Gs
-subunit
(Gs
) and L-type Ca2+ channels may be
responsible for at least some of the increased channel open probability
(17, 28, 32), independent of any increases in cAMP and
cAMP-dependent phosphorylation. Finally, extracellular ATP may interact
directly with the L-type Ca2+ channels to increase open probability.
To explore the possibility of a direct action of extracellular ATP on
L-type Ca2+ channels, we investigated the effect of ATP on
the activity of single cardiac L-type Ca2+ channels
reconstituted into planar lipid bilayers (26, 27). In this
simplified system, the extracellular and intracellular environments are
under good experimental control. Previous studies (26, 27)
have shown that reconstituted L-type Ca2+ channels are
virtually identical to those in intact myocytes with regard to
single-channel conductance, ion selectivity, voltage sensitivity,
activation and inactivation gating, and pharmacological sensitivity to
dihydropyridine and nondihydropyridine antagonists and pore blockers.
Extracellular ATP stimulated the activity of reconstituted porcine
cardiac L-type Ca2+ channels even though intracellular
signaling pathways were tightly controlled and changes in intracellular
cAMP or activated Gs
were extremely unlikely. Our
results suggest a direct stimulatory interaction between ATP and the
channel protein. Preliminary accounts of this study have appeared in
abstract form (19).
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METHODS |
Preparation of membrane vesicles and planar lipid bilayers, and
reconstitution of Ca2+ channels.
Sarcolemmal membrane fragments from porcine left ventricle were
prepared by homogenization, differential centrifugation, and a one-step
sucrose gradient fractionation (27). Planar lipid bilayers
were formed from decane solutions of
1-palmitoyl-2-oleoyl-phosphatidylethanolamine (15 mg/ml) and
1-palmitoyl-2-oleoyl-phosphatidylserine (5 mg/ml). Lipids were
obtained from Avanti Polar Lipids (Alabaster, AL). Aqueous solutions
contained 50 mM NaCl and 10 mM HEPES-NaOH (pH 7.0); BaCl2
was added to the cis chamber to a final concentration of 100 mM. We used Ba2+ rather than Ca2+ as the
current-carrying divalent cation because the conductance of L-type
Ca2+ channels is greater in Ba2+
(16) and because the influx of Ba2+ into the
trans chamber does not promote Ca2+-dependent
inactivation of Ca2+ channels (13). To ensure
that unambiguous channel activity could be recorded in the lipid
bilayers (26, 27), dihydropyridine agonist (+)202-791
(Novartis, Basel, Switzerland; 0.5 µM) was added to both
cis and trans solutions to increase channel open times (15); without the agonist, L-type Ca2+
channel openings are too small and brief to be resolved in bilayers. To
reduce the rate and extent of channel rundown during prolonged recordings, Gs
(5-6 nM), activated with guanosine
5'-O-(3-thiotriphosphate) (GTP
S), was added to the
trans chamber, as described previously (17,
32). Sarcolemmal vesicles were added to the cis
solution, and channel incorporation into the planar lipid bilayers via
vesicle fusion occurred spontaneously within a few minutes. Membranes were held at
60 or
70 mV (trans relative to
cis), and channels were activated by step depolarizations to
0 mV. L-type Ca2+ channels were identified by their
voltage-dependent activation during membrane depolarizations, current
amplitudes (1.1-1.3 pA at 0 mV in 100 mM external
Ba2+), and open and closed times. Typically, one or two
channels were incorporated in each bilayer, and later incorporations
were extremely rare. In the few cases of later additional fusion
events, the data were discarded. Channels were preferentially oriented
in the bilayers with the extracellular side facing the cis
solution; orientation was determined by the observation that
channel activity was evoked by step depolarizations from a negative
holding potential with the voltages defined as trans
relative to cis. Any channels that were incorporated with
the opposite orientation would experience a large positive holding
potential and would be inactivated (4, 13).
Chemicals.
ATP, ADP, adenosine, and uridine 5'-triphosphate (UTP) were obtained
from Sigma (St. Louis, MO), and ATP
S was obtained from Boehringer
Mannheim.
