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Cardiovascular Research 1999 41(1):166-174; doi:10.1016/S0008-6363(98)00214-4
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

P2 purinoceptors contribute to ATP-induced inhibition of L-type Ca2+ current in rabbit atrial myocytes

Taku Yamamotoa, Yoshizumi Habuchia,*, Manabu Nishiob, Junichiro Morikawab and Hideo Tanakaa

aDepartment of Laboratory Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602, Japan
bDepartment of Internal Medicine III, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602, Japan

* Corresponding author. Tel.: +81-75-251-5652; fax: +81-75-251-5678; e-mail: yhabuchi@Koto.Kpu-m.ac.jp

Received 4 February 1998; accepted 8 June 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Adenine compounds, including adenosine-5'-triphosphate (ATP) and adenosine (Ado), exert inhibitory effects on myocardium via P1 (subtype A1) purinoceptors. However, ATP per se is a potent activator of P2 purinoceptors. Our aim was to elucidate the respective roles of P1 and P2 purinoceptors in the actions of ATP on L-type calcium current (ICa) in rabbit atrial cells. Methods and Results: A whole cell clamp technique was used to record ICa in single atrial cells from the rabbit heart. ATP (0.1 µmol/l–3 mmol/l) produced an inhibitory effect on ICa prestimulated by isoproterenol (ISO, 30 nmol/l), even in the presence of Ado (1 mmol/l). Both 1,3-dipropyl-8-cyclopentylxanthine (A1 blocker) and suramin (P2 blocker) partially blocked the ATP-induced inhibition of ICa, while their co-application nearly completely abolished the effect of ATP. ATP-{gamma}S (30 µmol/l) inhibited ISO-stimulated ICa significantly, and this inhibition was completely blocked by suramin. {alpha},β-Methylene-ADP, an inhibitor of hydrolysis of AMP to Ado, eliminated the suramin-resistant component of ICa inhibition by ATP. Pretreatment with pertussis toxin (PTX) abolished the ATP inhibition of ICa. Both intracellular dialysis with 8Br cAMP and the application of forskolin plus 3-isobutyl-1-methylxanthine also eliminated the effect of ATP. Conclusions: Both P1 and P2 purinoceptors are involved in the ATP inhibition of ISO-stimulated ICa in rabbit atrial cells. The P1 stimulation by ATP results from hydrolysis of ATP to Ado. Both the P2- and the P1-mediated effects of ATP and Ado, respectively, involve a PTX-sensitive and cAMP-dependent pathway.

KEYWORDS ATP; L-type calcium current; Purinoceptor; Pertussis toxin; Rabbit; Atrial myocytes


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Adenine compounds cause various physiological responses by activating specific purinoceptors [1]. They are released from the cytoplasm in the heart during ischemia [1–3], and can cause disturbances in the electrical conduction and mechanical contraction [1–3]. ATP and Ado are used for the treatment of reentrant tachycardias involving the atrioventricular node, because of their negative dromotropic effect [2, 4].

The purinoceptors are classified into two major subtypes, P1 and P2, which are preferentially stimulated by Ado and ATP, respectively [5]. Activation of P1 purinoceptors by Ado inhibits L-type calcium current (ICa) and activates the muscarinic receptor-linked K+ current (IK,ACh) via the pertussis toxin (PTX)-sensitive GTP-binding (G) proteins [2, 6–8]. Accordingly, Ado causes negative inotropic, chronotropic and dromotropic effects on the heart [2, 9], and probably contributes to the ischemic preconditioning [10]. On the other hand, ATP has been reported both to increase and to decrease myocardial contractility [3]. Since ATP is rapidly degraded by ectoenzymes to Ado, the inhibitory effects of ATP can be ascribed to P1 purinoceptor activation by Ado [3, 11]. The P1-mediated inhibition of ICa involves intracellular cAMP and the cAMP-dependent protein kinase (PKA) [2]. Therefore, Ado inhibits ICa preferentially when the ICa is stimulated by β agonists. In addition to the P1-mediated mechanism, Qu et al. [12]showed that ATP inhibits ICa via P2-purinoceptors in ferret ventricular cells. In sharp contrast, an ATP-induced enhancement of ICa was shown by Yatani et al. [13]in frog atrial preparations. Vassort and his colleagues have demonstrated that in rat and frog heart cells, the potentiation of ICa by ATP is P2 purinoceptor-mediated [14–16]. These P2-mediated effects on ICa did not involve the cAMP–PKA pathway [12, 14–16].

