The M2-muscarinic receptor and the A1-adenosine receptor are directly coupled to the same population of K+ channels via pertussis toxin-sensitive G protein in atrial cell membrane(1). These K+ channels can be readily activated in the presence of acetylcholine (ACh) and thus have been referred to as ACh-activated K+(KACh) channels (1). The adenosine-induced activation of KACh plays an important role in the shortening of action potential and decreased twitch (negative inotropism) caused by adenosine in atrial myocytes (2,3).
The activation of P2-purinergic receptor by extracellular adenosine triphosphate (ATP) in rat cardiac myocytes has been shown to activate a Gs-type G protein that is directly coupled to the stimulation of L-type Ca2+ channels (4,5). However, it is still unknown how ATP causes shortening of action potential and activates K+ current in atrial myocytes. Because ATP can rapidly and sequentially be degraded to adenosine diphosphate (ADP) and this to adenosine monophosphate (AMP), which in turn is dephosphorylated by 5′-nucleotidase to adenosine, the electrophysiologic effect of ATP has been attributed to be in great part the result of its degradative product, adenosine (6-9). The results of the recent study of Matsurra et al. (10) indicated that extracellular ATP directly binds to P2-purinergic receptor of atrial myocytes and activates KACh channels. The direct coupling of the P2-receptor to the KACh channels was proposed to be mediated through pertussis toxin-sensitive G proteins (10). This finding suggested that the coupling mechanism of action of extracellular ATP was similar to the coupling of the M2-muscarinic receptor and of the A1-adenosine receptor to KACh channels. On the other hand, it was also recently reported that the ATP-induced activation of KACh channels could be the result of the direct binding of ATP to the A1-adenosine receptor (11).
Several distinct subtypes of purinergic receptor have been identified on the basis of the rank order of potency of various nucleotides to produce responses in numerous tissues and cells (12-14). It was recently reported that a separate subtype of purinergic receptor, the P2U-purinergic receptor, is activated by ATP and uridine 5′-triphosphate (UTP) with similar potency (13,15-18). This P2U-receptor subtype is functionally similar to, but pharmacologically distinct from, the P2Y-purinergic receptor. This subtype is known to be a phospholipase C-activating receptor, which is stimulated by both ATP and UTP (16). However, whether extracellular UTP can activate the KACh channels in atrial membranes remains unclear.
Therefore in this study, we investigated the electrophysiologic properties of the ATP-, adenyl compounds-, and UTP-activated K+ channels in rat atrial cells and sought to determine whether (a) the K+ channels activated by these purinergic agonists or their receptor-effector coupling mechanism or both are the same, and (b) the receptors that mediate the effects of these agonists are different.
MATERIALS AND METHODS
Preparation of single atrial myocytes
Single atrial myocytes were isolated from Sprague-Dawley rats weighing 350-400 g by an enzymatic dissociation method, as described previously (2,19). After the animals were killed, the excised hearts were retrogradely perfused at 35-37°C with oxygenated Tyrode's solution (100% O2; PO2 = 420 ± 10 mm Hg; n = 12). The hearts were then perfused with Ca2+-free Tyrode's solution for 10 min and subsequently with the enzyme solution for 20-30 min. To avoid possible proteolysis of cell-surface A1-adenosine or P2-purinergic receptors, the enzyme solution contained only collagenase (0.8 mg/ml, Sigma type II). A small piece of atrial tissue was dissected and gently agitated in the recording chamber filled with Tyrode's solution. This procedure consistently yielded an acceptable percentage of rod-shaped Ca2+-tolerant atrial myocytes.
In some experiments, isolated rat atrial myocytes were pretreated with pertussis toxin (500 ng/ml) for 5 h. The pretreatment with pertussis toxin did not abolish the isoproterenol-stimulated increase in the amplitude of L-type Ca2+ inward current in rat atrial myocytes(data not shown).