,
-Methylene-ATP (
,
-Me-ATP), 2-methylthio-ATP (2-MeS-ATP), suramin, reactive blue 2, and
pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) were
obtained from Research Biochemical International (Natick, MA). These
compounds were freshly prepared in deionized water immediately before use.
Bilayer voltage clamp.
Voltage clamp of the bilayers, recording of the currents, capacity
compensation, and analog and digital leak subtraction were performed as
described previously (13), except that a PC-501 patch-clamp amplifier (Warner Instruments, Hamden, CT) was used in
these studies. The bilayers were routinely held at
60 or
70 mV
(trans relative to cis) for 5.2 s between
800-ms pulses to depolarized test voltages (typically 0 mV). The
signals were filtered at 200 Hz (eight-pole Bessel, low pass) and
sampled at 1 kHz.
Data analysis.
Current amplitudes were measured by eye with computer-drawn horizontal
cursors. Channel open probability was determined from the time integral
of the current during the depolarization and normalized to the value
expected if the channel was open for the entire 800-ms depolarization
(27, 32). Ensemble averages were obtained by adding
leak-subtracted recordings and performing additional Gaussian digital
filtration (40 Hz) of the average. Results from different experimental
sets were compared using the Student's t-test (SigmaPlot, SPSS).
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RESULTS |
Extracellular ATP but not adenosine increases the open probability
of cardiac L-type Ca2+ channels.
The activity of two different reconstituted cardiac L-type
Ca2+ channels are shown in Fig.
1, A and C. Channel
openings (downward deflections) were triggered by membrane
depolarization from
60 mV to 0 mV. The amplitudes, open and closed
times, activation and inactivation kinetics, and pharmacological
sensitivity of the channels were characteristic of L-type
Ca2+ channels (26, 27). The level of channel
activity varied substantially among individual channels
(13), so we usually recorded about 3 min of "baseline"
activity before the addition of ATP. Although there was variability
between individual channels, the open probability of each channel was
relatively stable for at least 6 min due to the addition of activated
Gs
to the intracellular chamber (32).

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Fig. 1.
Single-channel currents recorded from L-type
Ca2+ channels incorporated into planar lipid bilayers.
Channel openings are downward deflections. The time of the
depolarization from the holding potential ( 60 mV) to the test
potential (0 mV) is indicated at the top. Recordings were
made from the same channel in the absence (A) or presence
(B) of 20 µM ATP added to the cis
(extracellular) chamber. Single-channel recordings were made in the
absence (C) or presence (D) of 50 µM adenosine
added to the cis chamber.
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The addition of 20 µM ATP to the cis (extracellular)
chamber (Fig. 1B) resulted in a substantial increase in the
channel open probability over control values (Fig. 1A). In
the presence of ATP there was a decrease in the number of
depolarizations that failed to evoke channel activity (blank or
"null" sweeps). In addition, there was an apparent slowing of
Ca2+ channel inactivation; ATP caused an increase in the
duration of channel activity during each depolarization so that channel openings occurred later in many depolarizations. Thus there was a
decrease in the number of long closed events that are responsible for
the inactivation of the channels during prolonged depolarizations (see
Fig. 5, below). There were no detectable changes in channel conductance, reversal potential, open-time durations, or short closed-time distributions (not shown). The kinetic effects of ATP could
arise if there was an increase in channel availability during
depolarizations (21) and a decrease in the rate of
time-dependent inactivation during prolonged depolarizations without
significant changes in the rate constants that govern transitions
between the open and short closed states. After stimulation by 20 µM
extracellular ATP, the reconstituted Ca2+ channels were
still blocked by the dihydropyridine antagonist nifedipine, suggesting
that ATP and dihydropyridines (either agonists or antagonists) act via
different molecular mechanisms.
Adenosine (50 µM) had no effect when added to the extracellular
compartment (Fig. 1, C and D). Adenosine neither
increased channel open probability above baseline (Fig. 1C)
nor changed the distribution of open channels during the
depolarization, indicating that the rate of channel inactivation was
not affected.
The effects on channel open probability of 20 µM extracellular ATP in
seven separate experiments are shown in Fig.
