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* Pat and Jim Calhoun Cardiology Center and the
Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut, USA
1 Correspondence: Pat and Jim Calhoun Cardiology Center, MC-3946, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030, USA. E-mail bliang{at}uchc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0 mV) further suggests a role for this receptor in the normal electrophysiological action of ATP in WT murine cardiac myocytes. The P2X antagonist suramin was only able to block partially the 2-meSATP-stimulated current in WT cells, implying that both P2X4 receptor and another yet-to-be-identified P2X receptor mediate this current.—Shen, J.-B., Pappano, A. J., Liang, B. T. Extracellular ATP-stimulated current in wild-type and P2X4 receptor transgenic mouse ventricular myocytes: implications for a cardiac physiologic role of P2X4 receptors.
Key Words: ion channels • membrane current • mouse • heart
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. There are two subfamilies of P2 receptors: ligand-gated ion channels and G-protein-coupled receptors, termed P2X and P2Y receptors, respectively (2)
. Seven subtypes of P2X (P2X1-7) receptor have been identified. Previous studies, including our own, have shown that ATP increased contractility and intracellular calcium transients in single cardiac myocytes and in intact murine and rat hearts (4
, 5)
. The identity of the P2 receptor subtype that mediates the ATP-stimulated positive inotropic effect is not clear. Among the various P2X receptors, P2X4 receptors are expressed in the heart, including the cardiac myocyte (6
, 7)
. Transgenic (TG) cardiac-specific overexpression of the human P2X4 receptor (hP2X4) in mice showed a phenotype of enhanced basal and 2-meSATP-stimulated heart contractility (7
, 8)
, suggesting P2X4 receptors may be important in enhancing cardiac performance.
The P2X4 receptor is a member of the P2X ligand-gated ion channel superfamily. Activation of P2X4 receptors leads to the opening of this nonselective cation channel permeable to Na+, K+, and Ca2+. Many electrophysiological studies have been carried out on recombinant P2X4 receptors expressed in human embryonic kidney cells (HEK293) or Xenopus oocytes (9
10
11)
. However, electrophysiological recordings of membrane currents through the P2X4 receptor in single cardiac myocytes are lacking. Although P2X4 receptors are detected in the mammalian cardiac myocyte by immunoblotting and immunocytochemistry (7
, 12)
, it is not known whether it has a physiologic function in that tissue. Thus, the principal objective of the present study was to investigate the ionic currents mediated by this receptor in cardiac myocytes and to determine whether it has any cardiac physiologic role. The cardiac-specific overexpression of P2X4 receptor provides an animal model to determine the ionic action of this receptor in the more physiologic system of cardiac myocytes. Comparison of the P2X4 receptor-mediated current in TG myocytes to that in wild-type (WT) myocytes provided insight on the cardiac physiologic role of P2X4 receptors. A preliminary account of these data has been presented in abstract form (13)
.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-MyHC expression vector and bred in B6SJL mice as described previously (7
, 8)
. Overexpression of the hP2X4 receptor in TG mice was verified by polymerase chain reaction (PCR) of the genomic DNA. Ventricular myocytes were obtained from 3-month-old WT and TG mice of either sex (26 WT, 47 TG) by an enzymatic dissociation procedure as described previously (7
, 5)
Briefly, the heart was rapidly excised from mice that had been anesthetized with pentobarbital and anticoagulated with 1000 U of heparin. The aorta was cannulated while visualized under a surgical microscope. The heart was perfused in a Langendorff apparatus with oxygenated (95% O2/5% CO2) Ca2+-free solution (37°C) for 5 min at a rate of 2.5 mL/min. The solution composition was (in mM): 126 NaCl, 4.4 KCl, 1.0 MgCl2, 18 NaHCO3, 11 glucose, 4 HEPES, and 3 BDM (2,3-butanedione monoxime), (pH 7.3 adjusted with NaOH). Thereafter, the perfusing solution was changed to one with 25 µM CaCl2 and liberase (70 µg/mL, Roche Molecular Biochemicals, Inc., Nutley, NJ, USA) for 8–10 min. The digested heart was then cut into small pieces, gently agitated, and incubated in Tyrode’s solution containing 3 mM BDM and 100 µM Ca2+. Cells were sedimented by gravity for 10–15 min, and the pellet was resuspended in 200 µM Ca2+-containing Tyrode’s solution (containing 3 mM BDM) and allowed to settle for at least 30–60 min at room temperature. A small aliquot of the cell suspension was placed in a 0.2 mL chamber mounted on the stage of an inverted microscope. After a settling period of 5–10 min, the cells were superfused with Tyrode’s solution containing (in mM): 135 NaCl, 5.4 KCl, 1.0 CaCl2, 1.0 MgCl2, 10 HEPES, and 10 dextrose (pH 7.4 adjusted with NaOH). The experiments were carried out at room temperature (22–23°C) and were completed within 4–6 h after myocyte isolation.