A few drops of cell suspension were transferred to the recording chamber, which was mounted on the stage of an inverted phase-contrast microscope (Diaphot-200; Nikon, Tokyo, Japan). The microscope was coupled to a video camera system with magnification ≤ ×1,500. Cells were bathed at room temperature (20-25°C) and superfused by gravity at a rate of ∼2-4 ml/min with Tyrode's solution. Only rod-shaped and quiescent atrial myocytes were used for the experiment. The GΩ seal-patch clamp technique was used in the "whole cell" or in the "cell-attached" patch configuration (20). To minimize dialysis of intracellular constituents with the pipette solution, a nystatin-perforated-patch whole-cell recording method was performed (21).
Currents were recorded by using a patch-clamp amplifier (RK-400; Biologic, Claix, France) and were amplified with a low-pass filter at 1-3 kHz. The patch pipettes were made from the capillary tubes (Kimax-51; Kimble Products, Vineland, NJ, U.S.A.) by using a vertical two-step electrode puller (PB-7; Narishige, Tokyo, Japan), and the tips were fire-polished with a microforge (MF-83; Narishige). The resistance of the patch pipette was 3-5 MΩ when it was immersed in normal Tyrode's solution. A hydraulic micromanipulator (WR-6; Narishige) mounted on the fixed stage of the inverted microscope was used to position the pipette near the cell. In experiments designed to construct the current-versus-voltage (I-V) relations, square or linear ramp command pulses were used and digitally generated at a rate of 0.2-0.5 Hz by the use of a programmable stimulator (SMP-311; Biologic).
Data recording and analysis
The signals consisting of voltage and current tracings were monitored on a digital storage oscilloscope (model 1602; Gould Instrument Systems, Inc., Valley View, OH, U.S.A.) and simultaneously recorded in a digital audiotape recorder (model 1204; Biologic). After the experiments, the stored data were then fed back and digitized at the sampling frequency of 5-10 kHz with a GPIB interface board (National Instruments Corp., Austin, TX, U.S.A.), which was controlled by a personal computer and DataVIEW software package(Biologic). Single-channel current records were analyzed by using a Biopatch program(Biologic). Channel activity in the patch was expressed as the number of functional channels in each patch (N) times open probability (Po). The event-detection threshold was set at ×0.5 the predetermined single-channel amplitude (22). All channel-opening events that exceeded the threshold level were then integrated numerically, yielding total current (I) for each record with a sufficiently large number of independent observations. N × Po for each record was calculated as total current (I) divided by unit amplitude of the single-channel current.
To estimate the concentration-dependent effects of adenosine, ATP, and UTP on the activation of KACh channels, the opening probability of channel current caused by ACh(10 μM) was taken as 100%. The curves were fitted to the Hill equation by using nonlinear regression analysis (23). The following form of the Hill equation was used: Equation (1) where x is the extracellular concentration of the agonist, y is pharmacologic response, ymax is the maximal response, c is the median effective concentration (EC50) value, and n is the Hill coefficient.
All data were reported as mean± standard error of the mean (SEM). The paired or unpaired Student's t test and Duncan's multiple-range test were used for the statistical analyses. The level of significance was taken at p < 0.05.
Drugs and solutions
Adenosine, R-N6-(2-phenylisopropyl)adenosine (R-PIA), adenosine 5′-triphosphate(ATP), adenosine 5′-diphosphate (ADP), adenosine 5′-monophosphate (AMP), uridine 5′-triphosphate (UTP), guanosine 5′-triphosphate (GTP), adenosine deaminase, nystatin, collagenase, and tetrodotoxin were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Glibenclamide, α,β-methylene-ATP (AMPCPP), 8-(p-sulfophenyl)theophylline (8-PST), 8-cyclopentyl-1,3-dipropylxanthine(CPX), and 2-[4-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamidoadenosine(CGS-21680) were obtained from Research Biochemicals (Natick, MA, U.S.A.). Dispase was purchased from Boehringer Mannheim (Indianapolis, IN, U.S.A.). Pertussis toxin was purchased from Biomol (Plymouth Meeting, PA, U.S.A.). The normal Tyrode's solution contained (in mM): NaCl, 136.5; KCl, 5.4; CaCl2, 1.8; MgCl2, 0.53; glucose, 5.5; and HEPES-NaOH buffer, 5.5 (pH 7.4). In conventional whole-cell clamp experiments, the pipettes were filled with solution (in mM): K-aspartate, 140; KH2PO4, 1; MgCl2, 1; EGTA-KOH, 5; Na2ATP, 3; Na2GTP, 0.1; and HEPES-KOH buffer, 5 (pH 7.3). In perforated-patch whole-cell recording experiments, nystatin was dissolved in dimethyl sulfoxide at a concentration of 50 mg/ml and then added to the internal pipette solution to yield a final nystatin concentration of 100 μg/ml (21). In the patch-clamp experiments, the composition of the pipette solution was (in mM): KCl, 145; CaCl2, 1; MgCl2, 1; and HEPES-KOH, 5 (pH 7.4).