2A. For each experiment, we
averaged the open probability of 30 successive depolarizations (3 min)
recorded before and after the addition of ATP. The data confirm that
there was a significant increase in open probability after addition of
ATP (P < 0.05). Adenosine (20-50 µM) had no
significant effect on channel open probability (Fig. 2B,
P > 0.1).

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Fig. 2.
Effect of ATP and adenosine on the open probability of a
population of L-type Ca2+ channels. A: effect of
20 µM ATP (cis) on average open probabilities. Open
probability from 30 depolarizations obtained before and after the
addition of ATP were averaged. Results are from 7 different
experiments. After addition of ATP, there was a significant increase in
open probability (P < 0.05, paired Student's
t-test). B: adenosine (Ado, 20 or 50 µM) had no
significant effect on the open probability of the channels
(P > 0.1).
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The increase in open probability by extracellular ATP is dose
dependent.
The effect of varying the extracellular concentration of ATP from 2 to
100 µM on L-type Ca2+ channel open probability is shown
in Fig. 3. The results were obtained by
normalizing the open probability of each Ca2+ channel in
the presence of extracellular ATP to that obtained prior to ATP
addition. To eliminate any possible desensitization of the effect, each
measurement was made on a single channel in a separate experiment. At
the highest ATP concentrations (50-100 µM), open probability was
increased ~2.6-fold. The concentration of ATP that produced a
half-maximal effect (EC50) was 3.9 µM, and the Hill
coefficient (nH) was 1.3.

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Fig. 3.
Dose-response relationship between the concentration of
extracellular ATP and Ca2+ channel open probability. The
average open probability after addition of ATP (cis) was
divided by the average open probability obtained before ATP addition
(30 depolarizations in each condition; holding potential = 60
mV, test potential = 0 mV). Results from 4-9 experiments were
averaged at each concentration. The curve represents the least-squares
fit of the data to a modified Hill equation:
Po = (Po,max 1)/[1 + (EC50/[ATP])nH] + 1, with an EC50 of 3.9 µM, a maximal open probability
(Po,max) of 2.6, and a Hill coefficient
(nH) of 1.3.
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As shown in Fig. 1B, the addition of extracellular ATP
caused an apparent decrease in the number of null depolarizations in which no channel openings were recorded. To evaluate the effect of
extracellular ATP on channel availability (21), we plotted the probability of observing a null depolarization (normalized to each
channel's control value) vs. the concentration of extracellular ATP (Fig. 4). The concentration of ATP
that gave a half-maximal inhibition (IC50) was 2.6 µM and
the nH was 1.0. These results demonstrated that
the increase in open probability (Figs. 2 and 3) was partially due to a
decrease in the number of null depolarizations.

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Fig. 4.
The frequency of "null" depolarizations (in which no
channel activity was evoked by the depolarization) vs. extracellular
ATP concentration. Null depolarization frequency was calculated after
addition of extracellular ATP and divided by the value obtained before
ATP addition (30 depolarizations in each condition). Data were from
4-9 experiments at each concentration of ATP. The curve was drawn
according to the equation: Pnull = (1 Pnull,min)/[1 + ([ATP]/IC50)nH]+
Pnull,min. The IC50 was 2.8 µM,
nH was 1.0, and the minimal null probability
(Pnull,min) was 0.32.
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ATP reduces the Ca2+ channel
inactivation rate but does not alter the voltage dependence of
inactivation.
As shown in Fig. 1, A and C, most cardiac L-type
Ca2+ channels enter a long-lasting closed state before the
end of the 800-ms depolarization, thus showing an obvious
voltage-dependent inactivation process (14, 21). The data
in Fig. 1B suggest that extracellular ATP reduced the rate
of inactivation during depolarization, causing channel activity to
persist longer in each depolarization. To characterize this effect more
fully, we analyzed ensemble averages of Ca2+ channel
activity. Figure 5 shows typical ensemble
averages obtained from 30 depolarizations before and after the addition
of 50 µM extracellular ATP. The increase in open probability is seen
as an increase in the magnitude of the average current. The addition of
extracellular ATP also slowed channel inactivation so that the time
constant for inactivation increased from ~400 ms before ATP to ~700
ms afterwards. The results shown in Fig. 5 are representative of the
seven experiments in which ensemble average analysis was performed.