Several biochemical studies have suggested that in platelets [17], hepatocytes [18]and smooth muscle cells [19], activation of P2-purinoceptors reduces the intracellular cAMP concentration via PTX-sensitive inhibitory G (Gi) proteins, whereas a pathway which positively links P2 purinoceptors with adenylate cyclase was reported in other cells (such as adrenal vascular endothelial cells [20]and neuroblastoma–glioma hybrid cells [21]). According to Yamada et al. [22], myocardial cells may have a pathway which links P2 purinoceptors with Gi proteins. Their findings give rise to the possibility that P1 and P2 purinoceptors synergically regulate ICa in the heart via Gi proteins. However, no study has shown that the PTX-sensitive pathway activated by P2 purinoceptors plays any physiological role in the heart. Alvarez et al. [15]showed an inhibitory effect of ATP on ISO-stimulated ICa in frog myocardial cells, although the receptors involved were not clarified. In this study, we aimed to elucidate the contribution of P2 purinoceptors to the ATP inhibition of ICa in rabbit atrial cells.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Cell isolation
The isolation of single cells and the measurement of the membrane currents were performed as reported previously from this laboratory [23]. The procedures conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Rabbits each weighing 2–2.5 kg were anesthetized by intravenous injection of 40 mg/kg pentobarbital sodium and 500 IU heparin. The excised heart was mounted quickly on a Langendorff apparatus and washed for 15 min with Ca2+-free, phosphate-buffered solution containing (in mmol/l): NaCl 136.9, KCl 5.4, MgCl2 1.0, NaH2PO4 0.33, Na2HPO4 2.24, and glucose 10 (pH 7.4). The solution was then replaced with one containing 0.02 mg/ml collagenase (Yakult, Tokyo, Japan) and 0.015 mg/ml protease (Type XIV, Sigma, St. Louis, MO, USA). The temperature was maintained at 37°C and the solutions were saturated with 100% O2. After superfusion with the enzyme solution, the right atrium was separated from the heart and minced. It was then agitated in a second enzyme solution which contained 0.8 mg/ml collagenase (Type H, Sigma, St. Louis, MO, USA). Isolated cells were collected by centrifugation at 70 g (3 min) and stored in stock solution containing (in mmol/l): potassium glutamate 75, oxalate 10, KCl 20, KH2PO4 10, MgSO4 1, taurine 20, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) 0.5, N-2-hydroxyethyl-piperazine-N'-2-ethanesulphonic acid (HEPES) 5, NaCl 5, creatine 5 and glucose 10 (pH 7.2 adjusted with KOH). The percentage of rod-shaped cells was 60–80% of the total cell number. The cells were stored in the stock solution containing 0.1% bovine serum albumin (Fraction 5, Sigma, St. Louis, MO, USA) at 4°C prior to experiments.

2.2 Electrical measurement and data analysis
For the electrical measurements, we used striated rod-shaped cells that had a clear border, were attached to the bottom of the chamber during control perfusion with normal Tyrode, and were apart from surrounding cells. The Tyrode solution contained (in mmol/l): NaCl 142, KCl 5.4, NaH2PO4 1, CaCl2 1.0, MgCl2 1, HEPES 5 and glucose 10 (pH=7.4 adjusted by NaOH). The recording chamber was 0.15 ml in volume, and the solutions were superfused at a rate of 5–6 ml/min.

The perforated-patch method was used for most of the voltage clamp experiments. The pipette solution contained (in mmol/l): CsCl 140, NaCl 6 and HEPES 5 (pH=7.2 adjusted with CsOH). The pipette tip was soaked in the amphotericin B-free solution and the pipette was backfilled with the amphotericin B-containing solution. Amphotericin B was dissolved in dimethylsulfoxide at a concentration of 80 mg/ml and diluted into the pipette solution to make a final concentration of 0.56 mg/ml. Pipettes had a tip resistance of 1–1.5 M{Omega}. After making the seal during perfusion with Tyrode solution, the cell was lifted up 0.5–1 mm from the bottom, to better expose it to the superfusion solution, and the perfusate was changed to an external test solution containing (in mmol/l): NaCl 135, CsCl 10, CaCl2 0.5, BaCl2 0.5, MgCl2 1.0, HEPES 5, glucose 10 (pH 7.4 adjusted with NaOH). Membrane permeabilization with a series resistance of 6.3±2.1 M{Omega} (SD, n=164) was attained 5–20 min after forming the seal. The series resistance was then electrically compensated by 20–50%. Test pulses were applied from a holding potential of –40 mV to 0 mV for 300 ms at 0.083 Hz, unless otherwise specified. The temperature during the experiments was 37°C.