Electrophysiological methods
The whole cell patch-clamp technique was used for the experiments. Electrodes were prepared from borosilicate glass pipette (1.2 mm i.d.) with a two-step pulling procedure and filled with pipette solution (see below). The pipette was connected via an Ag-AgCl wire to the head stage of an amplifier (List EPC-7, Medical Systems, Greenvale, NY) controlled by a computer and Axon pClamp software. For voltage clamp experiments, the electrodes were filled with a solution containing (in mM): 135 cesium aspartate, 5 NaCl, 5 Mg2ATP, 10 HEPES, and 10 EGTA (pH 7.3 adjusted with CsOH). Electrode resistances were 2–4 M
. As soon as electrical contact was established, the superfusion medium was changed to a modified Tyrode’s solution (5.4 mM KCl was omitted and 10 mM CsCl and 5 µM ouabain was added to Tyrode’s solution) to block K+ currents and the Na/K pump current, respectively
Two voltage clamp protocols were used. The first, a ramp voltage protocol, consisted of three stages: an initial 10 ms jump to –100 mV from the –80 mV holding potential, a second very slow depolarization to 50 mV over 5 s, then a third phase returning to the holding potential. The ramp voltage protocol was applied to cells at 20 s intervals. Three current traces from –100 mV to 50 mV were averaged to construct the I-V relationship. In the second protocol, membrane voltage was stepped from –80 mV holding potential to –40 mV for 300 ms, then to 10 mV for 500 ms to activate the L-type Ca current ICa(L), jumped to 50 mV for 300 ms, followed by an additional jump to –100 mV for 300 ms. The ICa(L) and the isochronal I-V relationship at –100, –80, –40, 10, 50 mV were obtained from this protocol.
Drugs and solutions
2-Methylthioadenosine 5'-triphosphate (2-meSATP), suramin, ivermectin, ZnCl2 were obtained from Sigma Chemical Co. (St. Louis, MO, USA) 2-meSATP was prepared just before each experiment. Stock solutions were prepared for suramin (in water) and for ivermectin (in DMSO) and added to the Tyrode’s solution to obtain the desired concentrations. For ivermectin, the final concentration of DMSO in Tyrode’s solution was less than 0.025%.
Data
The data are given as mean ± standard error of the mean (SE). Student’s t test for paired or unpaired samples was used to evaluate the effects of experimental interventions; P < 0.05 was taken as statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20-fold) P2X4 receptors than WT myocytes (7
, 8)
. If the enzymatic digestion disrupted similar number of this cell surface receptor on both cell types, TG myocytes would be less prone to damage. It is also possible that an uneven expression of the P2X receptor on individual myocytes could explain the lack of response in some cells. A typical recording of membrane current from a TG cell is shown in Fig. 1
A. Addition of 2-meSATP induced an inward current of 250pA in this cell held at –80 mV. Suramin (30 µM) slightly reduced this ATP-stimulated current that dissipated on washout of 2-meSATP. The increase in current by 2-meSATP (taken from traces where current marked at b minus that at a, as shown in Fig. 1A
, is the increase) is shown in Fig. 1B
. The 2-meSATP-stimulated current displayed a linear relation between –100 and +50 mV and reversed from inward to outward at 0 mV.