Various agents, such as acetylcholine, adenosine, AMP, ATP, AMPCPP, CPX, and 8-PST, were added to the normal Tyrode's solution in the whole-cell experiments and to the pipette solution in the cell-attached experiments.
Characterization of the effect of extracellular ATP on the activation of K+ current in rat atrial myocytes
The perforated whole-cell configuration of the patch-clamp technique was used to investigate macroscopic K+ currents in rat atrial myocytes. The membrane currents were evoked by the ramp command pulses at the voltage range between −100 and +40 mV, with a duration of 200 ms. These experiments were conducted with pipettes containing 140 mM K-aspartate, 3 mM ATP, and 0.1 mM GTP, and the cells were superfused with normal Tyrode's solution containing 0.5 mM CdCl2 and 10 μM tetrodotoxin. Within 1 min of exposing the cells to extracellular ATP, an outward membrane current was activated, and this current was readily reversed on washout of ATP (not shown). As depicted in Fig. 1, the current trace for the current-voltage relation of ATP-induced current was inwardly rectifying, and the direction of membrane current was reversed at ∼−75 mV. The addition of CPX (1 μM) to the superfusion medium, a potent A1-adenosine-receptor antagonist, only partially inhibited the ATP-induced current (n = 5 cells; Fig. 1A). The same current activated by adenosine was completely inhibited by CPX (1 μM; not shown). In contrast to the effect of CPX, addition of glibenclamide(30 μM) did not alter the magnitude of ATP-induced K+ current. The latter finding suggests that the K+ channels activated by extracellular ATP are not KATP channels.
In cell-attached experiments, the properties of the single-channel currents activated by adenosine and ATP were determined and compared. The cells were bathed in 5.4 mM K+-containing solution to settle the resting potential (Er) of the cells. Under this condition, the value of Er was ∼+72 ± 5 mV (n = 12). Figure 2 illustrates the single-channel currents activated by adenosine and ATP at various voltages. As expected, the amplitude of unitary current was increased as the membrane potential was hyperpolarized (n = 5 cells). The current-voltage relations of adenosine- and ATP-induced currents were almost superimposable (Fig. 2B). The single-channel conductance of the channel was 29 pS. Furthermore, the open-time histograms of the channel at the level of −20 mV could be fitted by a single exponential curve with a mean open time of 1.53 ms (data not shown). These values are similar to those of KACh identified in rat atrial myocytes (11). These results are also consistent with the finding that adenosine and ATP regulate the same population of K+ channels (i.e., KACh channels) in atrial myocytes (10,11). Addition of glibenclamide (30 μM) into the superfusion medium did not cause any change in the channel activity elicited by either adenosine or ATP (n = 5 cells).
The properties of the single-channel currents activated by ACh, adenosine, and adenine nucleotides also were compared. As shown in Fig. 3, single-channel currents activated by ACh, adenosine, AMP, or ATP at the level of −80 mV were recorded. In all cases, the amplitude of the unitary inward current was ∼4 pA. The addition of 8-PST (10 μM), an adenosine-receptor antagonist, fully suppressed adenosine-induced activity of channel openings. Even when adenosine deaminase (1 or 3 unit/ml) was present in the pipette solution, activation of KACh channels by AMP or ATP was observed (n = 5 cells for AMP and n = 6 cells for ATP; not shown). These findings suggest that AMP or ATP per se can directly activate KACh channels without having to be degraded to adenosine.