In some experiments (Fig. 6A), cells were incubated with PTX at a concentration of 1 µg/ml for 5–8 h in stock solution containing 0.1% bovine serum albumin. The temperature during the incubation was 37°C. Approximately 10% of the cells maintained a rod shape after this incubation. In these experiments, the incubation was judged to be successful when the ICa responded well to ISO (30 nmol/l) and acetylcholine (ACh, 1 µmol/l) failed to inhibit the ISO-stimulated ICa.


Figure 6
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  		Fig. 6 A, The inhibiton of ICa by ATP is sensitive to pertussis toxin (PTX). The cell was preincubated with PTX. The concentrations of ISO, ATP and ACh were 30 nmol/l, 30 µmol/l and 1 µmol/l, respectively. B, Absent effect of ATP in an 8Br cAMP-loaded cell. This experiment was carried out with the ruptured-patch method. The pipette contained 8Br cAMP at 100 µmol/l. ISO (30 nmol/l) and ATP (30 µmol/l) were perfused sequentially. C, Absent effect of ATP in a cell stimulated by forskolin (3 µmol/l) and 3-isobutyl-1-methylxanthine (IBMX, 100 µmol/l). D, Schematic illustration of the actions of ATP on purinoceptors. The dotted arrows denote inhibitory or antagonizing actions.

 
In other experiments, the membrane-ruptured patch-clamp method was employed to dialyze the cells with 8Br cAMP. The pipette solution contained (in mmol/l): CsCl 105, CaCl2 0.5, EGTA 10, Mg-ATP 5, Na2-phosphocreatine 5, Na3-GTP 0.2, HEPES 5 (pH=7.2 with CsOH). The external solution contained (in mmol/l): NaCl 135, CsCl 10, CaCl2 1.8, BaCl2 0.5, MgCl2 1.0, HEPES 5, glucose 10 (pH 7.4 with NaOH). 8Br cAMP was added to the pipette solution at a concentration of 100 µmol/l. Pipettes had a tip resistance of 2–3 M{Omega}. A liquid junction potential of 10 mV was corrected. Holding potential was –70 mV, and test pulses to 0 mV for 300 ms were applied following a conditioning pulse at –40 mV for 500 ms [23].

The amplifier used was a TM-1000 (ACT ME, Tokyo, Japan) or Axopatch 1-D (Axon Instrument, Foster, CA, USA). After the current signals were filtered at 2 kHz, they were monitored on a digital oscilloscope (Nicolet 310C, Madison, WI, USA) with a sampling time of 0.2 ms. Digitized data were subsequently analyzed on a computer (NEC 98, Tokyo, Japan). ICa was measured as the difference between the inward peak and the current at the end of the test pulse. In most experiments, we examined the effects of ATP and Ado in the presence of ISO at 30 nmol/l. The inhibitory effect of agonists (ATP and Ado) was expressed as percentage inhibition of the ISO-induced increase in ICa.

The data are reported as means±SEM. Statistical analyses were based on Student's t-test, and the differences were considered significant when P values were less than 0.05.

2.3 Ionized calcium concentration in the perfusate
Since ATP is negatively charged and binds with cations, high concentrations of ATP may reduce the free Ca2+, Mg2+, and Ba2+ concentrations in the perfusate. In some experiments, we used a perfusate containing 1 mmol/l CaCl2 and 1 mmol/l MgCl2, but without BaCl2. We measured the free Ca2+ concentration of this solution using a Ca2+- selective ion electrode. Application of ATP at 100, 300 and 1,000 µmol/l reduced the free Ca2+ concentration from 1 mmol/l to 0.98 (n=3), 0.94 (n=3) and 0.74 (n=3) mmol/l, respectively. Therefore, in the experiments wherein 1 or 3 mmol/l ATP was used, the external solution was titrated with CaCl2 (approximately 0.4 mmol/l for 1 mmol/l ATP, and 0.7 mmol/l for 3 mmol/l ATP) to adjust the free Ca2+ concentration to 1 mmol/l.