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Of cells that showed an induced current in response to 2-meSATP, the amplitude of 2-meSATP-stimulated current was significantly greater in cells from TG than from WT hearts (Fig. 1
C, P<0.05 except at the reverse potential of 0 mV). For example, the 2-meSATP-stimulated current was –2.25 ± 0.20 pA/pF at –100 mV and 1.95 ± 0.24 pA/pF at 50 mV in TG cells (N=58), but only –1.14 ± 0.14 pA/pF at –100 mV and 0.99 ± 0.12 pA/pF at +50 mV in WT cells (N=17). The 2-meSATP-stimulated current was significantly greater in TG than in WT cells at both potentials (P<0.01). The slope conductance was 12.03 ± 0.32 pS/pF in cells from WT mice and increased nearly 2-fold to 22.95 ± 0.69 pS/pF in those from TG mice overexpressing the hP2X4 receptor (P<0.01) (Fig. 1C
). The voltage-dependence of the 2-meSATP-stimulated currents from cells that responded to the agonist was compared in Fig. 1C
. The reversal potentials of the ATP-stimulated current were similar. In all subsequent experiments, only those 2-meSATP-responsive cells were further tested with the P2X receptor antagonist and activators.
Isochronal I-V relation and ICa(L)
We used the step pulse voltage clamp protocol to evaluate the effects of 2-meSATP on L-type Ca current ICa(L) and to obtain isochronal I-V relations. As in the ramp voltage clamp experiments, a membrane current induced by 3 µM 2-meSATP was observed in 23 of 29 cells from TG mice (79%) and in 13 of 31 cells from WT mice (42%). An example of the results from one cell of a TG heart is shown in Fig. 2
. End-of-pulse currents shifted inward at –100, –80, and –40 mV and shifted outward at 10 and 50 mV after applying 3 µM 2-meSATP. The end-of-pulse current recovered upon washout in ventricular cells from both the TG and WT animals. Of cells that showed an induced current in response to 2-meSATP, the agonist-evoked current shifts, representing current via the P2X receptor, were significantly larger in TG than in WT cells (P<0.05, Fig. 2B
), similar to results obtained with the ramp voltage protocol. These data, obtained with step pulse voltage clamp, further confirmed that the overexpressed hP2X4 receptor channel is capable of conducting the current in response to 2-meSATP.
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We next investigated whether overexpressing hP2X4 receptors in mouse hearts affects the function of the L-type calcium channel. In the experiment shown in Fig. 2A
, ICa(L) did not change as we measured the current from peak to end-of-pulse in the presence of the ATP analog. Note that there was a 2-meSATP-stimulated current superimposed on ICa(L). Basal ICa(L) was similar in cells from TG and WT hearts (not shown). The average ICa(L) was small (<4.5 pA/pF), possibly due to the low extracellular Ca2+ concentration (1 mM). In the P2X4R TG myocytes, the basal ICa(L) was –4.7 ± 0.63 pA/pF and the rundown in the absence of 2-meSATP was to 75.5 ± 3.4% of the basal current or –3.7 ± 0.72 pA/pF (mean±SE, n=8, P<0.05 vs. basal, paired t test). In another group of TG myocytes, basal ICa(L) was –4.6 ± 0.48 pA/pF, and decreased to 74.9 ± 9.2% of the basal current or –3.5 ± 0.42 pA/pF during the rundown when 2-meSATP was present (P<0.05 vs. basal, paired t test, n=26) (Fig. 2C
). The decrease in ICa(L) was similar whether or not 2-meSATP (3 µM) was present (P>0.1). Thus, the L-type Ca current ran down during experiments and P2X receptors did not alter its rundown.
Evidence for presence of suramin-sensitive and -insensitive 2-meSATP-induced current in WT cells
Suramin is reported to be an antagonist of all P2X receptors except the P2X4 receptor (1
, 2)
. We tested the effect of suramin on 2-meSATP-stimulated current in cells from WT and TG animals. The inhibitory effects of suramin on 2-meSATP-stimulated currents were compared at –100, –80, –40, and 50 mV. In cells (N=8) from WT mice, suramin (30 µM) reduced the amplitude of 2-meSATP-stimulated current significantly at all potentials (–100, –80, –40, and 50 mV; P<0.05). I-V relationships for 2-meSATP-stimulated current in the presence and the absence of suramin in a WT cell are shown in Fig. 3
A. These results suggest that in WT cells there are unidentified native P2X receptors, other than P2X4 receptors, that are also present and functional.