Because of the large channel-to-channel variability in inactivation
rates (14), we did not combine the ensemble averages from
different channels.

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Fig. 5.
Ensemble averages from 30 depolarizations before (light
trace) and after (bold trace) the addition of 50 µM ATP to the
extracellular (cis) chamber. Inactivation time constants
(obtained from exponential fits of the data to a single exponential
curve) increased from ~400 ms before addition of ATP to ~700 ms
afterwards.
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We next determined if extracellular ATP affected the steady-state
voltage-dependence of inactivation (Fig.
6). In these experiments, the holding
potential was varied between
70 and 0 mV and the open probabilities
of channel activity evoked by test depolarizations to 0 mV were
normalized by the open probability obtained at a holding potential of
70 mV. The data were fit by the Boltzmann equation (see legend to
Fig. 6); the voltage that produced half-maximal steady-state
inactivation (V1/2) was
18 mV and the slope
factor was 16 mV. Extracellular ATP (20 µM) had no effect on the
steady-state inactivation characteristics. Therefore, although
extracellular ATP substantially decreased the rate of inactivation of
cardiac L-type Ca2+ channels (Fig. 5), there was no
significant change in the steady-state voltage dependence of
inactivation (Fig. 6).

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Fig. 6.
Steady-state inactivation curves in the presence and
absence of ATP. Channel open probability at the test potential of 0 mV
was determined at different holding potentials and divided by the open
probability obtained at a holding potential of 70 mV. Data were from
20 experiments (at least 30 depolarizations each) before
( ) and after ( ) addition of 20 µM ATP
to the extracellular chamber. The curve represents the Boltzmann
equation Po = 1/[1 + exp(V V1/2)/ ] with a half-maximal steady-state
inactivation (V1/2) of 18 mV and a slope
factor ( ) of 16 mV.
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P2 receptor antagonists do not antagonize the effects of ATP.
Regulatory proteins such as receptors, G proteins, kinases, and
phosphatases can be closely associated with L-type Ca2+
channels and may co-reconstitute into planar lipid bilayers (17, 32). Therefore, it was possible that the observed effects of extracellular ATP were due to P2 receptors and/or G proteins that were
closely localized and associated with the Ca2+ channel
proteins. To determine whether a P2 receptor might be involved in the
ATP activation of reconstituted L-type Ca2+ channels, P2
receptor antagonists were added to the extracellular compartment before
ATP. Neither suramin (an antagonist of P2X, P2Y, and P2U receptors) nor
PPADS (a selective P2X receptor antagonist) had any effect on the
ATP-induced increase in open probability. After a 3 min incubation of
suramin (5-50 µM), extracellular ATP (5, 10, or 50 µM)
increased the normalized open probability by 1.63 ± 0.13 (n = 5), 2.48 ± 1.0 (n = 4), and
2.52 ± 0.40 (n = 2), respectively. These
increases were not different from those recorded in the absence of
antagonist (Fig. 3). After incubation with 50 µM PPADS, 5 µM
extracellular ATP caused an increase in open probability of 1.79 ± 0.10 (n = 3), a value that is essentially identical
to the 1.80 ± 0.19 increase observed with 5 µM ATP alone. Reactive blue 2, a nonselective P2Y antagonist, also did not alter the
stimulation by ATP (5 µM). We concluded that P2X, P2Y, and P2U
receptors were not responsible for the stimulation of reconstituted L-type Ca2+ channels by extracellular ATP.
Relative effects of nucleotides and nucleosides do not match that
of any know receptor subtype.