2.4 Materials
Na2-ATP, Mg-ATP, Na2-phosphocreatine, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), adenosine deaminase (Type VI), cAMP, {alpha},β-methylene-ADP (APCP), dimethylsulfoxide, amphotericin B and PTX were purchased from Sigma. ISO was purchased from Nacalai tesque (Kyoto, Japan). All other chemicals were from Wako Pure Chemicals (Osaka, Japan). ISO was dissolved in distilled water containing 1 mg/ml ascorbic acid as a 30 µmol/l stock solution. DPCPX and forskolin were dissolved in ethanol at a concentration of 100 µmol/l and 3 mmol/l, respectively. APCP, suramin, DIDS and 3-isobutyl-1-methylxanthine (IBMX) were directly added to each external solution.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effects of ATP on non-stimulated and stimulated ICa
We first tested the effects of ATP (30 µmol/l) on basal (non-stimulated) ICa in 20 cells. Fig. 1A represents the effects of ATP. ATP caused a small inhibition of the basal ICa (9.4±1.1%) in 11 cells. In 4 cells, as shown in Fig. 1A, this small inhibition was followed by an increment of the ICa amplitude, which exceeded the control baseline slightly. In 3 out of 20 cells tested, only a stimulatory effect on basal ICa was observed with ATP at this concentration, but the degree of the ATP-induced potentiation was small, 10% or less. Six of the 20 cells did not respond to 30 µmol/l ATP. Thus, the effect of ATP on basal ICa was small and inconsistent. In most cells, ATP slightly shifted the holding current inwardly at –40 mV (by 3.3–22.5 pA; data not shown). This shift disappeared quickly (<30 s) during exposure to ATP, presumably reflecting activation and desensitization of the non-specific cation current reported previously [24–26]. In contrast to the subtle effects of ATP on basal ICa, ATP showed a distinct, inhibitory effect on ICa when it was stimulated by ISO. As shown in Fig. 1B, ATP at 30 µmol/l reversibly inhibited the ISO-stimulated ICa. ISO at 30 nmol/l increased ICa by 200.9±8.2%, and 30 µmol/l ATP reduced the ISO-induced increase in ICa by 35.8±4.7% (n=11).


Figure 1
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  		Fig. 1 Effects of adenosine 5'-triphosphate (ATP) on L-type Ca2+ current (ICa) of rabbit atrial cells. Amplitudes of ICa are plotted against time. A, Small modulation by ATP (30 µmol/l) of basal ICa. B, Reversible inhibition by ATP (30 µmol/l) of ICa in the presence of isoproterenol (ISO, 30 nmol/l). Horizontal bars indicate the periods of exposure to ATP or ISO. Panel C shows the corresponding current traces indicated by arrows (a–c) in Panel B.

 
The effect of ATP on the current–voltage (I–V) relationship for the inward peak of ICa is shown in Fig. 2A. The data clearly show that ATP inhibited the ISO-stimulated ICa at all the test potentials ranging from –30 to +50 mV. However, as shown by the current traces in the inset, test depolarization between +20 and +40 mV produced a transient outward current (ITO). Under our experimental conditions using K+-free solutions and Ba2+ in the external solution, activation of the A-type ITO was unlikely. We thereby examined the effects of DIDS, a blocker of the Ca2+ activated ITO [27]. As shown in Fig. 2B, the ITO was potentiated by ISO and was completely abolished by DIDS (100 µmol/l). However, ITO was never observed in the other experiments where a test potential of 0 mV was used. The results shown in Fig. 2B, lower panel, indicate that DIDS affected neither the inward peak of ICa nor its inactivation time course at 0 mV in the presence of ISO. We confirmed the lack of the effect of DIDS at 0 mV either in the absence (n=8) or presence (n=5) of ISO. Since the cell membrane permeabilized by high concentrations of polyene antibiotics (amphotericin B and nystatin) carries Cl– comparably to monovalent cations [28, 29], the present results indicate that the intracellular milieu of these cells was sufficiently dialyzed with the high-Cl– (150 mmol/l) pipette solution, and that the contamination of this Cl– current did not distort our measurement of ICa when we used the test potential of 0 mV (equilibrium potential for Cl–). We then examined the effect of ATP on the I–V relationship for ICa in the presence of DIDS (100 µmol/l). As shown in Fig. 2C, the ICa inhibition by ATP (30 µmol/l) was not associated with a change in the voltage-dependent shape of the I–V relationship.


Figure 2
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  		Fig. 2 A, Effects of ATP on the current–voltage (I–V) relationship for ICa. 300 ms step pulses were applied from –40 mV to test potentials ranging from –30 to +50 mV. ICa was measured between the inward peak and the current at the end of the test pulse. The measurements were made during control, perfusion with ISO (30 nmol/l), and perfusion with ISO plus ATP (30 µmol/l) (n=4). The inset shows representative current traces at potentials ranging from 0 to +50 mV during perfusion with ISO. Note the transient outward current (ITO) at +20, +30 and +40 mV. B, Abolition of ITO by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The upper and lower panels show the membrane currents in response to depolarization to +30 and 0 mV, respectively. The cell was first exposed to ISO (30 nmol/l), and then exposed to the solution containing DIDS (100 µmol/l). Note that deporarization to 0 mV elicited no DIDS-sensitve current. C, I–V relationships in the presence of DIDS. The currents in response to test pulses ranging from –30 to +50 mV are shown on the left. DIDS (100 µmol/l) eliminated the contamination of ITO. The concentrations of ISO and ATP were same as in Panel A. n=5.