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In contrast, 30 µM suramin had only a small inhibitory effect on the 2-meSATP-stimulated current at any voltage in ventricular myocytes (N=20) from TG hearts. As noted in Fig. 1A
, suramin slightly reduced the agonist-stimulated current in a myocyte from a TG mouse. The inhibition of 2-meSATP-stimulated current by suramin averaged 12.9 ± 5.1% and 14 ± 4% at –100 mV and 50 mV, respectively (P<0.05 at both potentials) (Fig. 3B
). The % inhibition by suramin was significantly smaller in TG than in WT cells at both –100 mV and 50 mV (P<0.01) (Fig. 3B
). The smaller inhibitory effect of suramin in TG cells may be due to the blockade of other native non-P2X4 receptors (P2X1, P2X2, P2X5) that are present in TG cells since the overexpressed hP2X4 receptor is insensitive to blockade by suramin.
2-meSATP-stimulated current depends on extracellular Na+
Ion substitution experiments were done in cells from TG mice to ascertain the ion selectivity of the 2-meSATP-stimulated current. The ramp voltage clamp protocol was used. After the current had been induced, the extracellular medium was rapidly changed to a modified Tyrode’s solution in which 135 mM N-methyl-D-glucamine (NMDG) replaced external Na+ ([Na+]o) in the continued presence of 2-meSATP. The membrane I-V relation was obtained in the absence and presence of 3 µM 2-meSATP. A representative experiment is shown in Fig. 4
A. At a holding potential of –80 mV, 2-meSATP induced an inward current of -300 pA. When the current was steady, NMDG replaced Na+ in the bath in the continued presence of 2-meSATP. This was accompanied by an outward shift of current (200 pA above the 0 level) that reversed upon replacing NMDG with Na+. Differences in I-V relations for the 2me-SATP-stimulated current were taken in the presence and absence of Na+. The averaged I-V relation (Fig. 4B
) was linear in the presence of Na+ and reversed at 0 mV. On the assumption that the 2me-SATP-stimulated current is carried by monovalent cations, the apparent reversal potential can be estimated from the expression Erev = 58 log Na+o/Cs+i (14)
. With Na+o of 135 mM and Cs+i of 135 mM, the estimated Erev is 0 mV. If a modified Goldman-Hodgkin-Katz equation was used (15)
with equal permeabilities to Na+ and Cs+, the permeability of Ca2+ relative to Na+ is <0.006 and taken as negligible. When NMDG replaced external Na+, the current induced by 2-meSATP was rather linear and outward at all potentials from –100 to +50 mV. Replacing extracellular Na+ completely prevented inward 2-meSATP-stimulated currents in cells from TG hearts. When extracellular Na+ is removed, the estimated Erev is shifted to –124 mV (1 mM Na+o is assumed to remain in the bath). This is similar to the extrapolated estimate of Erev from the I-V relation (Fig. 4B
).
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Effects of ivermectin on 2-meSATP-stimulated currents
To target the P2X4 receptor specifically, we tested the effect of ivermectin on the 2-meSATP-stimulated current. Ivermectin, an antihelminthic agent, has a broad reaction spectrum on ligand-gated ion channels including glutamate-gated chloride channels, nicotinic receptors and the P2X4 receptor channel (16)
. However, the P2X4 receptor is the only member of the P2X receptor family that is sensitive to potentiation by ivermectin (1
, 16)
. Ivermectin was only applied to cells that were responsive to 2-meSATP. In cells from WT hearts, ivermectin significantly increased the amplitude of 2-meSATP-stimulated currents at all voltages tested (P<0.05) except at +50 mV (Fig. 5
). This indicates there are some native P2X4 receptors expressed in WT cells and that these native P2X4 receptors mediate the 2-meSATP-stimulated current. In TG cells, ivermectin facilitated a greater increase of 2-meSATP-stimulated currents at all potentials tested, including +50 mV (Fig. 5B
). The net potentiations of 2-meSATP-stimulated current by ivermectin in TG cells at –100, –80, and 50 mV were significantly greater than that in WT cells (P<0,05). In response to ivermectin, the basal 2-meSATP-induced current increased from 1.0 ± 0.17 to 1.5 ± 1.8 pA/pF at –100 mV and from 0.6 ± 0.15 to 0.8 ± 0.13 pA/pF at +50 mV in WT cells. In TG cells, ivermectin increased the 2-meSATP-induced current from 1.3 ± 0.31 to 2.5 ± 0.39 pA/pF at –100 mV and from 0.8 ± 0.18 to 1.8 ± 0.21 pA/pF at +50 mV. These data are consistent with a functional overexpression of the P2X4 receptor in TG myocytes.