Characterization of the effect of extracellular UTP on the activation of K+ current in rat atrial myocytes
In perforated whole-cell recording experiments, when cells were depolarized from a holding potential of −80 to various voltage steps with a voltage step of 200 ms, the instantaneous peak (i.e., transient outward) and late outward currents were elicited. On the other hand, the time-independent inward currents were elicited when hyperpolarizing pulses from −80 mV were applied. As illustrated in Fig. 4, the presence of UTP (100 μM) effectively increased the current amplitude. Figure 4B shows the current-voltage relation of the instantaneous and late currents. The UTP-induced current was taken as the difference between the current-voltage currents at the end of voltage steps in the control and that in the presence of UTP (Fig. 4C). In addition, neither CPX (1 μM) nor glibenclamide (30 μM) abolished UTP-induced K+ current.
The inactivation time constant of transient outward current was not affected by the presence of UTP. When cells were depolarized from −80 to +30 mV, the inactivation time constant of transient outward current in the absence and presence of UTP (100 μM) was 48 ± 3 ms (n = 8) and 49 ± 4 ms (n = 9), respectively. However, the absolute value of increases in instantaneous and late currents was significantly different. UTP (100 μM) increased the amplitudes of instantaneous and late currents to 690 ± 12 and 302 ± 9 pA from control values of 244 ± 8 and 160 ± 8 pA (n = 8). This result implies that both transient and late outward currents appear to be increased by UTP. However, in a separate set of experiments, when the cells were exposed to tetraethylammonium(10 mM), which blocks the components of late current, UTP (100 μM) did not cause any effect on instantaneous peak component of outward current.
In cell-attached mode, UTP (10-100 μM) added to the recording pipette enhanced single-channel activity, which occurred in rapid open-close transitions and in brief bursts (n = 5 cells; Fig. 5). The voltage dependence of the UTP-induced single-channel current is illustrated in the current-voltage plot of Fig. 5B. The channels were found to rectify in the inward direction at the membrane potentials more positive to the resting potential. The unitary events underlying the UTP-induced current had a single-channel conductance and open-time constant of ∼27 pS and 1.57 ms, respectively. These values are nearly identical to those of the K+ channels activated by adenosine or extracellular ATP.
Concentration-response relations for the effects of adenosine, ATP, and UTP
Concentration-response curves for the effects of extracellular adenosine, ATP, and UTP on the activation of KACh channels are shown in Fig. 6. Adenosine had a higher potency than ATP to activate KACh-channel current. The EC50 value for adenosine-induced channel activity was significantly(p < 0.05) smaller than that for ATP (4.5 vs. 10.3 μM). However, the observed maximally relative open probability of KACh channels in the presence of adenosine and ATP was nearly identical (0.42 vs. 0.41; (Fig. 6). On the other hand, UTP was less potent (EC50 = 28.5 μM) and efficacious than either adenosine or ATP in activating KACh-channel currents. The maximal opening probability (N × Po) in the presence of ATP was 1.5-fold greater than that in the presence of UTP, and the EC50 value of UTP was 2.8-fold greater than ATP. In addition, no cooperativity for the activation of K+ channel current by adenosine, ATP, or UTP in rat atrial membranes could be demonstrated.
Comparison of ATP, AMPCPP, and UTP effects on the KACh channel activity
Figure 7 shows the comparison of effects of ATP, AMPCPP, and UTP on the opening probability of KACh channels in rat atrial membranes. The cells were bathed in normal Tyrode's solution and held at the level of −20 mV. The opening probability(N × Po) obtained in the presence of ACh (10 μM) was considered to be 100%. Notably, CPX, a potent A1-adenosine-receptor antagonist, suppressed the ATP-induced channel current by ∼70%. CPX (1 μM) added to the pipette solution almost completely prevented AMPCPP-induced single KACh channel currents (Fig. 7). In contrast, the relative opening probability of UTP-induced KACh-channel activity was not significantly altered by the presence of CPX in the recording pipette. The latter finding indicates that the effect of UTP on KACh-channel activity was not mediated by the A1-adenosine receptor.