 
The inhibition of stimulated ICa by ATP may be mediated by P1 receptors, because adenine compounds are degraded into Ado (a potent P1 agonist) on the surface of single myocardial cells [30]. We compared the effects of ATP and Ado to attenuate ISO-stimulated ICa (Fig. 3A and B). Ado inhibited the stimulated ICa with an EC50 of 3.4 µmol/l. On the other hand, the effects of ATP were best fitted by assuming two reactions with different EC50s (1.0 and 151 µmol/l), indicating that at least two different pathways mediate the ICa inhibition by ATP. The maximal inhibition of the ISO-induced increase of ICa by ATP (1 and 3 mmol/l) was 63.8±2.6% (n=10), which was significantly larger than that induced by Ado at 1 and 3 mmol/l (50.3±2.6%, n=17). In Fig. 3C, the cell was superfused with a saturating concentration of Ado (1 mmol/l). Additional application of ATP (30 µmol/l) further reduced the ICa in all the 5 cells tested; i.e., Ado inhibited 50.3±3.3% of the 30 nmol/l ISO-induced increase of ICa, and co-application of ATP and Ado inhibited 78.9±8.7% (n=5) of the ISO-induced increase of ICa. Thus, ATP has some actions additional to P1 stimulation. When the effect of ISO was fully antagonized by ACh (10 µmol/l), ATP did not show any further inhibition of ICa (n=5, Fig. 3D).


Figure 3
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  		Fig. 3 A and B, Concentration-dependent inhibiton of ISO-stimulated ICa by ATP and adenosine (Ado). The effects of ATP and Ado are expressed as% inhibition of the 30-nmol/l ISO-induced increase in ICa (n=5–12 for each symbol). A single concentration of the agonist was used for one cell. The effect of Ado (Panel B) was expressed as:% decrease in ICa=Emax/(1+EC50/[Ado]), where Emax and EC50 represent the maximal response and the concentration eliciting half-maximal response, respectively. Emax=50.5% and EC50=3.4 µmol/l. The effects of ATP (Panel A) were expressed as:% decrease in ICa = Emax,1/(1+ EC50,1/[ATP]) + Emax,2/(1+ EC50,2/[ATP]), where Emax,1=27.4%, EC50,1=1.0 µmol/l, Emax,2=39.7% and EC50,2=150.6 µmol/l.C and D, Effects of ATP on ICa in the presence of Ado (C) and acetylcholine (D). ATP (30 µmol/l) was applied in the presence of a saturating concentration of Ado (1 mmol/l) or acetylcholine (ACh, 10 µmol/l). Note that ATP showed an additional inhibtion of ICa after Ado decreased the 30-nmol/l ISO-induced increase of ICa by 58%. The current records obtained at the time indicated by arrows (a–d) are shown in the insets.

 
3.2 Involvement of P1 and P2 purinoceptors
To determine the receptor types involved in the actions of ATP, we used suramin, a specific P2 blocker [31], and DPCPX, a specific A1 purinoceptor blocker [32]. A1 purinoceptor is a major subtype of P1 in the myocardium [2]. Fig. 4A represents the effect of DPCPX (100 nmol/l). DPCPX attenuated the inhibitory effect of ATP (30 µmol/l) on ISO-stimulated ICa from the control value of 35.8±4.7% (Fig. 1B) to a value of 22.2±3.2% (n=6), and suramin (100 µmol/l) also attenuated it to 11.6±1.1% (n=6; raw data, not shown); i.e., both DPCPX and suramin attenuated the effect of ATP partially. Co-application of suramin and DPCPX nearly completely abolished the effect of ATP (Fig. 4B). Thus, ATP inhibits ISO-stimulated ICa by activating both P1 and P2 purinoceptors. In the experiment shown in Fig. 4A, the effect of ATP decreased during a continuous exposure, suggesting a rapid desensitization of the P2-mediated response (see also Fig. 3C). At an ATP concentration of 30 µmol/l, the initial response of ICa to ATP declined by 61±14% (n=6) during the 2 min perfusion in the presence of DPCPX.