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Zinc potentiates the monovalent cation conductance induced by ATP at most P2X receptors (17
, 18)
. We also tested the effect of ZnCl2 (10 µM) on the 2-meSATP-stimulated current. In control experiments, 10 µM Zn2+ alone did not change the membrane currents in cells from either WT or TG hearts (not shown). Zn2+ was applied only after the 2-meSATP-stimulated current was induced in either WT or TG cells. When applied in the presence of 2-meSATP, Zn2+ significantly increased the membrane currents induced by 2-meSATP in both WT and TG cells. The I-V relations of the ATP-stimulated current show that Zn2+ potentiated the inward current at negative potentials and outward currents at positivepotentials (P<0.05, not shown). The reversal potential of the 2-meSATP-stimulated current did not change after Zn2+ application.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 7)
. Transgenic cardiac-specific overexpression of the human P2X4 (hP2X4) receptor showed an in vitro phenotype of increased cardiac performance (7)
. These data raised the possibility that the P2X4 receptor may be important in mediating the cardiac effect of extracellular ATP in mammalian cardiac myocytes, similar to a role of the P2X4 receptor in embryonic chick cardiac ventricular myocytes (6)
. However, evidence for a cardiac physiologic role of the P2X4 receptor in mammalian cardiac myocytes is lacking. Using a transgenic mouse with cardiac-specific overexpression of P2X4 receptors, this study characterized the ionic current mediated by this receptor and compared such current to that mediated by the native P2X receptor in WT murine cardiac myocytes.
We found that the P2X agonist 2-meSATP induced a membrane current in ventricular myocytes isolated from both WT mice and TG mice with cardiac-specific overexpression of hP2X4 receptor. The 2-meSATP-stimulated current was inwardly directed at negative potentials and reversed at around 0 mV in both WT and TG myocytes. Consistent with overexpression of the P2X4 receptor, the current magnitude at –100 mV was at least 2-fold greater in cells from TG mice. Desensitization kinetics of the P2X4 receptor, as well as that of other P2X receptors (P2X2, P2X5, P2X7), is classified as rather slowing desensitizing (2)
. In this study, the 2-meSATP-stimulated current persisted for the duration of agonist exposure in ventricular myocytes from WT and TG mice. Prolonged ATP application (for 3 min) to rat P2X4 receptor expressed in Xenopus laevis oocytes induced a membrane current consisting of two components: an initial transient current, which rapidly desensitized, and a maintained current, which is smaller but stable during the ATP-exposure time (17)
. This slower stable current component displays the pharmacological and biological characteristics of P2X4 receptor (17)
. Accordingly, the reported ATP-stimulated currents in our experiments likely represent the stable current through P2X4 receptors as we applied 2-meSATP for at least 3 min.
The current induced by 2-meSATP displayed a linear voltage dependence. This is similar to that seen when P2X4 receptors were heterologously expressed in HEK293 cells (10)
or in Xenopus oocytes (9
, 19)
. Activation of P2X4 receptors mediates the rapid nonselective passage of cations (Na+, K+, Ca2+) (1
, 9)
. Cs+, which replaced the K+, may be as permeable as K+. The reversal potential of the current of 0 mV lies between the Nernst potentials for Na+ and Cs+. In earlier experiments, a similar reversal potential was obtained (9
, 10
, 18
, 19)
. In the nominal absence of external Na+, the extrapolated reversal potential was more negative than –120 mV. This is consistent with Cs+ carrying outward current over the voltage range tested. For this system, we estimated the Erev of –124 mV; this agrees with the experimental data. The fact that replacing Na+ could eliminate the inward 2-meSATP-stimulated current at negative potentials in TG cells suggests that Na+ is the principal cation carrier in the presence of 1.0 mM extracellular Ca2+. Our finding is compatible with observation by others showing that Ca2+ is a minor component of the inward current via the P2X4 receptor under normal extracellular Ca2+ concentration (1.8 mM) (1)
.