To determine the selectivity of the channels for ATP, we tested the
relative efficacy of several nucleotides and adenosine on L-type
Ca2+ channels (Fig. 7). For
comparison, we used the same concentration (50 µM) for all compounds
except ADP (100 µM). The two stable ATP analogs, ATP
S
(n = 5), and
,
-Me-ATP (n = 7)
increased the activity of Ca2+ channels, but ATP
S was
much less effective than ATP or
,
-Me-ATP. Two other
nucleotides, 2-MeS-ATP (n = 6) and UTP
(n = 4), also stimulated the L-type Ca2+
channels. ADP had a minimal effect (~10% increase in open
probability). The relative magnitudes of the effect of the compounds
tested were ATP >
,
-Me-ATP > 2-MeS-ATP > UTP > ATP
S > ADP > adenosine. The results indicate
that several nucleotide triphosphates can increase Ca2+
channel activity. The relative efficacy did not match any known receptor subtype (9, 14), again indicating that these
receptors were unlikely to be involved in the effect of extracellular
ATP. The stimulatory effects of the nucleotides, including
,
-Me-ATP, did not desensitize during the experiments (3-10
min), also suggesting that purinergic receptors were not involved in
the stimulation of reconstituted L-type Ca2+ channels.

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Fig. 7.
The effect of extracellular ATP (50 µM),
, -methylene-ATP ( , -Me-ATP, 50 µM), 2-methylthio-ATP
(2-MeS-ATP, 50 µM), ATP S (50mM), UTP (50 µM), ADP (100 µM),
and adenosine (50 µM) on channel activity. The experiments were
performed as described in Fig. 2. Each nucleotide was tested 3-5
times.
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 |
DISCUSSION |
Summary and interpretation of current results.
L-type Ca2+ channels from sarcolemmal membranes were
reconstituted into lipid bilayers. Extracellular ATP increased the open probability of the channels by increasing channel availability and
decreasing channel inactivation rates but had no effect on single-channel conductance or ion selectivity. The data suggest that
ATP interacts directly with the extracellular surface of the L-type
Ca2+ channels or an associated membrane protein that can
regulate Ca2+ channel activity. Our data excludes the
involvement of purinergic receptors since receptor antagonists had no
effect on the ATP-induced increase in open probability. Our results
also exclude the involvement of intracellular signaling pathways,
because components of these pathways were effectively removed by the
procedures used to isolate and reconstitute cardiac sarcolemma. It is
possible that extracellular ATP could act via a membrane-delimited
signaling pathway that remained intact following extraction and
reconstitution into the planar lipid bilayers, but this possibility is
unlikely because ATP and GTP were excluded from the intracellular
chamber, preventing activation of membrane-associated kinases and G proteins.
We considered the possibility that the effect of ATP was mediated via a
P2 receptor rather than through a direct interaction with the
Ca2+ channel, even though membrane-delimited G protein
signaling was unlikely. The EC50s for ATP-dependent
increases in channel open probability and decreases in null sweep
probability (3.9 and 2.6 µM, respectively) were in the same the range
as ATP activation of P2 receptors (8). However,
antagonists of P2X and P2Y receptors (suramin, PPADS, and reactive blue
2) did not inhibit the effect of ATP on the reconstituted L-type
Ca2+ channels. Moreover, the order of efficacy of several
nucleotides (ATP >
,
-Me-ATP > 2-MeS-ATP > UTP > ATP
S > ADP > adenosine) did not match that
reported for any known P2 receptor (9, 14). The
observation that poorly hydrolyzable analogs of ATP such as
,
-Me-ATP, 2-MeS-ATP, and ATP
S also increase Ca2+
channel open probability suggests that ATP hydrolysis is probably not
necessary for the observed effects. Thus our results support a
mechanism for ATP-dependent stimulation of cardiac L-type
Ca2+ channels that does not depend on P2 receptor
activation, although novel types of purinoceptors and
membrane-delimited signaling pathways are theoretically possible.
The effects of ATP in our experiments are due to extracellular and not
intracellular ATP. First, the channels in the lipid bilayer were
oriented with the extracellular side of the channel facing the
cis chamber, as determined by their voltage dependence of
activation; any channels that may have been reconstituted with the
reverse orientation would be chronically inactivated by a positive
holding potential and by the large concentration of Ba2+ in
contact with the intracellular surface. Second, ATP was typically added
only to the extracellular chamber, and bilayer integrity was maintained
throughout the experiments so that the possibility of significant
accumulation of ATP in the intracellular chamber was remote. Third, in
a few experiments when ATP was added to the intracellular chamber,
Ca2+ channels could still be stimulated by the subsequent
addition of ATP to the extracellular chamber.