Effect of pertussis toxin on the activation of the K+ channels induced by extracellular UTP
To determine whether the effect of UTP on KACh-channel activity involves the activation of pertussis toxin-sensitive G protein(s), atrial myocytes were treated with pertussis toxin (500 ng/ml) for 5 h. When UTP (100 μM) was included in the recording pipette, cell-attached patch recording from pertussis toxin-treated atrial cells (n = 4; Fig. 8B) revealed only small-amplitude K+-channel currents, which underwent transitions at a very low frequency. In comparison, in cells that were not treated with pertussis toxin but maintained in medium for the same period (i.e., 5 h), the activity of UTP-induced KACh channels was clearly observed (n = 5 cells; Fig. 8A). These results indicate that UTP-induced activation of KACh channels is linked to pertussis toxin-sensitive G protein(s).
This study provides the evidence that extracellular ATP per se is capable of activating the inward-rectifier K+ current by directly opening KACh channels and that dephosphorylation of ATP to adenosine is not a prerequisite for the activation of these channels (11). In addition, because ATP-induced increase in K+ current was not affected by glibenclamide, a KATP-channel blocker, it is unlikely that the K+ channels activated by extracellular ATP are KATP channels (i.e., ATP-sensitive K+ channels). Because cytosolic ATP can be released when cardiac myocytes are metabolically compromised by hypoxia (24), ATP per se, like adenosine, may potentially act as an inhibitory metabolite to protect the myocardium from excessive work by reducing heart rate.
The results of our study demonstrate that the effect of extracellular ATP to activate KACh channels is mainly mediated by the binding of ATP to the A1-adenosine receptor, as reported previously (11). As shown in Fig. 6, when CPX, a potent A1-adenosine-receptor antagonist, was added into the recording pipette solution, the extra-cellular ATP-induced KACh-channel activity was reduced by as much as 70%. The remaining 30% of ATP-induced channel activity could thus be caused by the binding of ATP (or metabolite) to another purinergic-receptor subtype (25). AMPCPP, a nondegradable ATP analog and P2X-purinergic-receptor agonist, added to the recording pipette solution, was also capable of activating KACh channels in atrial membrane. The A1-adenosine antagonist completely prevented AMPCPP-induced activation of KACh channels. These findings suggest that although activation of P2-purinergic receptors with extracellular ATP in cardiac myocytes may facilitate Ca2+ influx and Ca2+ release from intracellular stores (4,25-28), the binding of P2X-purinergic receptor caused by AMPCPP does not appear to play a major role in mediating the activation of KACh channels caused by this nondegradable ATP analog.
Of interest, CGS-21680 (10 μM), an A2-adenosine-receptor agonist, caused no change in the activity of KACh channels (not shown). This finding suggests that A2-adenosine receptor does not play a role in the activation of KACh channels. These results could be interpreted to conflict with the previous observation that extracellular ATP plus theophylline in the recording pipette can interact with P2-purinergic receptor and almost fully activate the KACh-channel activity(10). However, it must be noted that theophylline is neither a specific, selective, nor potent adenosine-receptor antagonist. On the other hand, it is difficult to exclude the possibility that (a) P2-purinergic receptors or signal-transduction mechanisms or both of these receptors may be rendered nonfunctional during the cell isolation procedure, and (b) desensitization of P2-purinergic receptor or receptor-promoted signaling response by endogenous adenine nucleotides during cell preparation may occur. Nevertheless, a membrane patch on rat atrial myocytes under our experimental conditions should be sensitive enough to observe the alteration in extracellular ATP- or UTP-induced channel current.