Figure 4
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  		Fig. 4 Effects of P1 and P2 purinoceptor antagonists on ATP inhibition of ISO-stimulated ICa. A, Effects of P1 (A1)-receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (100 nmol/l). The concentrations of ISO and ATP were 30 nmol/l and 30 µmol/l, respectively. B, Lack of the effect of ATP in the presence of DPCPX plus suramin. The concentrations of ISO, DPCPX and ATP were as in Panel A. Suramin was applied at 100 µmol/l. C, Concentration-dependent inhibition of ISO-stimulated ICa by ATP in the presence of DPCPX or suramin. The control data (closed circles) were obtained from Fig. 3A. The concentrations of DPCPX and suramin were 100 nmol/l and 100 µmol/l, respectively. Adenosine deaminase (ADA, 0.1 unit/ml) did not change the effects of DPCPX. The fit was made to the data obtained with the ATP concentrations between 0.1 and 100 µmol/l. Emax=26% and EC50=1.7 µmol/l in the presence of DPCPX, and Emax=17% and EC50=20.8 µmol/l in the presence of suramin. n=5–11 for each symbol.

 
The P1- and P2-mediated effects of ATP are compared using the concentration-response curves in the presence of these blockers (Fig. 4C). As compared with the control concentration-response curve for ATP alone, suramin (100 µmol/l) reduced the effects of ATP at all the concentrations tested (P<0.05 with non-paired t-test). On the other hand, DPCPX (100 nmol/l) did not significantly alter the effects of ATP at low ATP concentrations (≤10 µmol/l), but reduced the effect of ATP at higher concentrations (30–300 µmol/l) (P<0.05). The inhibition by ATP at concentrations between 0.1 and 100 µmol/l in the presence of suramin or DPCPX was well expressed by the one-to-one stoichiometry equation. The EC50 for the ATP inhibition of ICa in the presence of DPCPX was 1.7 µmol/l. This value resembles the lower EC50 for the two-reaction fit made in Fig. 3A. The concentration-dependent inhibition of ICa in the presence of DPCPX was not affected by an addition of adenosine deaminase (0.1 unit/ml), indicating that DPCPX effectively blocked the Ado-mediated effects of ATP. Therefore, the inhibition by ATP at lower concentrations (0.1–10 µmol/l) is mediated preferentially by P2 purinoceptors. In Fig. 4C, ATP at 300 µmol/l inhibited the ICa more potently than expected from concentration-response curves in the presence of either DPCPX or suramin. At high concentration, ATP may have competed with these antagonists.

To elucidate whether ATP itself or Ado (produced from ATP) activates P1 purinoceptors, we examined the effect of ATP-{gamma}S, a non-hydrolyzable ATP analogue. ATP-{gamma}S at 30 µmol/l inhibited 32.5±4.1% of the ISO-stimulated component of ICa (Fig. 5A, n=6), which was completely blocked by suramin at 100 µmol/l (n=5, Fig. 5B); that is, ATP-{gamma}S has no effect on A1 purinoceptors. In the experiments shown in Fig. 5C, we used APCP (100 µmol/l), an inhibitor of ecto-5'-nucleotidase which hydrolyzes AMP to Ado [33]. In the presence of APCP, ATP (30 µmol/l) inhibited ISO-stimulated ICa by 21.9±1.9% (n=7; raw data, not shown). As shown in Fig. 5C, the inhibitory effect of ATP was markedly reduced by co-application of suramin and APCP (n=7). Fig. 5D shows a close resemblance between the effects of APCP and DPCPX on the ATP inhibition of ICa. These results clearly indicate that the P1-mediated effect results mostly from degradation of ATP to Ado.


Figure 5
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  		Fig. 5 A and B, Effects of ATP-{gamma}S on ISO-stimulated ICa. ATP-{gamma}S at 30 µmol/l inhibited the 30-nmol/l ISO-stimulated ICa by 38% (A). In Panel B, ATP-{gamma}S failed to affect the ISO-stimulated ICa in the presence of 100 µmol/l suramin. Subsequent application of ATP (30 µmol/l) to the same preparation caused an inhibition of the ICa. C, Involvement of the ectoenzyme in P1 purinoceptor stimulation by ATP. The inhibition of ICa by ATP (30 µmol/l) in the presence of suramin was abolished by {alpha},β-methylene-ADP (APCP, 100 µmol/l). D, Comparison of the effects of APCP with those of the P1 (A1) blocker DPCPX. The bars indicate the effect of 30 µmol/l ATP. The data in the presence of DPCPX were obtained from Fig. 4. n=6–11 for each bar.