In response to the allosteric enhancer ivermectin, the 2-meSATP-stimulated current increased significantly in both WT and TG cardiac myocytes. Since only the P2X4 receptor, among all known P2X receptors, can be facilitated by the P2X4-selective allosteric enhancer ivermectin (16)
, these data suggest an important role for the native P2X4 receptor in mediating this current. Micromolar concentration of Zn2+ was able to increase the 2-meSATP-induced current in both WT and TG cardiac myocytes. This action is characteristic of most P2X receptors except P2X7 receptor (1
, 2
, 19
, 20)
. The native P2X receptors in WT cells and those overexpressed hP2X4 receptors in TG cells can respond to the potentiation by Zn2+. The I-V relationship of 2-meSATP-stimulated current remained similar before and after the application of Zn2+ as Zn2+ only potentiates the cation conductance of P2X receptors. These findings further confirm that the 2-meSATP-induced current is mediated via the native P2X receptor or the overexpressed P2X4 receptor in the WT or the TG cardiac myocytes.
Suramin had a significantly more pronounced blocking effect on the 2-meSATP-stimulated current in myocytes from WT than in those from TG hearts. A distinguishing feature of the P2X4 receptor is its resistance to blockade by suramin, a potent antagonist at the other P2X receptor subtypes (1
, 11
, 21)
. As suramin can block all P2X receptors except the insensitive P2X4 receptor, the remaining unblocked ATP-stimulated current in WT cells may be mediated through native P2X4 receptors. The smaller inhibitory effect of suramin in TG myocytes is likely due to blockade at the native non-P2X4 receptor (1
, 2)
rather than at the overexpressed hP2X4 receptor because P2X4 receptors from mouse, rat, and human are all relatively suramin-insensitive with IC50’s > 100 µM (10)
. Taken together, these data suggest that in WT myocytes the 2-meSATP-stimulated current arose from actions at P2X4 and other, as yet unidentified P2X receptors while the current in TG myocytes was largely mediated by the overexpressed hP2X4 receptors.
2-meSATP had no effect on the magnitude of ICa(L) in cells from either WT or TG mice, indicating there is no direct connection between L-type calcium channels and P2X receptor channels. The basal ICa(L) was relatively small in our experiments, and is consistent with that observed in the presence of a reduced extracellular Ca2+ concentration (1 mM) (22)
. Thus, the ability of this ligand to increase contractions in intact heart (5
, 6
, 8)
and cell shortening in myocytes is not a result of increased Ca2+ entry through L-type channels. In rat heart, this analog was effective at increasing ICa(L) (23)
as well as inducing a nonselective cation current (24)
. The ATP effect on ICa(L) was attributed to the P2Y receptor and was sensitive to blockade by suramin (4
, 23)
. Whether the ionic current mediated by P2X4 receptors in TG myocytes is responsible for the enhanced contractility is not known and requires further investigation.
Overall, P2X4 receptors can mediate a significant voltage-dependent current in mammalian cardiac myocytes. In WT murine cardiac myocytes, P2X4 receptors and other yet-to-be identified P2X receptor(s) are responsible for this ATP agonist-stimulated current, which is inward at potentials negative to zero mV. In TG mice with cardiac-specific overexpression of the P2X4 receptor, a similar current is also identified that arose mainly from the overexpressed receptor. The overexpressed hP2X4 receptors maintain the pharmacological and biological characteristics of the P2X4 receptor, i.e., resistance to blockade by suramin and potentiation by Zn2+ and ivermectin. That the current in both WT and TG cells exhibits activation by the same agonist and shows similarities in voltage dependence, reverse potential, and potentiation by ivermectin further suggests the importance of P2X4 receptors in the WT cells. The P2X4 receptor TG cells represent a useful model to study the physiologic actions of this receptor in the heart.
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ACKNOWLEDGMENTS |
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Received for publication July 22, 2005. Accepted for publication October 12, 2005.
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