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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 > alpha ,beta -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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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) (ATPgamma 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 ATPgamma 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 alpha -subunit (Gsalpha ) 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 Gsalpha 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).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, Gsalpha (5-6 nM), activated with guanosine 5'-O-(3-thiotriphosphate) (GTPgamma 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 ATPgamma S was obtained from Boehringer Mannheim. alpha ,beta -Methylene-ATP (alpha ,beta -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).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Gsalpha 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.

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).

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.

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.

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.

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 (black-down-triangle ) addition of 20 µM ATP to the extracellular chamber. The curve represents the Boltzmann equation Po = 1/[1 + exp(- V1/2)/delta ] with a half-maximal steady-state inactivation (V1/2) of -18 mV and a slope factor (delta ) of 16 mV.

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, ATPgamma S (n = 5), and alpha ,beta -Me-ATP (n = 7) increased the activity of Ca2+ channels, but ATPgamma S was much less effective than ATP or alpha ,beta -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 > alpha ,beta -Me-ATP > 2-MeS-ATP > UTP > ATPgamma 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 alpha ,beta -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), alpha ,beta -methylene-ATP (alpha ,beta -Me-ATP, 50 µM), 2-methylthio-ATP (2-MeS-ATP, 50 µM), ATPgamma 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.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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 > alpha ,beta -Me-ATP > 2-MeS-ATP > UTP > ATPgamma 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 alpha ,beta -Me-ATP, 2-MeS-ATP, and ATPgamma 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), Gsalpha (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 beta -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 payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 September 2000; accepted in final form 15 December 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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