 
3.3 Coupling of P2 purinoceptors with PTX-sensitive pathways
Muscarinic and P1 receptors are characterized by the coupling with the PTX-sensitive G proteins [2, 8, 34]. Stimulation of this pathway reduces the intracellular cAMP concentration by inhibiting adenylate cyclase [34]or prompting the hydrolysis of cAMP [35, 36]. Fig. 6A shows that the pretreatment with PTX abolished the inhibitory effects of ATP (30 µmol/l) on ISO-stimulated ICa (n=6). ACh (1 µmol/l) also failed to inhibit the ICa, suggesting sufficient ribosylation of the G proteins by PTX. To clarify the role of cAMP in the inhibition of ICa by ATP, we examined the effect of ATP on the ICa, which was fully phosphorylated by PKA. In the experiments shown in Fig. 6B, the cells were dialyzed with the pipette solution containing 8Br cAMP (100 µmol/l), using the membrane-ruptured patch method. Under these conditions, sufficient dialysis was tested by applying ISO (30 nmol/l) in respective cells. ATP at 30 µmol/l did not affect the ICa or did not change the time course of the run-down (n=9). Without 8Br cAMP inside the pipette, ATP at 30 µmol/l inhibited the ISO (30 nmol/l)-stimulated component of ICa by 32.3±6.2% (n=6), similar to the cases with the perforated-patch experiments. In Fig. 6C, to saturate the intracellular cAMP concentration using the membrane perforated method, the cells were perfused with forskolin (3 µmol/l) and IBMX (100 µmol/l). In the presence of forskolin and IBMX, ATP at 30 µmol/l did not inhibit ICa significantly (n=5). Our these findings indicate that both the P1-and P2-purinoceptor stimulation by ATP inhibits ICa primarily by lowering the intracellular cAMP concentration via PTX-sensitive G proteins.


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 P2-mediated inhibition of ISO-stimulated ICa
In this study, we showed that ATP consistently inhibited ICa when ICa was stimulated by ISO. The concentration response curve indicated that plural mechanisms were involved. ATP significantly inhibited ICa when P1 purinoceptors were fully activated by a high concentration (1 mmol/l) of Ado. Both DPCPX and suramin partially blocked the ATP-induced inhibition in ICa, while co-application of DPCPX and suramin nearly completely abolished it. ATP-{gamma}S also inhibited ISO-stimulated ICa, which was abolished by suramin. These findings clearly indicate that not only P1 but P2 receptors contribute to the ATP inhibition of ICa. No significant change was observed with 30 µmol/l ATP when the cells were dialyzed with 8Br cAMP or stimulated by forskolin plus IBMX, or when the cells were pretreated with PTX. Thus, both the P1- and P2-mediated inhibitions of ISO-stimulated ICa involve the PTX-sensitive Gi proteins and the cAMP-PKA pathway. These actions of ATP suggested from the present experiments are schematically illustrated in Fig. 6D.

Yamada et al. [22]showed that P2 purinoceptor stimulation inhibits the accumulation of cAMP in response to ISO in mouse ventricular myocytes. Their biochemical results support our electrophysiological findings in that the P2-mediated inhibition of the cAMP accumulation was PTX-sensitive. Activation of PTX-sensitive G proteins by ACh and Ado leads to activation of IK,ACh [2, 8]. ATP also activates a K+ current having a similar channel conductance [37, 38]. Recently, Matsuura et al. [39]demonstrated that in guinea pig atrial cells, P2 purinoceptors and the PTX-sensitive G proteins mediate the ATP-induced activation of IK,ACh. This, together with our present findings regarding the effects of ATP on ICa, suggests that P2 purinoceptors couple with the pathways common, or similar, to those activated by muscarinic and P1 receptor stimulation.