Comparison to previous observations.
What is the mechanism by which ATP stimulates L-type Ca2+
channels? Is there more than one mechanism? The mechanisms are likely to be complex and, to some extent, species specific. Scamps and coworkers (30), studying single L-type Ca2+
channels in cell-attached patches from rat ventricular myocytes, also
found an upregulation of open probability, decrease in null probability, and no difference in unitary conductance. However, they
observed a change in the V1/2 of inactivation,
an effect we did not observe. In whole cells, the results are more
complex. There are several reports in which ATP increased L-type
Ca2+ channel activity in rat ventricular myocytes
(28-31), intracellular Ca2+ levels
(2, 5, 6, 7), and cardiac contractility (5, 20), but other reports indicate that extracellular ATP inhibits L-type Ca2+ current in ferret myocytes (23,
25). In frog ventricular myocytes, the effects of
extracellular ATP depend on the concentration range; concentrations of
ATP below 100 µM activate L-type Ca2+ channels but higher
concentrations are inhibitory (1). The concentrations of
ATP required to stimulate rat ventricular Ca2+ channels in
the whole cell configuration (1-10 µM; Ref. 29) are in the same
range as those reported here. The effect of ATP in intact cells may be
partially mediated by diffusible messengers (30),
Gs
(28), and/or protein kinases A and C
(22, 29-31). Our results suggest that in addition to
these messenger-dependent pathways, extracellular ATP could interact
directly with the channels to stimulate activity.
The responses of cells and tissues to ATP is complicated by the
ubiquitous presence of ecto-ATPases and other extracellular enzymes
that metabolize ATP to other active nucleotides and nucleosides including UTP, UDP, ADP, and adenosine (34). One advantage
of the planar bilayer approach over cellular electrophysiology is that
metabolism of ATP is extremely limited, because the extracellular enzymes that are responsible for the conversion of ATP are probably removed during the preparation of sarcolemmal membrane fragments. Thus
it is very likely that the stimulatory effects we observe are due to
ATP and not products of ATP metabolism. It is possible that some of the
apparently contradictory effects reported in intact cells could be due
to the action of cellular ecto-ATPases and the activation of inhibitory
receptors by ADP, AMP, and/or adenosine.
Physiological role.
Cardiac L-type Ca2+ channels play vital roles in setting
the strength of the heart beat, modulating the rate of pacemaker
depolarizations, and modulating the rate of slow conduction in nodal
cells. Extracellular ATP could augment
-adrenergic upregulation of
Ca2+ channels under physiological and pathophysiological
conditions, further increasing cardiac pace, contractility, and
conduction velocity in nodal regions. Even a small increase in L-type
Ca2+ channel activity by extracellular ATP might help
strengthen the heartbeat during cardiac ischemia. On the other
hand, small increases in cardiac L-type Ca2+ channels in
ischemic myocardium could promote reentry arrhythmias by
facilitating abnormal slow conduction in the ischemic border zone (10).
The action of extracellular ATP on L-type Ca2+ channels is
complex. ATP activates specific receptors (especially P2Y receptors) to
increase (1, 28-31) or decrease (23,
25) L-type Ca2+ channel activity. In addition,
ecto-ATPases and apyrases can create hydrolysis products, including
adenosine, that bind to A1 receptors which, in turn, inhibit
Ca2+ channels via Gi (24).
Now we have shown that ATP can interact directly with Ca2+
channels to increase their activity. The physiological effects of ATP
released by sympathetic nerves, ischemic myocytes, or blood cells, whether to increase Ca2+ channel activity (with
concomitant increases in contractility, pace, conduction velocity, and
potential reentry arrhythmogenesis) or inhibit Ca2+
channels (lowering contractility, pace, and the velocity of slow conduction), most likely depends on the number and type of receptors, channels, and extracellular ecto-ATPases present.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grants HL-49449 and HL-27430.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: R. L. Rosenberg, Dept. of Pharmacology, CB #7365, Univ. of North Carolina, Chapel Hill, NC 27599-7365 (E-mail: bobr{at}med.unc.edu).
The costs of publication of this
article were defrayed in part by the
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