The reason that UTP-induced increase in the amplitudes of instantaneous outward current is greater than that of late outward current (Fig. 4) is unclear, but it may be related to the time-dependent relaxation of UTP-induced current (1,2), the reduction of voltage-gated Ca2+ current (29), or both. However, UTP did not produce any effect on the peak component of instantaneous outward current under the conditions in which cells were exposed to tetraethylammonium chloride. UTP also did not affect the inactivation time course of instantaneous current. Thus the contributory role of UTP in increasing transient outward current would be minor.
This study also provides substantial evidence that extracellular UTP can directly activate KACh channels in rat atrial membrane. The single-channel conductance and mean open time of UTP-induced K+ channels were 27 pS and 1.57 ms, respectively. These values are almost identical to those of adenosine- or ATP-induced K+-channel activity (Figs. 2 and 5). This finding, showing that in pertussis toxin-treated atrial myocytes, UTP no longer activated KACh channels (Fig. 8), indicates that UTP binds to a receptor that is coupled to KACh channels via GK proteins, possibly similar to that of ACh, adenosine, and ATP (1,10). Furthermore, when UTP was added to the superfusion medium, instead of the recording pipette solution, the basal KACh-channel activity did not show any change (data not shown). It is thus unlikely that diffusible cytosolic messengers inside the cell (e.g., arachidonic acid metabolites or cyclic AMP) contribute to UTP-induced activation of KACh channels.
The single-channel conductance of KACh in atrial membrane of rats obtained in our experiments was 1.4-fold lower than that of other species(1,30). However, the open-time (1.5 ms), channel-bursting, and inward-rectifying properties were all nearly identical to those reported for atrial myocytes of other species. For instance, the conductance of KACh channels in rat atrial myocytes was 27-30 pS, with a mean open time of 0.9-1.5 ms (11), whereas the KACh channels in guinea-pig atrial myocytes had a conductance of 45 pS and a mean open time of 1.4 ms (1,10). This difference is currently unknown. However, it was previously reported that single-channel conductance of KACh channels is not the same in different regions [i.e., sinoatrial(SA) vs. atrial cells; 30]. In SA nodal cells of rabbit, the conductance of KACh channels activated by adenosine was noted to be ∼25 pS (29). Nevertheless, extracellular ATP or UTP can regulate the same population of K+ channels in atrial myocytes, and interspecies difference did not produce any change in the kinetic properties of KACh channels.
Because UTP activated KACh channels in the presence of A1-adenosine-receptor antagonist (Fig. 6), the effect of this nucleotide is likely to be mediated by its binding to the P2U-purinergic-receptor subtype (10,16). It is tempting to speculate that the CPX-insensitive component of the ATP-induced activation of KACh channels is also mediated by activation of P2U-purinergic receptors, a hypothesis that needs to be tested. Thus rat cardiac myocytes may express at least two subtypes of purinergic receptors for extracellular ATP, and activation of these receptor subtypes can rapidly elicit the opening of KACh channels. A similar phenomenon has been reported in other cell types(15,18,26). For instance, in rat osteoblastic cells, extracellular ATP can activate two subtypes of purinergic receptor (P2Y and P2U) and increase cytosolic Ca2+ concentration (26). Therefore the use of specific and selective blockers for P2Y- and P2U-purinergic receptors combined with the isolation or cloning or both of the various functionally defined purinergic-receptor types in myocardium will prove useful in helping to clarify this possibility.
In summary, the results of our study provide substantial evidence that extracellular ATP and UTP can activate KACh channels in rat atrial myocytes. The effect of ATP is mainly caused by its binding to the A1-adenosine receptor, whereas the effect of UTP appears to involve the activation of the P2U-purinergic receptor. Furthermore, activation of KACh channels by these purinergic ligands is linked to pertussis toxin-sensitive G protein.
Acknowledgment: This work was supported by grants from Veterans General Hospital-Kaohsiung (VGHKS-85-60) and National Science Council (NSC-85-2331-B-075B-012), Taiwan, ROC. We gratefully acknowledge Dr. Luiz Belardinelli for helpful discussion and critically reading the manuscript.
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