In contrast to the consistent inhibition by ATP of ISO-stimulated ICa, ATP only slightly changed basal ICa in rabbit atrial cells. The effects of ATP on basal ICa are reportedly variable with species and tissues. The stimulatory effects of ATP on ICa were extensively examined in rat and frog ventricular cells by Vassort et al. [14–16]. This stimulation was P2-mediated, and may involve the protein kinase C or inositol phosphates [14, 15]. They showed that P2 stimulation shifted the I–V relationship for ICa and the inactivation curve to more negative potentials [16], although no negative shift in the I–V relationship was observed in our experiments (Fig. 2C). In addition to the stimulatory effect, they showed that ATP inhibited ICa transiently before the stimulatory response developed. This inhibition also seems to be mediated by P2 purinoceptors [14, 16]. Qu et al. [12]found a more potent and persistent P2-mediated inhibition of basal ICa in ferret ventricular cells. They examined its intracellular mechanisms extensively, and found that this inhibition of ICa did not involve PTX-sensitive G proteins, intracellular Ca2+, cAMP, protein kinase C and inositol phosphates. Thus, the ATP-induced inhibition of ICa in these studies is by a different mechanism from that observed in our study, regarding the involvements of the PTX-sensitive G proteins and cAMP. An ATP-induced ICa inhibition, mediated by P2 purinoceptors but not by the cAMP-PKA pathway, was also reported recently in hamster ventricular cells [40]. Another undetermined mechanism underlying the ATP inhibition of ICa, which involves neither P1 nor P2 purinoceptors, is suggested in guinea pig sinoatrial node cells [41]. These complicated results suggest tissue- or species-dependent, differential distribution of various signal transduction pathways downstream of P2 purinoceptors [1, 3]. The slight effect of ATP on basal ICa in our experiments may be due to some of these pathways, although their roles seem very small in the rabbit heart.

4.2 Contributions of P1 and P2 receptors to inhibition of ICa
The mechanisms underlying P1 purinoceptor stimulation by adenine nucleotides in the heart have been studied extensively by Belardinelli et al. [2], who demonstrated that in the Langendorff-perfused guinea pig heart, more than 95% of ATP was degraded during the perfusion, and that the negative dromotropic effect of ATP was antagonized by aminophylline, a P1 purinoceptor antagonist [11]. They also showed that the activation of IK,ACh by AMP in isolated guinea pig atrial cells is ascribed to production of Ado and the resultant activation of P1 purinoceptors [30]. We also found that in rabbit atrial cells, APCP effectively eliminated the suramin-resistant component of ICa inhibition by ATP, and that ATP-{gamma}S showed no P1-mediated inhibition of ICa. Thus, when the cardiac cells are exposed to ATP at concentrations over 10 µmol/l, the degradation of ATP on the membrane surface is assumed to yield accumulation of Ado in the vicinity of the cell membrane, which is sufficient to activate P1 purinoceptors. Since ectoenzymes to degrade adenine nucleotides are present on vascular endothelial cells, intravenously injected ATP is rapidly degraded in the vessels as well as on the myocardial membrane surface [1, 3]. Therefore, the negative dromotropic effect of ATP in case of the therapeutic usage may be mainly due to P1 purinoceptor stimulation by Ado [1, 3]. On the other hand, when ATP is released within the tissue during ischemia, its diffusion to adjacent cells or surrounding tissues may inhibit ICa synergically with Ado.

The distribution of P1 (and probably P2) purinoceptors varies widely with tissues or species [2, 42]. In addition, the receptor-response coupling could vary similarly. Depending on the experimental conditions, the rates of adenosine formation and removal (degradation and uptake) can also affect the responses largely. For instance, Ado reportedly abolishes the ß agonistic effect of ISO completely, or nearly completely, in guinea pig atrial cells [43], human atrial cells [44]and rabbit sinoatrial and atrioventricular node cells [45, 46]. Whereas, saturating concentrations of Ado inhibited only 50% of the ISO-stimulated ICa in our experiments. The purinoceptor-mediated inhibition of ICa was not complete (approximately 80%) even when P1 and P2 receptors were simultaneously stimulated by ATP and Ado. In good agreement with our results, Endoh et al. [47]observed only 45% inhibition by Ado of the positive inotropic effect of ISO (3–30 nmol/l) in rabbit papillary muscles. In contrast, Hopwood et al. [48]reported no antiadrenergic effect of Ado in the same preparations. Recently, a partial inhibition by Ado of ISO stimulation of ICa was reported also in rabbit sinoatrial node cells [49]. Thus, the relative contribution of P1 and P2 purinoceptors to the ATP-induced inhibitory effects would largely depend on the preparations and experimental conditions.

In summary, we here showed that P2 purinoceptor stimulation by ATP inhibits ISO-stimulated ICa in rabbit atrial cells, synergically with P1 purinoceptor stimulation by Ado. Its physiological and clinical significance needs to be evaluated further.

Time for primary review 22 days.


   Acknowledgements
 
We would like to thank Profs. Manabu Yoshimura and Kei Kashima for their support and encouragement on this project.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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T. Yamamoto, Y. Habuchi, H. Tanaka, F. Suto, J. Morikawa, K. Kashima, and M. Yoshimura
EP receptor-mediated inhibition by prostaglandin E1 of cardiac L-type Ca2+ current of rabbits
Am J Physiol Heart Circ Physiol, October 1, 1999; 277(4): H1369 - H1374.
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